[Transcriber’s Note:
In this, the text file, italics delimited by underscores, and bold text
delimited by equal signs.]
[Illustration: RADIOLARIA.]
THE
MICROSCOPE
_ITS HISTORY, CONSTRUCTION, AND APPLICATION_
BEING A FAMILIAR INTRODUCTION TO THE USE OF
THE INSTRUMENT, AND THE STUDY OF
MICROSCOPICAL SCIENCE
BY JABEZ HOGG, M.R.C.S., F.R.M.S.,
FORMERLY AND FOR TWENTY-FIVE YEARS SURGEON TO THE ROYAL WESTMINSTER OPHTHALMIC
HOSPITAL; PAST PRESIDENT OF THE MEDICAL MICROSCOPICAL SOCIETY; HONORARY
FELLOW OF THE ACADEMY OF SCIENCES, PHILADELPHIA; OF THE MEDICO-LEGAL
SOCIETY, NEW YORK; OF THE BELGIAN MICROSCOPICAL SOCIETY, ETC.; AUTHOR
OF “ELEMENTS OF NATURAL PHILOSOPHY,” “A MANUAL OF
OPHTHALMOSCOPIC SURGERY,” ETC.
_WITH UPWARDS OF
NINE HUNDRED
ENGRAVED
AND COLOURED
ILLUSTRATIONS BY
TUFFEN WEST
AND
OTHER ARTISTS_
[Illustration: An 18th Century Microscope.]
_FIFTEENTH EDITION
RE-CONSTRUCTED,
RE-WRITTEN,
REVISED, AND
ENLARGED
THROUGHOUT_
LONDON AND NEW YORK
GEORGE ROUTLEDGE & SONS, LIMITED
1898
BRADBURY, AGNEW, & CO. I.D., PRINTERS,
LONDON AND TONBRIDGE.
PREFACE TO THE FIFTEENTH EDITION.
The First Edition of this work appeared in 1854, a time in the history
of the Microscope when the instrument, as an aid to original scientific
research, may be said to have been in its infancy. Then certainly it
was seldom employed in the laboratory or the medical schools. Now,
however, as I anticipated, it has asserted its proper position, and has
at length become one of the most important auxiliaries to science, and
a direct incentive to original work, while it has doubtless exercised
considerable influence over the student’s power of observation, and
materially assisted in his studies, let his ultimate object and
pursuits be what they may.
The greater use made of the Microscope has likewise conferred benefits
of untold value upon the arts and industries of the country, thereby
adding to the national prosperity in ways as manifold as unique. The
Microscope has also proved of immense value in the promotion of the
health of the community, and the art and science of healing, since
the theory of medicine has become a science, resting on the minute
microscopical examination of animal tissues.
The work of research in the sister sciences and by other methods has,
during the last decade, received a corresponding impetus, while it
has undoubtedly tended towards elaboration and specialisation in all
departments. In consequence, the progress of microscopical science
has become more dependent upon the specialist for gaining accurate
knowledge and for certain important details seen to be branching out
in many directions. There never was a time when the instrument was
so constantly and generally resorted to and with so much confidence
and advantage, as the present. It has shown itself equal to the task
imposed--that of teaching the eye to see things that are new, and also,
what is perhaps of more importance, to perceive things which had been
entirely overlooked. The older defects, perhaps, arose from two causes;
the want of more careful training of the organ of vision, and the want
of sufficient power and precision in the optical part of the Microscope
itself. Both of these obstacles have been to a considerable extent
removed, and all educational systems are looked upon as incomplete
without a knowledge of the Microscope.
A step has already been taken in another direction, that of furnishing
special forms of instruments, better adapted to the uses to which
they will hereafter be put, and purposely designed for chemical and
analytical processes, for petrological pursuits, the geometrical
measurement of crystals, for special work in connection with
manufacturing industries, for the dairyman, and the farmer. For the
detection of adulterations--that of butter, for example--a newer form
of instrument has been devised, namely, a “Butro-refractometer,” by the
help of which any adulteration of this universal article of diet will
at once be revealed. The form of instrument upon which the optician has
expended a greater amount of skill than perhaps on any other is the
Bacteriological Microscope, as may be inferred from the larger space
I have devoted to this important adjunct, since by original research,
there can be no doubt a still greater future is in store for science
in this special department of microscopy. But perfect success in this
direction remains very much with the practical optician, and the
further improvements made in the optical part of the instrument, since
it is admitted that the highest theoretical perfection has not yet been
reached.
It is a commonplace remark that every question solved is a step
towards new problems waiting solution. It is equally obvious that many
difficulties must be encountered by every author who uses his best
endeavours to supply a standard volume or even a fairly comprehensive
text-book on the Microscope, one that will remain a sure guide for any
lengthened period. Such a success I regard as scarcely possible. I
may, however, notice that my earlier work has met with a great amount
of appreciation, and its utility acknowledged in the past by a demand
almost unprecedented, edition after edition being called for.
It is hardly necessary to add that my task has been accomplished with
an earnest desire to assist in diffusing a love for an instrument which
has been my constant companion for upwards of sixty years.[1] Moreover,
I have a firm conviction of the real utility of the Microscope in the
work of education, its practical value in many branches of science,
art, and manufacturing industries. These are my chief reasons for
applying myself once more to the task of revision, rewriting, and
rearranging and bringing this book as far as possible into line with
the knowledge gained in chemical pathology and bacteriology.
It will be noticed that in the first part, my subjects have as far as
possible been treated from a historical point of view. This method has
enabled me to affix dates of introduction of special inventions and
improvements made in the instrument and its appliances. The enlargement
of my pages has enabled me to devote more space to bacteriological
processes, and by the further addition of plates and several hundred
illustrations to more fully elucidate the subject matter of my text. In
an Appendix I have introduced a selection of “Formulæ and Methods” of
staining, mounting, etc., also tables of the “Metrical System,” now in
general use in the laboratory; together with comparative thermometric
values, all of which I trust may prove of service to the student.
Before bringing these few prefatory remarks to a close, a pleasing
duty devolves upon me--that of tendering my thanks for cordial aid
received from Professor Dr. EDGAR CROOKSHANK in dealing with his
special subject, Bacteriology. From his valuable “Text-Book on
Bacteriology” I have extracted much useful matter. I am equally
indebted to Professor MARSHALL WARD, F.R.S., Cambridge, for much
information on “Economic Botany,” and the great advances made in the
knowledge of the uses of plants, and the industrial value of bacteria
in particular. My acknowledgments are also due to the Messrs. WARNE
for many illustrations placed at my disposal, and for useful facts
derived from their “Royal Natural History.” It will, however, be seen
that the results of a large amount of independent observation have
been consigned to my pages. As the references show, recourse has been
had to original sources for trustworthy, reliable information on many
subjects. These are constantly, almost daily, being added to, as is
made manifest by the numerous periodical publications of the day
devoted to this and kindred sciences; the foremost and most important
among which is that almost exclusively given to microscopical science,
“The Journal of the Royal Microscopical Society of London,” the perusal
of which I commend to my readers.
LONDON, JULY, 1898.
PREFACE TO THE FIRST EDITION.
The Author of this Publication entered upon his task with some
hesitation and diffidence; but the reasons which influenced him to
undertake it may be briefly told, and they at once explain his motives,
and plead his justification, for the work which he now ventures to
submit to the indulgent consideration of his readers.
It had been to him for some time a subject of regret that one of the
most useful and fascinating studies--that which belongs to the domain
of microscopic observation--should be, if not wholly neglected, at
best but coldly and indifferently appreciated by the great mass of
the general public; and he formed a strong opinion that this apathy
and inattention were mainly attributable to the want of some concise,
yet sufficiently comprehensive, _popular_ account of the Microscope,
both as regards the management and manipulation of the instrument, and
the varied wonders and hidden realms of beauty that are disclosed and
developed by its aid. He saw around him valuable, erudite, and splendid
volumes, which, however, being chiefly designed for circulation amongst
a special class of readers, were necessarily published at a price
that renders them practically unattainable by the great bulk of the
public. They are careful and beautiful contributions to the objects
of science, but they do not adequately bring the value and charm of
microscopic studies home, so to speak, to the firesides of the people.
Day after day, new and interesting discoveries, and amplifications of
truth already discerned, have been made, but they have been either
sacrificed in serials, or, more usually, devoted to the pages of
class publications; and thus this most important and attractive study
has been, in a great measure, the province of the few only, who have
derived from it a rich store of enlightenment and gratification: the
many not having, however, participated, to any great extent, in the
instruction and entertainment which always follow in the train of
microscopical science.[2]
The manifold uses and advantages of the Microscope crowd upon us in
such profusion, that we can only attempt to enumerate them in the
briefest and most rapid manner in these prefatory pages.
It is not many years since this invaluable instrument was regarded in
the light of a costly toy; it is now the inseparable companion of the
man of science. In the medical world, its utility and necessity are
fully appreciated, even by those who formerly were slow to perceive
its benefits; now, knowledge which could not be obtained even by the
minutest dissection is acquired readily by its assistance, which
has become as essential to the anatomist and pathologist as are the
scalpel and bedside observation. The smallest portion of a diseased
structure, placed under a Microscope, will tell more in one minute to
the experienced eye than could be ascertained by long examination of
the mass of disease in the ordinary method. Microscopic agency, in
thus assisting the medical man, contributes much to the alleviation
of those multiplied “ills which flesh is heir to.” So fully impressed
were the Council of the Royal College of Surgeons with the importance
of the facts brought to light in a short space of time, that, in 1841,
they determined to establish a Professorship of Histology, and to form
a collection of preparations of the elementary tissues of both animals
and vegetables, healthy and morbid, which should illustrate the value
of microscopical investigations in physiology and medical science. From
that time, histological anatomy deservedly became an important branch
of the education of the medical student.
In the study of Vegetable Physiology, the Microscope is an
indispensable instrument; it enables the student to trace the earliest
forms of vegetable life, and the functions of the different tissues in
the growth of plants. Valuable assistance is derived from its agency in
the detection of adulterations. In the examination of flour, an article
of so much importance to all, the Microscope enables us to judge of the
size and shape of the starch-grains, their markings, their isolation
and agglomeration, and thus to distinguish the starch-grains of one
meal from those of another. It detects these and other ingredients,
invisible to the naked eye, whether combined in atoms or aggregated
in crystals, which adulterate our food, our drink, and our medicines.
It discloses the lurking poison in the minute crystallisations which
its solutions precipitate. “It tells the murderer that the blood which
stains him is that of his brother, and not of the other life which he
pretends to have taken; and as a witness against the criminal, it on
one occasion appealed to the very sand on which he trod at midnight.”
The zoologist finds in the Microscope a necessary coadjutor. To the
geologist it reveals, among a multiplicity of other facts, “that our
large coal-beds are the ruins of a gigantic vegetation; and the vast
limestone rocks, which are so abundant on the earth’s surface, are the
catacombs of myriads of animal tribes, too minute to be perceived by
the unaided vision.”
By “conducting the eye to the confines of the visible form,” the
Microscope proves an effective auxiliary in defining the geometric
properties of bodies. Its influence as an instrument of research upon
the structure of bodies has been compared to that of the galvanic
battery, in the hands of Davy, upon Chemistry. It detects the smallest
structural difference, heretofore inappreciable, and, as an ally of
Chemistry, enables us to discover the very small changes of form and
colour effected by test-fluids upon solids; and dissects for us, so
to speak, the most multiplex compounds. It opens out to the mind an
extended and vast tract, opulent in wonders, rich in beauties, and
boundless in extent.
The Microscope not only assists studies, and develops objects
of profound interest, but also opens up innumerable sources of
entertainment and amusement, in the ordinary conventional acceptation
of these terms; disclosing to us peculiarities and attractions in
abundance; impressing us with the wonderful and beautifully skilful
adaptation of all parts of creation, and filling our minds with
additional reverence and admiration for the beneficent and Almighty
Creator.
The Author will conclude these prefatory observations with a few
words in explanation of his arrangements, by way of dealing with the
instrument and development of his subject. He has sought, in the volume
that he now lays before the public, to point out and elucidate at once
in a practical manner and in a popular style, the vast fund of utility
and amusement which the Microscope affords, and has endeavoured to
touch upon most of the interesting subjects for microscopic observation
as fully as the restrictions of a limited space, and the nature of the
succinct summary, would permit. To have dwelt upon each in complete
detail would have necessitated the issue of many expensive volumes--and
this would have entirely frustrated the aim which the writer had in
view; he has, therefore, contented himself with the humble, but, he
trusts, not useless, task of setting up a finger-post, so to say, to
direct the inquirer into the wider road. In the section of the work
devoted to the minuter portion of creation, he has ventured to dwell
somewhat longer, in the belief that that department is more especially
the province of the microscopist. He has arranged his topics under
special headings, and in separate chapters, for the sake of perspicuity
and precision; and has brought the ever-welcome aid of illustration to
convey his explanatory remarks more vividly to the minds of his readers.
Finally, it is the Author’s hope that, by the instrumentality of this
volume, he may possibly assist in bringing the Microscope, and its
valuable and delightful studies, before the general public in a more
familiar, compendious, and economical form than he found it at the
period of its publication, so that, in these days of a diffused taste
for reading and the spread of cheap publications, he may thus supply
further exercise for the intellectual faculties; contribute to the
additional amusement and instruction of the family circle, and aid the
student of nature in investigating the wonderful and exquisite works of
the Almighty. If it shall be the good fortune for this work, which is
now confided with great diffidence to the consideration of the public,
to succeed, in however slight a degree, in furthering this design,
the Author will feel fully repaid for the amount of time and labour
expended.
LONDON, _May_, 1854.
CONTENTS.
PART I.
PAGE
EARLY HISTORY OF THE MICROSCOPE 1
CHAPTER I.
LIGHT--ITS PROPAGATION--REFRACTION--SPHERICAL AND CHROMATIC
ABERRATION--HUMAN EYE--FORMATION OF IMAGES--VISUAL ANGLE--ABBÉ’S
THEORY OF MICROSCOPIC VISION--DEFINITION OF APERTURE--NUMERICAL
APERTURE--ABBÉ’S APERTOMETER--STEREOSCOPIC
BINOCULAR VISION 12
CHAPTER II.
SIMPLE AND COMPOUND MICROSCOPES--EVOLUTION OF THE MODERN
ACHROMATIC MICROSCOPE--IMPROVEMENTS IN THE MODERN MICROSCOPE:
THE ROSS-JACKSON, POWELL AND LEALAND, BECK’S, BAKER’S,
PILLISCHER, ZEISS, LEITZ, WATSON’S, VAN HEURCK’S EDINBURGH
STUDENTS’, SWIFT’S, AND BACTERIOLOGICAL 72
CHAPTER III.
APPLIED OPTICS--EYE-PIECES--ACHROMATIC OBJECTIVES AND
CONDENSERS--MAGNIFYING POWER OF EYE-PIECES AND OBJECTIVES--METHOD
OF EMPLOYING THE CONDENSER--ITS ILLUMINATION MIRROR--ACCESSORIES
OF THE MICROSCOPE--FINDERS--MICROMETERS--CAMERA-LUCIDA--MICROSCOPE
IN POSITION FOR DRAWING--ABBÉ’S
TEST-PLATE--MICRO-PHOTOGRAPHY--POLARISATION OF LIGHT--THE
MICRO-SPECTROSCOPE 139
CHAPTER IV.
PRACTICAL MICROSCOPY--MANIPULATION--MODE OF EMPLOYING THE
MICROSCOPE--WORKING ACCESSORIES--METHODS OF PREPARING,
HARDENING, STAINING, AND SECTION CUTTING--CUTTING HARD
STRUCTURES--INJECTING APPARATUS, MATERIAL, ETC., EMPLOYED IN
BACTERIOLOGICAL INVESTIGATIONS--WARM CHAMBERS AND INCUBATORS--PREPARING,
MOUNTING, CEMENTING, AND COLLECTING
OBJECTS 258
PART II.
CHAPTER I.
MICROSCOPIC FORMS OF LIFE--THALLOPHYTES, PTERIDOPHYTA,
PHANEROGAMÆ--STRUCTURE AND PROPERTIES OF THE CELL--PATHOGENIC
FUNGI AND MOULDS--PARASITIC DISEASES OF PLANTS, MAN, AND
ANIMALS--INDUSTRIAL USES OF FUNGI AND SACCHAROMYCETES--DESMIDIACEÆ
AND DIATOMACEÆ--LICHENS, MOSSES, FERNS,
FLOWERING PLANTS 353
CHAPTER II.
SUB-KINGDOM PROTOZOA--RHIZOPODA--GROMIA AND
FORAMINIFERA--INFUSORIA--CILIATA--ROTIFERA--PORIFERA--SPONGES 478
CHAPTER III.
ZOOPHYTES--CŒLENTERATA--MEDUSÆ--CORALS--HYDROZOA--MOLLUSCA--
ANNULOSA--WORMS--ENTOZOA--ANNELIDA--CRUSTACEA
515
CHAPTER IV.
ARTHROPODA--INSECTA--ARACHNIDA--ACARINA--IXODIDÆ--MITES AND
TICKS 583
CHAPTER V.
VERTEBRATA--INTERNAL AND EXTERNAL STRUCTURES 633
CHAPTER VI.
THE MINERAL AND GEOLOGICAL KINGDOMS 670
APPENDIX.
DR. MERCER ON ILLUMINATION, AND ABBÉ’S
THEORY--MICRO-SPECTROSCOPE--FORMULÆ,
METHODS, CEMENTING, CLEARING, HARDENING,
AND MOUNTING--TABLES, METRIC AND THERMOMETRIC 672
INDEX 691
DESCRIPTION OF PLATES,
COLOURED AND PLAIN.
FRONTISPIECE.
RADIOLARIA.
In this Plate _Fig._ 1 shows the elegant lattice-sphere of
Rhizosphæra; _Fig._ 2 represents Sphærozoum, whose skeleton
consists of loose spicules, arranged tangentially; Actinomma,
_Fig._ 3, possesses three concentric lattice-spheres, joined by
radiating spines; _Figs._ 4, 5, and 6, represent Lithomespilus,
Ommatocampe, and Carpocanium; _Fig._ 7 represents a deep-sea form
(Challengeria), whose oval case is formed of a regular, very
fine-meshed, network; _Fig._ 8 depicts the elegant lattice-sphere
of Heliosphæra; _Figs._ 9 and 10, Clathrocyclas and Dictyophimus.
PLATE I.--_Page_ 400.
PROTOPHYTA. THALLOPHYTES.
_Fig._ 1. Peziza bicolor--2. Truffle: _a._ ascus of spores; _b._
mycelium--3. Sphæria herbarum: _a._ piece of dead plant, with S.
herbarum natural size; _b._ section of same, slightly magnified;
_d._ Ascus with spores, and paraphyses more magnified--4. Peziza
pygmæa--5. Apical form of same--6. P. corpulasis: Ascus with
spores and paraphyses, merely given as a further illustration of
structure in Peziza--7. Yeast healthy--8. Yeast exhausted--9.
Phyllactinia guttata--10. Yeast with favus spores and mycelium
of fungus--11. Favus ferment, with oïdium and bacteria--12.
Puccinia spores, growing in a saccharine solution--13. Aerobic
bacteria--14. Spores and mycelia from eczema produced by yeast--15.
Volvox globator--16. Amœboid condition of portion of volvox--17.
Puccinia buxi--18. Ditto, more enlarged--(17 to 20 illustrate
Ascomycetes.)--19. Æcidium grossulariæ from transverse section of
leaf of currant: _a._ spermogones on upper surface; _b._ perithecia
with spores--20. Phragmidium bulbosum, development of--21. Palmella
parietina, trans. section through a spermogone, showing green
gonidia and spermatia escaping--22. Æcidium berberida, from leaf
of berberry--23. Vaucheria sessilis--24. Stephanosphæra pluvialis:
_a._ Full-grown example, germ cells spindle-shaped with flagella;
_b._ Resting-cell; _c._ division into four; _d._ Free-swimming
ciliated young specimen; _e._ Amœboid condition--25. _a_, _b_, _c_,
_d_, _e_, _f_ and _g_, Development of lichen gonidia--26. Palmella
stellaris (lichen), vertical section through apothecium, showing
asci, spores, and paraphyses, with gonidia and filamentous medulla:
_a._ Spermatophore with spermatia--27. Moss gonidia assuming
amœboid form.
Typical forms of Protophyta; 7 to 14, modes of development or
rudimentary conditions; Confervoideæ, 23; Vaucheria, Stephanosphæra,
24; Volvox, 15, &c.
PLATE II.--_Page_ 412.
PROTOPHYTA. ALGÆ.
_Fig._ 27. Ceramium acanthonotum--28. Closterium, Triploceras
gracilis--29. Cosmarium radiatum--30. Micrasterias denticulata--31.
Docidium pristidæ--32. Callithamnion plumula--33. Diatoma,
living: _a._ Licmophora splendida; _b._ Achnanthes longipes; _c._
Grammatophora marina. These figures are intended to show the
general character of the endochrome and growth of frustule--34.
Callithamnion refractum--35. Jungermannia albicans; _b._
representing elater and spores--36. Leaf with antheridia, or male
elements, represented more magnified at _a_ to the left of the
figure--37. Ceramium echinotum--38. Pleurosigma angulatum, side
view--39. Delesseria hypoglossum--40. Pleurosigma angulatum, front
view, endochrome not represented--41. Ceramium flabelligerum.
PLATE III.--_Page_ 479.
PROTOZOA.
_Figs._ 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. These figures are
from drawings made by Major Owen, to illustrate forms of living
Polycystina, sketched from life; these convey a faint idea of
the richly coloured appearance of the natural structure; _Figs._
48 to 52--53. Gregarina lumbricorum, round form--54. Gregarina
lumbricorum, the usual elongated form--55. Gregarina serpulæ--56.
Gregarina Sieboldii; illustration of septate form, with reflexed
hook-like processes--57. Gregarina lumbricorum, encysted--58.
Gregarina lumbricorum, more advanced and pseudo-navicellæ
forming--59. Gregarina lumbricorum, free pseudo-navicella of--60,
61. Gregarina lumbricorum, amœboid forms of--62. Cruciate
sponge-spicule--63. Astromma Humboldtii--64. Eözoon Canadense,
represents appearance of a portion of the natural size--65.
Eözoon Canadense, magnified, showing portions of cell-walls left
uncoloured, the animal sarcode inhabiting it coloured dark green as
in nature, and converted by fossilisation into a silicious mineral;
the narrow bands passing between these are processes (_stolons_)
of the same substance--66. Actinophrys sol, budding--67. Euglena
viridis: _a._ contracted; _b._ elongated form--68. Acineta
tuberosa--69. Œcistes longicornis (Davis)--70. Oxytricha gibba
(side view)--71. Oxytricha pellionella--72. Thuricola valvata,
expanded--73. Cyclidium (glaucoma)--74. Oxytricha scintillans--75
to 79, 80 to 85, illustrate types of Foraminifera discovered by
Major Owen, living--75. Globigerina acerosa, n. sp., broken open
to show interior--76. Globigerina, n. sp., broken open to show
interior--77. Globigerina hirsuta--78. Globigerina universa--79
and 81. G. Bulloides--80. Conochilus vorticella--82. Globigerina
inflata, sinistral shell--83. Pulvinulina Micheliniana--84. P.
Canariensis--85. P. Menardii.
PLATE IV.--_Page_ 514.
METAZOA. BRYOZOA.
_Fig._ 86. Hartea elegans--87. Side view of Synapta spicula--88.
Ophioglypha rosula (very immature specimen): _a._ Claw hooks;
_b._ palmate spicula. The development of this species is
described by G. Hodge, in “Transactions of Tyneside Naturalists’
Field-Club”--89. Spine of a star-fish, particularly interesting
as showing the reticular calcareous network obtaining in this as
in all other hard parts of the Echinodermata--90. Very minute
Spatangus, obtained from stomach of a bream: many of the spines
are gone, but the structure of the shell is intact and forms a
beautiful object, interesting in connection with the source whence
obtained--91. Ophioglypha neglecta: wriggling or brittle starfish.
The plate does not admit of a figure on a scale sufficient to show
the full beauty of this object--92. Tubularia Dumortierii--93.
Pedicellaria mandibulata from Uraster glacialis--94. Pedicellaria
forcepiforma, from the same--95. Cristatella mucedo; 96. Edge-view
of statoblast; 97. early stage in development of same--98. Lophopus
crystallinus--99. Plumatella repens with ova, on submerged
stem--100. Tænia echinococcus--101. Hydatids from human liver--102.
Bilharzia hæmatobia--103. Amphistoma conicum--104. Trichina
spiralis from fleshy part of Hambrc’ pork--105. Trichina spiralis
male, separated from muscle.--106, 107. Fasciola gigantea.
PLATE V.--_Page_ 556.
MOLLUSCA.
_Fig._ 108. Velutina lævigata, portion of lingual membrane--109.
Velutina lævigata, part of mandible--110. Hybocystis blennius,
portion of palate--111. Sepia officinalis, portion of palate--112.
Aplysia hybrida, part of mandible--113. Loligo vulgaris, part
of palate--114. Haliotis tuberculatus, part of palate--115.
Cistula catenata, part of palate--116. Patella radiata, part of
palate--117. Acmæa virginea, part of palate--118. Cymba olla, part
of palate--119. Scapander ligniarius--120. Oneidoris bilamellata,
part of palate--121. Testacella Maugei, part of palate--122.
Pleurobranchus plumula, part of mandible--123. Turbo marmoratus,
part of palate.
Lingual membranes of Mollusca; drawings made from specimens in the
collection formed by F. E. Edwards, Esq., now in the British Museum.
Typical examples of the numerous forms of Odontophors met with in
Gasteropod and Cephalopod Mollusca.
PLATE VI.--_Page_ 582.
INSECTA.
_Fig._ 124. Egg of Caradrina morpheus, mottled rustic moth--125.
Egg of tortoise-shell butterfly, Vanessa urticæ--126. Egg of
common footman, Lithosia complanula--127. Egg of shark moth,
Cucullia umbratica--128. Maple-aphis--129. Egg shell of acarus,
empty--130. Egg of house-fly--131. Mouth of Tsetse-fly, Glossina
morsitans--132. Vapourer moth, Orgyia antiqua: antenna of
male--133. Vapourer moth: antenna of female; _a_. branch more
magnified to show rudimentary condition of the parts--134.
Tortoise-shell butterfly; head in profile, showing large compound
eye, one of the palpi, and spiral tongue--135. Tortoise-beetle,
Cassida viridis; under surface of left fore-foot, to show the
bifurcate tenent appendages, one of which is given at _a_ more
magnified. This form of appendage is characteristic of the family.
“West on Feet of Insects,” Linn. Trans. vol. xxiii. tab. 43-136.
Egg of blue argus butterfly, Polyommatus argus--137. Egg of
mottled umber, Erannis defoliaria--138. Egg of Ennomos erosaria,
thorn-moth--139. Egg of Aspilates gilvaria, straw-belle--140.
Blow-fly, Musca vomitoria: left fore-loot, under-surface, to show
tenent hairs; _a_ _b_ more magnified; _a_ from below, _b_ from the
side--141. House-fly larva--142. Amara communis: left fore-foot,
under-surface, to show form of tenent appendages, of which one
is given more magnified at _a_. These, in ground beetles, are
met with only in the males, believed to be used for sexual
purposes. These appendages are carefully protected when not in
use, as explained by West--143. Ephydra riparia: left fore-foot,
under-surface. This fly is met with sometimes in immense numbers
on the water in salt-marshes; it has no power of climbing on
glass, as seen by the structure of the tenent hairs; the central
tactile organ also is peculiar, the whole acting as a float, one
attached to each foot, enabling the fly to rest on the surface of
the water; _a._ an enlarged external hair--144. Egg of bot-fly, the
larva just escaping--145. Egg of parasite of pheasant--146. Egg
of Scatophaga--147. Egg of parasite of magpie--148. Egg of Jodis
vernaria, small emerald moth.
PLATE VII.--_Page_ 633.
VERTEBRATA.
_Fig._ 149. Toe of mouse, integuments, bone of foot, and
vessels--150. Tongue of mouse, showing erectile papillæ and
muscular layer--151. Brain of rat, showing vascular supply--152.
Vertical section of tongue of cat, fungi-form papillæ and capillary
loops passing into them, vessels--153. Kidney of cat, showing
Malpighian turfts and arteries--154. Small intestine of rat, with
villi and layer of mucous membrane exposed--155. Nose of mouse,
showing vascular supply to roots of whiskers--156. Vascular supply
to internal gill of tadpole, during one phase of development--157.
Section through sclerotic coat and retina of cat’s eye, showing
vascular supply of choroid vessels cut cross-ways--158. Interior of
fully-developed tadpole, exhibiting heart, vascular arrangement and
vascular system throughout body and tail.
This plate is designed to show the value, in certain cases, of
injected preparations in the delineation of animal structures. By thus
artificially restoring the blood and distending the tissues, a better
idea is obtained of the relative condition of parts during life.
PLATE VIII.--_Page_ 220.
POLARISCOPE OBJECTS.
_Fig._ 158. New Red Sandstone--159. Quartz--163. Granite--161.
Sulph. Copper--162. Saliginine--163. Sulph. Iron and Cobalt,
crystallized in the way described by Thomas--164. Borax--165.
Sulph. Nickel and Potash--166. Kreatine--167. Starch granules--168.
Aspartic Acid--169. Fibro-cells, orchid.--170. Equisetum
cuticle--171. Holothuria spicula, Australia--172. Holothuria
spicula, Port Essington--173. Deutzia scabra; upper and under
surface--174. Cat’s tongue, process--175. Prawn shell, exuvia with
crystals of lime--176. Grayling scale--177. Scyllium caniculum
scale--178. Rhinoceros horn, transverse section--179. Horse
hoof--180. Dytiscus, elytra with crystals of lime.
PLATE IX.--_Page_ 362.
TYPICAL PLATE OF BACTERIA AND SCHIZOMYCETES.
_Fig._ 1. Cocci, singly, and varying in size--2. Cocci in chains or
rosaries (streptococcus)--3. Cocci in a mass (staphylococcus)--4
and 5. Cocci in pairs (diplococcus)--6. Cocci in groups
of four (merismopedia)--7. Cocci in packets (sarcina)--8.
Bacterium termo--9. Bacterium termo × 4000 (Dallinger and
Drysdale)--10. Bacterium septicæmiæ hæmorrhagicæ--11. Bacterium
pneumoniæ crouposæ--12. Bacillus subtilis--13. Bacillus
murisepticus--14. Bacillus diphtheriæ--15. Bacillus typhosus
(Eberth)--16. Spirillum undula (Cohn)--17. Spirillum volutans
(Cohn)--18. Spirillum choleræ Asiaticæ--19. Spirillum Obermeieri
(Koch)--20. Spirochæta plicatilis (Flügge)--21. Vibrio rugula
(Prazmowski)--22. Cladothrix Försteri (Cohn)--23. Cladothrix
dichotoma (Cohn)--24. Monas Okenii (Cohn)--25. Monas Warmingii
(Cohn)--26. Rhabdomonas rosea (Cohn)--27. Spore-formation of
Bacillus alvei--28. Spore-formation (Bacillus anthracis)--29.
Spore-formation in bacilli cultivated from rotten melon (Fränkel
and Pfeiffer)--30. Spore-formation in bacilli cultivated from
earth (Fränkel and Pfeiffer)--31. Involution-form of Crenothrix
(Zopf)--32. Involution-forms of Vibrio serpens (Warming)--33.
Involution-forms of Vibrio rugula (Warming)--34. Involution-forms
of Clostridium polymyxa (Prazmowski)--35. Involution-forms of
Spirillum choleræ Asiaticæ--36. Involution-forms of Bacterium aceti
(Zopf and Hansen)--37. Spirulina-form of Beggiatoa alba (Zopf)--38.
Various thread-forms of Bacterium merismopedioides (Zopf)--39.
False-branching of Cladothrix (Zopf).
PLATE X.--_Page_ 420.
DESMIDIACEÆ.
_Fig._ 1. Euastrum oblongum--2. Micrasterias rotata--3. Desmidium
quadrangulatum--4. Didymoprium Grevillii--5. Micrasterias,
sporangium of--6. Didymoprium Borreri--7. Cosmarium Ralfsii--8,
9. Xanthidiæ--10. X. armatum--11. Cosmarium crenatum--12. C.
Sphærozosma vertebratum--13, 17. Sporangia of Cosmarium--14.
X. fasiculatum--18. Staurastrum hirsutum--19. Arthrodesmus
convergens--15. Staurastrum tumidum--16. Staurastrum dilitatum--21.
Penium--22. Euastrum Didelta--23. Docidium clavatum--24.
Pediastrum biradiatum--25. Closterium, showing conjugation or
self-division--26. Volvox, parent cell about to break up--27.
Penium Jennerii--28. Aptogonum desmidium--29. Pediastrum
pertusum--30. Ankistrodesmus falcatus--31. Parent cell of
Closterium--32. Staurastrum gracilis.--33. Conjugation of Penium
margaritaceum--34. Spirotænia--35. Closterium
PLATE XI.--_Page_ 428.
DIATOMACEÆ.
_Fig._ 1. Arachnoidiscus--2. Actinocyclus (Bermuda)--3. Cocconeis
(Algoa Bay)--4. Coccinodiscus (Bermuda)--5. Isthmia enervis--6.
Zygoceros rhombus--7. Campilodiscus clypeus--8. Biddulphia--9.
Gallionella sulcata--10. Triceratium, found in Thames mud--11.
Gomphonema geminatum, with their stalk-like attachments--12.
Dictyocha fibula--13. Eunotia--14. Cocconema--15. Fragilaria
pectinalis--16. Meridion circulare--17. Diatoma flocculosum.
PLATE XII.--_Page_ 438.
MICRO-PHOTOGRAPH OF TEST DIATOMS.
Taken with Zeiss’s 3 mm. N.A. 1·40 by Mr. A. A. Carvell for the Author.
_Fig._ 1. Portion of Surirella gemma, magnified × 1,000--2. Broken
Frustule of Pleurosigma angulatum, × 750--3 and 5. Triceratium
favus ×--1,000--4. Navicula rhomboides × 1,300--6. Pleurosigma
formosum, showing black dots--7. P. formosum, showing white dots, ×
750.
PLATE XIII.--_Page_ 454.
PHANEROGAMIÆ--ELEMENTARY TISSUE OF PLANTS.
_Fig._ 1. Elementary ovid cells--2. Branching tissue--2A and 3.
Spiral vessels from Opuntia vulgaris--4. Stellate tissue, section
of rush--5. Mushroom spawn--6. Starch from _Tous-les-mois_--7.
Starch from sago--8. Starch from rice--9. Wheat-starch--10. Rhubarb
starch in isolated cells--11. Maize-starch--12. Oat-starch--13.
Barley-starch--14. Section of Potato cells, filled with healthy
starch--15. Potato starch more highly magnified--16. Section of
Potato with nearly all starch absent--17. Potato with starch
destroyed by fungoid disease--18. Ciliated spermagones--19. Hairs
of stinging-nettle--20. Section of cellular parenchyma of ripe
strawberry.
PLATE XIV.--_Page_ 472.
STELLATE AND CRYSTALLINE TISSUE.
_Fig._ 1. Epidermis of husk of wheat, spiral vessels and silicious
crystals--2. Section of cane, silicious cell walls, internal
portion filled with granular bodies--3. Cuticular layer of the
onion, showing crystals of calcium carbonate and oxalate--4. Cells
of garden rhubarb, with crystalline bodies and raphides--4_a_.
Another layer filled with starch grains--5. Section of pear, testa,
sclerogenous and granular tissue--6. Stellate hairs, sinuous
cells and silicious parenchyma of leaf of Deutzia scabra, under
surface--7. Silicious cuticle layer of grass, Pharus cristatus.
PLATE XV.--_Page_ 482.
RHIZOPODA.--GROMIA.--FORAMINIFERA.
_Fig._ 1. Astrorhiza limicola--2. Lieberkühnia paludosa--3.
Micro-gromia socialis undergoing fission--4. A colony of Hertwig’s
Micro-gromia socialis--5. G. Lieberkühnia--6. Egg-shaped Gromia,
G. oviformis, with pseudopodia extended, magnified 500 diameters.
“Hertwig Ueber Micro-gromia, archiv. für Mickr. Anat. bdx.”
PLATE XVI.--_Page_ 510.
SPONGE SPICULES.
_Fig._ 1. A portion of sponge, Halichondria simulans, showing
silicious spicula imbedded in the sarcode matrix--2. Spicula
divested of its matrix by acid--3. Gemmule Spongilla fluviatallis
enclosed in spicula--4. Birotulate spicula from same--5. Gemmule
after being steeped in acid showing reticulated coating of
birotulate spicula--6. Gemmules of Geodia--7. Gemmule in more
advanced stage of growth--8. Skeleton of the acerate form covered
by rows of spines--9. Showing rings of growth and horny covering,
and bundles of spicula of the genus Verongia--10. Sphero-stellate
spicula of Tethya--11. Tricuspidanchorate and sphero-stellate
spicula--12. Acuate-bi-clavate and other forms of spicula from
Geodia--13. Clavate spicula covered with short spines.
PLATE XVII.--_Page_ 518.
ZOOPHYTES, ASTEROIDS, NUDIBRANCHS, AND ECHINOIDS.
_Fig._ 1. _a._ Astrophyton scutatum--_b._ Doris pinnatifida, back
and side view--_c._ Æquorea Forbesina--_d._ Medusæ bud--_e._
Thaumantias corynetes--_f._ Echinus in an early free stage--_g._
Echinus sphæra--_h._ Cydippe pyleus--_i._ Ascidiæ--_k._ Botryllus
violaceus, on a Fucus--_l._ Corystes cassivelaunus--_m._ Eurynome
aspera--_n._ Ophiocoma rosula--_o._ Pagurus Prideauxii--_p._ Ebalia
Permantii.
PLATE XVIII.--_Page_ 558.
SHELLS OF MOLLUSCA.
_Fig._ 1. Transverse section of spine of Echinus--2. Another
section of Echinus, showing reticulated structure, the calcareous
portion dissolved out by acid--3. Horizontal section of shell of
Haliotis splendens, showing stellate pigment--4. Shell of crab with
granules in articular layer--5. Another section of same shell,
showing hexagonal structure--6. Horizontal section of coach-spring
shell, Terebratulata rubicunda, showing radiating perforations--7.
Transverse section of shell of the Pinna ingens--8. Crystals of
carbonate of lime, from oyster shell.
PLATE XIX.--_Page_ 636.
VERTEBRATA.
_Fig._ 1. _a._ Spheroidal epithelium cells, filled with central
nuclei and granular matter; _b._ mucous membrane of stomach,
showing cells, with open mouths of tubes at the bottom of each,
magnified 50 diameters--2. _a._ Diagram of a portion of the
involuted mucous membrane, showing continuation of its elements
in the follicles and villi, with a nerve entering the submucous
tissue. The upper surface of one villus is covered with cylindrical
epithelium; the other denuded, and with dark line of basement
membrane running around it; _b._ epithelium cells, separated and
magnified 200 diameters, a central nucleus, with a nucleolus,
seen in centre; _c._ pavement epithelium cells, from the mucous
membrane of bronchial or air tubes with nuclei, and nucleoli in
some; _d._ vibratile or ciliated epithelium, nuclei visible, and
cilia at the upper free surface, magnified 200 diameters--3. _a._
is one of the tubular follicles from a pig’s stomach, cut obliquely
to display upper part of cavity, and the cylindrical epithelium
forming its walls, a few cells detached; _b._ shows a section of a
lymphatic, with capillary blood-vessels, distributed beneath the
mucous surfaces--4. Cells of adipose tissue, or fat, magnified
100 diameters--5. a single fat-cell separated, and magnified 250
diameters--6. A capillary of blood-vessels distributed through
tissue--7. Section of the Tendo-Achillis as it joins the cartilage,
showing stellate cells of tendon, seen to be gradually coalescing
to form round or oval cells of cartilage--8. A vertical section of
cartilage, with clusters of cells arranged in columns previous to
their conversion into bone--9. A small transverse section of the
same, showing the gradual change of the cartilage cells at _a._
into the true bone cells, _lacunæ_, at _b._ with characteristic
canaliculi--10. A stellate nerve corpuscle, with tubular processes
issuing forth, at _a._ filled with corpuscles containing black
pigment, above which is a corpuscle the nucleus of which is seen
to have nucleoli; at _b._ a corpuscle enclosed within sheath,
and filled with granular matter taken from the root of a spinal
nerve--11. The continuity of muscle, the upper portion, with
connective tissue of the lower portion, from the tongue of a
lamb--12. Branched muscle, ending in stellate connective cells,
from the upper lip of the rat--13. Choroidal black pigment-cells
from the human eye.
PLATE XX.--_Page_ 658.
BONE STRUCTURE.
_Figs._ 1. and 2. Transverse section of the human clavicle (collar
bone), showing Haversian canals, concentric laminæ, and concentric
arrangement of bone cells--3. Transverse section of the femur of
an ostrich--4. Transverse section of humerus (fore-arm) bone of a
turtle, Chelonia mydas--5. Horizontal section of the lower jaw-bone
of a conger eel, in which no Haversian canals are present--6. A
portion of the cranium of a siren, Siren lacertina--7. Portion of
bone taken from the shaft of humerus of a Pterodactyle, showing
elongated bone-cells characteristic of the order Reptilia--8.
Horizontal section of a scale, or flattened spine, from the skin of
a Trygon (sting-ray), showing large Haversian canals, numerous wavy
parallel tubes, also bone-cells with canaliculi communicating as in
dentine.
ERRATA.
Prefaces, page vi., line 22 _Insert_ “a” _into_ “admitted.”
" " x., line 13 _Insert_ “the” _before_ “assistance.”
" " xii. _Insert_ “for” at commencement of
line 24.
Page 33, line 13 _For_ “Rabbit” _read_ “Kitten.”
" 486, 4th line from bottom _Strike out_ “The late.”
" 511, two lines from bottom _For_ “Plate XIII.” _read_ “Plate XVI.”
" 584, 5th line from bottom _Insert_ “Stalk-Eyed” _before_
“crustaceans.”
" 624, line 12 _For_ “or” _read_ “and.”
" 633, Plate VII. Numbering of figures--
_For_ “152” _read_ “158.”
_For_ “152a” _read_ “152.”
(Professor Abbe, erroneously referred to more than once as “the late”
is, the author is happy to say, in excellent health).
THE MICROSCOPE.
PART I.
Early History of the Microscope.
The instrument known as the Microscope derives its designation from two
Greek words, μικρὸς (_mikros_), _small_, and σκοπέω (_skopeo_), _to see
or observe_; and is an optical instrument by means of which objects
are so magnified that details invisible or indistinct to the naked eye
are clearly seen. Its origin, so far as yet can be traced back, seems
to be of a doubtful nature. It is tolerably certain the ancients had
little or no conception of the magnifying power of lenses; this may be
surmised from their writings. The elder Pliny incidentally states that
the physicians of his day cauterised by means of “a globe of crystal.”
The learned Greek physician, Galen, however, demonstrates conclusively
that in the first and second centuries of our era the use of magnifying
lenses was quite unknown either to Greek or Roman. Moreover, the
writings of Archimedes, Ptolemy, and other learned men, show that,
although they had some idea of the action of refraction at plane
surfaces, as of water, yet of the refraction at curved surfaces they
had formed no conception. Indeed, they refer quite indiscriminately to
the spherical form, or the disc, or the plane surface of the water,
but not one of them speaks of the lenticular form, or the curvature of
their surfaces.
As to the more powerful optical instruments, the telescope and
microscope, although it would appear that Alhazen in the 10th or 11th
century, Roger Bacon in the 13th, and Fracastoro and Baptist Porta in
the 16th, had formed some idea that lenses might be made and combined
so that distant objects might be seen clearer, or near ones magnified
beyond the power of normal vision; yet we hold with Kepler, that no
instrument analogous to our telescope was known before the early part
of the 17th century.
The combination of lenses associated with the name of Galileo, was,
he tells us, of Dutch origin, and of a date anterior to that of his
telescope, constructed by him in 1609; and this would appear to be the
probable origin of the microscope consisting of a combination of a
convex object lens with a concave eye lens.[3]
It now appears almost impossible to assign the exact date of the
first production of the microscope (as distinguished from the simple
magnifying lens), but those who have made a special investigation,
agree that it must have been invented between 1590 and 1609, and that
either of the three spectacle-makers of Middelburg, Holland, Hans
Janssen, his son Zacharias Janssen, and Hans Lippershey, may have been
the inventor, the probabilities being in favour of the Janssens, and
there the question must remain.
The history of the modern microscope, like that of nations and arts,
has had its brilliant periods, in which it shone with uncommon
splendour, and was cultivated with extraordinary ardour; these periods
have been succeeded by intervals marked with no discovery, and in which
the science seemed to fade away, or at least to lie dormant, till some
favourable circumstance--the discovery of a new object, or some new
improvement in the instruments of observation--awakened the attention
of the curious, and reanimated the spirit of research. Thus, soon after
the invention of the microscope, the field it presented to observation
was cultivated by men of the first rank in science, and who enriched
almost every branch of natural history by the discoveries made by means
of this instrument.
The Modern Microscope.
To the celebrated Dr. Hooke belongs the honour of publishing an account
of the compound instrument in 1665 in his “Micrographia.” His first
claim, however, is founded on the application of a lamp adjustable on a
pillar, together with a glass globe of water and a deep plano-convex
condensing lens. By means of this arrangement, he says, “The light can
be directed more directly on the object under examination.” In the
further description given of his microscope, he explains: “It has four
draw-tubes for lengthening the body, and a third lens to the optical
combination.” This, it would appear, was only brought into use when
he wished to see the whole object at once: “The middle-glass lens,
conveying a very great company of radiating pencils (of light) which
would stray away; but when I had occasion to examine the small parts of
a body, I took out the middle glass and made use of one eye-glass with
the object-glass.”
From Hooke’s description I gather that he also introduced the
ball-and-socket movement into the construction of the body of his
instrument. This has found many imitators since his day; some of
them have gone so far as to claim the invention as one quite new.
For small accessories, where the leverage need not be considered,
the ball-and-socket has proved convenient enough; but not, however,
if applied to the stand of the microscope. Hooke, in his early work,
expressed dissatisfaction with the English-made lenses he had in use.
He complains of the “apertures of the object-glasses, which are so
small that very few rays are admitted; none will admit a sufficient
number of rays to magnifie the object beyond a determinate bigness.”
So we may take it that he thus early discovered the great importance
of an increase in the aperture of his microscope. Other improvements
of importance were made, and he was the first to describe a useful
method of estimating the magnifying power of his lenses, and the
difficulty of distinguishing between a prominence and a depression in
the object under investigation, which he was made more fully aware
of when preparing drawings for the illustration of his “Micrographia
Illustrata”; this would be in 1664, if not earlier. His book created
no little sensation on its first appearance, and it soon became
scarce. Hooke (says Mr. Mayall) “must undoubtedly be credited with the
first suggestion of immersion lenses.” Nevertheless, in his “Lectures
and Collections,” published in 1676, he appears to be no longer
enthusiastic over his double microscope, and once more he reverts to
the simpler instrument of his earlier days. Whether this change of
opinion was due to the publication of Leeuwenhoek’s observations with
his simple microscopes it is impossible to say.
As early as 1673 Leeuwenhoek communicated some important discoveries
made by a simple microscope of his own construction to the Royal
Society; he, however, gave no particulars of the construction of the
instrument. Dr. Adams, writing to his friend (Sir) Hans Sloane, says:
“They appear to be spherules lodged between two plates of gold or
brass, in a hole whose diameter appears to be no bigger than that of a
small pin’s head.” At his death he bequeathed to the Royal Society a
cabinet containing twenty-six of these microscopes; the cabinet and the
microscopes long ago disappeared, but not before they were carefully
examined and described by Mr. Henry Baker, F.R.S. In his report to the
Royal Society, he says: “They consisted of a series of convex-lenses,
ranging in power from 1·20 to 1·5, and magnifying from 160 to 40
diameters.” This must now be regarded as an eventful period in the
history of the microscope, since Leeuwenhoek’s discoveries created
a great sensation throughout Europe. And all further improvements
in compound instruments appear to have been laid aside for some
considerable period in consequence: and the pocket instrument of
Wilson, together with that of his scroll standard (seen on the cover of
this book), and which was one of the first simple microscopes with a
mirror mounted on the base in a line with the optic axis.
The discoveries once more made, and at a much later period (1738), by
Dr. Nathaniel Lieberkuhn with his simple microscopes, and by means of
which he discovered the minute structure of the mucous membrane of the
alimentary canal, and which alone would have immortalised his name
had we not preserved in use to this day an important adjunct of every
modern instrument, the Lieberkuhn reflector.
In the Museum of the Royal College of Surgeons of England, there is
a small cabinet of two drawers, containing a set of twelve of his
simple microscopes, each being provided with an original injection. The
form of the instrument is shown in Figs. 1 and 2. _a b_ represents a
piece of brass tubing about an inch long and an inch in diameter and
provided with a cap at each extremity. The one at _a_ carries a small
double-convex lens of half an inch focal length; while at _b_ there is
fixed a condensing lens three-quarters of an inch in diameter. In Fig.
2 the instrument is seen in section, and explains itself. It is held by
the handle in such a position that the rays of light, from a lamp or
a white cloud, may fall on the condenser _b_, and concentrate on the
speculum _l_. This again further condenses the rays on the disc _c_,
where the object is held, and its adjustment made by the milled-head
screw _d_, so as to bring it within the focus of the lens _a_.
[Illustration: Fig. 1.]
From this digression I pass on to the evolution of the compound
microscope. The earliest workable form known was that designed by
Eustachio Divini, who brought it to the notice of the Royal Society
in 1668. It consisted of two plano-convex lenses, combined with their
convex surfaces retained in apposition. His idea was subsequently
improved upon by a London optician. Not long afterwards, Philip
Bonnani published an account of his improved compound microscope; and
we are certainly indebted to him for two or more forms of the movable
horizontal microscopes, and for the compound condenser fitted with
focussing gear for illuminating transparent objects by transmitted
light. I must, however, pass by the many changes made in the structure
and form of the instrument by the celebrated Dr. Culpeper, Scarlet,
Cuff, and many other inventors.
[Illustration: Fig. 2.--Lieberkuhn’s Microscope.]
=Benjamin Martin’s Microscope.=--Benjamin Martin, about 1742, was
busily engaged in making improvements in the microscope, and I may
say he was certainly the first to provide accurate results for
determining the exact magnifying power of any object-lens, so that
the observer might state the exact amplification in a certain number
of diameters. He devised numerous improvements in the mechanism and
optical arrangements of the instrument; the rack and pinion focussing
adjustments; the inclining movements to the pillar carrying the stage;
and the rectangular mechanical motions to the stage itself. He was
familiar with the principles of achromatism, since it appears he
produced an achromatic objective about 1759, and he is said to have
sent an achromatic objective to the Royal Society about that date. But
an ingeniously constructed microscope by Martin found its way to George
the Third, the grandfather of our Queen, and afterwards came into the
possession of the late Professor John Quekett, of the Royal College
of Surgeons, who presented it to the Royal Microscopical Society of
London. This microscope will ever associate Martin’s name with the
earliest and best form of the instrument, even should he not receive
full recognition as the inventor of the _achromatic microscope_. On
this account I introduce a carefully made drawing of so singularly
perfect a form of the early English microscope to the notice of my
readers. (Fig. 3.) The description given of it by the late Professor
Quekett is as follows:--“It stands about two feet in height, and is
supported on a tripod base, A; the central part of the stem, B, is of
triangular figure, having a rack at the back, upon which the stage,
O, and frame, D, supporting the mirror, E, are capable of being moved
up or down. The compound body, F, is three inches in diameter; it is
composed of two tubes, the inner of which contains the eye-piece, and
can be raised or depressed by rack and pinion, so as to increase or
diminish the magnifying power. At the base of the triangular bar is a
cradle joint, G, by which the instrument can be inclined by turning
the screw-head, H (connected with an endless screw acting upon a
worm-wheel). The arm, I, supporting the compound body, is supplied with
a rack and pinion, K, by which it can be moved backwards and forwards,
and a joint is placed below it, upon which the body can be turned into
the horizontal position; another bar, carrying a stage and mirror, can
be attached by a screw, L N, so as to convert it into a horizontal
microscope. The stage, O, is provided with all the usual apparatus for
clamping objects, and a condenser can be applied to its under surface;
the stage itself may be removed, the arm, P, supporting it, turned
round on the pivot, C, and another stage of exquisite workmanship
placed in its stead, the under surface of which is shown at Q.”
[Illustration: Fig. 3.--Martin’s Universal Microscope. 1782.]
This stage is strictly a micrometer one, having rectangular movements
and a fine adjustment, the movements being accomplished by the
fine-threaded screws, the milled heads of which are graduated. The
mirror, E, is a double one, and can be raised or depressed by rack and
pinion; it is also capable of removal, and an apparatus for holding
large opaque objects, such as minerals, can be substituted for it.
The accessory instruments are very numerous, and amongst the more
remarkable may be mentioned a tube, M, containing a speculum, which can
take the place of the tube, R, and so form a reflecting microscope.
The apparatus for holding animalcules or other live objects, which is
represented at S, as well as a plate of glass six inches in diameter,
with four concave wells ground in it, can be applied to the stage, so
that each well may be brought in succession under the magnifying power.
The lenses belonging to this microscope are twenty-four in number;
they vary in focal length from four inches to one-tenth of an inch;
ten of them are supplied with Lieberkuhns. A small arm, capable of
carrying single lenses, can be supplied at T, and when turned over,
the stage of the instrument becomes a single microscope; there are
four lenses suitable for this purpose, their focal length varying
from one-tenth to one-fortieth of an inch. The performance of all the
lenses is excellent, and no pains appear to have been spared in their
construction. There are numerous other pieces of accessory apparatus,
all remarkable for the beauty of their workmanship.[4]
In addition to the movements described by Quekett, the body-tube with
its support can be moved in an arc concentrically with the axis of the
triangular pillar, on the top of which it is fitted with a worm-wheel
and endless-screw mechanism, actuated by the screw-head, T, below.
It must therefore be admitted that Martin led the way far beyond his
contemporaries, both in the design and the evolution of the microscope.
Furthermore, in his “New Elements of Optics,” 1759, he dealt with
the principle of achromatism, by the construction of an achromatic
telescope.
At a somewhat later period there lived in London a philosophical
instrument maker of some repute, George Adams, who published in 1746
a quarto book, entitled “Micrographia Illustrata, or the Knowledge of
the Microscope Explained.” This work fairly well describes “the nature,
uses, and magnifying powers of microscopes in general, together with
full directions how to prepare, apply, examine, and preserve minute
objects.” Adams’ book was the first of the kind published in this
country, and it contributed in no small degree to the advancement of
microscopical science. Adams writes: “We owe the construction of the
variable microscope to the ingenuity and generosity of a noble person.
The apparatus belonging to it is more convenient, more certain, and
more extensive than that of any other at present extant; consequently,
the advantage and pleasure attending the observations in viewing
objects through it must be as extensive in proportion.” This is
believed to apply to Martin’s several microscopes, and that especially
constructed for the king, afterwards improved upon by Adams. Another
early form of microscope, Wilson Simple Scroll (1746), stamped on the
cover of this book, and has thus become familiar to microscopists, was
also made by Adams.
We now closely approach a period fertile in the improvement of the
microscope, and in the discoveries made by its agency. The chief of
those among the honoured names of the time we find Trembley, Ellis,
Baker, Adams, Hill, Swammerdam, Lyonet, Needham, and a few others.
Adams somewhat sarcastically observes “that every optician exercises
his talents in improving (as he calls it) the microscope, in other
words, in varying its construction and rendering it different in
form from that sold by his neighbour; or at the best rendering it
more complex and troublesome to manage.” There were no doubt good
reasons for these and other strictures upon inventors as well as
makers of microscopes, even in the Adams’ day. In the year 1787 the
“Microscopical Essays” of his son were published, in which he described
all the instruments in use up to that period.
Looking back, and taking a general survey of the work of nearly two
centuries in the history of the microscope, it cannot be said that
either in its optical or mechanical construction any great amount of
progress was made. This in part may have arisen from the fact that
no pressing need was felt for either delicate focussing or higher
magnification. At all events, it was not until the application of
achromatism to the instrument that new life was infused into its use,
and a great impetus was given to its development, both optically and
mechanically.
In the year 1823 a strong desire became manifest for improved forms of
the instrument, in France by M. Selligue, by Frauenhofer in Munich,
by Amici in Modena, by M. Chevalier in Paris, and by Dr. Goring, Mr.
Pritchard, and Mr. Tully in London. The result was that in 1824 a new
form of achromatic object-glass was constructed of nine-tenths of
an inch focal length, composed of three lenses, and transmitting a
pencil of eighteen degrees; and which, as regards accurate correction
throughout the field, was for some years regarded as perfect.
Sir David Brewster was the first to suggest the great importance
of introducing materials of a more highly refracting nature into
the construction of lenses. He wrote: “There can be no essential
improvement expected in the microscope unless from the discovery
of some transparent substance which, like the diamond, combines a
high refractive with a low dispersive power.” Having experienced the
greatest difficulty in getting a small diamond cut into a prism in
London, he did not conceive it practicable to grind, polish, and form
it into a lens.
Mr. Pritchard, however, was led to make the experiment, and on the
1st of December, 1824, “he had the pleasure of first looking through
a diamond microscope.” Dr. Goring also tried its performance on
various objects, both as a single microscope and as an objective of
a compound instrument, and satisfied himself of its superiority over
other kinds of lenses. But here Mr. Pritchard’s labours did not end. He
subsequently found that the diamond used had many flaws in it, which
led him to abandon the idea of finishing it. Having been prevented
from resuming his operations on this refractory material for a time
he made a third attempt, and met with another unexpected defect; he
found that some lenses, unlike the first, gave a double or triple image
instead of a single one, in consequence of some of their parts being
either harder or softer than others. These defects were found to be
due to polarisation. Mr. Pritchard having learned how to decide whether
a diamond is fit for a magnifier or not, subsequently succeeded in
making two planoconvex lenses of adamant; these proved to be perfect
for microscopic purposes. “One of these, of one-twentieth of an inch
in focal length, is now in the possession of his Grace the Duke of
Buckingham; the other, of one-thirtieth of an inch focus, is in his own
hands.”
“In consequence of the high refracting power of a diamond lens over
a glass lens, the former material may be at least one-third as thin
as that of the latter, and if the focal length of both be equal, say,
one-eightieth of an inch, the magnifying power of the diamond lens
will be 2,133 diameters, whereas that of glass will be only 800.” At
a date (1812) before Brewster proposed diamond lenses he demonstrated
a simple method of rendering both single and compound microscopes
achromatic. “Starting,” he says, “with the principle that all objects,
however delicate, are best seen when immersed in fluid, he placed
an object on a slip of glass, and put above a drop of oil, having a
greater dispersive power than the single concave lens, which formed the
object-glass of the microscope. The lens was then made to touch the
fluid, so that the surface of the fluid was formed into a concave lens,
and if the radius of the outward surface was such as to correct the
dispersion, we should have a perfect achromatic microscope.” Here we
have the immersion system foreshadowed. Shortly after these experiments
of Brewster’s were in progress, Dr. Goring is said to have discovered
that the structure of certain bodies could be readily seen in some
microscopes and not in others. These bodies he named test objects.
He then examined these tests with the achromatic combinations of the
Tullys, and was led to the discovery that “the penetrating power of the
microscope depends upon its angle of aperture.”
“While these practical investigations were in progress,” writes Andrew
Ross, “the subject of achromatism engaged the attention of some of
the most profound mathematicians in England, Sir John Herschel, and
Professors Airy and Barlow. Mr. Coddington and others contributed
largely to the theoretical examination of the subject; and although the
results of their labours were not applicable to the microscope, they
essentially promoted its improvement.”
About this period (1812) Professor Amici, of Modena, was
experimentally engaged in the improvement of the achromatic
object-glass, and he invented a reflecting microscope superior to
those of Newton, Baker, or Smith, made as early as 1738, and long ago
abandoned. In 1815 Amici made further experiments, and introduced the
immersion system; while Frauenhofer, of Munich, about the same time
constructed object-glasses for the microscope of a single achromatic
lens, in which the two glasses, although placed in juxtaposition, were
not cemented together.
Dolland, it has been said, introduced achromatic lenses; but although
he constructed many achromatic telescopes, he did not apply the
same principle to microscopes, and those which he sold were only
modifications of the compound microscope of Cuff.
Dr. Wollaston employed a new form of combination in a microscope
constructed for his own use, and by which “he was able to see
distinctly the finest markings upon the scales of the _Lepisma_ and
_Podura_, and upon those of the gnat’s wing.” His doublet is still
employed, and to which I shall refer under “Simple Microscopes.”
[Illustration: Fig. 3_a_.--Sir David Brewster’s Microscope, of the
early part of the century, recently presented to the British Museum.]
CHAPTER I.
Elementary Optics.
Value of Inductive Science--Light: Its Propagation, Refraction,
Reflection--Spherical and Chromatic Aberrations--Human Eye,
formation of Images of External Objects in--Visual Angle
increased--Abbe’s Theory of Microscopic Vision.
The advances made in physics and mechanics during the 17th and 18th
centuries fairly opened the way to the attainment of greater perfection
in all optical instruments. This has been particularly exemplified
with reference to the invention of the microscope, as briefly sketched
out in the previous chapter. Indeed, in the first half of the present
century the microscope can scarcely be said to have held a position
of importance among the scientific instruments in frequent use. Since
then, however, the zoologist and botanist by its aid have laid bare
the intimate structure of plants and animals, and thereby have opened
up a vast kingdom of minute forms of life previously undreamt of; and
in connection with chemistry a new science has been founded, that of
bacteriology.
For these reasons it will be of importance to the student of microscopy
to begin at the beginning, and it will be my endeavour to introduce to
his notice such facts in physical optics as are closely associated with
the formation of images, and, so to speak, systematise such stepping
stones for work hereafter to be accomplished. Elementary principles
only will be adduced, and without attempting to involve my readers
in intricate mathematical problems, and which for the most part are
unnecessary for the attainment of the object in view. I therefore
pass at once to the consideration of the propagation of light through
certain bodies.
The microscope, whether simple or compound, depends for its magnifying
power on the influence exerted by lenses in altering the course of the
rays of light passing through them being REFRACTED. _Refraction_ takes
place in accordance with two well-known laws of optics. When a ray of
light passes from one transparent medium to another it undergoes a
change of direction at the surface of separation, so that its course
in the second medium makes an angle with its course in the first. This
change of direction is a resultant of refraction. The broken appearance
presented by a stick partly immersed in water, and viewed in an oblique
position, is an illustration of the law of refraction. Liquids have a
greater refractive power than air or gases. As a rule, with some few
exceptions, the denser of the two substances has the greater refractive
power; hence it is customary in enumerating some of the laws of optics
to speak of the denser medium and the rarer medium. The more correct
designation would be the more refractive and the less refractive.[5]
[Illustration: Fig. 4.--Law of Refraction.]
Let R I (Fig. 4) be a ray incident at I on the surface of separation
of two media, and let I S′ be the course of the ray after refraction.
Then the angles which R I and I S make with the normal are the _angle
of incidence_ and the _angle of refraction_ respectively, and the
first law of refraction is that these angles lie in the same plane,
or the _plane of refraction_ is the same as the _plane of incidence_.
The law which connects the magnitudes of these angles, and which was
discovered by Snell, a Dutch philosopher, can only be stated either
by reference to a geometrical construction, or by using the language
of trigonometry. Describe a circle about the point of incidence, I as
a centre, and drop perpendiculars from the points where it cuts the
rays on the normal. The law is that these perpendiculars, R′ P′, S′ P,
will have a constant ratio, or the sines _of the angles of incidence
and refraction are in a constant ratio_; that is, so long as the media
through which the ray first passes, and by which it is afterwards
refracted, remain the same, and the light also of the same kind, then
it is referred to as the law of sines.
Indices of Refraction.
The ratio of the sine of the angle of incidence to the sine of the
angle of refraction, when a ray passes from one medium to another is
termed the relative index of refraction. When a ray passes from vacuum
into any medium, this ratio is always greater than unity, and is called
the _absolute index of refraction_, or simply the index of refraction
for the medium in question.
The absolute index of air is so small that it may be neglected in
comparison with those of solids and liquids; but strictly speaking, the
relative index for a ray passing from air into a given substance must
be multiplied by the absolute index of the air, in order to obtain the
true index of refraction.
[Illustration: Fig. 5.--Vision through a Glass Plate.]
=Critical Angle.=--It will be seen from the law of sines that, when
the incident ray is in the less refractive of the two media, to
every possible angle of incidence there is a corresponding angle of
refraction. The angle referred to is termed the _critical angle_, and
is readily computed if the relative index of refraction be given. When
the media are air and water, this angle is about 48° 30′. For air and
ordinary kinds of glass its value varies from 38° to 41°.
The phenomenon of total reflection may be observed in several familiar
instances. For example, if a glass of water, with a spoon in it, is
held above the level of the eye, the under side of the surface is
seen to shine like a mirror, and the lower part of the spoon is seen
reflected in it. Effects of the same kind are observed when a ray
of sunlight passes into an aquarium--on the other hand rays falling
normally on a uniform transparent plate of glass with parallel faces
keep their course; but objects viewed obliquely through the same
are displaced from their true position. Let S (Fig. 5) be a luminous
point which sends light to an eye not directly opposite to it, on the
other side of a parallel plate. The emergent rays which enter the
eye are parallel to the incident rays; but as they have undergone
lateral displacement, their point of concourse is changed from S to
S′, and this is accordingly the image of S. The rays in such a case
which compose the pencil that enters the eye will not exactly meet in
any one point; there will be two focal lines, just as in the case of
spherical mirrors. The displacement produced, as seen in the figure
referred to above, increases with the thickness of the plate, its index
of refraction, and the obliquity of incidence. This furnishes one of
the simplest means of measuring the index of refraction of a glass
substance, and is thus employed in Pichot’s refractometer (“Deschanel”).
[Illustration: Fig. 6.--Refraction through a Prism.]
=Refraction through a Prism.=--A prism is a portion of a refracting
medium bounded by two plane surfaces, inclined at a definite angle
to one another. The two plane surfaces are termed the _faces_ of the
prism, and their inclination to one another is the refracting angle
of the prism. A prism preserves the property of bending rays of light
from their original course by refraction. A cylinder may be regarded
as the limit of a prism whose sides increase in number and diminish in
size indefinitely: it may also be regarded as a pyramid whose apex is
removed to an indefinite distance.
Let S I (Fig. 6) be an incident ray in the plane of the principal
section of the prism. If the external medium be air, or other substance
of less refractive power than the prism, the ray on entering the same
will be bent nearer to the normal, taking such a course as I E, and on
leaving the prism will be bent away from the normal, taking the course
E B. The effect of these two refractions is, therefore, to turn the ray
away from the edge (or refracting angle) of the prism. In practice,
the prism is usually so placed that I E, the path of the ray through
the prism, makes equal angles with the two faces at which refraction
occurs. If the prism is turned very far from this position, the course
of the ray may be altogether different from that represented in the
figure; it may enter at one face, be internally reflected at another,
and come out at the third.
It is evident, therefore, that the minimum number of sides, _i.e._, the
bounding faces, exclusive of the ends, which a prism can have is three.
In this form, it constitutes a most valuable instrument of research in
physical optics. A convex lens is practically merely a curved form of
two prisms combined, their bases being brought into contact; on the
other hand the concave lens is simply a reversal of the position of
the apices brought into contact, as shown in Fig. 11. Both convex and
concave lenses are therefore closely related to the prism.
=Reflection.=--The laws that govern the change of direction which a ray
of light experiences when it strikes upon the surfaces of separation
of two media and is thrown back into the same medium from which it
approached is as follows:--When the reflecting surface is plain the
direction of the reflected ray makes with the normal to the surface the
same angle which the incident ray makes with the same normal; or, as it
is usually expressed, the angles of reflection and incidence are equal.
When the surfaces are curved the same law holds good. In all cases of
reflection the energy of the ray is diminished, so that reflection must
always be accompanied by absorption. The latter probably precedes the
former. Most bodies are visible by light reflected from their surfaces,
but before this takes place the light has undergone a modification,
namely, that which imparts colour peculiar to the bodies viewed. When
light impinges upon the surface of a denser medium part is reflected,
part absorbed, and part refracted. But for a certain angle depending
upon the refractive index of the refracting medium no refraction takes
place. This angle is termed the angle of total reflection, since all
the light which is not absorbed is wholly reflected.
Multiple images are produced by a transparent parallel plate of
glass. If the glass be silvered at the back, as it usually is in the
microscope-mirror, the second image is brighter than the first, but as
the angle of incidence increases the first image gains upon the second;
and if the luminous object be a lamp or candle, a number of images,
one behind the other, will be visible to an eye properly placed in
front. This is due to the fact that the reflecting power of a surface
of glass increases with the angle of incidence.
[Illustration: Fig. 7.--Conjugate Foci of Curved Surfaces.]
=Concave Surfaces.=--Rays of light proceeding from any given point in
front of a concave spherical mirror, are reflected so as to meet in
another point, and the line joining the two points passes through the
centre of the sphere. The relation between them is or should be mutual,
hence they are termed _conjugate foci_. By a _focus_ in general is
meant a point in which a number of rays of light meet, and the rays
which thus meet, taken collectively, are termed _a pencil_. Fig. 7
represents two pencils of rays whose foci, S s, are conjugate, so that,
if either of them be regarded as an incident pencil, the other will be
the corresponding reflected pencil. Each point, in fact, sends a pencil
of rays which converge, after reflection, to the conjugate focus. _The
principal focal distance is half the radius of curvature._ But it will
not escape attention that concave mirrors have two reflecting surfaces,
a front and a back. This, however, does not practically disturb its
_virtual focus_, since the achromatic condenser when brought into use
collects and concentrates the light received from the mirror upon an
object for the purpose of rendering it more distinctly visible to the
eye when viewing an object placed on the stage of the microscope. The
images seen in a plane mirror are always virtual, and any spherical
mirror, whether concave or convex, is nearly equivalent to a plane
mirror when the distance of the object from its surface is small in
comparison with the radius of curvature.
Lenses.
=Forms of Lenses.=--A lens is a portion of a refracting medium bounded
by two surfaces which are portions of spheres, having a common axis,
termed the _axis of the lens_. Lenses are distinguished by different
names, according to the nature of their surfaces.
[Illustration: Fig. 8.--Converging and Diverging Lenses.]
Lenses with sharp edges (thicker at the centre) are _convergent_ or
_positive_ lenses. Lenses with blunt edges (thinner at the centre) are
_divergent_ or _negative_ lenses. The first group comprises:--(1) The
bi-convex lens; (2) the plano-convex lens; (3) the convergent meniscus.
The second group:--(4) The concave lens; (5) the plano-concave lens;
(6) the divergent meniscus (Fig. 8).
=Principal Focus.=--A lens is usually a solid of revolution, and the
axis of revolution is termed the principal axis of the lens. When the
surfaces are spherical it is the line joining the centre of curvature.
From the great importance of lenses, especially convex lenses, in
practical optics, it will be necessary to explain their properties
somewhat at length.
[Illustration: Fig. 9.--Principal Focus of a Convex Lens.]
=Principal Focus of Convex Lens.=--When rays which were originally
parallel to the principal axis pass through a convex lens (Fig. 9),
the effect of the two refractions which they undergo, one on entering
and the other on leaving the lens, is to make them all converge
approximately to one point F, which is called the principal focus.
The distance A F of the principal focus from the lens is called the
principal focal distance, or more briefly and usually, the focal length
of the lens. The radiant point and its image after refraction are known
as the conjugate foci. In every lens the right line perpendicular to
the two surfaces is the _axis_ of the lens. This is indicated by the
line drawn through the several lenses, as seen in the diagram (Fig. 8).
The point where the axis cuts the surface of the lens is termed the
_verte_.
Parallel rays falling on a _double-convex_ lens are brought to a focus
in the centre of its diameter; conversely, rays diverging from that
point are rendered parallel. Hence the focus of a _double-convex_
lens will be at just half the distance, or half the length, of the
focus of a _plano-convex_ lens having the same curvature on one side.
The distance of the focus from the lens will depend as much on the
degree of curvature as upon the refracting power (termed the index
of refraction) of the glass of which it may be formed. A lens of
crown-glass will have a longer focus than a similar one of flint-glass;
since the latter has a greater refracting power than the former.
For all ordinary practical purposes we may consider the _principal
focus_--as the focus for parallel rays is termed--of a double-convex
lens to be at the distance of its radius, that is, in its centre of
curvature; and that of a plano-convex lens to be at the distance of
twice its radius, that is, at the other end of the diameter of its
sphere of curvature. The converse of all this occurs when divergent
rays are made to fall on a convex lens. Rays already converging are
brought together at a point nearer than the principal focus; whereas
rays diverging from a point within the principal focus are rendered
still more diverging, though in a diminished degree. Rays diverging
from points more distant than the principal focus on either side, are
brought to a focus beyond it: if the point of divergence be within the
circle of curvature, the focus of convergence will be beyond it; and
_vice-versâ_. The same principles apply equally to a _plano-convex
lens_; allowance being made for the double distance of its principal
focus; and also to a lens whose surfaces have different curvatures; the
principal focus of such a lens is found by multiplying the radius of
one surface by the radius of the other, and dividing this product by
half the sum of the radii.
[Illustration: Fig. 10.--Principal Focus of Concave Lens.]
In the case of a concave lens (Fig. 10), rays incident parallel to the
principal axis diverge after passing through; and their directions,
if produced backwards, would approximately meet in a point F; this is
its _principal focus_. It is, however, only a virtual focus, inasmuch
as the emergent rays do not actually pass through it, whereas the
principal focus of a converging lens is real.
[Illustration: Fig. 11.--Principal Centre of Lens.]
=Optical Centre of a Lens.=--_Secondary Axes._--Let O and O′ (Fig. 11)
be the centres of the two spherical surfaces of a lens. Draw any two
parallel radii, O I, O′ E, to meet these surfaces, and let the joining
line I E represent a ray passing through the lens. This ray makes equal
angles with the normals at I and E, since these latter are parallel by
construction; hence the incident and emergent rays S I, E R also make
equal angles with the normals, and are therefore parallel. In fact, if
tangent planes (indicated by the dotted lines in the figure) are drawn
at I and E, the whole course of the ray S I E R will be the same as if
it had passed through a plate bounded by these planes.
Let C be the point in which the line I E cuts the principal axis, and
let R, R′ denote the radii of the two spherical surfaces. Then from
the similarity of the triangles O C I, O′ C E, we have (O C)/(C O′) =
R′/R; which shows that the point C divides the line of centres O O′ in
a definite ratio depending only on the radii. Every ray whose direction
on emergence is parallel to its direction before entering the lens,
must pass through the point C in traversing the lens; and conversely,
every ray which in its course through the lens traverses the point C,
has parallel directions at incidence and emergence. The point C which
possesses this remarkable property is called the _centre_, or _optical
centre_, of the lens.
This diagram may also be taken to prove my former proposition, that the
convex lens is practically a form of two prisms combined.
[Illustration: Fig. 12.--Conjugate Foci, one Real, the other Virtual.]
=Conjugate Foci, one Real, one Virtual.=--When two foci are on the same
side of the lens, one (the most distant of the two) must be virtual.
For example, in Fig. 12, if S, S′ are a pair of conjugate foci, one of
them S being between the principal focus F and the lens, rays sent to
the lens at a luminous point at S, will, after emergence, diverge as
if from S′; and rays coming from the other side of the lens, if they
converge to S′ before incidence, will in reality be made to meet in
S. As S moves towards the lens, S′ moves in the same direction more
rapidly; and they become coincident at the surface of the lens.
=Formation of Real Images.=--Let A B (Fig. 13) be an object in front
of a lens, at a distance less than the principal focal length. It will
have a real image on the other side of the lens. To determine the
position of the image by construction, draw through any point A of the
object a line parallel to the principal axis, meeting the lens in A′.
The ray represented by this line will, after refraction, pass through
the principal focus, F, and its intersection with the secondary axis,
A O, determines the position of _a_, the focus conjugate to A. We can
in like manner determine the position of _b_, the focus conjugate to B,
another point of the object; and the joining line _a b_ will then be
the magnified image of the line A B. It is evident that if _a b_ were
the object, A B would be the image.
[Illustration: Fig. 13.--Real and Magnified Image.]
The figures 12 and 13 represent the cases in which the distance of the
object is respectively greater and less than twice the focal length of
the lens.
The focal length of a lens is determined by the convexity of its
surfaces and the refractive power of the material of which it is
composed, being shortened either by an increase of refractive power,
or diminution of the radii of curvature of the faces of the lens. The
increase or decrease of spherical aberration is determined by the
shape or curvature of the lens; it is less in the bi-convex than in
other forms. When a lamp or other source of light is placed at the
focus of the rays constituting that portion of its light which falls
upon the lens, the light is so refracted as to become parallel. Should
the source of light be brought nearer to the lens than the focus the
refracted rays are still divergent, though not to the same extent;
on the other hand, if the source be beyond the focus, the refracted
rays are rendered convergent so as to meet at a point which is
mathematically related to the distance of the luminous source from the
focus. The former arrangement is that with which we are most familiar,
since it is the ordinary magnifying glass.
Concave Lenses.
The refracting influence of a _concave_ lens (Fig. 14) will be
precisely the opposite of that of a convex. Rays which fall upon it in
a parallel direction will be made to diverge as if from the principal
focus, which is here called the _negative_ focus. This will be, for a
_plano-concave_ lens, at the distance of the diameter of the sphere of
curvature; and for a _double-concave_, in the centre of that sphere.
[Illustration: Fig. 14.--A Virtual Image formed by Concave Lens.]
In Fig. 14 A B is the object and _a b_ the image. Rays incident from A
and B parallel to the principal axis will emerge as if they came from
the principal focus F; hence, the points _a b_ are determined by the
intersections of the dotted lines in the figure with the secondary
axis, O A, O B. An eye on the other side of the lens sees the image _a
b_, which is always virtual, erect and diminished.
In the construction of the microscope, either simple or compound, the
curvature of the lenses employed is usually spherical. Convergent
lenses, with spherical curvatures, have the defect of not bringing all
the rays of light which pass through them to one and the same focus.
Each circle of rays from the axis of the lens to its circumference
has a different focus, as shown in Fig. 15. The rays _a a_, which
pass through the lens near its circumference, are seen to be _more
refracted_, or come to a focus at a shorter distance behind it than
the rays _b b_, which pass through near its centre or axis, and are
_less refracted_. The consequence of this defect of lenses with
spherical curvatures, which is called _spherical aberration_, is that
a well-defined image or picture is not formed by them, for when the
object is focussed, for the circumferential rays, the picture projected
to the eye is rendered indistinct by a halo or confusion produced by
the central rays falling in a circle of dissipation, before they have
come to a focus. On the other hand, when placed in the focus of the
central rays, the picture formed by them is rendered indistinct by the
halo produced by the circumferential rays, which have already come to
a focus and crossed, and now fall in a state of divergence, forming a
circle of dissipation. The grosser defects of spherical aberration are
corrected by cutting off the passage of the rays _a a_, through the
circumferences of the lens, by means of a stop diaphragm, so that the
central rays, _b b_, only are concerned in the formation of the image.
This defect is reduced to a minimum, by using the meniscus form of
lens, which is the segment of an ellipsoid instead of a sphere.
[Illustration: Fig. 15.--Spherical Aberration of Lens.]
The ellipse and the hyperbola are forms of lenses in which the
curvature diminishes from the central ray, or axis, to the
circumference _b_; and mathematicians have shown that spherical
aberration may be practically got rid of by employing lenses whose
sections are ellipses or hyperbolas. The remarkable discovery of
these forms of lenses is attributed to Descartes, who mathematically
demonstrated the fact.
If _a l_, _a l′_, for example (Fig. 16) be part of an ellipse whose
greater axis is to the distance between its foci _f f_ as the index of
refraction is to unity, then parallel rays _r l′_, _r′′ l_ incident
upon the elliptical surface _l′ a l_, will be refracted by the single
action of that surface into lines which would meet exactly in the
farther focus _f_, if there were no second surface intervening between
_l a l′_ and _f_. But as every useful lens must have two surfaces, we
have only to describe a circle _l a′ l′_ round _f_ as a centre, for the
second surface of the lens _l′ l_.
[Illustration: Fig. 16.--Converging Meniscus.]
As all the rays refracted at the surface _l a l′_ converge accurately
to _f_, and as the circular surface _l a′ l′_ is perpendicular to every
one of the refracted rays, all these rays will go on to _f_ without
suffering any refraction at the circular surface. Hence it should
follow, that a meniscus whose convex surface is part of an ellipsoid,
and whose concave surface is part of any spherical surface whose centre
is in the farther focus, will have no appreciable spherical aberration,
and will refract parallel rays incident on its convex surface to the
farther focus.
[Illustration: Fig. 17.--Aplanatic Doublet.]
The spherical form of lens is that most generally used in the
construction of the microscope. If a true elliptical or hyperbolic
curve could be ground, lenses would very nearly approach perfection,
and spherical aberration would be considerably reduced. Even this
defect can be further reduced in practice by observing a certain ratio
between the radii of the anterior and posterior surfaces of lenses;
thus the spherical aberration of a lens, the radius of one surface of
which is six or seven times greater than that of the other, will be
much reduced when its more convex surface is turned forward to receive
parallel rays, than when its less convex surface is turned forwards. It
should be borne in mind that in lenses having curvatures of the kind
the object would only be correctly seen in focus at one point--the
mathematical or geometrical axis of the lens.
=Chromatic Aberration.=--We have yet to deal with one of the most
important of the phenomena of light, CHROMATIC ABERRATION, upon the
correction of which, in convex lenses in particular, the perfection
of the objective of the microscope so much depends. Chromatism arises
from the unequal refrangibility and length of the different coloured
rays of light that together go to make up white light; but which, when
treated of in optics, is always associated with _achromatism_, so that
a combination of prisms, or lenses, is said to be _achromatic_ when
the coloured rays arising from the dispersion of the pencil of light
refracted through them are combined in due proportions as they are in
perfectly white light.
A lens, however, of uniform material will not form a single white
image, but a series of images of all colours of the spectrum, arranged
at different distances, the violet being nearest, and the red the most
remote, every other colour giving a blurred image; the superposition
of these and the blending of the different elementary rays furnishing
a complete explanation of the beautiful phenomenon of the rainbow.
Sharpness of outline is rendered quite impossible in such a case, and
this source of confusion is known as _chromatic aberration_.
In order to ascertain whether it is possible to remedy this evil
by combining lenses of two different materials, Newton made some
trials with a compound prism composed of glass and water (the latter
containing a little sugar of lead), and he found it impossible by any
arrangement of these two, or by other substances, to produce deviation
of the transmitted light without separation into its component colours.
If this ratio were the same for all substances, as Newton supposed,
achromatism would be impossible; but, in fact, its value varies
greatly, and is far greater for flint than for crown glass. If two
prisms of these substances, of small refracting angles, be combined
into one, with their edges turned in opposite directions, they will
achromatise each other.
The chromatism of lenses may, however, be somewhat further reduced by
stopping out the marginal rays, but as the most perfect correction
possible is required when lenses are combined for microscopic uses,
other means of correction are resorted to, as will be seen hereafter.
I shall first proceed to show the deviations which rays of white light
undergo in traversing a lens.
If parallel rays of light pass through a double-convex lens the violet
rays, the most refrangible of them, will come to a focus at a point
much nearer to the lens than the focus of the red rays, which are the
least refrangible; and the intermediate rays of the spectrum will be
focussed at points between the red and the violet. A screen held at
either of these foci will show an image with prismatic fringes. The
white light, A A′′ (Fig. 18), falling on the marginal portion of the
lens is so far decomposed that the violet rays are brought to a focus
at C, and crossing there, diverge again and pass on to F F′, while the
red rays, B B′′, do not come to a focus until they reach the point D,
and cross the divergent violet rays, E E′. The foci of the intermediary
rays of the spectrum (red, green, and blue) are intermediate between
these extremes. The distance, C D, limiting the blue or violet, and the
red is termed the longitudinal chromatic aberration of the lens. If the
image be received upon a screen placed at C, violet will predominate
and appear surrounded by a prismatic fringe, in which violet will
predominate. If the screen be now shifted to D, the image will have a
predominant red tint, surrounded by a series of coloured fringes in an
inverted order to those seen in the former experiment. The line E E′
joins the points of intersection between the violet and red rays, and
this marks the mean focus, the point where the coloured rays will be
least apparent.
[Illustration: Fig. 18.--Chromatic Aberration of Lens.]
In the early part of this century the optical correction of chromatic
aberration was partially brought about by combining a convex lens of
crown-glass with a concave lens of flint-glass, in the proportion of
which these two kinds of glass respectively refract and disperse rays
of light; so that the one medium may by equal and contrary dispersion
neutralise the dispersion caused by the other, without at the same
time wholly neutralising its refraction. It is a curious fact that the
media found most available for the purpose should be a combination of
crown and flint-glass, of _crown-glass_ whose index of refraction is
1·519, and dispersive power 0·036, and of _flint-glass_ whose index
of refraction is 1·589, and dispersive power 0·0393. The focal length
of the convex crown-glass lens must be 4-1/3 inches, and that of the
concave flint-glass lens 7-2/3 inches, and the combined focal length 10
inches. The diagram (Fig. 19) shows how rays of light are brought to a
focus, nearly free from colour. The small amount of residual colour in
such a combination is termed the _secondary spectrum_; the violet ray
F Y, crossing the axis of the lens at V, and going to the upper end P
of the spectrum, the red ray F B going to the lower end T. But as the
flint-glass lens _l l_, on the prism A _a_ C, which receives the rays
F V, F R, at the same points, is interposed, these rays will unite at
_f_, and form a small circle of white light, the ray S F being now
refracted without colour from its primitive direction S F Y into the
direction F _f_. In like manner, the corresponding ray S F′ will be
refracted to _f_, and a white colourless image be the result.
[Illustration: Fig. 19.--Correction of Chromatic Aberration.]
The achromatic aplanatic objective constructed on the optical formula
enunciated, did not meet all the difficulties experienced by the
skilled microscopist, in obtaining resolution of the finest test
objects, and whereby the intrinsic value of the objective (in his
estimation) must stand or fall. There were other disturbing residuary
elements besides those of the secondary spectrum, and which at a later
period were met by the practical skill of the optician, who applied the
screw-collar, and by means of which the back lens of the objective is
made to approach the front lens, thus more accurately shortening the
distance between the eye-piece, where the image is eventually formed,
and the back lens of the objective.
In this diagram L L is a _convex_ lens of _crown-glass_, and _l l_ a
_concave_ one of _flint-glass_. A convex lens will refract a ray of
light (S) falling at F on it exactly in the same manner as the prism A
B C, whose faces touch the two surfaces of the lens at the points where
the ray enters, and quits. The ray S F, thus refracted by the lens L
L, or prism A B C, would have formed a spectrum (P T) on a screen or
wall, had there been no other lens.
[Illustration: Fig. 20.--Virtual Image formed by Convex Lens.]
=Formation of Virtual Images.=--The normal eye possesses a considerable
power of adjusting itself to form a distinct image of objects placed
at varying distances; the nearer, within a certain limit, the larger
it appears, and the more distinctly the details are brought out.
When brought within a distance of two or three inches, the images
become blurred or quite indistinct, and when brought closer to the
eye, cannot be seen at all, and it simply obstructs the light. Now
the utility of a convex lens, when interposed between the object and
the eye, consists in reducing the divergence of the rays forming the
several pencils which issue from it, and send images to the retina in
a state of moderate divergence, that is, as if they had issued from an
object beyond the nearest point of distinct vision, and so that a more
clearly defined image may reach the sensitive membrane of the eye. But,
not only is the course of the several rays in each pencil altered as
regards the rest, but the course of the pencils themselves is changed,
so that they enter the eye under an angle corresponding with that
under which they would have arrived from a larger object situated at a
greater distance, and thus the picture formed by any object corresponds
in all respects with one which would have been made by the same
object increased in its dimensions and viewed at the smallest ordinary
distance of distinct vision. For instance, let an object A B (Fig. 20)
be placed between a convex lens and its principal focus. Then the foci
conjugate to the points A B are virtual, and their positions can be
found by construction from the consideration that rays through A, B,
parallel to the principal axis, will be refracted to F, the principal
focus on the other side. The refracted rays, if produced backwards,
must meet the secondary axis O A, O B in the required points. An eye
placed on the other side of the lens will accordingly see a virtual
image erect, magnified, and at a greater distance from the lens than
the object. This is the principle of the simple microscope.
The Human Eye.
To gain a clear insight into the mode in which a single lens serves to
magnify objects, it will be necessary to revert to the phenomena of
ordinary vision. An eye free from any defect has a considerable power
of adjusting itself to very considerable distances. One of the special
functions of the eye is bringing the rays of light, by a series of
dioptric mechanisms, to a perfect focus on its nervous sensitive layer,
the retina. The eye in this respect has been compared to a photographic
camera. But this is not quite correct. The retina is destined simply
to receive the images furnished by the dioptric apparatus, and has no
influence upon the formation of these images. The luminous rays are
refracted by the dioptric apparatus; the images would be formed quite
as well--indeed, even better in certain cases--if the retina were not
there. The dioptric apparatus and its action are absolutely independent
of the retina.
The same laws with regard to the passage of the rays of light into the
human eye hold good, as those already enunciated in the previous pages.
As to change of direction when rays are passing obliquely from a medium
of low density to that of a higher density, _i.e._, it changes its
course, and is bent towards the perpendicular. On leaving the denser
for the rarer medium it is bent once more from the perpendicular.
Again, by means of a convex lens, the rays of light from one source
will be refracted so as to meet at a point termed _the principal focus_
of vision.
In the eye there are several surfaces separating the different media
where refraction takes place. The refractive index of the aqueous
humour and the tears poured out by the lachrymal gland is almost equal
to that of the cornea. We may, therefore, speak of the refracting
surfaces as three, viz.: Anterior surface of cornea, anterior surface
of lens, and posterior surface of lens; and also of the refracting
media as three--the aqueous humour, the lens, and vitreous humour.
These several bodies are so adapted in the normal eye that parallel
rays falling on the cornea are converged to a focus at the most
sensitive spot (the _yellow spot_, or _fovea centralis_) in the retina,
a point representing to the principal focus of the eye. A line drawn
from this point through the centre of the cornea is called the optic
axis of the eye-ball.
[Illustration: Fig. 21.--Nerve and Stellate Cell Layer of Cornea,[6]
stained by chloride of gold; magnified 300 diameters. _a_, Nerve cells.
_b_, Stellate cells.]
[Illustration:
Fig. 22.--Anterior section of Eye, showing changed form of lens during
the act of accommodation, a voluntary action in the eye. M, Ciliary
muscle; I, Iris; L, Lens; V, Vitreous Humour; A, Aqueous Humour; C,
Cornea and optic axis.]
But as we are able to form a distinct image of near objects, and as
we notice when we turn our gaze from far to near objects there is a
distinct feeling of muscular effort in the eyes, there must be some
means whereby the eye can readily adapt itself for focussing near and
distant objects. In a photographic camera the focus can be readily
altered, either by changing the lenses, employing a lens of greater
or less curvature, or by altering the distance of the screen from the
lens. The last method is obviously impossible in the rigid eye-ball,
and therefore the act of focussing for near and distant objects is
associated with a change in the curvature of the lens, a faculty of the
eye termed _accommodation_ (Fig. 22), a change chiefly accomplished
by the ciliary (muscle) processes, which pull the lens forwards and
inwards by virtual contracting power of the ciliary muscle, and by
which its suspensory ligament is relaxed, and the front of the lens
allowed to bulge forward. In every case, however, accommodation is
associated with contraction of the iris, the special function of which
is that of a limiting diaphragm (an iris-diaphragm), Fig. 23.
In an ordinary spherical bi-convex lens, as already pointed out, the
rays of light passing through the periphery of the lens come to a focus
at a nearer point than the rays passing through the central portion.
In this way a certain amount of blurring of the image takes place, and
which, in optical language, is termed _spherical aberration_. This
defect of the eye is capable of correction in three possible ways,
and which it may be well to repeat: 1. By making the refractive index
of the lens higher at its centre than at its circumference; (2) By
making the curvature of the lens less near the circumference than
at the centre; (3) By stopping out the peripheral rays of light by a
diaphragm. The two latter methods are those resorted to in most optical
instruments.
[Illustration: Fig. 23.--1. Equatorial section of Eyeball, showing
Iris and Ciliary Processes, after washing away the pigment, × three
diameters.
2. Nerves of the Cornea of Kitten’s Eye, stained with iodine.
3. Fibres or Tubules of Lens, × 250, seen to be made up of superimposed
crenated layers, and is therefore not homogeneous in structure, but
made up of a number of extremely fine tubules, whose curvatures are
nearly spherical.]
In the human eye an attempt is made to apply all these methods, but
the most important is the third, that of applying the diaphragm formed
by the iris, a circular semi-muscular curtain lying just in front of
the anterior surface of the lens. The iris is also furnished with a
layer of pigmental cells which effectually stop out all peripheral
rays of light that otherwise would pass into the eye, creating circles
of diffusion of a disturbing nature to perfect vision. This delicate
membrane, then, is kept in constant action by a two-fold nerve supply,
derived from five or six sources, which it is unnecessary to describe
at length. But the eye, with all its marvellous adaptations, has an
obvious defect, that of secondary or uncorrected chromatic aberration.
=Chromatic Aberration of the Eye.=--White light, as previously
explained, is composed of different wave lengths; and accordingly as
these undulations are either longer or shorter, so do they produce
on the eye the impression of different colours. We have seen how a
pencil of white light may, by means of a prism, be decomposed into a
multi-coloured band. In an ordinary magnifying reading-glass these
coloured fringes are always seen around the margins. In practical
optics chromatic aberration is partially corrected by employing two
different kinds of glass in the construction of certain combined
lenses. In the human eye chromatism cannot be corrected in this way;
hence a blue light and a red light placed at the same distance from the
eye appears to be unequally distant: the red light requiring greater
accommodation in the eye than the blue, and this accordingly appears to
be the nearer of the two.
This visual error may be experimentally shown and explained. There is
a kind of glass which at first sight appears dark blue or violet, but
which really contains a great deal of red. Take an ordinary microscope
lamp, having a metal or opaque chimney, and drill a circular hole in
it, about 3 mm. in diameter. This opening should be just at the height
of the flame; cover it over with a piece of ground glass and a piece of
the red-blue glass. Thus will be formed a luminous point whose light is
composed of red and blue, _i.e._, of colours far apart from each other
in the spectrum.
[Illustration: Fig. 24.--Chromatic Aberration of Eye, showing the wave
differences of the _blue and red rays of light_ (_Landolt_).]
If rays coming from this point enter the eye, the blue rays (Fig. 24),
being more strongly reflected than the red, will come to a focus sooner
than the latter. The red rays, on the contrary, will be brought to a
focus later than the blue, while the latter, past their focus, are
diverging. Let A B C D (Fig. 24) be the section of a pencil of rays
given off from a red-blue point sufficiently distant so that these rays
may be regarded as parallel. The focus of the blue is at _b_, that of
the red at _r_.
An eye is adapted to the distance of the luminous point when the circle
of diffusion, received upon the retina, is at its minimum. This is the
case when the sentient layer of the retina lies between the two foci
E. In this case the point will appear as a small circle, composed of
the two colours, that is to say--violet. If the retina be _in front_
of this point, at the focus of the _blue_ rays for instance, the eye
will perceive a _blue point surrounded by a red circle_, the latter
being formed by the periphery of the luminous cone of red rays, which
are focussed only after having passed the retina. The blue point will
become a circle of diffusion larger in proportion as the retina is
nearer the dioptric system, or as the focus for blue is farther behind
it. But the blue circle will always be surrounded by a red ring. If,
on the contrary, the retina is _behind the focus for red_, the blue
cone will be greater in diameter than the red, and we shall have a _red
circle of diffusion_, larger in proportion as the retina is farther
from the focus, but always _surrounded by a blue ring_ M. If the
blue-red point is five metres, or more, distant, the _emmetropic_[7]
eye will evidently see it more distinctly, _i.e._, as a small _violet
point_; the _hyperopic_ eye, whose retina is situated _in front_ of the
focus of its dioptric system, will see a _blue circle, surrounded by
red_; the _myopic_ eye, whose retina is _behind_ its focus, will see
a _red circle, surrounded by blue_. The size of these circles will be
either larger or smaller when the principal focus of the eye is either
in _front_ of or _behind the retina_.[8]
The refractive surfaces of a perfectly formed eye are very like an
ellipsoid of revolution with two axes, one of which, the major axis of
the ellipse, is at the same time the optic axis and that of rotation;
the other is perpendicular to it, and is equal in all meridians. Eyes,
however, perfectly constructed are rarely met with. The curvature of
the cornea is nearly always greater in one meridian than in another.
Its surfaces then cannot be regarded as entirely belonging to an
ellipsoid of revolution, since the solid figure, of which the former
would constitute a part, has not only two axes, but three, and these
unequal. This irregularity is not, however, always great enough to
produce discomfort and it is therefore disregarded. But in other cases
the difference of curvature in the different meridians of the eye
attain to a higher degree, and vision falls far below the average.
[Illustration: Fig. 25.--Lines as seen by the Astigmatic.]
The refractive anomaly alluded to is termed _astigmatism_ (from the
Greek, α privative, στιγμα, a point--inability to see a point). The
way in which objects appear to such a person will mainly result from
the way in which he sees _a point_. Take, for example, the vertical to
be the most, and the horizontal to be the least, refractive meridian:
place a vertical line (Fig. 25, I) at a stated distance before the
eye, and the line will appear elongated, owing to the diffusion image
of each of the points composing it. It will also seem to be somewhat
broadened, as at II. If the _vertical_ meridian is adapted to the
distance of the vertical, the line will appear very diffuse and
broadened, as at III. All these little diffusion lines overlap each
other, and give the line an elongated appearance. Hence a straight line
is seen distinctly by an astigmatic eye only when the meridian to which
it is perpendicular is perfectly adapted to its distance. A _vertical
line_ is seen distinctly when the _horizontal meridian_ is adapted to
its distance. It appears _indistinct_ when its image is formed by the
vertical meridian. The way in which an astigmatic person sees points
and lines led to the discovery of this remarkable irregularity in the
refraction of the eye. The late Astronomer Royal, Sir George Airy,
suffered for some years until, indeed, he discovered how it could be
corrected. This anomaly of curvature of the refractive surfaces of the
eye is now known to prevail largely among the more civilised races of
mankind. It is, then, of very great importance when using high powers
of the microscope. In most persons the visual power of both eyes is
rarely quite equal; on the other hand, the mind exerts an important
influence, dominates, as it were, the eye in the interpretation of
visual sensations and images. An example of this is presented in
Wheatstone’s pseudoscope, known to produce precisely the opposite
effect of his stereoscope--conveys, in fact, the _converse of relief_
produced by the latter and better known instrument.
=Visual Judgment.=--The apparent size of an object is determined by
the magnitude of the image formed on the retina, and this is inversely
proportional to the distance. Thus the size of an image on the retina
of an object two inches long at a distance of a foot, is equal to the
image of an object four inches long at a distance of two feet. An
object can be seen if the visual angle subtended by it is not less than
sixty seconds. This is equivalent to an image on the _fovea centralis_
of the retina of about 4 µ[9] across, and which corresponds to the
diameter of a cone: so that while we have had under consideration the
optical and physical conditions of human vision, we have likewise
taken a lesson on the action of lenses used in the construction of the
microscope.
The Theory of Microscopical Vision.
It has been said that no comparison can be instituted between
microscopic vision and macroscopic; that the images formed by minute
objects are not delineated microscopically under ordinary laws of
diffraction, and that the results are dioptrical. This assertion,
however, cannot be accepted unconditionally, as will be seen on more
careful examination of the late Professor Abbe’s masterly exposition
of “The Microscopical Theory of Vision,” and also his subsequent
investigations on the estimation of aperture and the value of
wide-angled immersion objectives, published in the “Journal of the
Royal Microscopical Society.”
The essential point in Abbe’s theory of microscopical vision is
that the images of minute objects in the microscope are not formed
exclusively on the ordinary _dioptric_ method (that is, in the same
way in which they are formed in the camera or telescope), but that
they are largely affected by the peculiar manner in which the minute
construction of the object breaks up the incident rays, giving rise to
_diffraction_.
The phenomena of diffraction in general may be observed experimentally
by plates of glass ruled with fine lines. Fig. 26 shows the appearance
presented by a single candle-flame seen through such a plate, an
uncoloured image of the flame occupying the centre, flanked on either
side by a row of coloured spectra of the flame, which become dimmer as
they recede from the centre. A similar phenomenon may be produced by
dust scattered over a glass plate, and by other objects whose structure
contains very minute particles, or the meshes of very fine gauze wire,
the rays suffering a characteristic change in passing through such
objects; that change consisting in the breaking up of a parallel beam
of light into a group of rays, diverging with wide angle and forming
a regular series of maxima and minima of intensity of light, due to
difference of phase of vibration.[10]
[Illustration: Fig. 26.]
In the same way, in the microscope, the diffraction pencil originating
from a beam incident upon, for instance, a diatom, appears as a fan of
isolated rays, decreasing in intensity as they are further removed from
the direction of the incident beam transmitted through the structure,
the interference of the primary waves giving a number of successive
maxima of light with dark interspaces.
When a diaphragm opening is interposed between the mirror, and a plate
of ruled lines placed upon the stage such as Fig. 27, the appearance
shown in Fig. 27_a_, will be observed at the back of the objective on
removing the eye-piece and looking down the tube of the microscope. The
centre circles are the images of the diaphragm opening produced by the
direct rays, while those on the other side (always at right angles to
the direction of the lines) are the diffraction images produced by the
rays which are bent off from the incident pencil. In homogeneous light
the central and lateral images agree in size and form, but in white
light the diffraction images are radially drawn out, with the outer
edges red and the inner blue (the reverse of the ordinary spectrum),
forming, in fact, regular spectra the distance separating each of which
varies inversely as the closeness of the lines, being for instance with
the same objective twice as far apart when the lines are twice as close.
[Illustration: Fig. 27.]
[Illustration: Fig. 27_a_.]
The influence of these diffraction spectra may be demonstrated by some
very striking experiments, which show that they are not by any means
accidental phenomena, but are directly connected with the image which
is seen by the eye.
The first experiment shows that with the central beam, or any one of
the spectral beams alone, only the contour of the object is seen, the
addition of at least one diffraction spectrum being essential to the
visibility of the structure.
[Illustration: Fig. 28.]
[Illustration: Fig. 28_a_.]
When by a diaphragm placed at the back of the objective, as in Fig.
28, we cover up all the diffraction spectra of Fig. 27_a_, and allow
only the central rays to reach the image, the object will appear to be
wholly deprived of fine details, the outline alone will remain, and
every delineation of minute structure will disappear, just as if the
microscope had suddenly lost its optical power, as in Fig. 28_a_.
This experiment illustrates a case of the _obliteration_ of structure
by obstructing the passage of the diffraction spectra to the eye-piece.
The next experiment shows how the appearance of fine structure may be
_created_ by manipulating the spectra.
[Illustration: Fig. 29.]
[Illustration: Fig. 29_a_.]
When a diaphragm such as that shown in Fig. 29 is placed at the back of
the objective, so as to cut off each alternate one of the upper row of
spectra in Fig. 27_a_, that row will obviously become identical with
the lower one, and if the theory holds good, we should find the image
of the upper lines identical with that of the lower. On replacing the
eye-piece, we see that it is so, the upper set of lines are doubled in
number, a new line appearing in the centre of the space between each
of the old (upper) ones, and upper and lower set having become to all
appearance identical, as seen in Fig. 29_a_.
[Illustration: Fig. 30.]
[Illustration: Fig. 30_a_.]
In the same way, if we stop off all but the outer spectra, as in Fig.
30, the lines are apparently again doubled, as seen in Fig. 30_a_.
A case of apparent creation of structure, similar in principle to the
foregoing, though more striking, is afforded by a network of squares,
as in Fig. 31, having sides _parallel_ to this page, which gives the
spectra shown in Fig. 31_a_, consisting of vertical rows for the
horizontal lines and horizontal rows for the vertical ones. But it is
readily seen that two diagonal rows of spectra exist at right angles to
the diagonals of the squares, just as would arise from sets of lines
in the direction of the diagonals, so that if the theory holds good
we ought to find, on obstructing all the other spectra and allowing
only the diagonal ones to pass to the eye-piece, that the vertical and
horizontal lines have disappeared and are replaced by two new sets of
lines at _right angles to the diagonals_.
[Illustration: Fig. 31.]
[Illustration: Fig. 31_a_.]
[Illustration: Fig. 32.]
[Illustration: Fig. 32_a_.]
On inserting the diaphragm, Fig. 32, and replacing the eye-piece, we
find in the place of the old network the one shown in Fig. 32_a_, the
squares being, however, smaller in the proportion of 1 : √2, as they
should be in accordance with the theory propounded.
An object such as _Pleurosigma angulatum_, which gives six diffraction
spectra arranged as in Fig. 33, should, according to this theory, show
markings in a hexagonal arrangement. For there will be one set of lines
at right angles to _b_, _a_, _e_, another set at right angles to _c_,
_a_, _f_, and a third at right angles to _g_, _a_, _d_. These three
sets of lines will obviously produce the appearance shown in Fig. 33_a_.
[Illustration: Fig. 33.]
[Illustration: Fig. 33_a_.]
[Illustration: Fig. 34.]
A great variety of appearances may be produced with the same
arrangement of spectra. Any two adjacent spectra with the central beam
(as _b_, _c_, _a_) will form equilateral triangles and give hexagonal
markings. Or by stopping off all but _g_, _c_, _e_ (or _b_, _d_, _f_),
we again have the spectra in the form of equilateral triangles; but
as they are now further apart, the sides of the triangles in the two
cases being as √3 : 1, the hexagons will be smaller and three times as
numerous. Their sides will also be arranged at a different angle to
those of the first set. The hexagons may be entirely obliterated by
admitting only the spectra _g_, _c_, or _g_, _f_, or _b_, _f_, etc.,
when new lines will appear at right angles, or obliquely inclined, to
the median line. By varying the combinations of the spectra, therefore,
different figures of varying size and positions are produced, all
of which cannot, of course, represent the true structure. Not only,
however, may the appearance of particular structure be obliterated or
created, but it may even be _predicted_ before being seen under the
microscope. If the position and relative intensity of the spectra in
any particular case are given, the character of the resultant image,
in some instances, may be worked out by mathematical calculations. A
remarkable instance of such a prediction is to be found in the case
recorded by Mr. Stephenson, where a mathematical student who had
never seen a diatom, worked out the purely mathematical result of the
interference of the six spectra _b-g_ of Fig. 33 (identical with _P.
angulatum_), giving the drawing copied in Fig. 34. The special feature
was the small markings between the hexagons, which had not, before this
time, been noticed on _P. angulatum_. On more closely scrutinizing a
valve, stopping out the central beam and allowing the six spectra only
to pass, the small markings were found actually to exist, though they
were so faint they had previously escaped observation until the result
of the mathematical deduction had shown that they _ought_ to be seen.
These experiments seem to show that diffraction plays a very essential
part in the formation of microscopical images, since dissimilar
structures give identical images when the differences of their
diffractive effect is removed, and conversely similar structures
may give dissimilar images when their diffractive images are made
dissimilar. Whilst a purely dioptric image answers point for point
to the object on the stage, and enables a safe inference to be drawn
as to the actual nature of that object, the visible indications of
minute structure in a microscopical image are not always or necessarily
conformable to the real nature of the object examined, so that nothing
more can safely be inferred from the image as presented to the eye,
than the presence in the object of such structural peculiarities as
will produce the particular diffraction phenomena on which these images
depend.
Further investigations and experiments led Abbe to discard so much of
his theoretical conclusions relating to superimposed images having
a distinct character as well as a different origin, and as to their
capability of being separated and examined apart from each other.
In a later paper he writes: “I no longer maintain in principle the
distinction between the absorption image or direct dioptrical image and
the diffraction image, nor do I hold that the microscopical image of
an object consists of two superimposed images of different origin or a
different mode of production. Thus it appears that both the absorption
image and the diffraction image he held to be equally of diffraction
origin; but while a lens of small aperture would give the former with
facility, it would be powerless to reveal the latter, because of its
limited capacity to gather in the strongly-deflected rays due to the
excessively minute bodies the microscopical objective has to deal
with.”[11]
Abbe’s theory of vision has been questioned by mathematicians, and
since his death Lord Rayleigh went more deeply into the question of
“the theory of the formation of optical images,” with special reference
to the microscope and telescope. He has shown that two lines cannot
be fairly resolved unless their components subtend an angle exceeding
that subtended by the wave-length of light at a distance equal to the
aperture; also, that the measure of resolution is only possible with
a square aperture, or one bounded by straight lines, parallel to the
lines resolved.
Lord Rayleigh’s Theory of the Formation of Optical Images, with Special
Reference to the Microscope.[12]
Of the two methods adopted, that of Helmholtz’s consists in tracing
the image representative of a mathematical point in the object, the
point being regarded as self-luminous; that of Abbe’s the typical
object was not, as we have seen, a luminous _point_, but a _grating_
illuminated by plane waves of light. In the latter method, Lord
Rayleigh argues that the complete representation of the object
requires the co-operation of all the spectra which are focussed in the
principal focal plane of the objective; when only a few are present
the representation is imperfect, and wholly fails when there is only
one. He then proceeds to show, by the aid of diagrams and mathematical
formula, how the resolving power can be adduced.
On further criticism of the Abbe spectrum theory, he observes “that
although the image ultimately formed may be considered to be due to
the spectra focussed to a given point, the degree of conformity of
the image to the object is another question. The consideration of the
case of a very fine grating, which might afford no lateral spectra at
all, shows the incorrectness of the usually accepted idea that if all
the spectra are utilised the image will still be incomplete, so that
the theory (originally promulgated by Abbe) requires a good deal of
supplementing; while it is inapplicable when the incident light is
not parallel, and when the object is, for example, a double point and
not a grating. Even in the case of a grating, the spectrum theory is
inapplicable, if the grating is self-luminous; for in this case no
spectra can be formed since the radiations from the different elements
of the grating have no permanent phase-relations.” For these reasons
Lord Rayleigh advises that the question should be reconsidered from
the older point of view, according to which the typical object is a
point and not a grating. Such treatment will show that the theory of
resolving power is essentially the same for all instruments. The
peculiarities of the microscope, arising from the divergence-angles
not being limited to be small, and from the different character of
the illumination, are theoretically only differences of detail. These
investigations can be extended to gratings, and the results so obtained
confirm for the most part the conclusions of the spectrum theory.
Furthermore, that the function of the condenser in microscopic practice
in throwing upon the object the image of the lamp-flame is to cause
the object to behave, at any rate in some degree, as if it were
self-luminous, and thus to obviate the sharply-marked interference
bands which arise when permanent and definite phase-relations are
permitted to exist between the radiations which issue from various
points of the object. This is capable of mathematical proof; and in
the case where the illumination is such that each point of the row or
of the grating radiates independently, the limit to resolution is seen
to depend only on the width of the aperture, and thus to be the same
for all forms of aperture as for those of the rectangular. That Abbe’s
theory of microscopic vision is fairly open to the criticisms passed on
it by Lord Rayleigh must be taken for granted.
Definition of Aperture; Principles of Microscopic Vision.
It must be well within the last half-century that the achromatic
objective-glass for the microscope was brought to perfection and its
value became generally recognised. Prior to the discovery of the
achromatic principle in the construction of lenses it was assumed that
the formation of the microscopic image took place (as we have already
seen) on ordinary dioptric principles. As the image is formed in the
camera or telescope, so it was said to be in the microscope. This
belief existed, it will be remembered, at a time when dry objectives
only were in favour and the use of the term _angle of aperture_ was
misunderstood, when it was supposed that the different media with
diffraction-indices were used; and the angle of the radiant pencil was
believed not only to admit of a comparison of two apertures in the same
medium, but likewise to admit of a standard of comparison when the
media were entirely different in their refractive qualities.
It was during my tenure of office as secretary of the Royal
Microscopical Society (1867 to 1873), that the aperture question,
and also that of _numerical aperture_, came under discussion, both
being met by the majority of the Fellows of the Society and practical
opticians by a _non-possumus_.
Opticians alleged, that is, before the value of aperture became fully
recognised (1860), that the achromatic objective had reached a stage of
perfection, beyond which it was not possible to go; indeed, not only
opticians, but physicists of high standing, as Professor Helmholtz,
who made many important contributions to the theory of the microscope,
and who, after duly weighing all the known physical laws on which the
formation of images can be explained, emphatically stated that in
his opinion “the limit of possible improvement of the microscope as
an instrument of discovery had been very nearly reached.” A quarter
of a century ago I ventured to throw a doubt upon so questionable a
statement. I determined, if possible, to submit the aperture question
to an exhaustive examination. My views were accordingly submitted to
two of the highest authorities in this country--Sir George Airy, the
then Astronomer Royal, and Sir George Stokes, Professor of Physics at
Cambridge University--both of whom agreed with me that the possible
increase of aperture would be attended with great advantage to the
objective, and open the way to an extension of power resolution in the
microscope.[13] The discussion afterwards took a warm turn, as will be
seen on reference to “The Monthly Microscopical Journals” of 1874, 1875
and 1876.
The confusion into which the aperture question at this period had
lapsed was no doubt due to the fact that its opponents had not yet
grasped the true meaning of the term _aperture_. It was believed to be
synonymous with “angular aperture,” much in use at the time. It will,
however, appear quite unaccountable that even the older opticians
should have confounded the latter with the former; and so entirely
disregarded the fact that the angles of the pencil of light admitted by
the objective cannot serve as a measure of its _aperture_, and that
high refractive media can greatly reduce the value length of waves of
light.
When the medium in which the objective works is the same as air, it
is not that a comparison can be made by the angles of the radiant
pencils only, but by their sines. For example, if two dry objectives
admit pencils of 60° and 180°, their real apertures are not as 1 : 3,
but as 1 : 2 only. Aperture in fact is computed by mathematicians by
tracing the rays from the back focus through the system of lenses to
the front focus, the front focus being the point at which the whole
cone of rays converge as free as may be from aberration. If the front
focus be in air, no pencil greater than 82°, “double the angle of
total reflection,” can _emerge_ from the plane front of the lens; and,
obviously, if no greater cone can emerge to a focus one way, neither
can any greater cone enter the body of the lens from the radiant. This
angle, then, of 82°, must be regarded as the limit for dry lenses or
objectives.
This limit, it will be seen on more careful examination, is very nearly
the maximum angle that can be computed for a lens to have a front
focus in air. This can be proved by the consideration of the angle
of the image of rays, as they are radiated from the object itself in
balsam: for although this angle of image rays viewed as nascent from a
self-luminous object capable of scattering rays in all directions, may
be 180° in the substance of the balsam and cover-glass, of the 180°
only 82° of the central portion will emerge into air--all rays beyond
this limit are internally reflected at the cover-glass. This cone,
then, of 82° becomes 180° in air, and a large part must necessarily be
lost by reflection at the first incidence on the plane front of the
lens. But with a formula permitting the use of a water medium between
the front lens and the cover-glass, the aperture of the image rays may
reach 126°--double the critical angle from glass to water; and with an
oil medium, the aperture will be found to be limited only by the form
of the front lens that can be constructed by the optician.
To sum up, then, the effect of the immersion system, greatly assists
in the correction of aberration, gives increased magnification and
angular aperture, increase of working distance between the objective
and object, and renders admissible the use of the thicker glass-cover.
The aperture question would in all probability have remained unsolved
many years longer (ten or twelve years elapsed after I brought the
question under discussion before opticians gave way), but for the
fortunate circumstance that the eminent mathematical and practical
optician, Professor Abbe, of Jena, was about to visit London. This
came off in the early part of the seventies, when the late Mr. John
Mayall and myself had the good fortune to interview him. The subject
discussed was naturally the increase of aperture and the theory
of microscopical vision. He readily at our request undertook to
re-investigate the question in all its bearings on the microscope. It
is almost unnecessary to add that the conclusions he came to, and the
results obtained, have proved of inestimable value to the microscopist
and practical optician, and it may well seem necessary to explain
somewhat at greater length the conclusions the learned Professor came
to, and by the adoption of which the microscope has been placed on a
more scientific basis than it had before attained to. Several papers
were published _in extenso_ in the “Journal of the Royal Microscopical
Society,” and I am greatly indebted to Mr. Frank Crisp, LL.D., for an
excellent _resumé_ of Abbe’s Monograph.[14]
The essential step in the consideration of aperture is, as I have
said, to understand clearly what is meant by the term. It will at
once be recognised that its definition must necessarily refer to its
primary meaning of _opening_, and must, in the case of an optical
instrument, define its capacity for receiving rays from the object, and
transmitting them to the image received at the eye-piece.
In the case of the telescope-objective, its capacity for receiving and
transmitting rays is necessarily measured by the expression of its
absolute diameter or “opening.” No such absolute measure can be applied
in the case of the microscope objective, the largest constructed lenses
of which having by no means the largest apertures, being, in fact,
the lower powers of the instrument, whose apertures are for the most
part but small. The capacity of a microscope objective for receiving
and transmitting rays is, however, as will be seen, estimated by its
_relative_ opening, that is, its opening in relation to its focal
length. When this relative opening has been ascertained, it may be
regarded as synonymous with that denoted in the telescope by _absolute_
opening. That this is so will be better appreciated by the following
consideration:--
In a single lens, the rays admitted within one meridional plane
evidently increase as the diameter of the lens (all other circumstances
remaining the same), and in the microscope we have, at the back of the
lens, the same conditions to deal with as are in front in the case
of the telescope; the larger or smaller number of emergent rays will
therefore be measured by the clear diameter, and as no rays can emerge
that have not first been admitted, this will give the measure of the
admitted rays under similar circumstances.
If the lenses compared have different focal lengths but the same clear
“openings,” they will transmit the same number of rays to equal areas
of an image at a definite distance, because they would admit the same
number if an object were substituted for the image; that is, if the
lens were used as a telescope-objective. But as the focal lengths are
different, the amplification of the images is different also, and equal
areas of these images correspond to different areas of the object from
which the rays are collected. Therefore, the higher power lens with
the same opening as the lower power, will admit a _greater_ number of
rays in all from the same object, because it admits the _same_ number
as the latter from a _smaller_ portion of the object. Thus, if the
focal lengths of two lenses are as 2 : 1, and the first amplifies N
diameters, the second will amplify 2 N with the same distance of the
image, so that the rays which are collected _to_ a given field of 1
mm. diameter of the image are admitted _from_ a field of 1/N mm. in
the first case, and of 1/(2N) mm. in the second. As the “opening” of the
objective is estimated by the diameter (and not by the area) the higher
power lens admits _twice_ as many rays as the lower power, because
it admits the same number from a field of half the diameter, and, in
general, the admission of rays by the same opening, but different
powers, must be in the inverse ratio of the focal lengths.
In the case of the single lens, therefore, its aperture is determined
by the ratio between the clear opening and the focal length. The same
considerations apply to the case of a compound objective, substituting,
however, for the clear opening of the single lens the diameter of
the pencil at its emergence from the objective, that is, the clear
utilised diameter of the back lens. All equally holds good whether the
medium in which the objective is placed is the same in the case of the
two objectives or different, as an alteration of the medium makes no
difference in the power.
[Illustration: 180° Oil Angle. (Numerical Aperture 1·52.)
Illustration: 180° Water Angle. (Numerical Aperture 1·33.)
Illustration: 180° Air Angle. 96° Water Angle. 82° Oil Angle.
(Numerical Aperture 1·00.)
Illustration: 97° Air Angle. (Numerical Aperture ·75.)
Illustration: 60° Air Angle. (Numerical Aperture ·50.)
Fig. 35.--Relative diameters of the (utilized) back lenses of various
dry and immersion objectives of the same power (1/4-in.) from an air
angle of 60° to an oil angle of 180°.]
Thus we arrive at a general proposition for all kinds of objectives:
1st, when the power is the same, the admission of rays (or aperture)
varies with the diameter of the pencil at its emergence; 2nd, when the
powers are different, the same aperture requires different openings in
the ratio of the focal lengths, or conversely with the same opening the
aperture is in inverse ratio to the focal lengths. We see, therefore,
that just as in the telescope the absolute diameter of the object-glass
defines its _aperture_, so in the microscope _the ratio between the
utilised diameter of the back lens and the focal length_ of the
objective defines its aperture also, and this is clearly a definition
of aperture in its primary and only legitimate meaning as “opening;”
that is, the capacity of the objective for admitting rays from the
object and transmitting them to the image.
If, by way of illustration, we compare a series of dry and
oil-immersion objectives, and commencing with small air angles,
progress up to 180° air angle, and then take an oil-immersion of 82°
and progress again to 180° oil angle, the ratio of opening to power
progresses also, and attains its maximum, not in the case of the air
angle of 180° (when it is exactly equivalent to the oil angle of only
82°), but is greatest at the oil angle of 180°. If we assume the
objectives to have the same power throughout we get rid of one of the
factors of the ratio, and we have only to compare the diameters of the
emergent beams, and can represent their relations by diagrams.
Fig. 35 illustrates five cases of different apertures of 1/4-in.
objectives, viz.: those of dry objectives of 60°, 97°, and 180° air
angle, a water-immersion of 180° water angle, and an oil-immersion of
180° oil angle. The inner dotted circles in the two latter cases are of
the same size as that corresponding to the 180° air angle.
A dry objective of the maximum air angle of 180° is only able to
utilise a diameter of back lens equal to twice the focal length, while
an immersion lens of even only 100° utilises a _larger_ diameter,
_i.e._, it is able to transmit more rays from the object to the image
than any dry objective is capable of transmitting. Whenever the angle
of an immersion lens exceeds twice the critical angle for the immersion
fluid, _i.e._, 96° for water or 82° for oil, its aperture is in excess
of that of a dry objective of 180°.
[Illustration: Fig. 36.]
This excess will be _seen_ if we take an oil-immersion objective
of, say 122° balsam angle, illuminating it so that the whole field
is filled with the incident rays, and use it first on an object not
mounted in balsam, but dry. We then have a _dry objective_ of nearly
180° angular aperture, for, as will be seen by reference to Fig. 36,
the cover-glass is virtually the first surface of the objective, as
the front lens, the immersion fluid, and the cover-glass are all
approximately of the same index, and form, therefore, a front lens
of extra thickness. When the object is close to the cover-glass the
pencil radiating from it will be very nearly 180°, and the emergent
pencil (observed by removing the eye-piece) will be seen to utilise as
much of the back lens of the objective as is equal to twice the focal
length, that is, the _inner_ of the two circles at the head of Fig. 35.
If now balsam be run in beneath the cover-glass so that the angle of
the pencil taken up by the objective is no longer 180°, but 122° only
(that is, _smaller_), the diameter of the emergent pencil is _larger_
than it was before, when the angle of the pencil was 180° in air, and
will be approximately represented by the _outer_ circle of Fig. 35. As
the power remains the same in both cases, the larger diameter denotes
the greater aperture of the immersion objective over a dry objective of
even 180° angle, and the excess of aperture is made plainly visible.
Having settled the principle, it is still necessary, however, to find a
proper _notation_ for comparing apertures. The astronomer can compare
the apertures of his various objectives by simply expressing them in
inches, but this is obviously not available to the microscopist, who
has to deal with the ratio of two varying quantities.
In consequence of a discovery made by Professor Abbe in 1873, that a
general relation existed between the pencil admitted into the front of
the objective and that emerging from the back of the objective, he was
able to show that the ratio of the semi-diameter of the emergent pencil
to the focal length of the objective could be expressed by the formula
_n_ Sin _u_, _i.e._, by the sine of half the angle of aperture (_u_)
multiplied by the refractive index of the medium (_n_) in front of the
objective (_n_ being 1·0 for air, 1·33 for water, and 1·52 for oil or
balsam).
When, then, the values in any given cases of the expression _n_ Sin
_u_ (which is known as the “numerical aperture”) has been ascertained,
the objectives are instantly compared as regards their aperture, and,
moreover, as 180° in air is equal to 1·0 (since _n_ = 1·0 and the
sine of half 180° = 1·0) we see, with equal readiness, whether the
aperture is smaller or larger than that corresponding to 180° in air.
Thus, suppose we desire to compare the apertures of three objectives,
one a dry objective, the second a water immersion, and the third an
oil immersion; these would be compared on the angular aperture view
as, say 74° air angle, 85° water angle, and 118° oil angle, so that
a calculation must be worked out to arrive at the actual relation
between them. Applying, however, the _numerical_[15] notation, which
gives ·60 for the dry objective, ·90 for the water immersion, and
1·30 for the oil immersion, their relative apertures are immediately
recognised, and it is seen, for instance, that the aperture of the
water immersion is somewhat less than that of a dry objective of 180°,
and that the aperture of the oil immersion exceeds that of the latter
by 30%.
The advantage of immersion, in comparison with dry objectives, becomes
at once apparent. Instead of consisting merely in a diminution of the
loss of light by reflection or increased working distance, it is seen
that a wide-angled immersion objective has a larger aperture than a dry
objective of maximum angle, so that for any of the purposes for which
aperture is essential an immersion must necessarily be preferred to a
dry objective.
That pencils of identical angular extension but in different media are
different physically, will cease to appear in any way paradoxical if we
recall the simple optical fact that rays, which in air are spread out
over the whole hemisphere, are in a medium of higher refractive index
such as oil _compressed_ into a cone of 82° round the perpendicular,
_i.e._, twice the critical angle. A cone exceeding twice the critical
angle of the medium will therefore embrace a _surplus_ of rays which do
not exist even in the hemisphere when the object is in air.
The whole aperture question, notwithstanding the innumerable
perplexities which heretofore surrounded it, is in reality completely
solved by these two simple considerations: First, that “aperture” is
to be applied in its ordinary meaning as representing the greater or
less capacity of the objective for receiving and transmitting rays;
and second, that when so applied the aperture of an objective is
determined by the ratio between its opening and its focal length; the
objective that utilises the larger back lens (or opening) relatively
to its focal length having necessarily the larger aperture. It would
hardly, therefore, serve any useful purpose if we were here to discuss
the various erroneous ideas that gave rise to the contention that 180°
in air must be the maximum aperture. Amongst these was the suggestion
that the larger emergent beams of immersion objectives were due to
the fact that the immersion fluid abolished the refractive action of
the first plane surface which, in the case of air, prevented there
being any pencil exceeding 82° within the glass. Also the very curious
mistake which arose from the assumption that a hemisphere did not
magnify an object at its centre because the rays passed through without
refraction. A further erroneous view has, however, been so widespread
that it seems to be desirable to devote a few lines to it, especially
as it always appears at first sight to be both simple and conclusive.
[Illustration: Fig. 37.]
[Illustration: Fig. 37_a_.]
If a dry objective is used upon an object in air, as in Fig. 37, the
angle may approach 180°, but when the object is mounted in balsam,
as in Fig. 37_a_, the angle at the object cannot exceed 82°, all
rays outside that limit (shown by dotted lines) being reflected back
at the cover-glass and not emerging into air. On using an immersion
objective, however, the immersion fluid which replaces the air above
the cover-glass allows the rays formerly reflected back to pass through
to the objective, so that the angle at the object may again be nearly
180° as with the dry lens. The action of the immersion objective was,
therefore, supposed to be simply that it repaired the loss in angle
which was occasioned when the object was transferred from air to
balsam, and merely restored the conditions existing in Fig. 37_a_ with
the dry objective on a dry object.
As the result of this erroneous supposition, it followed that an
immersion objective could have no advantage over a dry objective,
except in the case of the latter being used upon a balsam-mounted
object, its aperture then being (as was supposed) “cut down.” The
error lies simply in overlooking the fact that the rays which are
reflected back when the object is mounted in balsam (Fig. 37_a_) are
not rays which are found when the object is in air (Fig. 37), but are
_additional and different_ rays which do not exist in air, as they
cannot be emitted in a substance of so low a refractive index.
Lastly, it should also be noted that it is numerical and not angular
aperture which measures the quantity of light admitted to the objective
by different pencils.
[Illustration: Fig. 38.]
[Illustration: Fig. 38_a_.]
First take the case of the medium being the same. The popular notion
of a pencil of light may be illustrated by Fig. 38, which assumes that
there is equal intensity of emission in all directions, so that the
quantity of light contained in any given pencils may be compared by
simply comparing the contents of the solid cones. The Bouguer-Lambert
law, however, shows that the quantity of light emitted by any bright
point varies with the obliquity of the direction of emission, being
_greater_ in a perpendicular than in an oblique direction. The rays are
less intense in proportion as they are more inclined to the surface
which emits them, so that a pencil is not correctly represented by Fig.
38, but by Fig. 38_a_, the density of the rays decreasing continuously
from the vertical to the horizontal, and the squares of the sines of
the semi-angles (_i.e._, of the numerical aperture) constituting the
true measure of the quantity of light contained in any solid pencil.
If, again, the media are of different refractive indices, as air
(1·0), water (1·33), and oil (1·52), the total amount of light emitted
over the whole 180° from radiant points in these media under a given
illumination is not the same, but is _greater_ in the case of the
media of greater refractive indices in the ratio of the squares of
those indices (_i.e._, as 1·0, 1·77 and 2·25). The quantity of light
in pencils of different angle and in different media must therefore
be compared by squaring the product of the sines and the refractive
indices, _i.e._ (_n_ Sin _u_^2), for the square of the numerical
aperture.
The fact is therefore made clear that the aperture of a dry objective
of 180° does not represent, as was supposed, a maximum, but that
aperture increases with the increase in the refractive index of the
immersion fluid; and it should be borne in mind that this result has
been arrived at in strict accordance with the ordinary propositions of
geometrical optics, and without any reference to or deductions from the
diffraction theory of Professor Abbe.
There still remains one other point for determination, namely, the
proper function of aperture in respect to immersion objectives of large
aperture. The explanation of the increased power of vision obtained by
increase of aperture was, that by the greater obliquity of the rays
to the object “shadow effects” were produced, a view which overlooked
the fact, first, that the utilisation of increased aperture depends
not only on the obliquity of the rays sent to the _object_, but also
to the _axis of the microscope_; and exactly as there is no acoustic
shadow produced by an obstacle, which is only a few multiples of the
length of the sound waves, so there can be no shadow produced by minute
objects, only a few multiples from the light waves, the latter then
passing completely _round_ the object. The Abbe diffraction theory,
however, supplies the true explanation of this, and shows that the
increased performance of immersion objectives of large aperture is
directly connected (as might have been anticipated) with the larger
“openings” in the proper sense of the term, which, as we have already
explained, such objectives really possess. Furthermore, in order that
the image exactly corresponds with the object, all diffracted rays
must be gathered up by the objective. Should any be lost we shall
have not an actual image of the object, but a spurious one. Now,
if we have a coarse object, the diffracted rays are all comprised
within a narrow cone round the direct beam, and an objective of small
aperture will transmit them all. With a minute object, however, the
diffracted rays are widely spread out, so that a small aperture can
admit only a fractional part--to admit the whole or a very large
part, and consequently to see the minute structure of the object, or
to see it truly, a large aperture is necessary, and in this lies the
value of _aperture_ and of a _wide-angled immersion objective_ for the
observation of minute structures.
Numerical Aperture.
=Measure of Apertures of Objectives. N.A.=--Numerical aperture, as it
is termed, is measured by the scale of measurement calculated by the
late Professor Abbe, and which has since been generally recognised and
adopted. He showed that even in lenses made for the same medium (as
air) their comparative aperture as compared with their focus was not
correctly measured by the angle of the rays grasped, but by the actual
diameters of the pencil of rays transmitted, which depend, as already
seen, more upon the back of the lens than the front. To get a geometric
measure for comparison, he took the radii, or half diameters (whose
relative proportions would be the same), and which geometrically are
the sines of the semi-angle of the outermost rays grasped. Abbe further
showed that if this sine of half the outside angle were multiplied by
the refractive index of the medium used we should have a number which
would give the comparative _aperture_ of any lens, whatever the medium.
This number, then, determines both the numerical aperture and the
resolving power of the objective.
The following table of numerical apertures shows the respective angular
pencils which they express in air, water and cedar oil, or glass.[16]
The first column gives the numerical apertures from 0·20 to 1·33; the
second, third, and fourth, the air, water and oil (or balsam) angles of
aperture from 23° 4′ air angle to 180° balsam angle. The theoretical
resolving power in lines to the inch is shown in the sixth column; the
line E of the spectrum being taken from about the middle of the green,
the column giving “illuminating power” being of less importance; while
in using that of penetrating power, it must be remembered that several
data beside that of 1/_a_ go to make up the total depth of vision with
the microscope.
ABRIDGED NUMERICAL APERTURE TABLE.
=====+=========================+==========================+======+======
| Corresponding Angle |Limit of Resolving Power, | |
| (2 _u_) for | in Lines to an Inch. | |
+--------+-------+--------+--------+-------+---------+ |
(1) | (2) | (3) | (4) | (5) | (6) | (7) | (8) | (9)
-----+--------+-------+--------+--------+-------+---------+------+------
1·33 | ... |180° 0′|122° 6′| 128,225| 138,989|168,907 | 1·769| ·752
1·32 | ... |165° 56′|120° 33′| 127,261| 137,944|167,637 | 1·742| ·758
1·30 | ... |155° 38′|117° 35′| 125,333| 135,854|165,097 | 1·690| ·769
1·28 | ... |148° 42′|114° 44′| 123,405| 133,764|162,557 | 1·638| ·781
1·26 | ... |142° 39′|111° 59′| 121,477| 131,674|160,017 | 1·588| ·794
1·24 | ... |137° 36′|109° 20′| 119,548| 129,584|157,477 | 1·538| ·806
1·22 | ... |133° 4′|106° 45′| 117,620| 127,494|154,937 | 1·488| ·820
1·20 | ... |128° 55′|104° 15′| 115,692| 125,404|152,397 | 1·440| ·833
1·18 | ... |125° 3′|101° 50′| 113,764| 123,314|149,857 | 1·392| ·847
1·16 | ... |121° 26′| 99° 29′| 111,835| 121,224|147,317 | 1·346| ·862
1·14 | ... |118° 0′| 97° 11′| 109,907| 119,134|144,777 | 1·300| ·877
1·12 | ... |114° 44′| 94° 55′| 107,979| 117,044|142,237 | 1·254| ·893
1·10 | ... |111° 36′| 92° 43′| 106,051| 114,954|139,698 | 1·210| ·909
1·08 | ... |108° 36′| 90° 34′| 104,123| 112,864|137,158 | 1·166| ·926
1·06 | ... |105° 42′| 88° 27′| 102,195| 110,774|134,618 | 1·124| ·943
1·04 | ... |102° 53′| 86° 21′| 100,266| 108,684|132,078 | 1·082| ·962
1·02 | ... |100° 10′| 84° 18′| 98,338| 106,593|129,538 | 1·040| ·980
1·00 | 180° 0′| 97° 31′| 82° 17′| 96,410| 104,503|126,998 | 1·000| 1·000
0·98 | 157° 2′| 94° 56′| 80° 17′| 94,482| 102,413|124,458 | ·960| 1·020
0·96 | 147° 29′| 92° 24′| 78° 20′| 92,554| 100,323|121,918 | ·922| 1·042
0·94 | 140° 6′| 89° 56′| 76° 24′| 90,625| 98,223|119,378 | ·884| 1·064
0·92 | 133° 51′| 87° 32′| 74° 30′| 88,697| 96,143|116,838 | ·846| 1·087
0·90 | 128° 19′| 85° 10′| 72° 36′| 86,769| 94,053|114,298 | ·810| 1·111
0·88 | 123° 17′| 82° 51′| 70° 44′| 84,841| 91,963|111,758 | ·774| 1·136
0·86 | 118° 38′| 80° 34′| 68° 54′| 82,913| 89,873|109,218 | ·740| 1·163
0·84 | 114° 17′| 78° 20′| 67° 6′| 80,984| 87,783|106,678 | ·706| 1·190
0·82 | 110° 10′| 76° 8′| 65° 18′| 79,056| 85,693|104,138 | ·672| 1·220
0·80 | 106° 16′| 73° 58′| 63° 31′| 77,128| 83,603|101,598 | ·640| 1·250
0·78 | 102° 31′| 71° 49′| 61° 45′| 75,200| 81,513| 99,058 | ·608| 1·282
0·76 | 98° 56′| 69° 42′| 60° 0′| 73,272| 79,423| 96,518 | ·578| 1·316
0·74 | 95° 28′| 67° 37′| 58° 16′| 71,343| 77,333| 93,979 | ·548| 1·351
0·72 | 92° 6′| 65° 32′| 56° 32′| 69,415| 75,242| 91,439 | ·518| 1·389
0·70 | 88° 51′| 63° 31′| 54° 50′| 67,487| 73,152| 88,899 | ·490| 1·429
0·68 | 85° 41′| 61° 30′| 53° 9′| 65,559| 71,062| 86,359 | ·462| 1·471
0·66 | 82° 36′| 59° 30′| 51° 28′| 63,631| 68,972| 83,819 | ·436| 1·515
0·64 | 79° 36′| 57° 31′| 49° 48′| 61,702| 66,882| 81,279 | ·410| 1·562
0·62 | 76° 38′| 55° 34′| 48° 9′| 59,774| 64,792| 78,739 | ·384| 1·613
0·60 | 73° 44′| 53° 38′| 46° 30′| 57,846| 62,702| 76,199 | ·360| 1·667
0·58 | 70° 54′| 51° 42′| 44° 51′| 55,918| 60,612| 73,659 | ·336| 1·724
0·56 | 68° 6′| 49° 48′| 43° 14′| 53,990| 58,522| 71,119 | ·314| 1·786
0·54 | 65° 22′| 47° 54′| 41° 37′| 52,061| 56,432| 68,579 | ·292| 1·852
0·52 | 62° 40′| 46° 2′| 40° 0′| 50,133| 54,342| 66,039 | ·270| 1·923
0·50 | 60° 0′| 44° 10′| 38° 24′| 48,205| 52,252| 63,499 | ·250| 2·000
0·45 | 53° 30′| 39° 33′| 34° 27′| 43,385| 47,026| 57,149 | ·203| 2·222
0·40 | 47° 9′| 35° 0′| 30° 31′| 38,564| 41,801| 50,799 | ·160| 2·500
0·35 | 40° 58′| 30° 30′| 26° 38′| 33,744| 36,576| 44,449 | ·123| 2·857
0·30 | 34° 56′| 26° 4′| 22° 46′| 28,923| 31,351| 38,099 | ·090| 3·333
0·25 | 28° 58′| 21° 40′| 18° 56′| 24,103| 26,126| 31,749 | ·063| 4·000
0·20 | 23° 4′| 17° 18′| 15° 7′| 19,282| 20,901| 25,400 | ·040| 5·000
=====+========+========+=======+========+========+========+======+======
(1) Numerical Aperture. (_n_ sin _u_ = _a_.)
(2) _Air_ (_n_ = 1·00).
(3) _Water_ (_n_ = 1·33).
(4) _Homogeneous Immersion_ (_n_ = 1·52).
(5) White Light. (λ = 0·5269 μ, Line E.)
(6) Monochromatic (Blue) Light.(λ = 0·4861 μ, Line F.)
(7) Photography. (λ = 0·4000 μ, Near Line _h__k_.)
(8) Illuminating Power (a^2.)
(9) Penetrating Power (1/a.)
Abbe’s Apertometer.
[Illustration: Fig. 39.--Abbe’s Apertometer.]
The apertometer is an auxiliary piece of apparatus invented by Abbe,
for testing the fundamental properties of objectives and determining
their numerical and angular apertures. This accessory of the microscope
involves the same principles as that of Tolles, which the late Mr. J.
Mayall and myself brought to the notice of the Royal Microscopical
Society of London in 1876. Abbe’s apertometer (Fig. 39) consists of a
flat cylinder of glass, about three inches in diameter, and half an
inch thick, with a large chord cut off, so that the portion left is
somewhat more than a semicircle; the part where the segment is cut
is bevelled from above downwards, to an angle of 45°, and it will be
seen that there is a small disc with an aperture in it denoting the
centre of the semicircle. To use this instrument the microscope is
placed in a vertical position, and the apertometer is placed upon the
stage with its circular part to the front and the chord to the back.
Diffused light, either from the sun or lamp, is assumed to be in front
and on both sides. Suppose the lens to be measured is a dry one-quarter
inch; then with a one-inch eye-piece having a large field, the centre
disc, with its aperture on the apertometer, is brought into focus.
The eye-piece and the draw-tube are now removed, leaving the focal
arrangement undisturbed, and a lens supplied with the apertometer is
screwed into the end of the draw-tube. This lens, with the eye-piece
in the draw-tube, forms a low-power compound microscope. This is now
inserted into the body-tube, and the back lens of the objective whose
aperture we desire to measure is brought into focus. In the image of
the back lens will be seen stretched across, as it were, the image of
the circular part of the apertometer. It will appear as a bright band,
because the light which enters normally at the surface is reflected
by the bevelled part of the chord in a vertical direction, so that in
reality a fan of 180° in air is formed. There are two sliding screens
seen on either side of the figure of the apertometer; they slide on
the vertical circular portion of the instrument. The images of these
screens can be seen in the image of the bright bands. _These screens
should now be moved so that their edges just touch the periphery of the
back lens._ They act, as it were, as a diaphragm to cut the fan and
reduce it, so that its angle just equals the aperture of the objective
and no more.
This angle is now determined by the arc of glass between the screens;
thus we get an angle in _glass_ the exact equivalent of the aperture of
the objective. As the numerical apertures of these arcs are engraved
on the apertometer, they can be read off by inspection. A difficulty
is not infrequently experienced from the fact that it is not easy to
determine the exact point at which the edge of the screen touches
the periphery of the back lens, or rather the limit of the aperture.
Zeiss, to meet this difficulty, made a change in the form of the
apparatus--furnished a glass disc mounted on a metal plate, with a slot
for the purpose of its more accurate adjustment.[17]
Stereoscopic Binocular Vision.
Professor Wheatstone’s remarkable discovery of stereoscopic vision
led, at no distant period, to the application of the principle to
the microscope. It may therefore prove of interest to inquire how
stereoscopic binocular vision is brought about. Indeed, the curious
results obtained in the stereoscope cannot be well understood without
a previous knowledge of the fundamental optical principles involved
in this contrivance, whereby two slightly dissimilar pictures of any
object become fused into one image, having the actual appearance of
relief. The invention of the stereoscope by Sir Charles Wheatstone,
F.R.S., 1838, and improved by Brewster, was characterised by Sir John
Herschel as “one of the most curious discoveries, and beautiful for
its simplicity, in the entire range of experimental optics,” led to
a more general appreciation of the value of the conjoint use of both
eyes in conveying to the mind impressions of the relative form and
position of an object, such as the use of either eye singly does not
convey with anything like the same precision. When a near object having
three dimensions is looked at, a different perspective representation
is seen with each eye. Certain parts are seen by the right eye, the
left being closed, that are invisible to the left eye, the right
being closed, and the relative positions of the portions visible
to each eye in succession differ. These two visual impressions are
simultaneously perceived by both eyes, and combined in the brain into
one image, producing the effect of perspective and relief. If truthful
right-and-left monocular pictures of an object be so presented to the
two eyes that the optic axes when directed to them shall converge at
the same angle as when directed to the object itself, a solid image
will be at once perceived. The perception of relief referred to is
closely connected with the doubleness of vision which takes place
when the images on corresponding portions of the two retina are not
similar. But, if in place of looking at the solid object itself we look
with the right and left eyes respectively at pictures of the object
corresponding to those which would be formed by it on the retina of the
two eyes if it were placed at a moderate distance in front of them, and
these visual pictures brought into coincidence, the same conception of
a solid form is generated in the mind just as if the object itself were
there.
Professor Abbe, however, contended that the method by which dissimilar
images are formed in the binocular microscope differs materially from
that of ordinary stereoscopic vision, and that the pictures are united
solely by the activity of the brain, not by the prisms which ordinarily
give rise to sensations of _solidity_. This can be only partially true,
as binocularity in the microscope is due to difference of projection
exhibited by the different parallax displacement of the images, and
also to the perception of depth imparted by the instrument.
Wheatstone was firmly convinced that his stereoscopic principle could
be applied to the microscope, and he therefore applied first to Ross
and then to Powell to assist him in its adaptation. But whether either
of these opticians made any attempt to give effect to his wishes
and suggestions is not known. In the year 1851 Professor Riddell, of
America, succeeded in constructing a binocular microscope by employing
two rectangular prisms behind the objective. M. Nachet also constructed
a binocular with two body-tubes and a series of prisms. But neither
Riddell’s nor Nachet’s instrument was ever brought into use; they were
either too complicated or too costly.
It will be understood, however, that the binocular stereoscope combines
two dissimilar pictures, while the binocular microscope simply enables
the observer to look with both eyes at images which are essentially
identical. Stereoscopic vision, to be effective, requires that the
delineating pencil shall be equally separated, so that one portion
of the admitted cone of light is conducted to one eye, and the other
portion to the other eye.
Select any object lying in an inclined position, and place it in the
centre of the field of view of the microscope; then, with a card held
close to the object-glass, stop off alternately the right or left
hand portion of the front lens: it will then appear that during each
alternate change certain parts of the object will change their relative
positions.
[Illustration: Fig. 40.--Portions of Eggs of Cimex.]
To illustrate this, Fig. 40 _a_, _b_ are enlarged drawings of a portion
of the egg of the common bed-bug (_Cimex lecticularis_), the operculum
which should cover the opening having been forced off at the time the
young was hatched. The figures exactly represent the two positions that
the inclined orifice will occupy when the right- and left-hand portions
of the object-glass are stopped off. This object is viewed as an
opaque object, and drawn under a two-thirds object-glass of about 28°
aperture. If this experiment is repeated, by holding the card over the
eye-piece, and stopping off alternately the right and left half of the
ultimate emergent pencil, exactly the same changes and appearances will
be observed in the object under view. The two different images just
produced are such as are required for obtaining stereoscopic vision.
It is therefore evident that if instead of bringing them confusedly
together into one eye we can separate them so as to bring together
_a_, _b_ into the left and right eye, in the combined effect of the
two projections we obtain at once all that is necessary to enable us
to form a correct judgment of the solidity and distance of the several
parts of the object.
Nearly all objectives from the one inch upwards of any considerable
aperture give images of the object seen from a different point of
view with the two opposite extremes of the margin of the cone of
rays; the resulting effect is that there are a number of dissimilar
perspectives of the object blended together at one and the same time on
the retina. For this reason, if the object under view possesses bulk,
a more accurate image will be obtained by reducing the aperture of the
objective.
[Illustration: Fig. 41.]
Diagram 3, Fig. 41, represents the method employed by Mr. Wenham for
bringing the two eyes sufficiently close to each other to enable them
both to see through the double eye-piece at the same moment. _a a a_
are rays converging from the field lens of the eye-piece; after passing
the eye-lens _b_, if not intercepted, they would come to a focus at
_c_; but they are arrested by the inclined surfaces, _d d_, of two
solid glass prisms. From the refraction of the under incident surface
of the prisms, the focus of the eye-piece becomes elongated, and falls
within the substance of the glass at _e_. The rays then diverge, and
after being reflected by the second inclined surface _f_, emerge from
the upper side of the prism, when their course is rendered still more
divergent, as shown by the figure. The reflecting angle given to the
prisms is 47-1/2°, to accommodate which it is necessary to grind away
the contact edges of the prisms, as represented, otherwise they
prevent the extreme margins of the reflecting surfaces from coming into
operation, which are seldom made quite perfect.
[Illustration: Fig. 42.--Professor Abbe’s Stereoscopic Eye-pieces.]
Fig. 42 represents a sectional view of Abbe’s stereoscopic eye-pieces,
which consist of three prisms of crown glass, _a_, _b_ and _b′_, placed
below the field-glass of the two eye-pieces; the tube _c_ is slipped
into the tube or body like an ordinary eye-piece. The two prisms _a_
and _b_ are united so as to form a thick plate with parallel sides,
inclined to the axis at an angle of 38·5°. The cone of rays from
the objective is thus divided into two parts, one being transmitted
and the other reflected; that transmitted passing through _a b_ and
forming an image of the object in the axial eye-piece B. Adjustment
for different distances between the eyes is effected by the screw
placed to the right-hand side of the figure, which moves the eye-piece
B′, together with the prism _b′_, in a parallel direction. The tubes
can also be drawn out, if greater separation is required. The special
feature of this instrument is that on halving the cone of rays by
turning the caps, an orthoscopic or pseudoscopic effect is produced.
This double-eyed piece arrangement of Abbe’s has not been at all
brought into use in this country; this is partly owing to its original
adaptation for use with the shorter Continental body-tube of 160 mm.,
and not for our 10-inch body.
The most perfect method of securing pleasing satisfactory stereoscopic
vision of objects is that devised by Mr. Wenham. In his binocular
microscope an equal division of the cone of rays, after passing through
the objective is secured and again united in the eye-pieces, which
act as one, so that each eye is furnished with an appropriate and
simultaneous view of the object. The methods contrived by the earlier
experimenters not only materially interfered with the definition of
the objective and object, but also required expensive alterations and
adaptations of the microscope, and sometimes separate stands for their
employment. Mr. Wenham’s invention, on the contrary, offers no such
obstacle to its use, and the utility of the microscope as a _monocular_
is in no way impaired either when using the higher powers.
[Illustration: Fig. 43.]
The most important improvement, then, effected by Wenham consists in
the splitting up or dividing the pencil of rays proceeding from the
objective by the interposition of a prism of the form shown in Fig.
43. This is placed in the body or tube of the microscope so as to
interrupt only one-half (_a c_) of the pencil, the other half (_a b_)
proceeding continuously to the field-glass, eye-piece, of the principal
body. The interrupted half of the pencil on its entrance into the
prism is subjected to very slight refraction, since its axial ray is
perpendicular to the surface it meets. Within, the prism is subjected
to two reflections at _b_ and _c_, which send it forth again obliquely
on the line _b_ towards the eye-piece of the secondary body, to the
left-hand side of the figure; and since at its emergence its axial
ray is again perpendicular to the surface of the glass, it suffers no
further refraction on passing out of the prism than on entering. By
this arrangement, the image sent to the right eye is formed by rays
which have passed through the left half of the objective; whilst the
image sent to the left eye is formed by rays which have passed through
the right half, and which have been subjective to two reflections
within the prism, and passing through two surfaces of glass. The prism
is held by the ends only on the sides of a small brass drawer, so that
all the four polished surfaces are accessible, and should slide in so
far that its edge may just reach the central line of the objective, and
be drawn back against a stop, so as to clear the aperture of the same.
[Illustration: Fig. 44.--Sectional view of the Wenham Binocular.]
The binocular, then (Fig. 44), consists of a small prism mounted in
a brass box A, which slides into an opening immediately above the
object-glass, and reflects one-half of the rays which form an image of
the object, into an additional tube B, attached at an inclination to
the ordinary body C. One half of the rays take the usual course with
their performance unaltered; and the remainder, though reflected twice,
show no loss of light or definition worthy of notice, if the prism be
well made.
As the eyes of different persons are not the same distance apart, the
first and most important point to observe in using the binocular is
that each eye has a full and clear view of the object. This is easily
tried by closing each eye alternately without moving the head, when
it may be found that some adjustment is necessary by racking out the
draw-tubes D, E, of the bodies by means of the small milled head near
the eye-pieces; this will increase the distance of the centres; and, on
the contrary, the tubes, when racked down, will suit those eyes that
are nearer together.
If the prism be drawn back till stopped by the small milled head, the
field of view in the inclined body is darkened, and the rays from the
whole aperture of the object-glass pass into the main body as usual,
neither the prism nor the additional body interfering in any way with
the use of the instrument as a monocular microscope.
The prism can be withdrawn altogether for the purpose of being wiped:
this should be done frequently, and very carefully, on all four
surfaces, with a perfectly clean cambric or silk handkerchief or a
piece of wash-leather; but no hard substance must be used. During this
process the small piece of blackened cork fitted between the prism and
the thick end of the brass box may be removed; but it must be carefully
replaced in the same position, as it serves an important purpose in
stopping out extraneous light.
As the binocular microscope gives a real and natural appearance to
objects, this effect is considerably increased by employing those
kinds of illumination to which the naked eye is accustomed. The most
suitable are all the opaque methods where the light is thrown down
upon the surface; but for those objects that are semi-transparent, as
sections of bone or teeth, diatomaceæ, living aquatic animalcules, &c.,
the dark-field illumination by means of the parabolic reflector will
give an equally good result.
For perfectly transparent illumination, it is much better to diffuse
the light by placing under the object various substances, such as
tissue-paper, ground glass, very thin porcelain, or a film of yellow
bees’ wax, run between two pieces of thin glass.
To ensure the full advantage and relief to both eyes in prolonged
observations with high as well as low powers, and with objectives of
large aperture, Mr. Wenham devised a compound prism for use with his
binocular microscope, the body tubes of which are also made expressly
to suit the prism, as extreme accuracy is necessary to bring them
into proper position. The main prism somewhat resembles in form the
ordinary Wenham prism. Over the first reflecting surface is placed a
second smaller prism, the top plane of which is parallel with the base
of the first, so that direct rays pass through without deviation, but
at the two inclined surfaces of the prisms (nearly in contact) there
is a partial reflection from each, which, combined, give as much light
as in the direct tube. The reflected image from these two surfaces
is directed up into the inclined tube as usual. A somewhat later
improvement is that of Dr. Schroeder, the high power prism, by means of
which the whole of the rays emanating from the objective pass through
it, and the full aperture of any power is thereby effectively utilised.
Furthermore, Messrs. Ross have also constructed a right- and left-hand
pair of eye-pieces, which ensure greater perfection of the image. It
was, in fact, noticed that the size of the image in the left-hand field
glass slightly differed from that of the right when examined by the
ordinary Huyghenian eye-pieces. To compensate for this difference, the
left-hand eye-piece has been carefully calculated, and its focus is
now so accurately adjusted that the position of each eye in observing
is brought into one plane of the binocular. The pairs of the several
series of eye-pieces A, B and C have also been altered, and the effect
is to greatly improve the image and give increased comfort to the
observer.
Dr. Carpenter, who warmly espoused the binocular, and constantly
employed it in his work, very truly said of it: “The important
advantages I find it to possess are in penetrating power, or focal
depth, which is in every way superior to that of the monocular
microscope, so that an object whose surface presents considerable
inequalities is very much more distinctly seen with the former than
with the latter.”
This difference may in part be attributed to the practical modification
in the angle of aperture of the objective, produced by the division
of the cone of rays transmitted through the two halves, so that the
picture or image received through each half of the objective of 60° is
formed by rays diverging at an angle of only about 30°. He confesses,
however, that this does not satisfactorily explain the fact that the
binocular brings to the _mind’s_ eye the _solid_ image of the object,
and thus gives to the observer a good idea of its form and which could
hardly be obtained by the monocular microscope. Carpenter cites in
support of his views the wing of a little-known moth, _Zenzera Œsculi_,
which has an undulating surface, whereon the scales are set at various
angles instead of having the usual imbricated arrangement, a good
object for demonstrating; the general inequality of surface and the
obliquity of its scales, which are at once seen by the binocular with a
completeness not obtained by the monocular instrument.
To one unaccustomed to work with the binocular the views expressed by
Dr. Carpenter as to the extreme value of the instrument for ordinary
work may appear somewhat exaggerated, but from my own experience,
having long had in constant use a Ross-Zentmayer binocular, furnished
with a special prism, constructed for working with a 1/8 dry objective
or a 1/10 immersion, the perfection of picture obtained was in every
case quite equal to that of the monocular microscope. The relief to the
eyes can hardly be over-estimated; the slight inequality of the pencil
rays may be regarded rather as a part of the welcome rest afforded when
a prolonged examination is made; it certainly appears to me to equalise
the slight physiological difference known to exist between the eyes
of most people. If one image is seen a little clearer by the stronger
eye, the weaker eye assists rather more the stereoscopic effect of the
object under observation. The advantage gained by the binocular is
perhaps more appreciated when opaque objects are under examination,
as the eggs of insects, and the tongue of the blow-fly, specimens of
mosses, lichens, parasites (vegetable and animal), whose planes and
inequalities of surface require penetration, and which usually demand
more time for their observation.
[Illustration: Fig. 45.--Swift-Stephenson’s Erecting Binocular.]
No variation or change of any kind proposed either in the form of the
instrument or the prism has proved of sufficient value or importance
to bring it into use, and therefore Wenham’s instrument is scarcely
likely to be superseded. It must be admitted that the improvement
effected in the eye-piece form by Mr. Tolles, of Boston, U.S., is
an exception to the rule laid down. It consists in mounting the
prisms in a light material, vulcanite, made to fit into the monocular
microscope body, thus taking the place of the ordinary eye-piece.
The image transmitted by the objective is brought to a focus on the
face of the first equilateral triangular prism by the intervention of
an erector-eye-piece inserted beneath it. The second set of prisms
have a rack and pinion movement to adjust them to any visual angle.
The illumination of both fields in this eye-piece is nearly equal in
brightness. Mr. Stephenson’s erecting binocular (Fig. 45) has proved
to be of some practical value. It has the advantage of being of equal
use with high and low powers, and with little loss of definition. When
used for dissecting purposes it gives an erect image of the object.
It is equally useful as a working microscope, for arranging diatoms
and botanical specimens of every kind. The sub-stage tube will receive
a diaphragm or illuminating apparatus; the eye-pieces have a sliding
adjustment for regulating the widths between eyes.
[Illustration: Fig. 46.--An early form of the Ross-Wenham Binocular;
nose-piece and prism-holder detached.]
CHAPTER II.
Simple and Compound Microscope.
Microscopes are known as simple and compound. The simple microscope
may, for convenience, be divided into two classes; those used in the
hand (hand magnifiers), and those provided with a stand (mounted, as
it is termed) for supporting the object to be viewed, together with an
adjustment for the magnifying power, and a mirror for reflecting the
light through the object.
[Illustration: Fig. 47.--Visual Angle.]
A _simple microscope_, mounted, is preferable to a single lens, being
usually composed of two or more lenses separated by a small distance
on a common axis; the increase of the size of an object being the
angle it subtends to the eye of the observer, or the angle formed by
the combination drawn from the axis of vision to the extremity of the
object, as in Fig. 47. The lines drawn from the eye to a and r form
an angle, which, when the distance is small, is nearly twice as large
as the angle from the eye to o w, formed by lines drawn at twice the
distance. This is called the angle of vision, or the visual angle.
Now, the utility of a convex lens interposed between a near object and
the eye consists in its reducing the divergence of the rays forming
the several pencils issuing from it, so that they enter the eye in a
state of moderate divergence, as if they were issuing from an object
beyond the near point of distinct vision, and a well-defined image is
thereby formed upon the retina. In the next Fig. (48), a double-convex
lens illustrates the action of the _simple microscope_, the small
arrow being the object brought under view, and the large arrow the
magnified image. The rays having first passed through the lens are
bent into nearly parallel lines, or pencils diverging from some point
within the limits of distinct vision. Thus altered, the eye receives
rays precisely as if they had emanated directly from a larger arrow
placed about ten inches away from it. The difference between the real
and the imaginary object represents the magnifying power of the lens.
The object in this case is magnified nearly in the proportion the focal
distance of the lens bears to the distance of the object when viewed by
the unassisted eye; and this is due to the object being more distinctly
viewed so much nearer to the eye than it otherwise could be without the
lens.[18]
[Illustration: Fig. 48.--Virtual Image formed by Convex Lens.]
It should be remembered that the shorter the focus and the nearer the
eye the magnifying lens is placed the smaller will be the diameter
of the sphere of which it forms a part, and unless its aperture be
proportionally reduced, the distinctness of the image will be destroyed
by the spherical and chromatic aberrations of its high curvature.
Nevertheless, it was by the use of lenses so constructed that the older
microscopists--of whom Leeuwenhoek was the more eminent--were enabled
to do so much excellent work.
The various kinds of simple pocket lenses for the most part consist of
a double-convex, or a plano-convex, or a combination of both, varying
in focal length from a quarter of an inch to two inches. Sometimes
they are set in pairs with a hole, a small diaphragm, cut in the piece
of horn placed between them. These are extremely useful for carrying in
the waistcoat pocket; to the anatomist and field botanist for examining
various objects and preparations.
[Illustration: Fig. 49.--Wollaston’s Doublet.]
Perhaps the most important improvement effected in this form of the
simple microscope was that ascribed to the celebrated Dr. Wollaston,
who devised a doublet of two plano-convex lenses having their focal
lengths, in the proportion of one to three, mounted with their convex
side directed towards the eye of the observer, and the lens of shorter
focal length next the object. The explanation given of the correction
thus effected in Dr. Wollaston’s doublet will be best understood
on reference to the annexed diagram, _l l′_, in Fig. 49, being the
object for a segment of the cornea of the eye, and _d d′_ the stop or
diaphragm. Now, it will be seen that each pencil of light proceeding
from _l l′_, the object, is rendered excentrical by the limiting
aperture or the diaphragm _d d_; consequently, they pass through the
lenses on opposite sides of their common axis _o p_; thus each becomes
affected by opposite errors, which to some extent balance and correct
each other. To take the pencil _l_, for instance, as it enters the eye
at _r b_; _r b_ is bent to the right at the first lens, and to the left
at the second; and as each bending alters the direction of the blue
ray more than the red, and as the blue ray falls nearer the margin of
the second lens, where the refraction is greater than that nearer the
centre, and compensates to some extent for the greater focal length of
the second lens, the blue rays will emerge very nearly parallel, and
colourless to the eye. At the same time, its spherical aberration has
been diminished, since the side of the pencil as it proceeds through
one lens passes nearer the axis, and in the other nearer the margin.
This must be taken to apply to pencils farthest from the centre of
the object. Central rays, it is obvious, would pass both lenses
symmetrically, the same portions of rays occupying nearly the same
relative places in both lenses. The blue ray would enter the second
lens nearer its axis than the red; and being thus less refracted than
the red by the second lens, some amount of compensation would take
place, differing in principle, and inferior in degree, to that which
is found in the excentrical pencils. In the intermediate spaces the
corrections are still more imperfect and uncertain; and this explains
the cause of aberrations which must of necessity exist even in the
best-made doublet. It is, however, infinitely superior to a single
lens, and will transmit a pencil of an angle of from 35° to 50°.
The next step towards improving the simple microscope was in relation
to the eye-piece, and was effected by Holland. It consisted in
substituting two lenses for the first in the doublet, and placing a
stop between them and the third. The first bending of the pencils
of light being effected by two lenses instead of one, produces less
spherical and chromatic aberration, which are more nearly balanced or
corrected at the second bending, and in the opposite direction, by the
third lens.
Another form of simple lens was devised by Dr. Wollaston, the
“Periscopic.” This combination consists of two hemispherical lenses
cemented together by their plane faces, with a stop between them to
limit the aperture. A similar proposal, made by Sir David Brewster
in 1820, is known as the Coddington lens,[19] shown at Fig. 50:
this has a somewhat larger field, and is equally balanced in all
directions, as is made evident, the pencils _a b_ and _b a_ passing
through under precisely the same circumstances. Its spherical form
has the further advantage of rendering the position in which it is
held of comparatively little consequence. It is still used as a hand
magnifier, although its definition is certainly not so good as that
of a well-made doublet. It is usually set in a folding case, as
represented in the figure, and so contrived as to be admirably adapted
for the waistcoat-pocket. It is usually sold with the small _holder_,
Fig. 50_a_, for holding and securing small objects during examination.
Browning’s Platyscopic Pocket Lens is a useful form of pocket lens for
the botanist and mineralogist. Its focus is nearly three times longer
than that of the Coddington, and allows of opaque objects being more
easily examined; it has also a magnifying power of 15, 20, and 30
diameters.
[Illustration: Fig. 50.--The Coddington Lens.]
[Illustration: Fig. 50_a._]
One of the best combinations of the hand or pocket form of lens is
that known as _Steinheil’s aplanatic lens_ (Fig. 51); it consists
of a bi-convex lens cemented between two concavo-convex lenses,
giving a relatively long focal distance and a large flat field. The
higher powers of this lens are much used for dissecting purposes.
This handy magnifier appears to have suggested a later combination,
the apochromatic of Zeiss. No hand lens can compare with Steinheil’s
“_loups_.”
[Illustration: Fig. 51.--Steinheil’s Aplanatic Lens.]
[Illustration: Fig. 52.--Simple Microscope.]
When the magnifying power of a lens is considerable, or when its focal
length is short, or it is wished to use it with greater precision and
steadiness, it should be mounted on a short stand with a tubular stem,
with rack-work focussing movement and mirror illumination. Fig. 52
represents a simple dissecting microscope, with a glass circular stage,
4-1/2 inches in diameter, supported on three legs--a handy and useful
form of instrument for many purposes.
The Compound Microscope.
The compound microscope differs from the simple, inasmuch as the image
is formed by an object-glass, and further magnified by one or more
lenses forming an eye-glass. For a microscope to be a compound one,
its essential qualification is that it should have an object-glass
or objective, and an eye-glass or eye-piece, so called because they
are respectively near the object and the eye of the observer when the
instrument is in use. The microscope consists of a tube or _body_,
and a _stand_, an arrangement for carrying the _body_, combined with
which is a _stage_ for holding the object, and a _mirror_ for its
illumination. To the more modern instrument has been added a substage,
to carry a condenser and other accessories.
The _body_ of a microscope, which carries the system of magnifying
lenses, must be placed at one particular distance from the object,
termed the _focus_, in order that a clear image may be obtained. For
the purpose of _focussing_ two motions are supplied, the one for
_coarse adjustment_, with lower powers; the other for higher powers,
termed the _fine adjustment_. It is in this wise that the magnifying
power of the compound microscope is turned to good account.
There are, however, limits to the use to which lenses can be put with
advantage in the direction of magnifying the object, just as there are
in varying the magnifying power of the eye-glass. Defects in either,
although not first seen, that is, when the image is but moderately
enlarged, are brought into prominence by greater amplification. In
practice, therefore, it is found to be of advantage to vary the power
by employing object-glasses of different values (foci). In whatever
way increase of amplification is brought about, two things will always
result from the change: the proportion of surface of the object of
which an image can be formed must be diminished, and the amount of
light spread over the image proportionally lessened.
In addition to the two lenses mentioned, it was found to be of
considerable advantage to introduce a third lens between the
object-glass and the image formed by it at eye-piece, the purport of
which is to change the course of the rays (bend in the pencil) so that
the image may not be found of too great a dimension for the whole to
be brought within the circumference of the eye-glass. This, it will be
readily seen, allows more of the object to be viewed at the same time
by the _field-glass_, as the eye-piece of the microscope is termed.
[Illustration: Fig. 53.]
Fig. 53 represents the body of an ordinary compound microscope with
its triplet object-glasses; o is an object, above it is the triple
achromatic object-glass, in connection with the eye-piece _e e, f
f_ the plano-convex lenses; _e e_ being the eye-glass, and _f f_
the field-glass, between which, at _b b_, the arrow represents the
diaphragm. The course of the light is shown by three rays drawn from
the centre, and three from each end of the object _o_; these rays,
if not prevented by the lens _f f_, and the diaphragm _b b_, would
form an image at _a a_; but here, as they meet with the lens _f f_ in
their passage, are converged by it at _b b_, the diaphragm at _b b_
intercepting a portion of peripheral rays, permitting only those to
pass that are necessary for the formation of the image, the further
magnification of which is, however, here brought about by the eye-glass
_e e_, precisely as if it were that of the original object under
examination. It will be apparent, then, that the field-lens _f f_
belongs in principle to the object-glass, or objective, taking a share
in the image-forming rays, although this is taken to be a part of the
eye-piece.
Evolution of the Modern Achromatic Microscope.
The great advances made in the optical arrangements of the modern
microscope necessitated important changes and improvements in its
several mechanical parts. Indeed, as the apertures of objectives became
increased, and focal planes became correspondingly shallower, it was
absolutely necessary to apply a more sensitive system of focussing than
that for many years past commonly in use. The leading manufacturers
at once grasped the situation, and in a short space of time the older
model microscopes were discarded, and replaced by instruments better in
workmanship and finish, and in every way more suitable for the student
and the promotion of original scientific research.
From an early period English amateurs appear to have bestowed greater
attention on the improvement of the microscope than those of any other
country. Between 1820 and 1835 Tully, Pritchard, Dolland, James Smith,
Andrew Ross, and Hugh Powell, encouraged by Wollaston, Brewster,
Goring, Herschel, and Lister, worked out innumerable combinations
of single and compound lenses to be employed as simple microscopes,
explained in a previous chapter.
The theories propounded about this time for the improvement of lenses
and the various combinations for amateurs were not of lasting value.
Nevertheless, they were not wholly made in vain, as during the last
twenty years they have indirectly borne good fruit, inasmuch as by
working in another direction Professor Abbe was led to the discovery
of new and better kinds of glass, by which the secondary spectrum has
been so nearly eliminated, and the optical parts of the microscope
so materially improved. In pursuing this subject I would not have it
supposed that Continental opticians were either idle or supine. On the
contrary, Oberhäuser, Fraunhofer, Chevalier, Nachet, Hartnach, and
others took an active part in the work.
The compound microscope made for anatomists by the first-named
optician about 1825 has not been entirely superseded. He was the
first to make a rotating stage, to apply mechanism to focussing, and
to introduce the system of direct push or pull of the condenser tube
within the sub-stage socket. Nachet made other improvements on the
Oberhäuser microscope by applying under the stage a tail-piece having a
dove-tailed groove in which a slide carrying the sub-stage was moved by
a stud-pin. More recently the lever movement was superseded by American
opticians, who made other changes. Hartnach ultimately very much
improved Oberhäuser’s model, and this remains with us.
The English modern compound microscope, together with the achromatic
objective, we owe to a mind teeming with scientific inventions,
Joseph Jackson Lister, F.R.S., who in 1826 supplied Mr. Tully, a
well-known London optician of that period, with original drawings for
the important improvements in its mechanical details and accessory
apparatus which followed so soon afterwards.
Among the many ingenious novelties enumerated in his published papers
we find the graduated lengthening of the body-tube of the microscope; a
stage-fitting for clamping and rotating the object; a subsidiary stage;
a dark-well, and a large disc to incline and rotate opaque objects;
a ground-glass light moderator; a live-box with bevelled flat-glass
plate; an erector-eye-piece; an adapter for using Wollaston’s camera
lucida for microscopical drawing; and, above all, a combination of
lenses to act as a condenser under the object (evidently the first
approach to the present achromatic sub-stage condenser). The value of
the erector-eye-piece for facilitating dissections under the microscope
is not even yet sufficiently appreciated. Tully published a descriptive
account of Lister’s microscope, the first one of which he made, and
acknowledged his indebtedness to “Mr. Lister’s ingenuity and skill.”
Shortly afterwards Lister made known his discovery of the two aplanatic
foci in a double achromatic object glass, and gave verbal directions
to the three principal makers of microscopes in London, James Smith,
Andrew Ross, and Hugh Powell, for the future construction of the
achromatic objective, all of whom were intent on the improvement of
their several models. To the latter the Society of Arts awarded, in
1832, a medal for his improved mechanical stage movements, on the
“Turrell system,” which Powell first constructed for Edmund Turrell.
This stage was made to rotate completely on its optic axis by means of
an obliquely-placed pinion acting on a bevelled rack on the inner face
of the stage-ring supporting the mechanism. In 1834 Powell once more
received a Society of Arts medal, “the Iris,” for improvements in the
application of a new form of fine adjustment.
About the same date (1835) Andrew Ross introduced the socket-carrier of
the body-tube of the microscope on a strong stem, with rack bent in the
middle, thus affording space for a larger stage. He likewise devised
the hollow cross-bar, placed at right angles to the rack-stem, whereby
he was enabled to use a new system of fine adjustment, consisting of a
delicate screw with large milled head, acting by a point on the long
arm of a lever, the short arm of which ends in a fork in contact with
a stud placed on either side of a cylindrical sliding tube forming the
nose-piece of the body-tube, and into which the objective is screwed.
A spiral spring presses down the nose-piece, and against this the screw
and lever act.
This appears to have been the first really sensitive focussing method
applied to the nose-piece; it was, and probably is, one of the most
delicate systems ever applied to the microscope. It has enjoyed a
long period of popularity, and I believe it still survives in Powell
and Lealand’s instruments, which are very generally admitted to be
of superior excellence for all purposes where extreme delicacy of
focussing is an essential element.
The rival system of fine adjustment--the short lever and screw applied
externally to the body-tube--known as the Lister-Jackson system, which
appears to have been contrived to allow the body-tube to be supported
more firmly on the limb or stem, has had its merits ably realised in
the microscopes of Smith and Beck and their successors, but, except as
modified by the successors of Andrew Ross (Schrœder’s form), it is, I
believe, admitted that it has been superseded by other modifications
lately introduced into the Ross-Jackson instrument.
The year 1830 was, however, a propitious period in the history of the
modern microscope, as in January of that year Mr. Lister published his
epoch-making paper, “On the Improvement of the Achromatic Microscope.”
This appeared together with certain personal practical directions (for
no man was ever more anxious to communicate his knowledge than Mr.
Lister) to the before-mentioned opticians, which led up to changes
lasting until 1840, when, by the efforts of this gentleman and his
personal friends, “The Microscopical Society of London” came into
existence. Among the more prominent members of the Society was Mr.
George Jackson, a name still well known to microscopists, and who,
jointly with Mr. Lister, gave us the Jackson-Lister form of microscope.
This was forthwith accepted as a perfect model. Soon after Andrew
Ross effected a further change in the instrument, shown in Fig. 54 in
its complete form as left by this optician. It is here represented as
having a bar movement, with a claw foot bolted to two uprights to carry
the trunnions with the body and stage. This base, is insufficiently
wide and extended to carry so large an instrument with its centre of
gravity so high. The coarse adjustment bar also was rectangular, and
the fine adjustment a lever, with the milled head in the middle of
the bar, which involved a certain amount of tremor; withal it was an
instrument of excellent workmanship, and its defects were not regarded
as irremediable. Messrs. Ross, however, preferred to construct an
entirely new model designed by Zentmayer, the “Ross-Jackson-Zentmayer,”
to which I shall refer presently. A later model, however, has to
some extent taken its place, “the Histological and Bacteriological
Microscope,” Fig. 55.
[Illustration: Fig. 54.--An early Ross-Jackson Microscope.]
My reference to the older form of instrument is chiefly with the view
of directing attention to the sensitive focussing system, applied in
the first instance to the nose-piece; now placed below the coarse
adjustment. It certainly is a delicate form of fine adjustment. This
model possesses other points of interest well worth preserving, which
fully entitle it to occupy the prominent place given in the list of the
house of Ross. In the Ross-Jackson “Histological and Bacteriological
Microscope” much attention seems to have been given to eliminate
certain weak points in the earlier Ross-Jackson model--defects still
extant in stands of certain English and foreign makers--while retaining
the more practical improvements of both constructions. Steadiness is
secured by an extension of the tripod or claw-foot and the shorter and
more solid uprights that sustain the whole weight of the instrument.
[Illustration: Fig. 55.--The Ross-Jackson Histological Microscope.]
[Illustration:
Fig. 56.--Powell and Lealand’s Students’ Microscope, with Amici prism
arranged for oblique illumination, the Sub-stage and Condenser being
detached.]
The Ross-Jackson, then, survives, together with the original tripod
stand of Hugh Powell’s, upon which he expended all the resources of the
practical optician, and applied the early principles involved in the
Lister-Jackson instrument, but from different points of view. However,
there is hardly a choice between one and the other in workmanship, both
opticians having furnished microscopes of a typical class and very high
order. The firm of Powell and Lealand have but one form of stand, from
which they have never been tempted to deviate. It is supported on a
true tripod base, forming a solid and substantial support to the body,
which is of such a length as to give as nearly as possible the standard
optical interval of 10 inches between the posterior principal focus of
the objective and the anterior focus of the eye-piece; the variation
in the optical tube length does not exceed a quarter of an inch with
objectives of 1/2 inch and upwards. The arm on which the body is fixed
is 5-3/4 inches long, which not only gives a clearance of 3-1/2 inches
from the optic axis, but also permits of the introduction of a long
fine-adjustment lever.
[Illustration: Fig. 57.--Powell’s larger No. 2 Instrument.]
[Illustration:
Fig. 58.--Powell and Lealand’s Students’ Microscope arranged for direct
illumination. _A._ Secondary or Sub-stage racked up to bring the
Achromatic Condenser close to the object.]
The cross arm encloses the lever mechanism for the fine adjustment,
as originally devised by Andrew Ross. This cross arm is longer than
that used by Ross, and carries the body more forward, so as to provide
radial space for the complete rotation of the stage and the optic axis,
and at the same time the lever of the adjustment is lengthened, and
delicacy of motion secured. The stage retains the mechanical movements
invented by E. Turrell, and first applied by Hugh Powell. It also
rotates completely by means of an obliquely placed pinion acting on
a bevelled rack on the inner face of the stage-ring supporting the
mechanism. Finders are engraved on the plates, and the main support of
the stage-ring is graduated for angle measuring, a pointer on the ring
marking the unit of motion in arc.
The _sub-stage_ is carried by rack-work, and has rectangular centring
movements, supporting an inner socket that can be rotated by rack and
pinion, and which carries the several sub-stage accessories. A fine
adjustment, by screw-cone and stud, is applied by means of an extra
slide.
The _stage_ is attached to the sheath of the stem by a special
arrangement of screws, by which the rotation in the optic axis can
be centred; sliding spring clips and a movable and a removable and
adjustable angle-piece to hold the slides are applied on the upper
surface. The body-tube is pivoted to move laterally on the top of the
stem, and an adjustable steel stud beneath serves to stop the movement
in the axis. Such is Powell’s present instrument, and it represents the
results of sixty years’ steady devotion to secure perfection, and at
the same time embody the best ideas of mechanical design by Andrew Ross.
A cheaper form of students’ microscope is furnished by Powell and
Lealand, with 3/4-inch stage movement, coarse and fine adjustments to
body, plane and concave mirrors, revolving diaphragm, two eye-pieces,
and Lister’s dark wells. These makers also adopt a gauge of tubing, the
size being such that it will take in a binocular body, a Huyghenian 2
inch eye-piece having the largest field-glass possible. The tube of
the sub-stage is the same size, so as to secure one gauge of tubing
throughout. This allows of a Kellner or other eye-piece to be used as a
condenser.
Ross’s Microscopes.
Messrs. Ross have more recently introduced several changes and
modifications in the Zentmayer stand, all tending to improve it,
so that the Ross-Zentmayer model takes its place as a first-class
microscope.
Messrs. Ross have lately manufactured other forms of microscopes; one
especially designed for those commencing the study of bacteriology
(Fig. 59). This instrument is one of the steadiest among those lately
constructed for high-class work. The circular foot and short stout
pillar support the whole instrument, and a substantial knee-joint
sustains the full weight in the upright or inclined positions, while
the centre of gravity is by no means disturbed, and absolute steadiness
secured. The stage is of the horse-shoe form, which affords convenient
space for the fingers to lift the slide up while the oil is placed
in contact with the objective. The fine adjustment is extremely
sensitive, working smoothly and direct; this is entirely covered, to
prevent injury by dust. The micrometer screw works directly in the
centre of its fittings, the milled head being divided to read to 1/500
of an inch. The sub-stage is fitted with a new centring coarse and fine
adjustment, so that when using high powers with the Abbe condenser
accurate focus can be secured with the least amount of trouble.
[Illustration: Fig. 59.--Ross’s “Bacteriological and Histological”
Microscope.]
The amount of activity shown during the last few years by opticians
in the manufacture of new forms of microscopes renders it somewhat
difficult to keep pace with improvements, some of which are novel. A
further source of congratulation is that economy has all along been
studied; so much so, that the instruments in question are within the
reach of persons of moderate means. Messrs. Ross and Co. have taken
a new departure in this respect, and their _“Eclipse” Microscope_ is
an entirely new form of stand with a ring foot. This microscope has
been produced for the especial use of students, and can be purchased
for a moderate sum. It will be seen at a glance (Fig. 60) how steady
this form of stand must necessarily be, since the centre of gravity
is secured in every direction and inclination. The body-tube carries
eye-pieces, numbered, of the Continental size and optical tube-length
(160 mm.), for which the object glasses are adjusted, and a draw-tube
extending to eight inches.
[Illustration: Fig. 60.--Ross’s Rigid Pattern “Eclipse” Microscope.]
The fine adjustment is independent of set screws, and not subject to
derangement. It is extremely sensitive and direct in action, and from
its construction is equal in perfection of working to the best that can
be made. Its fitting, by a new contrivance, is completely covered at
all points, being thus preserved from disturbance or injury by dust.
The Eclipse is furnished with two eye-pieces, 1′′ and 1/4′′ object
glasses of highest excellence and large angular aperture, both adjusted
to a double nose-piece, so that they focus in the same plane; and a
swinging mirror and stage iris diaphragm.
In “Wenham’s Radial” Microscope the chief aim has been directed towards
providing a very considerable range of effects, both in altitude and
azimuth. The leading principle followed throughout in the construction
of this form of stand is that of facilitating the work of the
microscopist and of obtaining the maximum range of oblique illumination
in all directions. This is fairly well attained by causing all the
movements of inclination and rotation to radiate from the object as a
common centre. Thus it has been found possible to combine seven radial
motions, so that when the instrument is inclined backwards, as in Fig.
61, or placed in the horizontal, as in Fig. 62 or rotated from in the
brass plate, a pencil of light from a fixed source shall always reach
the object and pass to the objective. The stage is made to rotate
completely, and its rectangular motions are effected by milled heads
acting entirely within the circumference. The sub-stage is mounted
on the Zentmayer system, with two centring screws, by means of which
the optic axis is secured. It is also provided with rectangular and
rotating motions. The coarse adjustment is that of the Ross-Jackson
form--a spiral pinion and diagonal rackwork, while the fine is on an
entirely new principle designed by Dr. H. Schrœder.
[Illustration: Fig. 61.--Ross’s Wenham Radial Microscope.]
The “Ross-Zentmayer Microscope” is a thoroughly substantial and
practical instrument, combining elegance of appearance with strength
and firmness.
[Illustration: Fig. 62.--The Ross-Wenham Radial Microscope.]
It is a true tripod model, consisting of a triangular base with two
pillars rising from a cross-piece, which carries the trunnions. The
slow movement is obtained by a second slide close behind the first; but
to avoid the friction of rubbing surfaces, hardened steel rollers are
inserted between them, which give a frictionless fine motion, amenable
to the slightest touch of the milled-head screw situated conveniently
at the back of the limb, through which a steel lever passes which
actuates the slow motion slide. The body of the instrument is therefore
not touched during the fine focussing, so that all lateral movement
is avoided. The mechanical stage rotates axially, and the outer edge
of the lower plate is divided into degrees, in order to register the
angles; a simple mode of adjustment is provided for setting the centre
of rotation exactly coincident with the focal point of the objective.
As the plates of the stage have no screw or rackwork between them
(these are placed externally), they are brought close together, thus
affording the advantage of a thin substantial stage, and ensuring
rigidity where most required; phosphor-bronze being used in its
construction. The stage is attached to the limb by a conical stem, with
a screw and clamp nut at the back, so that it can be easily removed for
the substitution of a simple plate or other stage; by turning the stem
in the socket the stage may be tilted sideways at any angle required.
A feature in the Ross-Zentmayer stand is the swinging sub-stage and
bar carrying the mirror, having its axis of rotation situated from an
axial point in the plane of the object, which consequently receives
the light without requiring alteration of focus in any position of
the bar; by this means facilities are afforded for the resolution
of objects requiring oblique light and for the development of their
structure. Rays are thus obtained from any angle and indicated by the
graduated circle round the top of the swing-bar, and many troublesome
and expensive pieces of sub-stage apparatus dispensed with. The value
of this arrangement was long ago recognised in Grubb’s “Sector Stand,”
the movement of which was obtained in a far less efficient manner.
[Illustration: Fig. 63.--The Improved Ross-Zentmayer Model.]
The base or foot of the Ross-Zentmayer instrument is made in one piece.
Preference must be given to the double pillar support, as this is
firmer, and allows the sub-stage to swing free while the microscope
is in a vertical position, as in working with fluid preparations. The
sub-stage is provided with screws for centring, and, when determined,
secured by a clamping screw.
The sub-stage, with its apparatus in place, can be instantly removed,
by being drawn out sideways, so as to use the mirror alone, which is a
great convenience.
The mechanical movements of this instrument are perfect, and well
adapted to their purpose.
Messrs. Ross have other typical forms of microscopes. Their _“New
Industrial” Microscope_, for the use of farmers, horticulturists,
textile and other trades, for the examination of produce and raw
materials, is a surprisingly cheap one, and deserving of commendation.
The great utility of microscopical research to purposes of advanced
agriculture is fully recognised, and a less costly instrument than that
usually supplied for more complex investigations was much needed. It
is provided with a broad square stage for the purpose of receiving a
glass dish to contain liquids or manifold objects, and which may be
moved on the stage to bring the various particles under observation.
A fitting beneath the stage carries a plate with diaphragm apertures
for modifying the light, and as seeds, textile fibres, and other opaque
objects form a large portion of those to be examined, this sub-stage
plate has a space between the perforations which, when brought into
position, provides a dark ground by preventing the passage of light
from underneath. A condensing lens is, however, provided for the better
lighting of opaque objects. Here we have a microscope which combines
efficiency with stability, while its very simplification allows of a
really good and effective instrument for the small sum of £3 3_s._
[Illustration: Fig. 64.--Ross’s “New Industrial” Microscope.]
Messrs. Beck’s Microscopes.
Messrs. Beck have adopted what may be termed a rival system of fine
adjustment in their modern microscopes. The short lever and screw
applied externally to the body tube is peculiar, I may say, to the
Ross-Jackson system, and was originally devised to allow of the body
tube being supported somewhat more firmly on the limb. This change
had its merits fully realised in the early microscopes of Smith and
Beck. To their successors, R. & J. Beck, the microscope owes much,
and very many important improvements, while all their instruments and
accessories are excellent examples of good workmanship and finish. In
their _Pathological Microscope_ we have a movement originally found in
Tolles’ microscopes: a vertical disc, by which the centre can be raised
or depressed to correspond with the thickness of the slide. The stage
can also be brought into an inverted position by rack and pinion. Their
fine adjustment has been greatly improved, as we shall presently see,
whereby it has been made more sensitive and delicate of adjustment.
The general construction of their microscopes as a rule possess the
following advantages: the stands are strong, firm, and yet not too
light or too heavy, the instruments cannot alter from the position in
which they are placed, as, unfortunately, will occasionally happen when
joints work loose; in every position the heavier part of the stand
maintains the centre of gravity.
Beck’s _Pathological Microscope_ (Fig. 65) is a nearly perfect
instrument, furnished with a firm triangular foot, which ensures
great steadiness in any position. It has a well adapted joint for
placing the instrument at any angle of inclination; coarse adjustment
by spiral rack and pinion; fine adjustment by delicate lever and
micrometer screw motion; rack and pinion focussing and screw centring
sub-stage, made to carry all condensers and other sub-stage apparatus;
mechanical stage with horizontal and vertical traversing motions. The
stage is attached to the instrument by two screws and can therefore be
removed at pleasure, leaving a large square flat glass stage for the
culture-plate. It is likewise provided with finder divisions, and as it
always fits on to the same place, any particular portion of the object
can be recorded and found at any moment. The triple nose-piece is a
convenient addition, and a very acceptable one to the student while
diligently engaged in histological research.
[Illustration: Fig. 65.--Beck’s Pathological Microscope, with square
and removable stage.]
[Illustration: Fig. 66.--Beck’s Large “Continental Model” Microscope.]
_Beck’s Large “Continental Model” Microscope_ is of superior finish.
It is provided with a substantial horse-shoe foot, which gives support
to the strong, well-balanced body, jointed for giving the microscope
any angle of inclination. The body is provided with a draw-tube which
can be racked down to the Continental measurement. It has a spiral
rack and pinion coarse adjustment, and a fine adjustment of the most
perfect workmanship, which will be described in detail presently. It
has a large square stage with vulcanite top plate to receive culture
preparations. The sub-stage is of the most approved form for centring,
and carries an achromatic or Abbe condenser, iris diaphragm, &c. The
double mirror can be swung out of place for direct illumination and
micro-photography. Altogether, this instrument is in every way fitted
for critical or class-room work.
[Illustration: Fig. 67.--Beck’s “New Fine Adjustment.”]
To return to the fine adjustment of this, as of other forms of Messrs.
Beck’s microscopes, the applied mechanism of which is believed to be
one of the most sensitive and delicate character yet contrived. It
is constructed as shown in the accompanying figure. The body of the
instrument is supported upon the barrel D D; this barrel is accurately
and smoothly fitted to the triangular core E E. At the top of barrel D
D is screwed the cap G, to which is attached the rod C; this rod passes
through the centre of the core E E and connects with the lever arm A at
B. The action of the spring J, which is wrapped spirally around the rod
C, raises the body of the microscope and holds the lever arm A tightly
against the screw arm F. The slightest motion, therefore, of the screw
F is communicated through the lever A and the rod C to the body of the
microscope.
The great delicacy of this arrangement will be appreciated when it
is noticed that the distance from I H is double the distance of I B,
therefore any motion at B is only half that at H. This adjustment is
one of the most delicate made for use with high powers.
[Illustration: Fig. 68.--Beck’s National Binocular Microscope.]
In the construction of Beck’s Binocular National Microscope, the body
is held in a sliding fitting in the limb, and is moved up or down
by means of a rack and pinion motion. This constitutes the coarse
focussing adjustment. The fine adjustment is effected by the milled
head, which acts upon the body by means of a lever inside the limb.
The upper circular surface of the stage is made of glass, and carries
the object holder, which is provided with a ledge and spring to hold
the object by means of the pressure of an ivory-tipped screw, so that
it can be moved about readily and smoothly. The pressure of the screw
is adjusted by the milled head, which permits of more or less pressure
being made upon the edge of the object.
[Illustration: Fig. 69.--Beck’s Star Microscope.]
When the stage is required for other purposes the object holder can be
unscrewed and removed. Beneath the stage there is a cylindrical fitting
for the reception of a diaphragm, a polariser, or other apparatus. The
mirror, besides swinging in a rotatory semi-circle, is made to slide
up or down the stem. The microscope is supported by a firm pillar on a
tripod base, and the body can be inclined at any angle convenient for
working. A sub-stage can be added at any time for the reception of an
achromatic condenser fitted with concentric screws--a necessity for
more delicate microscopical research work.
_Beck’s Star Microscope_ is in every sense a students’ or class-room
instrument. It is firm and well made, with joint for inclination,
large square stage, sliding coarse adjustment and fine adjustment by
micrometer screw, draw-tube, iris diaphragm, double mirror on swinging
crank arm, A or B eye-piece, a one-inch and quarter-inch objective, the
magnifying power of which ranges from 38·5 to 183.
[Illustration: Fig. 70.--Beck’s Binocular Dissecting Microscope.]
An early binocular microscope for dissecting purposes was devised
by the late Mr. R. Beck. (Fig. 70.) This took the form of a simple
instrument built up on a square mahogany base A raised about four
inches upon four brass supports B B, having a large circular stage
plate made to revolve on a second plate, on which the object is placed
and brought under the eye for dissection. On the left hand side is a
milled head rack and pinion K, which acts upon a horizontal bar I for
focussing the magnifying lens. Another bar, R, carries the prism P and
a pair of eye-pieces arranged on the principle of M. Nachet’s binocular
microscope. Mr. Beck preferred to adopt Wenham’s method of arranging
these prisms; that is, by allowing half the cone of rays to proceed to
one eye without interruption, while the other half is intercepted by
the prisms and transmitted to the other eye. Beneath the stage is the
ordinary mirror L. The condensing lens M is supported on a separate
brass holder let into one of the supports of the stand. In practice,
however, this arrangement was found inconvenient, and the microscope
has therefore not been brought into general use.
Messrs. Watson’s Microscopes.
Among London opticians, the various microscopes manufactured by Messrs.
Watson, of Holborn, are of high finish and good workmanship. Those
specially designed for the use of students possess merits of their
own in their mechanical construction, and also embody a provision,
as indeed do all their instruments, whether for students or more
pretentious work, whereby wear and tear in their frictional parts
can be compensated for by the user himself. This is effected in a
simple but efficient manner. The fittings are sprung, and screws set
just outside the dove-tails. The very slightest turn of the screws
compresses the dove-tails, and a very large amount of wear can in this
way be prevented.
I am glad to notice that Messrs. Watson have adopted certain standard
sizes recommended some time ago by the Royal Microscopical Society for
the diameters of eye-pieces. It would be a great advantage if the same
standard became generally recognised and brought into use, since it is
a matter of much importance to microscopists.
_Watson’s Edinburgh Students’ Microscope_ (Fig. 71) is a thoroughly
efficient one for all practical purposes, great care having been
bestowed upon its smallest details, and it is not difficult to perceive
the reason of its popularity among students. The tripod form of foot
ensures great steadiness and firmness; the body carries the smaller
0·92 eye-piece, and with draw-tube closed is of the Continental length.
The draw-tube is graduated to millimetres, and when fully extended the
body measures 10 inches. The stage is provided with mechanical and
rotary movements; the compound sub-stage with centring screws, rack
and pinion to focus, and a means of lifting the condenser out of the
optical axis when not required for use. Notwithstanding, none of the
movements are at all cramped; a clear distance is maintained beneath
the stage, affording plenty of room for manipulating the mirror. Both
coarse and fine adjustments work with smoothness, the latter being on
Watson’s latest improved principle--one revolution of the milled head
moves the body 1/300 of an inch. The stage is of extra large size,
to allow of the use of large culture-plates. No Continental stand of
higher price compares with the Edinburgh microscope. Its height when
placed in the vertical position is 11-1/2 inches.
[Illustration: Fig. 71.--Watson’s Edinburgh Students’ Microscope.]
[Illustration:
Fig. 72.--Sub-stage of Edinburgh Students’ Microscope. This view of
underside of stage of students’ instrument shows the mirror set at an
angle for oblique illumination, and sub-stage turned aside.]
The various sizes of oculars adopted by opticians and at present in
vogue cause considerable confusion. A standard size is specially
needed for students’ and small microscopes. The standard long used by
Continental manufacturers is 0·92 of an inch. The adoption of this size
would place the eye-piece in the same position as that of the universal
screw for the objective, formulated by the Royal Microscopical Society
many years ago. The desirability of using standard sizes has been fully
recognised by Messrs. Watson and they are now adapted to most of their
microscopes. The English diameter, 1·35 of an inch, known as the “Ross”
size, is retained in all their microscopes of large size.
Watson’s Mechanical Draw-tube.
[Illustration: Fig. 73.--Watson’s Mechanical Draw-tube (full-size).]
An important feature in connection with the body-tube of Watson’s
Edinburgh Students’ Microscope (as, indeed, in all their fully
furnished instruments) is that they are provided with two draw-tubes;
one moved by rack-work, the other sliding inside the body-tube. The
advantage is, that the body can be made very short or extremely long,
while sufficient latitude can be given to objectives corrected for
either Continental or English tube-lengths, and to adjusting the same
for thickness of cover-glass by variation of tube length. Should the
cover-glass be thicker than that for which the objective is corrected,
a shorter tube-length is necessary; if thinner, the body must be
lengthened. This is effected by means of the rackwork draw-tube. The
length of the body when closed is 142 millimetres (5-5/8 inches), and
when the two draw-tubes are extended, 305 millimetres (12 inches),
being, therefore, shorter than the Continental and longer than the
English tube lengths. Both draw-tubes are divided into millimetres,
and on the rackwork draw-tube a double scale is engraved, reading
continuously from the sliding draw-tube when fully drawn out, or
giving the body length when the rackwork draw-tube alone is in use.
The utility of this mechanical draw-tube is that it permits of quick
manipulation with perfect results.
[Illustration: Fig. 74.--Watson’s Histological Microscope. Stand
“A.”--Height, when placed vertically and tube pushed home, 9-1/2
inches.]
The inside top of the draw-tube is smaller than the remainder, the
former making a fitting for the eye-piece about 1 inch long, permitting
of the tube being blackened inside up to this fitting, thus minimising
reflection. The end of the draw-tube has the universal screw for using
the apertometer, &c.
_Watson’s Histological Microscope_ (Fig. 74) is a somewhat cheaper
form of instrument, designed for the student; although of plainer
construction it is quite as well made as the costlier model. It is
provided with spiral rack and pinion coarse adjustment, and with this
motion the greatest smoothness is preserved. There is no backlash, the
teeth of the pinion never leaving the rack; so effective is it that a
high power can be perfectly focussed by its means. It is also furnished
with their universal pattern of fine adjustment. This can be had for £3
3_s._
[Illustration: Fig. 75.--Watson’s Semi-Mechanical Stage.]
Messrs. Watson have among other accessories of value introduced in
connection with their several microscopes a semi-mechanical stage,
whereby they are enabled to reduce the cost of manufacture. Fig. 75 is
an outline sketch of the same.
This stage is of the horse-shoe shape, with cut-out centre, constructed
of 1/4-inch brass plate, and measures over all 5-1/4 inches wide by 4
inches deep. Fitting on the edges of the main stage is a frame which is
actuated vertically by means of a double rack and pinion from beneath,
giving 3/4-inch of movement, having controlling heads on either side
of the stage; on the edges of this mechanical frame a sliding bar is
fitted, consequently movement may be imparted either by rackwork or
by hand. The mechanical movement, however is in one direction only;
but as the bar carries the object, the worker can easily move the
object out horizontally with the finger. The advantage of this stage
is that the whole surface is perfectly flush, and the pinion heads are
below its level, so that culture plates or continuous sections may be
conveniently examined.
[Illustration: Fig. 76.--New Centring Underfitting for Microscope.]
Another addition of considerable value is the centring underfitting for
students’ microscopes.
This fitting places in the hands of student workers a means of
accurately centring the sub-stage condenser, at a low cost. It consists
of the usual underfitting tube, having a flange at the top which is
fitted in a box between two plates. The centring is effected by means
of two screws, which press the flange against a spring, as in the
ordinary sub-stage centring movement. The fitting can be adapted to any
form of Messrs. Watson’s and most other makers of students’ microscopes.
_Watson’s Bacteriological Improved Van Heurck’s Microscope_ (Fig.
77) is in every way a superior instrument, and it at once conveys a
favourable impression to the practical worker. When set up for use its
many convenient points--its excellence of workmanship and the precision
of its movements--seem to imply its special adaptation for the
bacteriological laboratory and for other high-class work where absolute
reliance has to be placed in the results obtained. Every detail of
the instrument is carried out in the best possible manner. The coarse
adjustment is effected by means of a diagonal rack and spiral pinion,
which ensures the smoothest possible motion; while the fine, the most
important movement in the instrument, is made with an extra long lever,
a specialty of Messrs. Watson’s, and which imparts an extremely slow
action: this is now one of the most delicate and reliable forms of
fine adjustment. By its means the entire body is raised or lowered
by means of a milled head fixed to a screw having a hardened steel
point acting on a lever against a point attached to the body slide, in
a dove-tailed fitting about 2-1/2 inches long. Owing to the position
of the controlling milled head on the limb, it can be worked with
either hand. Another feature of importance is that, in using the fine
adjustment the distance between the eye-piece and objective remains
unaltered. All the frictional parts of the microscope have spring slots
to the dove-tailed fittings, in which compensating screws are fitted.
These are some few of the more important points, to which much thought
and attention have been given. The body permits also of the use of
objectives of any other optician, since its total length when the draw
tubes are closed up is only 143 mm.; when extended, a total length of
320 mm. is available. By this means an ample margin is left for the
correction for cover-glass thickness, whether the objective used be
intended for the 160 mm. or 250 mm. tube length. The height of the
microscope when placed in the vertical position is 13-1/8 inches.
[Illustration: Fig. 77.--Watson’s Improved “Van Heurck Bacteriological”
Microscope.]
_The Stage._--A somewhat new design has been used in building this up
so as to reduce vibration to a minimum. The bracket carrying the stage,
instead of being screwed on to the front of the limb, as is usually
done, is made in a solid casting, taking the sub-stage beneath, and
passing into the joint at the top of the foot. The joint bolt goes
through the whole (limb and stage bracket), rendering the limb stage
and sub-stage as firm as if it were one piece; a point of considerable
importance.
The mirrors, which are plain and concave, are mounted on a swing arm,
so that they may be turned aside when direct illumination of the object
is required. On the right hand side also there is a steel clamping bar
for fixing the microscope at any angle of inclination. The tripod foot,
which has superseded most other forms, is adopted. At the points of
contact with the table the feet are provided with cork pads, which give
increased firmness and prevent vibration to some extent.
The sub-stage is provided with a fine adjustment of similar design to
that employed for the focussing of the objective. It has become needful
to embody such a refinement, in order that sub-stage condensers of
large aperture, such as are in constant use for critical high-power
work, may be adjusted with the same facility and precision as the
objective--they, in fact, require it if the best work is to be got
out of them. No pains have been spared by Messrs. Watson to render it
absolutely perfect.
_Watson’s Portable Microscope._--This instrument is similar in general
detail to the Histological Microscope, but the foot, mirror stem,
&c., are made to fold up in exceedingly compact form, and when set up
for use the stand is perfectly rigid. Portable microscopes are, as a
rule, but makeshifts. This, however, is a thoroughly sound, practical
instrument and capable of best work with the highest power objectives,
having good adjustments and universal size fittings throughout, so that
the objectives and apparatus made for the larger instruments can be
employed with it.
[Illustration: Fig. 78.--Watson’s Portable Microscope. Height of
instrument when placed vertically and racked down is 9-3/8 inches.]
_Watson’s Petrological Microscope_ (Fig. 79) is a modification of their
Edinburgh Students’ pattern, and designed specially for petrological
and mineralogical work.
[Illustration: Fig. 79.--Students’ Petrological Microscope.]
A polariscope having prisms of large size is supplied with it, the
analyser being fitted in the body, and the polariser in the under-stage
fitting. The latter has a divided circle and a spring catch at every
quarter circle. By removing the polariser and withdrawing the analyser,
for which provision is made, the microscope can be used for purposes
of ordinary research. A Klein’s quartz plate is fitted beneath the
analyser, also in the body of the microscope.
The stage, which has a glass surface, rotates concentrically, and has
a divided circumferential edge reading by the verniers. The eye-piece
has cross webs to the diaphragm, and when it is desired, an analyser,
having a divided circle fitted with a calc-spar plate, can be used
above the eye-piece, and condenser lenses attached to the polariser for
stereoscopic purposes. All the fittings have the universal thread, and
are interchangeable.
[Illustration: Fig. 80.--Swift’s Histological and Physiological
Microscope.]
Messrs. Swift’s Microscopes.
_Messrs. Swift’s Microscopes_ have a well-established reputation for
quality and good workmanship, and therefore can in no way suffer
by comparison when placed beside those of other opticians. One of
the characteristics of Messrs. Swift’s microscopes--and this runs
through the whole series--is that they are all made to a _standard_
gauge, so that the several parts of the instruments, as well as their
accessories, are interchangeable; the cheaper forms, with those of the
first quality and finish. Should the student, then, start with a No. 1
model, he can at any time build it up, as it were, with the accessories
designed for a No. 3 or 4, that is, for an instrument of double the
price he started with. The optical centre is preserved throughout the
whole series of microscopes.
[Illustration: Fig. 81.--Swinging Leg Attachment of Swift & Son’s
Four-Legged Microscope Stand.]
The tripod foot has, it appears, taken the place of some of their other
forms of instruments, while their four-legged _tripod_, if it can be so
designated, is a novelty of quite an unusual character.
The swing leg is attached to the framework of the tripod by the screw
(Fig. A), which is provided with a powerful steel spiral spring,
compressed between two steel collets when the screw is driven home, as
shown in Fig. B.
The expansion of this spring will obviously take up and compensate
automatically any wear and tear that is likely to occur between the
bearing surfaces, and it is therefore impossible for the fitting to get
loose.
_Swift’s Four-legged Microscope_ (Fig. 80) is one possessing great
stability in whatever position it may be placed; the body being
supported on a horse-shoe platform, from which its four legs spring,
the two front legs being fixed, while the hind legs are pivoted to
the platform. This arrangement of pivoting the hind legs enables
the microscope to adapt itself to any uneven surface, thus keeping
it always in a steady position, while it also reduces the danger of
being upset by any lateral movement of an accidental nature. The feet
are studded with corks, an additional aid to steadiness and fixity
for microphotography. The length of the body from the ocular to the
nose-piece is 6-1/2 inches, and can be extended to 9 or 10 inches by
means of the draw-tube, which has a millimetre graduation. The stage,
which is of horse-shoe shape, is provided with spring clips, to which
a movable mechanical stage can at any time be attached. The sub-stage
partakes of two forms, one being an ordinary fitting, taking an
ordinary condenser; the other, the regular rack and pinion achromatic
condenser with centring adjustments. It has a diagonal rack and pinion
coarse adjustment, the fine adjustment being made by micrometer screw
of the finest character.
[Illustration: Fig. 82.--Swift’s Spiral Rack and Pinion Coarse
Adjustment.]
Fig. 82 is intended to illustrate the advantage of the spiral rack
and pinion which Messrs. Swift fit to their microscopes, in place of
the ordinary conventional horizontal rack and pinion movement. The
advantage will at once be seen, since there is more gearing contact
between rack and pinion, thus ensuring durability and reducing loss of
time or back lash to a minimum, with less wear and tear. The leaves of
the pinion also roll into the teeth of the rack by degrees, ensuring a
very much smoother action, which, if properly made and fitted, prevents
the gearing of the two being felt by the hand whilst focussing.
[Illustration: Fig. 83.--Graduated Supplementary Draw-Tube.]
Fig. 83 is a supplementary draw-tube with rack and pinion movement,
which can be adapted to any of Swift’s microscopes in place of
the ordinary draw-tube, the size of the thread being of the same
diameter, so as to render all draw-tubes, as well as other parts
of these instruments, interchangeable. The draw-tube being divided
into millimetres can be extended from 160 to 250 millimetres. One
advantage of this arrangement is that the correct adjustment of any
objective with each eye-piece is easily found and recorded for future
observations with the same combination.
Messrs. Swift’s Three-legged Tripod Microscope (Fig. 86). In most
respects the description already given of the four-legged instrument
is applicable to this stand. Although of an apparently different form,
it can be built up, as already explained, into one of a higher class.
It is suitable in every way for histological investigations. The
horse-shoe platform in this, as in the preceding stand, is extremely
serviceable, as it allows the pillar of the instrument to rest firmly
upon it, thus rendering the stand very rigid.
_Swift’s Bacteriological Microscope_ (Fig. 84), designed by Professor
Wright, of the Army Medical School, Netley, a sufficient warranty of
its excellency and perfect adaptation for bacteriological high-class
work. One of the advantages connected with this microscope is the
facility with which it can be adapted for either high or low power
investigation, without the necessity of adding or detaching any part.
The objectives, arranged on a triple nose-piece, are approximately in
focus when revolved into position for immediate use, thus effecting
a saving of time in changing the objective. Moreover, the nose-piece
carrying the objectives is of new construction, and fitted in such a
way that the entry of dust is rendered impossible.
[Illustration: Fig. 84.--Swift’s Army Bacteriological Microscope.]
[Illustration: Fig. 85. Under-Stage of same.]
The Abbe condenser, fitted with an iris diaphragm, is mounted on
an eccentric arm, so that it can readily be thrown out of the axis
of the microscope when not required, without having to re-arrange
the focus when again brought into position. The condenser must be
turned aside when plate cultivations and preparations of unstained
bacteria are being looked over for selection of colonies for mounting,
in which case an arm carrying a quadrant with three apertures is
brought into position in place of the condenser, the apertures being
severally centred by a spring catch and used with oblique light. This
arrangement, shown in Fig. 85, is seen from the under surface of the
stage. The stage is sufficiently large, so that when Petrie plates
are being examined at the extreme edges there is little fear of their
overbalancing.
[Illustration: Fig. 86.--Swift’s Histological Students’ Microscope.]
The fine adjustment is the Swift’s Patent Campbell Differential Screw,
which offers great facilities for delicate focussing with the highest
power objectives. The stand is of the most substantial and rigid form,
and thus ensures the microscope from vibration.
The under-stage of microscope (Fig. 85) is seen to be of the most
approved form.
[Illustration: Fig. 87.--Swift’s Advanced Students’ Microscope.]
_Swift’s Advanced Students’ Microscope._--In this microscope (Fig. 87)
we have a superior instrument for the use of the advanced student,
which may be described as of high mechanical excellence, well suited
for every requirement of work. The stand is the well-known tripod
form of their Challenger Microscope, and admits of the instrument
being placed at any angle of inclination; the body is short enough
to work with objectives of Continental makers, and is provided with
a draw-tube, to elongate it to the standard of 10 inches, with a
diameter of 1-3/16 inch to take the same eye-pieces as the larger
stands. The coarse adjustment is by spiral rack and pinion; the fine,
by a carefully made differential screw motion for delicate focussing.
The stage is of the horse-shoe pattern, to which a mechanical stage
can at any time be adapted, as well as an achromatic condenser to the
sub-stage seen beneath. Here the student will find the foundation for a
superior instrument.
Messrs. Baker’s Microscopes.
Of Messrs. Baker’s larger stands, the Improved “Nelson Model,” No. 2
(Fig. 88) stand is selected in preference to their more elaborate No.
1, and their simpler form, No. 3, as a high-class instrument, and one
well suited for fine critical work; the former being somewhat better,
only from having extra adjustments; the latter possessing no superior
advantage over the “Advanced Students’” Microscope. This microscope
is mounted on a solid tripod foot, which insures stability, whether
placed in a vertical, horizontal, or inclined position; the front
toes are slotted, so that they may be clamped to the base plate of a
photo-micrographic apparatus, first introduced for photo-micrographic
work, and will also be found convenient in ordinary work; as the fine
adjustment milled head is placed at the bottom of the pillar, instead
of at the top, the more usual place. For photo-micrographic work the
advantage is that the strain of the pulley in such apparatus actuates
the fine adjustment, and is less liable to cause vibration of the
instrument. The advantage when the instrument is used for ordinary
work lies in the fact that the weight of the hand is rested on the
top of the tripod, thus admitting of steadier movement of the milled
head. The fine adjustment is obtained by a “Campbell” differential
screw, each revolution of which is equal to 1/200 m.m. The draw-tubes
being graduated in m.m., allow of either short or long tube objectives
being used, closing up to 150 m.m. and extending to 280 m.m., the rack
and pinion adjustment to the lower tube affording a ready means of
correction for cover-glass thicknesses. The eye-piece gauge, as will
be seen from its dimensions, is of large size, being the same as
that adopted by Zeiss for his long tube compensating oculars; smaller
eye-pieces can, however, be adapted at any time.
[Illustration:
Fig. 88.--Baker’s Improved “Nelson Model” Microscope.
Dimensions.--Height when in vertical position and body racked down,
11′′; Height of stage, 4-1/8′′; Height of optic axis when in horizontal
position, 8-1/2′′; Spread of tripod foot, 8 × 8-1/2′′; Diameter of
mirrors, 2-3/8′′; Internal diameter of draw-tube, 1-3/10′′.]
The mechanical rotating stage is divided on brass to 1/100 inch, with
clamping bars and stop, by which a specimen can always be brought back
to a certain position for registration. The sub-stage has rack-work
focussing adjustment, and centring screws; a fine adjustment is added,
if desired. On the whole, the instrument is suitable for special
critical work, and is equally well suited for photo-micrography.
[Illustration:
Fig. 89.--Baker’s Advanced Students’ Microscope. Dimensions.--Height
when in vertical position and body racked down, 11-1/4′′; Height of
stage, 4-3/4′′; Width of stage, 4′′; Height of optic axis when in
horizontal position, 6-1/2′′; Spread of foot, 6′′ × 6′′; Diameter of
mirrors, 1-3/4′′; Internal diameter of draw-tube, 11/12′′.
Explanatory lettering of instrument: A, Huyghenian eye-piece;
B, draw-tube graduated in millimetres; C, nose-piece; D, coarse
adjustment; E, fine adjustment with millimetre screw; F, horse-shoe
sliding stage, graduated with sliding bar in vertical and horizontal
directions _for use as finder_; G, sub-stage rack and pinion screw;
H and I, centring screws to sub-stage; J, carrier for condenser; K,
mirror with movable arm supported on solid tripod foot.]
The points of difference between this stand and the No. 1 model are
that in the latter the fine adjustment carries the body only, and
not the rack adjustment; the limb carrying both the body and the
sub-stage is in one piece, giving, if possible, still greater rigidity;
the rotation of the mechanical stage, which is divided on silver, is
complete, and can be actuated by hand or rack work; it has a clamping
screw and fine adjustment to sub-stage.
_Baker’s Advanced Students’ Microscope_ (Fig. 89) may be described as
a typical instrument, equally suitable for histological work and that
of the advanced student. The intention of the maker in simplifying
the adjustments and reducing the instrument in size, was to furnish a
well-finished portable instrument at a moderate cost. This object has
not been attained by supplying adjustments of second-rate quality, but
by reducing their number to a minimum.
[Illustration: Fig. 90.--The Mayall Removable Mechanical Stage.]
The tripod foot of the “Nelson Model” is replaced by a claw foot,
which is in effect a tripod, as it rests on three points; it has
not the same wide spread, but this, far from being a disadvantage,
renders the instrument more portable. It has rack and pinion coarse
and Campbell differential screw fine adjustments, draw-tube graduated
in m.m., extending to 180 m.m., eye-piece gauge the same as the
Continental size, large square open stage to afford the greater freedom
of manipulation; sliding bar with graduations on bar and stage, which
suffice for registering any given field under a low power; holes are
also drilled in the stage ready to receive an attachable mechanical
stage should it be thought advisable to add one at a later date.
The sub-stage is of the universal size with rack-work focussing,
adjustment, and centring screws.
[Illustration:
Fig. 91.--Baker’s Model Histological Microscope. Dimensions.--Height
when in vertical position and body racked down, 10-1/2′′; Height of
stage, 4′′; Width of stage, 3-1/2′′; Height of optic axis when in
horizontal position, 5-1/4′′; Spread of foot, 5-1/4′′; Diameter of
mirrors, 1-5/8′′; Internal diameter of draw-tube, 11/12′′.]
Messrs. Baker have recently introduced a similar instrument with
swing-out sub-stage and adjustments for compensating for wear and tear
of rack. The stage is also somewhat larger from back to front.
These stands are very suitable for bacteriological research, and for
amateurs wishing to obtain a stand which will carry all the apparatus
they are likely to need, without going to the expense of the larger
models, no better instrument could be desired.
Their “Removable Mechanical Stage” (Fig. 90) is a modification of the
pattern designed by the late Mr. J. Mayall. The vertical movement is
by rack and pinion, giving a range of 1-1/8 inch. The horizontal motion
of 1-1/2 inch is accomplished by means of a quick-acting screw. The
object is pressed tightly to the stage of the microscope by means of
three points, and the whole of the mechanical part is firmly clamped
by two thumb screws which can be readily removed. The stage is made to
carry slides of any size less than 1-3/4 inch wide.
_Baker’s Histological Microscope_ (Fig. 91) is of a different type
to the preceding, and is intended to represent one of medium power,
affording magnification of about × 400 as a maximum. It is supplied
with a diaphragm beneath the stage, without other illuminating
apparatus than that of the mirror. But if the adjustments of such a
stand are good, there is no reason why some form of sub-stage condenser
should not be added, to make the instrument somewhat more serviceable.
There is, however, a rather too limited space beneath the stage of an
instrument of this kind to admit of a sub-stage condenser, consequently
it cannot be said to be suitable for critical work. For all ordinary
students’ work this microscope is certainly available.
[Illustration: Fig. 92.--Rousselet’s Tank Microscope.]
The stand of the Model Histological Microscope has the same form of
foot as the more advanced student’s stand. It is somewhat lighter, and
more portable, a matter of consideration in a student’s microscope,
which often has to be carried to and from a class-room. It is provided
with rack and pinion coarse adjustment, and a Campbell differential
screw fine adjustment, draw-tube, and diaphragm; the diaphragm carrier
being of the universal size, so that it can be replaced by an Abbe
condenser at any time. With the additions suggested, this instrument
can be made equal to those of a higher standard.
Rousselet’s Tank Microscope (Fig. 92), for rapidly looking over pond
water and weeds, consists of a jointed arm moving parallel to the side
of the tank to carry an aplanatic lens; the arm is focussed by means of
rack and pinion fixed to the upright of a mahogany stand, upon which
the tank can be placed, or it can be clamped directly to the tank by
means of a screw. This handy form of pond microscope is made by Messrs.
Baker.
Pillischer’s Microscopes.
Mr. Pillischer (New Bond Street) is favourably known for the excellency
of his instruments. He has lately brought out several microscopes of
an improved form. His larger model, the “New International,” consists
of a solid, well-built, firm tripod stand of the Ross-Jackson pattern,
which appears to be quite in the ascendant among London opticians;
rack and pinion coarse adjustment, and a superior micrometer fine
adjustment; sub-stage with centring screws and rack and pinion
focussing adjustment; a new form of sliding pin-hole diaphragm and
iris diaphragm; B and C eye-pieces; 5/8 and 1/7 objectives; Abbe
condenser, N.A. 1·20; in every respect a perfect model, neatly packed
in a mahogany case, for a very moderate sum. Mr. Pillischer’s No. 2
(Fig. 93) “International” Microscope, being the _Army pattern_ as well
as the _student’s_, is well adapted for clinical work. A firm tripod
stand supports two dark bronze uprights, with rack and pinion coarse
adjustment, _e_, and fine adjustment, _d_, the stage, _i_, is wide and
suitable for clinical work, and large enough for dissecting upon. The
whole instrument is well made; the coarse adjustment is so good that
the one-eighth inch can be focussed with ease, and without using the
fine adjustment.
For a few shillings extra, a mechanical stage can be added, consisting
of levers, having an action similar to the movements of a parallel
ruler, which is so easy of adjustment that it can be worked under
the eighth-inch objective with the hands--an advantage in a clinical
microscope.
[Illustration: Fig. 93.--Pillischer’s “International” Microscope.]
The following reference letters serve to explain the general
construction of the microscope (Fig. 93):--_a_, the eye-piece; _b_, the
draw-tube; _c_, the sliding-tube; _d_, micrometer or fine adjustment;
_e e_, the coarse adjustment; _g_, the mirror arm and mirror; _h_,
sub-stage carrying Abbe condenser; _i_, the stage with spring-clips;
_j_, objectives screwed into place and double nose-piece.
The “Kosmos” is Pillischer’s cheaper model. The stand of this somewhat
novel and original microscope is framed entirely of brass and
gun-metal. The fine adjustment is very sensitive and perfectly steady,
admitting of the highest immersion objectives being used. The optical
parts are constructed upon principles consistent with the latest
improvements. It has a claw-foot stand with a semi-circular arm, which
carries the body, with sliding-tube coarse adjustment, and micrometer
screw fine adjustment, with a large square stage diaphragm and mirror.
The instrument is neatly packed in a mahogany box, together with the
A or B eye-piece, 1-inch and 1/5-inch objectives of good defining and
penetrating power, magnifying from 30 to 380 diameters, in mahogany
cabinet, for the moderate sum of £5.
Pillischer’s Binocular Microscope (Fig. 94) is constructed on a plan
somewhat intermediate between that of Beck’s and Ross’s well-known
patterns, and in point of finish is equal to any student’s microscope
in use. The semi-circular form given to the arm carrying the body
increases the strength and solidity of the instrument, although it
is doubtful whether it adds to its steadiness when placed in the
horizontal position. The straight body rests for a great part of its
length upon a parallel bar of solid brass ploughed into which is a
groove for the reception of the rack attached to the body, the groove
being of such a form that the rack is held firmly while the pinion
glides smoothly through it. A steady, uniform motion is thus obtained,
which almost renders the fine adjustment unnecessary. The binocular
bodies are inclined at a smaller angle to one another than in most
instruments; nevertheless, the range of motion given to the eye-pieces
by the rack and pinion enables those whose eyes are widely separated
to use the instrument with comfort. The prism is so well set that it
illuminates both fields with equal intensity. The stage is provided
with rectangular traversing movements to the extent of an inch and a
quarter in each direction. The milled heads which effect these are
placed on the same axis, instead of side by side, one of them--the
vertical one--being repeated on the left of the stage, so that the
movements may be communicated either by the right hand alone or by both
hands acting in concert. The stage-plate has the ordinary vertical and
rotatory motions, but to a much greater extent than usual; and the
platform which carries the object is provided with a spring clip to
secure the object when the stage is placed in the vertical position.
A new form of sub-stage with centring screws is made to carry the Abbe
achromatic condenser, diaphragm, polarising and other apparatus.
[Illustration: Fig. 94.--Pillischer’s Binocular Microscope.]
Continental Microscopes.
_Continental Microscopes._--The better known among continental
opticians are Zeiss, Leitz, Seibert, Reichert and Hartnack. All seem
to have vied with each other in the attainment of perfection in the
manufacture of the most useful forms of microscopes. The late Carl
Zeiss did more for the modern microscope than either of the opticians
referred to above. I therefore take a medium typical model of his from
a long series of highly-finished instruments for my illustration.
Zeiss’s successors have of late endeavoured to perfect the mechanical
details of their instruments in three or four directions, i.e.,
fundamental features of the stand, stage arrangements, means of
focussing, and illumination.
_The Stand._--The general form of the stand still partakes too much of
the original sameness of type introduced by Oberhäuser, and modified
and improved by Hartnack; the “Babuchin” stand being still in favour
with some few makers. The greater firmness and steadiness of Zeiss’s
stand (Fig. 95) is secured by the horse-shoe form of foot, which,
for the most part, is massive and well adapted to carry the stout
uprights, which support a well-balanced, substantial body-tube and a
graduated draw-tube, circular stage with a vulcanite disc, 4 inches in
diameter; a sub-stage with centring arrangement for Abbe’s illuminating
apparatus, and iris diaphragm and other diaphragms for use when the
condenser is thrown aside. The mirror is full-sized, plane and concave.
The coarse adjustment is regulated by a rack and pillion movement so
perfect that objectives of medium power can be focussed by it alone.
The fine adjustment is made by micrometer screw, the force exercised by
which is transferred to the movable body by a single contact between
two hardened steel surfaces. This ensures extremely delicate and
uniform motion of the body which carries the tube.
The divisions in the milled head of the screw furnish a means for the
registration of the vertical movements of the tube. In the latest
stands, each division corresponds to an elevation or depression of
the tube in the direction of the optic axis of 0·01 mm. By this means
measurements of thicknesses may be made with a considerable degree of
accuracy, the upper and lower surfaces of the object being successively
focussed, and the amount read off on the milled-head, by the fixed
index. In doing this, care must be taken to make both adjustments by a
rotation of the screw in the same direction. The thickness of an object
in air is then equal to the difference between the two readings. By
this means the thickness of any other substance may be measured--that,
for instance, of the cover-glass of the object.
[Illustration: Fig. 95.--Zeiss’s Medium Stand Microscope.]
The medium tube-length of the microscope is 160 mm. from the attachment
of the objective to the eye-piece end. The draw-tube admits of the
length being increased or diminished, and this may be read off by means
of the millimetre scale engraved on the tube. My description of this
model also applies to the higher class microscopes, which will be found
in every way well finished and adapted to biological and scientific
research.
[Illustration: Fig. 96.--E. Leitz’s Medium-sized Microscope.]
_E. Leitz’s of Wetzlar Microscopes._--This optician publishes a
series of twelve high-class forms of instruments. By preference, the
horse-shoe form of stand (Fig. 96) is adopted in the whole of this
maker’s models, the body being supported on a hinge joint and clamped
over, and fitted with a circular revolving centred mechanical stage,
attached to the ordinary stage by means of a set pin, which fixes the
stage in position. By removing the screw, the stage can be detached;
in this way, the stage serves for searching over large surfaces and
registering the results.
[Illustration: Fig. 97.--Leitz’s Dissecting Microscope.]
The coarse adjustment is made by rack and pinion, and the fine
adjustment by micrometer screw, the head of which is provided with
a scale reading 1/100 mm. The draw-tube is also cut and ruled to
millimetre scale. The sub-stage has rack and pinion movement, and is
arranged for the Abbe condenser and iris diaphragm. This is attached
to the upper stage by means of a set pin, which fixes and retains it
in position after perfect centring. By removing the pin, the sub-stage
can be either detached or swung aside by pressing a button. In short,
this microscope is in all respects well furnished and fitted with
the requisite complex mechanism necessitated by modern high-class
technicological work.
Leitz’s students’ microscope, with sliding body, micrometer screw fine
adjustment, concave mirror, two eye-pieces and two objectives, 3/4 inch
and 1/8 inch, in mahogany case, costs £3 10_s._ Leitz’s dissecting
microscope, with a heavy foot and rests, is fitted with two aplanatic
lenses, magnifying × 10, × 20 diameters.
_Reichert and Seibert_ adhere to the same model as that of Zeiss,
and therefore require only a brief notice. Their microscopes are
characterised by substantial workmanship, suitable construction, and
exact centring. The coarse adjustment is obtained in the usual way by
rack and pinion, the fine by micrometer screws, which work easily, and
are protected against wear and tear by having their working surfaces
hardened. The stands of the better class instruments have micrometer
screws graduated, and draw-tubes cut to millimetre scale. Their
mechanical stages and sub-stages and accessories are in every way well
finished; stage forceps, tests, and an assortment of cover glasses
and slides being added. Their first-class microscopes are sent out in
mahogany boxes.
On going through the continental makers’ catalogues, it will be noticed
that their well-equipped microscopes are rather more costly than that
of their English _confreres_. It is understood Messrs. Baker and Watson
are the constituted agents for these opticians.
_Nachet’s Microscope_, a new form of which was first seen at the
Antwerp Exhibition 1892, is very solidly built, and has all the
qualities necessary for histological work. The stage rotates about the
optic axis, and carries a movable slide holder. The coarse adjustment
is by rack and pinion movement, the fine by the new system of
micrometer screw (described in the journal of the Royal Microscopical
Society of 1886), with divided head indicating the 1/400 part of a mm.
The plane and convex mirror is mounted on a jointed arm. The draw-tube
is divided into millimetres. The illuminating system, consisting of a
wide-angled Abbe condenser (N.A. 1·40) with iris diaphragm, is raised
or lowered by rack and pinion screws. The iris diaphragm, being mounted
on a wheel, is worked by a tangent screw, which by a very slight
movement causes the aperture of the diaphragm to pass from the centre
to the periphery of the condenser. Altogether the arrangement of the
sub-stage is novel, and the instrument is extremely well arranged and
adapted to modern requirements.
Nachet and Hartnack, of Paris, hold an almost equal rank as makers of
first-class microscopes, and in point of excellence of workmanship
fairy rival those of our English makers.
[Illustration: Fig. 98.--Nachet’s Class Demonstrating Microscope.]
There are very many other London and Continental makers of microscopes
besides those especially mentioned, who have well-sustained reputations
as opticians, and who, from want of space, I have been obliged to pass
over. Messrs. Newton’s Students’ Microscope must be mentioned with
respect. It is a good and useful instrument, has a firm stand with a
reversible (rotatory) body movement, which seems to ensure steadiness
when brought into the horizontal position for micro-photographic
purposes. There are other opticians whose microscopes have stood the
test of time--Messrs. Collins, Crouch, &c. It may, however, be taken as
a well-established fact that those opticians known to manufacture the
more highly-finished models also produce the more serviceable forms of
students’ class-room, and other microscopes.
The Bacteriological Microscope.
The microscope required for bacteriological studies should be perfect
in all its parts. With regard to the choice of an instrument, it is
very much a matter of price, since the most perfect is usually the
most costly; I shall therefore proceed to give a typical example of
the instrument in use in a bacteriological laboratory. The microscope
should possess the following qualifications, all of which are
absolutely necessary for the study of such minute objects as bacteria
and other micro-organisms.
“The typical bacteriological microscope should be well equipped with
objectives of sufficiently high magnifying power, and with a special
form of illuminating apparatus; while the mechanical arrangements for
focussing should act with the greatest smoothness and precision; the
stage, also, should be wide enough to admit of the examination of plate
cultivations.”
We will consider these several points and recommendations _seriatim_,
commencing with the stand.
Messrs. Watson & Sons’ Van Heurck model stand so well answers the
several conditions laid down by an experienced teacher of bacteriology,
that I have no hesitation in presenting it to my readers as a typical
instrument, one in every way worthy of the high praise it has already
received from those who have worked with it, and whose judgment may
be relied upon in every way. The microscope is fully described among
Messrs. Watson’s instruments, page 108.
_The Stand._--A good firm stand is undoubtedly of the first importance
for all high-class work. The steadiness of the instrument and its
entire freedom from vibration depends largely upon the form of the
stand. I am glad to find Dr. Crookshank in accord with me as to the
Ross-Jackson model, one which, in my opinion, has not been entirely
superseded by models of a more recent date. Indeed, the latest
improvement effected in the Ross-Jackson form, in which attention has
been given to the spreading-out of the feet, has converted it into as
solid and firm a stand as Powell’s; it is equally free from vibration
when placed in the horizontal position.
There are, however, four different forms of stands--the tripod; the
plate with double columns; the single column ending in a plate or a
bent claw; and the horse shoe. The tripod stand, with cork feet, is
by far the steadiest form of model. The single upright pillar support
should unquestionably be condemned, as it admits of considerable
vibration, and is most inconvenient for laboratory work. The heavy
horse-shoe form is compact and firm, and the weight of it can hardly be
considered an objection.
_The Tubular Body_ is from eight to ten inches in length, to which is
added a draw-tube with an engraved millimetre scale. By extending the
draw-tube greater magnification is obtained, but since this is at the
cost of definition it should hardly ever be employed in the examination
of bacteria. _A Triple Nose-piece_ is doubtless a convenience, saving
time which is otherwise spent in replacing objectives of different
magnifying powers; there is also less risk of injuring them. _Focus_
should be obtained by means of a rack and pinion coarse adjustment,
together with the most approved kind of fine adjustment. The sliding
tube cannot be recommended, as the motion may be stiff, encouraging the
use of force, which in turn may result in the objective being brought
violently into contact with the specimen, thus doing injury to the lens
or damage to the preparation; or it may get too loose and readily slip
out of focus.
_The Stage_ should be flat and rigid, either rectangular or circular,
so long as it is sufficiently large to accommodate plate cultivation. A
removable mechanical stage is of great advantage for working with high
powers, as a motile bacterium can be constantly kept in view, while one
hand is engaged in working the fine adjustment; it may also be employed
as a finder, if engraved with a longitudinal and vertical scale, and
provided with a stop. The mechanical stage must be removable, so that
the stage proper may be free from any attachments when required for the
examination of cultures.
_Diaphragms._--The plan of using a series of separate discs of
different sizes should be avoided, as they are easily lost, and
bacteriological investigations may have to be made under conditions in
which it is difficult to replace them. A better plan is a revolving
plate with apertures of different sizes, but the most convenient form
is the _iris diaphragm_.
_The Sub-stage Condenser_ is as necessary in biological work as in
the objective--in fact, the condenser and the objective should be
considered as forming one piece of optical apparatus; the microscope
must be regarded as incomplete without it.
It is by the _sub-stage condenser_ that the rays of light are
concentrated at one point, or on one particular bacterium; for the best
definition it is essential that there should be mechanical arrangements
for accurately centring and focussing the condenser. All this will be
explained and enlarged upon under “Practical Optics.”
In the historical review presented to my readers on the evolution of
the modern microscope, I have for the most part relied upon my long
and close association, extending over a period of upwards of half a
century, with microscopy. I need hardly say I could have very much
extended my remarks with pleasure and profit had space permitted, and
thereby much increased the number of names of manufacturers, who have
well-established reputations for the quality of their work, and whose
instruments, more or less complete in design, realise the wants of
students and of that large class of present-day workers engaged in
microscopical pursuits to whom economy of outlay is almost a first
consideration. No valid reason, however, can be assigned for splitting
up, as some writers do, the several forms of microscopes into some six
different classes, which implies inferiority in mechanical details or
finish, whereas the difference wholly consists in luxurious appliances
to save time, and in accessories for special work or original research.
Before bringing these remarks to a close, it is my wish to direct the
student’s attention to one or two points of importance in connection
with the use of the instrument, viz.: variations in body-lengths of
microscopes, especially between those of English and of Continental
manufacture. The _optical-standard_ measurement adopted in this country
for the body-tube-length is 10 inches; and for its _mechanical_, 8-3/4
inches. That of Continental opticians is, optical-tube-length 7·08
inches, or 180 mm.; the mechanical, 6·3 inches = to 168 mm.
Professor Abbe constructed an apochromatic immersion objective
especially for the English optical tube-length of 10·6 inches (= to
270 m.m.), and mechanical tube-length somewhat less in measurement.
This may be taken to mean a slight increase in the standard value of
the tube, and therefore the addition of the rack-and-pinion to the
draw-tube, now generally made a part of the microscope, is certainly
of some practical value. This difference, however, when working with
the English body-tube of 10 inches, may be discarded; it is, in
fact, only where the shorter Continental body is in use, that so
small a difference of tube-length exercises a disturbing effect over
adjustment. Moreover, an object placed on the stage of the shorter
body microscope will not be seen with the same distinctness by the
draughtsman should he wish to make use of the _camera lucida_.
The _optical_ tube-length of the body is measured from the back lens of
the objective to the front lens or principal focus of the eye-piece;
the _mechanical_ tube-length from the end of nose-piece of objective to
the top lens of the eye-piece.
[Illustration: The Hartnach Students’ Model Microscope.]
CHAPTER III.
Applied Optics:--Eye-pieces; Achromatic Objectives; Condensers.
It is almost unnecessary to say that the eye-piece forms a most
important part of applied optics in the microscope. It is an optical
combination designed to bring the pencil of rays from the objective to
assist in the formation of a real or virtual image before it arrives
at the eye of the observer. Greater attention has been given of late
years to the improvement of the eye-piece, since flatness of field
much depends upon it. Opticians have therefore sought to make it both
achromatic and compensatory.
There are several forms of eye-pieces in use, some of which partake of
a special character, and these will receive attention in their proper
places. It is, however, customary among English opticians to denote
the value of their several eye-pieces by Roman capitals, A, B, C, D,
and E. Continental opticians, on the other hand, have a preference
for numerals, 1, 2, 3, 4, 5 and 6, or more, and by which they are
recognised.
The eye-piece in more general use is that known as the _Huyghenian_
(Fig. 99); this came into use upwards of two centuries ago. It was
constructed by Christian Huyghens, a Dutch philosopher and eminent man
of science, secretary to William III.
It was made for the eye-piece of a telescope he constructed with his
own hands, and it has been in constant use as the eye-piece of the
microscope for nearly two centuries. It consists of two plano-convex
lenses, with their plane surfaces turned towards the eye, and divided
at a distance equal to half the sum of their focal lengths--in other
words, at half the sum of the focal length of the eye-glass and of the
distance from the field-glass at which an image from the object glass
would be formed, a stop, or diaphragm, being placed between the two
lenses for the reason about to be explained. Huyghens himself appears
to have been quite unaware of the value of an eye-piece so cleverly
constructed.
[Illustration:
Fig. 99.--Huyghenian Eye-piece A, the dotted lines show position of
lenses.]
It was reserved for Boscovich to point out that, by this important
arrangement, he had corrected a portion of the chromatic aberration
incidental to the earlier form of eye-pieces. Let Fig. 100 represent
the Huyghenian eye-piece of a microscope, _f f_ being the field-glass,
and _e e_ the eye-glass, and _l m n_ the two extreme rays of each of
the three pencils emanating from the centre and ends of the object,
of which, but for the field-glass, a series of coloured images would
be formed from _r r_ to _b b_; those near _r r_ being red, those
near _b b_ blue, and the intermediate ones green, yellow, and so on,
corresponding with the colours of the prismatic Spectrum.
The effect described, that of projecting the blue image beyond the red,
over-correcting the object-glass as to colour, is purposely produced;
it is also seen that the images _b b_ and _r r_ are curved in the wrong
direction to be seen distinctly by the convex eye-lens; this then is a
further defect of the compound microscope made up of two lenses. But
the field-glass, at the same time that it bends the rays and converges
them to foci at _b′ b′_ and _r′ r′_, also reverses the curvature of the
images as here shown, giving them the form best adapted for distinct
vision by the eye-glass _e e_. The field-glass has at the same time
brought the blue and red images closer together, so that they produce
an almost colourless image to the eye. The chromatic aberration of
lenses has been clearly explained in a previous chapter. But let it
be supposed that the object-glass had not been over-corrected, that
it had been perfectly achromatic; the rays would then have appeared
coloured as soon as they had passed the field-glass; the blue rays
of the central pencil, for example, would converge at _b′′_, and the
red rays at _r′′_, which is just the reverse of what is required of
the eye-lens; for as its blue focus is also shorter than its red, it
would require that the blue image should be at _r′′_, and the red at
_b′′_. This effect is due to over-correction of the object-glass,
which removes the blue foci _b b_ as much beyond the red foci _r
r_ as the sum of the distances between the red and the blue foci of
the field-lens and eye-lens; so that the separation _b r_ is exactly
taken up in passing through those two lenses, and the several colours
coincide, so far as focal distance is concerned, as the rays pass the
eye-lens. So that while they coincide as to distance, they differ in
another respect--the blue image is rendered smaller than the red by
the greater refractive power of the field-glass upon the former. In
tracing the pencil _l_, for instance, it will be noticed that, after
passing the field-glass, two sets of lines are drawn, one whole and
one dotted, the former representing the red, and the latter the blue
rays. This accidental effect in the Huyghenian eye-piece was pointed
out by Boscovich. The separation into colours of the field-glass is
like the over-correction of the object-glass--and opens the way to
its complete correction. If the differently-coloured rays were kept
together till they reached the eye-glass, they would still be coloured,
and present coloured images to the eye. The separating effected by the
field-glass causes the blue rays to fall so much nearer the centre of
the eye-glass, where, owing to its spherical figure, the refractive
power is less than at the margin, so that spherical error of the
eye-lens may be said to constitute a nearly equal balance to the
chromatic dispersion of the field-lens, and the blue and red rays _l′_
and _l′′_ emerge nearly parallel, presenting a fairly good definition
of a single point to the eye. The same may be said of the intermediate
colours of the other pencils. The eye-glass thus constructed not only
brings together the images _b′ b′_, _r′ r′_, but it likewise has the
most important effect of rendering them flatter, and assisting in the
correction of chromatic and spherical aberration.
[Illustration: Fig. 100.--Huyghenian Eye-piece.]
[Illustration: Fig. 101.--Ramsden’s Eye-piece.]
The later form of the Huyghenian eye-piece is that of the late Sir
George Airy, the field-glass of which is a meniscus with the convex
side turned towards the objective, and the eye-lens a crossed convex
with its flatter side towards the eye. Another negative eye-piece is
that known as the _Kellner_, or orthoscopic eye-piece. It consists
of a bi-convex field-glass and an achromatic doublet eye-lens. This
magnifies ten times, but it in no way compares with the Huyghenian in
value. Neither does it afford the same flatness of field.
The _Ramsden_, or positive eye-piece, is chiefly employed as a
micrometer eye-piece for the measurement of the values of magnified
images. The construction of this eye-piece is shown in Fig. 101, a
divided scale being cut on a strip of glass in 1/100ths of an inch,
every fifth of which is cut longer than the rest to facilitate the
reading of the markings, and at the same time that of the image of the
object, both being distinctly seen together, as in the accompanying
reduced micro-photograph of blood corpuscles, Fig. 102.
The value of such measurements in reference to the real object, when
once obtained; is constant for the same objective. It becomes apparent,
then, that the value of the divisions seen in the eye-piece micrometer
must be found with all the objectives used, and carefully tabulated.
It was Mr. Lister who first proposed to place on the stage of the
microscope a divided scale of a certain value. Viewing the scale as a
microscopic object, he observed how many of the divisions on the scale
attached to the eye-piece corresponded with one or more of a magnified
image. If, for instance, ten of those in the eye-piece correspond with
one of those in the image, and if the divisions are known to be equal,
then the image is ten times larger than the object, and the dimensions
of the object ten times less than that indicated by the micrometer.
If the divisions on the micrometer and on the magnified scale are not
equal, it becomes a mere rule-of-three sum; but in general this trouble
is taken by the maker of the instrument, who furnishes a table showing
the value of each division of the micrometer for every object-glass
with which it will be employed.
[Illustration: Fig. 102.--Blood Corpuscles and Micrometer, magnified
1·3500.]
Mr. Jackson’s simple and cheap micrometer is represented in Fig. 103.
It consists of a slip of glass placed in the focus of the eye-glass,
with the divisions sufficiently fine to have the value of the
ten-thousandth part of an inch with the quarter-inch object-glass, and
the twenty-thousandth with the eighth; at the same time the half, or
even the quarter of a division may be estimated, thus affording the
means of attaining considerable accuracy, and may be used to supersede
the more complicated and expensive screw-micrometer, being handier to
use, and not liable to derangement in inexperienced hands.
The positive eye-piece affords the best view of the micrometer, the
negative of the object. The former is quite free from distortion, even
to the edges of the field; but the object is slightly coloured. The
latter is free from colour, and is slightly distorted at the edges. In
the centre of the field, however, to the extent of half its diameter,
there is no perceptible distortion, and the clearness of the definition
gives a precision to the measurement which is very satisfactory.
[Illustration: Fig. 103.--Jackson’s Eye-piece Micrometer.]
Short bold lines are ruled on a piece of glass, _a_, Fig. 103, to
facilitate counting, the fifth is drawn longer, and the tenth still
longer, as in the common rule. Very fine levigated plumbago is rubbed
into the lines to render them visible; they are then covered with a
piece of thin glass, cemented by Canada balsam, to prevent the plumbago
from being wiped out. The slip of glass thus prepared is secured in a
thin brass frame, so that it may slide freely into its place.
Slips are cut in the negative eye-piece on each side, so that the brass
frame may be pressed across the field in the focus of the eye-glass,
as at _m_; the cell of which should have a longer screw than usual, to
admit of adjustment for different eyes. The brass frame is retained
in its place by a spring within the tube of the eye-piece; and in
using it the object is brought to the centre of the field by the stage
movements; the coincidence between one side of it and one of the long
lines is made with great accuracy by means of the small screw acting
upon the slip of glass. The divisions are then read off as easily as
the inches and tenths on a common rule. The operation, indeed, is
nothing more than the laying of a rule across the body to be measured;
and it matters not whether the object be transparent or opaque, mounted
or unmounted, if its edges can be distinctly seen, its diameter can be
taken.
Previously, however, to using the micrometer, the value of its
divisions should be ascertained with each object-glass; the method of
doing this is as follows:--
Place a slip of ruled glass on the stage; and having turned the
eye-piece so that the lines on the two glasses are parallel, read off
the number of divisions in the eye-piece which cover one on the stage.
Repeat this process with different portions of the stage-micrometer,
and if there be a difference, take the mean. Suppose the hundredth of
an inch on the stage requires eighteen divisions in the eye-piece to
cover it; it is plain that an inch would require eighteen hundred,
and an object which occupied nine of these divisions would measure
the two-hundredth of an inch. Take the instance supposed, and let
the microscope be furnished with a draw-tube, marked on the side
with inches and tenths. By drawing this out a short distance, the
image of the stage micrometer will be expanded until one division is
covered by twenty in the eye-piece. These will then have the value
of two-thousandths of an inch, and the object which before measured
nine will then measure ten; which, divided by 2,000, gives the decimal
fraction ·005.
Enter in a table the length to which the tube is drawn out, and the
number of divisions on the eye-piece micrometer equivalent to an
inch on the stage; and any measurements afterwards taken with the
same micrometer and object-glass may, by a short process of mental
arithmetic, be reduced to the decimal parts of an inch, if not actually
observed in them.
In ascertaining the value of the micrometer with a deep objective, if
the hundredth of an inch on the stage occupies too much of the field,
then the two-hundredth or five-hundredth should be used and the number
of the divisions corresponding to that quantity be multiplied by two
hundred or five hundred, as the case may be.
The micrometer should not be fitted into too deep an eye-piece, as it
is essential to preserve good definition. A middle-power Kellner or
Huyghenian is frequently employed; at all events, use the eye-piece of
lower power rather than impair the image.
The eye-lens above the micrometer should not be of shorter focus than
three-quarters of an inch, even with high-power objectives.
_The Ramsden Eye-piece._--The cobweb micrometer is the most efficient
piece of apparatus yet brought into use for measuring the magnified
image. It is made by stretching across the field of the eye-piece two
extremely fine parallel wires or cobwebs, one or both of which can be
separated by the action of a micrometer screw, the trap head of which
is divided into a hundred or more equal parts, which successively pass
by an index as the milled head is turned, shown in Fig. 104. A portion
of the field of view is cut off at right angles to the filaments by a
scale formed of a thin plate of brass having notches at its edges, the
distances between which correspond to the threads of the screw, every
fifth notch (as in the previous case) being made deeper than the rest,
to make the work of enumeration easier. The number of entire divisions
on the scale shows then how many complete turns of the screw have been
made in the separation of the wires, while the number of index points
on the milled head shows the value to the fraction of a turn, that
may have been made in addition. A screw with one hundred threads to
the inch is that usually employed; this gives to each division in the
scale in the eye-piece the value of 1/100th of an inch. The edge of the
milled head is also divided into the same number of parts.
[Illustration: Fig. 104.--Ramsden Screw Micrometer Eye-piece.]
In Watson’s Ramsden screw micrometer, Fig. 104, the micrometer scale
(seen detached) is ruled on a circular piece of glass, and this, by
unscrewing the top, is dropped into its place, and one of the wires,
both being fixed, is set a little to the side of the field, the teeth
of the screw being cut to 1/100ths, and the drum giving the fractional
space between the teeth to 1/100ths, so that the 1/10000th of an inch
can be read off. This micrometer eye-piece is constructed entirely of
aluminium, a decided advantage, being so much lighter than brass to
handle.
In the screw micrometer of other makers, other modifications are found.
An iris diaphragm being placed below the web to suit the power of
the eye-piece employed, a guiding line at right angles to the web is
sometimes added. Care should be taken to see that when the movable web
coincides exactly with the fixed web, the indicator on the graduated
head stands at zero.
_The Compensating Eye-piece._--The very important improvements effected
in the construction of the objective naturally led up to an equally
useful change for the better in the eye-piece.
All objectives of wide aperture, from the curvature of their
hemispherical front lenses, show a certain amount of colour defect in
the extra-axial portion of the field, even if perfectly achromatic in
the centre. Whether an image be directly projected by the objective, or
whether it be examined with an aplanatic eye-piece, colour fringes may
be detected, possibly in an increasing degree towards the periphery.
This residual chromatic aberration has at length been very nearly
eliminated by the aid of the compensating eye-piece.
The construction of compensating eye-pieces is somewhat remarkable,
since they have an equivalent error in an opposite direction--that is,
the image formed by the red rays is greater than that corresponding
to the blue rays; consequently, eye-pieces so constructed serve to
compensate for the unequal magnification produced by different coloured
rays, and images appear free from colour up to the margin of the field.
Zeiss’s compensating eye-pieces are so arranged that the lower focal
points of each series lie in the same plane when inserted in the
body-tube of the microscope; no alteration of focus is therefore
required on changing one eye-piece for another. This of itself is
not only an advantage but also a saving of time, while the distance
between the upper focal point of the objective and the lower one of the
eye-piece, which is the determining element of magnification, remains
constant.
[Illustration:
Fig. 105.--A sectional view of Zeiss’s Compensating series of
Eye-pieces, 1/2 the full size.
A.--Plane of the upper edge of the tube.
B.--Lower focal plane of eye-pieces, with their lenses _in situ_.]
The ordinary working eye-pieces, Huyghenian and others, commencing
with a magnification of four diameters, are so constructed that they
can be conveniently used, as we are accustomed to use them in England,
with high powers, Zeiss’s Nos. 12 and 18 compensating eye-pieces
being adapted for use with his lower power apochromatic lenses of 16
and 8 mm. The numbering of the eye-pieces is carried out on the plan
originally proposed by Professor Abbe--that is, the number denotes
how many times an eye-piece, when employed with a given tube-length,
increases the initial magnifying power of the objective, and at the
same time furnishes figures for their rational enumeration. It is on
this basis that the German compensating eye-pieces have been arranged
in series, and in agreement with their magnifying power and distinctive
numberings of 2, 4, 6, 8, 12, 18. Of these several eye-pieces, 12 is
found to be the most useful. The magnification obtained by combining
a compensating eye-piece with any apochromatic objective is found by
multiplying its number by the initial magnification of the objective,
as given in the following proof:--An objective of 3·0 mm. focus, for
example, gives in itself a magnification of 83·3 (calculated, for the
conventional distance of vision, 250 mm.); eye-piece 12 therefore gives
with this objective a magnification of 12 × 83·3 = 1000 diameters.
The classification, however, of these eye-pieces, as furnished by
Abbe, is dependent upon increase in the total magnifying power of the
microscope obtained by means of the eye-piece as compared with that
given by the objective alone. The numbering, then, denotes how many
times an eye-piece increases the magnifying power of the objective
when used with a given body-tube; the proper measure of the eye-piece
magnification; and, at the same time, the figures for rational
enumeration.
[Illustration: Fig. 106.--B and C Achromatic Eye-pieces.]
Compensating eye-pieces have been introduced for the correction of
certain errors in high-power objectives--those made with hemispherical
fronts. All such lenses, whether apochromatic or not, are greatly
improved by the compensating eye-piece, but the dry objective and the
lower powers are certainly deteriorated. The lower power compensating
eye-pieces are Huyghenian, the higher are combinations, with no
field-lens, and therefore in working act as a single or positive
eye-piece. This is of importance to those who work with low powers--the
older forms of objectives.
Messrs. Watson and Swift have adopted a new formula for their series
of _achromatic eye-pieces_, whereby their magnification and flatness
of field are improved. These also bear a constant ratio to the initial
power of their objectives.
The compensating eye-pieces of these makers are constructed on the same
principle as those of Zeiss’s for the correction of errors of colour in
the marginal portion of the field, and consequently are in every way
as effective as those of Continental manufacture. Figs. 106, 107, and
108 show in dotted outline the form and position of the several lenses
combined in these eye-pieces.
_Projection Eye-pieces_ are chiefly used in micro-photography, and for
screen demonstrations. The cap of this eye-piece is provided with a
spiral adjustment for focussing, the diaphragm being placed in front
of the eye-lens, an essential arrangement for obtaining an accurate
focus. The ring seen below the cap, Fig. 108, is graduated so that the
rotation for distance of screen may be carefully recorded.
[Illustration: Fig. 107.--The Compensating Eye-piece.]
[Illustration: Fig. 108.--Projection Eye-piece.]
Schmidt’s goniometer positive eye-piece, for measuring the angles of
crystals, is so arranged as to be easily rotated within a large and
accurately graduated circle. In the focus of the eye-piece a single
cobweb is drawn across, and to the upper part is attached a vernier.
The crystals being placed in the field of the microscope, care being
taken that they lie _perfectly flat_, the vernier is brought to zero,
and then the whole apparatus turned until the line is parallel with
one face of the crystal; the frame-work bearing the cobweb, with the
vernier, is now rotated until the cobweb becomes parallel with the next
face of the crystal, and the number of degrees which it has traversed
may then be accurately read off.
_Goniometer._--If a higher degree of precision is required, then, the
double-refracting goniometer invented by the late Dr. Leeson must be
substituted. With this goniometer (Fig. 109) the angles of crystals,
whether microscopic or otherwise, can be measured. It has removed
the earlier difficulties incident to similar instruments formerly in
use. Among other advantages, it is capable of measuring opaque and
even imperfect crystals, beside microscopic crystals and those in the
interior of other transparent media. It is equally applicable to the
largest crystals, and will measure angles without removing the crystal
from a specimen, provided only the whole is placed on a suitable
adjusting stage. The value of the goniometer depends on the application
of a doubly refracting prism, either of Iceland spar or of quartz, cut
of such a thickness as will partially separate the two images of the
angle it is proposed to measure.
Dr. Leeson strongly insisted on the importance of the microscope in
the examination of the planes of crystals subjected to measurement,
as obliquity in many cases arises from not only conchoidal fractures,
but also from imperfect laminæ elevating one portion of a plane, and
yet allowing a very tolerable reflection when measured by the double
refracting goniometer.
[Illustration: Fig. 109.--Leeson’s Goniometer.]
Microscopes for crystallographic and petrological research are now
specially constructed for measuring the angles of crystals.
Erector eye-pieces and erecting prisms are employed for the purpose
of causing the image presented to the eye to correspond with that
of the object. They are also helpful in making minute dissections
of structure; the loss of light, however, by sending it through two
additional surfaces is a drawback, and impairs the sharpness of the
image. Nachet designed an extremely ingenious arrangement whereby the
inverted image became erect; he adapted a simple rectangular prism to
the eye-piece. The obliquity which a prism gives to the visual rays
when the microscope is used in the erect position, as for dissecting,
is an advantage, as it brings the image to the eye at an angle very
nearly corresponding to that of the inclined position in which the
microscope is ordinarily used.
The Achromatic Objective.
[Illustration: Fig. 110.--Pan-aplanatic Achromatic Objectives.]
_The Achromatic Objective_, of all the optical and mechanical adjuncts
to the microscope, is in every way the most necessary, as well as the
most important. The ideal of perfection aimed at by the optician is
a combination of lenses that shall produce a perfect image--that is,
one absolutely perfect in definition and almost free from colour. The
method resorted to for the elimination of spherical and chromatic
aberration in the lens has been fully explained in a former chapter. It
will now be my endeavour to show the progressive stages of achromatism
and evolution of the microscope throughout the present century.
It is almost as difficult to assign the date of the earliest
application of achromatism to the microscope as to that of the
inception and many modifications of the instrument in past ages;
indeed, the question of priority in every step taken in its improvement
has been the subject of controversy.
Among the earlier workers in the first decade of this century will be
found the name of Bernardo Marzoni, who was curator of the Physical
Laboratory of the Lyceum of Brescia. He, an amateur optician, it
has come to light, in 1808 constructed an achromatic objective, and
exhibited it at Milan in 1811, when he obtained the award of a silver
medal for its merits, under the authority of the “Institute Reale
delli Scienzo.” Through the good offices of the late Mr. John Mayall
one of Marzoni’s objectives, which had been carefully preserved, was
presented to the Royal Microscopical Society of London in 1890.[20]
This objective is a cemented combination, with the plane side of the
flint-lens presented to the object. This was an improvement of a
practical kind, and of which Chevalier subsequently availed himself.
In 1823 Selligue, a French optician, is credited with having first
suggested the plan of combining two, three, or four plano-convex
achromatic doublets of similar foci, one above the other, to increase
the power and the aperture of the microscope. Fresnel, who reported
upon this invention, preferred on the whole Adam’s arrangement, because
it gave a larger field. Selligue subsequently improved his objective by
placing a small diaphragm between the mirror and the object.
In this country, Tully was induced by Dr. Goring to work at the
achromatic objective, and his first efforts were attended with a
success quite equal to that of Chevalier’s. Lister on examining these
lenses said:--“The French optician knows nothing of the value of
aperture, but he has shown us that fine performance is not confined to
triple objectives.” Amici, the amateur optician of Modena, visited this
country in 1827 and brought his achromatic microscope and objectives,
which were seen to give increase of aperture by combining doublets with
triplets. The most lasting improvement in the achromatic objective was
that of Joseph Jackson Lister, F.R.S., the father of Lord Lister, and
one of the founders of the Royal Microscopical Society of London.
Lister’s discoveries at this period (1829) in the history of the
optics of the microscope were of greater importance than they have
been represented to be. That he was an enthusiast is manifest, for,
being unable to find an optician to carry out his formula for grinding
lenses, he at once set to work to grind his own, and in a short time
was able to make a lens which was said to be the best of the day.
Lister, in a paper contributed to the proceedings of the Royal Society
the same year, pointed out how the aberrations of one doublet could be
neutralised by a second. He further demonstrated that the flint lens
should be a plano-concave joined by a permanent cement to the convex
crown-glass. The first condition, he states, “obviates the risk of
error in centring the two curves, and the second diminishes by one half
the loss of light from reflection, which is very great at the numerous
surfaces of every combination.” These two conditions then--that the
flint lens shall be plano-concave, and that it shall be joined by some
cement (Canada balsam) to the convex--may be taken as the basis for
the microscopic objective, provided they can be reconciled with the
correction of spherical and chromatic aberration of a large pencil.
Andrew Ross was not slow to perceive the value of Lister’s suggestions
and in 1831 he had constructed an object-glass on the lines laid
down by Lister, Fig. 112; _a a′_ representing the anterior pair, _m_
the middle, and _p_ the posterior, the three sets combined forming
the achromatic objective, consisting of three pairs of lenses, a
double-convex crown-glass, and a plano-concave of flint.
[Illustration: Fig. 111.--Lister’s double-convex crown and
plano-concave flint cemented combination.]
[Illustration: Fig. 112.--Andrew Ross’s 1/4-inch Objective.]
Lister proposed other combinations, and himself made an object-glass
consisting of a meniscus pair with a triple middle, and a back
plano-convex doublet. This had a working distance of ·11 and proved to
be so great a success that other opticians--Hugh Powell, 1834; James
Smith, 1839--made objectives after the same formula.
The publication of Lister’s data proved of value in another direction:
it stimulated opticians to apply themselves to the further improvement
of the achromatic objective. Andrew Ross was one of the more earnest
workers in giving effect to Lister’s principles and a short time
afterwards found that a triple combination, with the lenses separated
by short intervals, gave better results. In the accompanying diagram
the changes made in the combination of the objective from 1831, and
extending over a period of about twenty years from this date, are
shown.
Each objective, from the 1/2-inch to the 1/12-inch, is seen to be built
up of at least six or eight different fronts, the back combinations
being a triplet formed of two double-convex lenses of crown glass with
an intermediary double concave lens of flint-glass.
[Illustration: Fig. 113.--Combinations of Early Dry Objectives.
_A_, Double-convex lens; _B_, Plano-concave; _C_, Bi-convex and
plano-concave united; shown in their various combinations, as at _D_,
form the 3-in., 2-in. or 1-1/2-in.; at _E_, 1-in. and 2/3-in.; and at
_F_, the 1/2-in., 4/10-in., 1/4-in. and 1/25-in. objectives.
Combination _D_ was for many years known as the Norfolk Objective.]
[Illustration: Fig. 114.--Lister’s Correction Collar, (in section).]
No sooner had Ross constructed 1/4-inch achromatic objectives on
Lister’s formula than he discovered an error which had hitherto escaped
attention, viz., that the thinnest cover-glass of an object produced
a considerable amount of refractive disturbance. A marked difference
was observed in the image when viewed with or without a cover-glass.
This difficulty was first met by the addition of a draw-tube to the
microscope body. But as this also impaired the image, Lister overcame
the difficulty by mounting the front lens of the objective in a
separate tube made to fit over a second tube carrying the two pairs of
lenses. This arrangement led up to his invention of the _screw-collar
adjustment_, the mechanism for applying which is shown in Fig. 114. The
anterior lens _a_ at the end of the tube is enclosed in a brass-piece
_b_ containing the combination; the tube _a_, holding the lens nearest
the object, is then made to move up or down the cylinder _b_, thus
varying the distance, according to the thickness of the glass covering
the object, by turning the screw ring _c_, thus causing the one tube
to slide over the other, and clamping them together when properly
adjusted. An aperture is made in the tube _a_, within which is seen
a mark engraved on the cylinder, on the edge of which are two marks,
a longer and a shorter, engraved upon the tube. When the mark on the
cylinder coincides with the longer mark on the tube, the adjustment
is made for an uncovered object; and when the coincidence is with
the shorter mark, the proper distance is obtained to balance the
aberrations produced by a cover-glass the hundredth of an inch thick;
such glass covers are now supplied. The adjustment should be tested
experimentally by moving the milled edge which separates or closes the
combinations, and at the same time using the fine adjusting screw of
the microscope. The difficulty associated with the cover-glass of old
has, by the introduction of the homogeneous immersion system, been very
nearly eliminated. There still remains, however, a disturbing amount
of residual colour aberration in the achromatic dry objective, and for
the correction of which Zeiss proposed mounting the several lenses on a
method somewhat different to that so long in use in this country. Fig.
115 shows an objective in which the screw-collar ring _b b_ is made
to adjust the exact distance between the two back lenses placed at _a
a_. The value of the screw-collar is not questioned. It is difficult
to obtain at all times cover-glasses of a perfectly uniform thickness;
they will vary, and therefore perfect definition must be obtained, as
heretofore, by adjusting for each separate preparation while the object
is under examination.
[Illustration: Fig. 115.--The Continental Screw-collar Adjustment.]
As early as 1842 the excellence of Andrew Ross’s achromatic objectives
were acknowledged, and his formula for their construction was
generally followed. No doubt many of these early objectives of his
manufacture are still regarded as treasures. I possess a 1/2-inch
and a 1/4-inch, which I believe to be comparable with any achromatic
objectives of the same apertures of the present day. These I have
always found most serviceable for histological work.
In 1850 Mr. Wenham produced an achromatic objective of considerable
achromatic value. This consisted of a single hemispherical front
combination, shown in the accompanying enlarged diagram, Fig. 116.
Wenham’s formula seems to have been generally adopted by Continental
opticians, who sold these lenses at a reduction of price. In Paris,
Prazmowski and Hartnack--I have had one of Hartnack’s earliest
immersions in use for many years--brought this form of objective to
greater perfection, and in 1867 Powell and Lealand adopted the single
front combination system in their early water-immersion objective,
whereby the focal distance was said to be “practically a constant
quantity, while reduction of aperture by making the front lens
thinner ensures a much greater working distance without affecting the
aberrations, since the first refraction takes place at the posterior
or curved surface of the front lens, the removal of any portion of
thickness at the anterior or plane surface simply cuts off zones of
peripheral rays without altering the distance--any space being filled
by the homogeneous immersion fluid, or by an extra thickness of
cover-glass.”[21]
[Illustration: Fig. 116.--A Single Front Combination formulated by
Wenham for Messrs. Ross (enlarged).]
Great improvements were brought about by R. B. Tolles, of Boston,
1874, in the objective, as well as in the optical and mechanical parts
of the microscope, most of which, however, must be ascribed to the
criticisms and suggestions of amateur workers skilled in the exhibition
of test-objects--the late Dr. Woodward of Washington, for example,
whose series of photographs of the more difficult frustules of diatoms
have rarely been surpassed. Such results were due to improvements made
in the optical part of the microscope at his suggestion. He came to the
conclusion, arrived at about the same time by mathematical scientists,
that increase of power in the microscope was only possible in two
directions, the qualitative and the quantitative.
It was now that microscopists turned to the late Professor Abbe for
assistance in perfecting the objective in the dioptric direction. This,
he pointed out, must be looked for in further improvements in the art
of glass-making.
A series of experiments ultimately brought to light a mineral
substance, _Fluorite_, which, when combined in the proper proportion,
one part to two of German crown and flint glass, was found to have
the qualities looked for, and to possess different relations of a
dispersive and refractive power. From Professor Abbe’s researches,
begun in 1876, we have had the aperture of the objective greatly
enlarged, and the homogeneous system brought into general use.
Previous to this date the best made objective merely approximated to
colour correction. Undoubtedly the chief object to be obtained was
the removal or diminution of the secondary colour aberration. This,
together with other residual errors Abbe pointed out in 1880, led
to the improvement of the optical quality of the glass used in the
manufacture of all optical instruments, the chief difficulties being
surmounted in the Jena glass factory, whereby a complete revolution
was effected in the microscopic objective. The apochromatic glasses
of Zeiss, Powell, Beck, Ross, Watson, Swift, and other makers, in
which the secondary spectrum has been totally eliminated, or only a
negligible tertiary spectrum remains--that is to say, the objectives
of these makers--are now corrected for three spectrum rays, and not
two, as in the older objectives; and only those who look forward for
making further discoveries in the intimate structure of bacilli or for
resolving the finest diatom markings can be said to fully appreciate
the importance and value of the investigations of the late Professor
Abbe, and which have, so to speak, entirely changed old empirical
views as to the value of high aperture, and demonstrated that high
amplification, unless associated by proportionally high aperture,
necessarily produces untrue images of minute structures. It was he also
who introduced a practically perfect system of estimating apertures,
known as the “numerical aperture notation,” by which not only can an
accurate comparison be made of the relative apertures of any series
of objectives, whether dry or immersion, but their resolving power
under the various conditions of the kind of light employed. Their
penetrating power and their illuminating power can now be estimated
with mathematical exactness.
[Illustration: Fig. 117.--Diagram of an Apochromatic Combination.]
The practical advantages, then, secured by the adoption of the
homogeneous system were, on the whole, greater than any before made or
believed to be possible, and when taken into account in connection with
the improvement of the eye-piece (also due to Abbe), almost perfect
achromatism and homogeneity between objective, object, and eye-piece
is secured, together with a sharp definition of the image over the
whole visual field. These, with an increase of working distance between
the object and the objective, and other important results, have been
placed within the reach of the microscopist by men of science, and the
outcome is the general adoption of the homogeneous system, termed by
Carl Zeiss, a fellow-worker with Abbe, the[22] apochromatic system of
constructing objectives.
Relative Merits of the English and German Objectives.
As to the relative merits of German-made objectives, no superiority can
be claimed for them over those made by English opticians.
The Continental form of the 1/12-inch oil-immersion objective, shown in
Fig. 118, on the scale of 6 to 1, consists of four systems of lenses,
namely, the front, a deep hemispherical crown lens of high refractive
index; the second front of the system, an achromatic lens of such a
form that it gathers the light from the hemispherical front; the middle
lens, a single meniscus; and the back an achromatised lens, the second
front of the back being connected in such a way as to compensate for
the spherical and chromatic aberrations of the front lens.
The first homogeneous immersion objective which came under my
observation was manufactured in the well-known Jena workshop of Carl
Zeiss, December, 1877. This had a very considerable increase of
_numerical aperture_, upwards of 50 per cent.; a clear gain, as an
oil angle of even 110° proved to be of greater value than an angle of
180° in air, while the resolving power of the objective was increased
in like proportion. There does not at present appear to be a bar to
the construction of objectives of yet higher power, with increase of
aperture. The available course open in this direction is the further
discovery of another vitreous material and a suitable immersion fluid
with an index of 1·8 or 1·9, and glass with a corresponding index,
so as to ensure homogeneity of the combination. Zeiss asserts that
in the more difficult departments of microscopical research the
apochromatic lenses will supplant the older objectives, yet there are
many problems in microscopy awaiting solution which do not demand
the highest attainable degree of perfection in the objective, and in
the majority of cases the older achromatic objective is all that is
needful, provided it is good of its kind. The achromatic objectives
and eye-pieces of the older type have still an advantage, as, owing to
their simpler construction, really good lenses of the class required
can be purchased at considerably lower prices than the objectives of
the new series. These, from being more complicated in construction,
involve a greater amount of skilled manual labour.
[Illustration: Fig. 118.--The Continental 1/12-in. Oil-immersion
Combination (enlarged diagram).]
The German glasses of to-day afford satisfactory evidence both of
skill and workmanship displayed in their production. Their cost is
greater, then, for the reason given, as will be seen on reference to
Continental catalogues. The dry series of objectives cost somewhat
less, a 1/2-inch (numerical aperture 0·30) can be had for £1 10s., and
a 1/6-inch (numerical aperture 0·65) for £2. On the other hand, the
apochromatic series rapidly increase in price as the numerical aperture
approaches the limit of numerical aperture 0·40. The best of Zeiss’s
series are the 12 mm. (1/2-inch) and the 3 mm. (1/8-inch), numerical
aperture 1·4, both of which possess the optical capacity assigned to
them. These objectives are undoubtedly the finest to be met with in
the workshop of any optician. Achromatic objectives of Continental
manufacture have been as much improved as those of English make by the
introduction of the newer varieties of glass, as already explained,
while a new nomenclature has sprung up in consequence. We now have
semi-apochromatic and parachromatic. The German opticians have followed
Zeiss’s lead, since almost the same series of objectives are given
in the catalogues of Leitz, Reichert, and Seibert, while the quality
of both dry and immersion objectives is found to be much the same.
The low price of Reichert’s immersion objectives should be noted, as
their performance is quite perfect. A 1/12-inch (numerical aperture
1·30) of Leitz’s, with which I have worked at _bacteria_, has given
me much satisfaction; supplied by Watson and Baker at £5. A 1/12-inch
dry objective by the same maker (numerical aperture 0·87) costs £3,
and a water immersion 1/12-inch (numerical aperture 1·10) £3 5s. Leitz
reminds me that it requires a good lens of from six to seven hundred
magnifying power for the examination of bacteria. For this reason he
has constructed a new form of lens, a 1/10-inch oil-immersion of 2·5
mm. focus, for the purpose of adding to the resources of bacteriology.
This lens necessarily has a lower magnification than his former
1/12-inch oil-lens, but as it is less costly to manufacture it is
sold at a smaller price. The before-mentioned 1/12-inch, with a No. 3
compensating eye-piece, gives a magnification of over seven hundred
or eight hundred diameters. To secure the best results in using the
higher powers of Leitz’s, from No. 5 upwards, a cover-glass of 0·17 mm.
in thickness should be used, and care taken to make the length of the
draw-tube equal to 170 mm. This length of tube should be adhered to
in the use of this optician’s oil-immersion lenses. If the microscope
be provided with a nose-piece, the draw-tube should be drawn out to
160 mm.; in its absence it should be set at 170 mm., a deviation of
10 mm. or more from the correct tube-length deteriorates from the
value of Leitz’s oil-immersion objectives as of other opticians. It is
suggested that the German apochromatic combination of three cemented
lenses is that adopted by Steinheil long before, in the construction
of his well-known hand-magnifier (see page 77, Fig. 51). Zeiss’s 3 mm.
objective has a triple front, balanced by two triple backs--in all
nine lenses--a somewhat amplified diagram of which is represented in
Fig. 118. The formula for this combination was furnished by Tolles,
of Boston, America, and it at once secured increase of aperture
(the value of this optician’s many contributions to microscopy has
since his death been generally acknowledged). The metrical equivalent
focus assigned by Zeiss to his series of dry achromatic objectives is
given in somewhat ambiguous terms, which tend to confuse rather than
classify them; for instance, two lenses of the same aperture--24 mm.
and 16 mm.--corresponding to the English 1-inch and 2/3-inch, each have
assigned to them an aperture of 0·30; a 12 mm. and 8 mm., corresponding
to the English 1/2-inch and 1/3-inch, have an aperture of 0·65; while
a 6 mm. = 1/4-inch, and a 4 mm. = 1/4-inch and 1/6-inch, have each an
aperture of 0·95.
Nachet exhibited at the Antwerp Exhibition a fine 1/10-inch
oil-immersion, which was highly praised by the jurors.
It is necessary, to make the fact perfectly clear, that dry and
immersion lenses having the same angular aperture have also a similar
defining power. The pencil of rays, however, differs in intensity
and density as the rays emerging from the cover-glass of the object
into air are very considerably deflected, and the cone suffers a
corresponding loss of brightness. On this important point, then,
I believe it will prove of value to interpolate a clear and full
exposition of the change brought about by the cover-glass.
It is not difficult, then, to perceive the importance of Amici’s
discovery as to the value of a drop of water inserted between the
object and the objective, and it now seems somewhat surprising it
should have been so long neglected by opticians, since it is at once
seen to diminish the reflection which takes place in the incidence of
oblique light. The film of water not only gives increased aperture,
but also greater cleanness and sharpness to the image. The film, then,
as already shown, collects the straying away of peripheral rays of
light, and sends them on to the eye-piece, and greatly assists in
rendering the image more perfect, and materially aids in the removal
of residuary secondary aberrations; while with air, or dry objectives,
a certain amount of aberration takes place, sufficient to affect the
pencils on their passage from the radiant to the medium of the front
lens, adding a considerable ratio to the total spherical aberration
with the objective, which, in the case of wide angles, increases
disproportionately from the axis outwards. This can only be corrected
by a rough method of balancing; that is, by introducing an excess of
opposite aberration in the posterior lens. An uncorrected residuum,
rapidly increasing with larger apertures, is then left, and this
appears in the image amplified by the total power of the objective, so
that with a non-homogeneous medium there is a maximum angular aperture
which cannot be surpassed without undergoing a perceptible loss of
definition, provided working distance is required. If we abolish the
anterior aberration for all colours, by an immersion fluid which is
equal to cover-glass in refractive and dispersive power, the difficulty
is at once overcome. If, for instance, we have an objective of 140° in
glass (= 1·25 N.A.) and water as the immersion fluid, the aberration in
front would affect a pencil of 140°. Substituting a homogeneous medium,
the same pencil, contracted to the equivalent angle in that medium
of 112°, will be admitted to the front lens without any aberration,
and may be made to emerge from the curved surface also without any
disturbing aberration, but contracted to an angle varying from 70° to
90°. The first considerable spherical aberration of the pencil then
occurs at the anterior surface of the _second_ lens, where the maximum
obliquity of the rays is already considerably diminished.
[Illustration: Fig. 119.]
[Illustration: Fig. 119_a_.]
Figs. 119 and 119_a_ will doubtless make this clearer. If the objective
of 140° works with water (Fig. 119), there would be a cone of rays
extending up to 70° on both sides of the axis, _and this large cone
would be submitted to spherical aberration at the front surface a_.
But with homogeneous immersion (Fig. 119_a_) the whole cone of 112°
is admitted to the front lens without any aberration, there being no
refraction at the plane surface; and as the spherical surface of the
front lens is without notable spherical aberration, the incident pencil
is brought from the focus F to the conjugate focus F′, and contracted
to an angle of divergence of 70°-90° _without having undergone any
spherical aberration at all_.
The problem of correcting a very wide-angled objective has thus been
reduced by the homogeneous oil-immersion system, both in theory and
practice.[23]
Abbe’s Test-plate.
Abbe designed the test-plate (Fig. 120) for testing the spherical and
chromatic aberrations of objectives, and estimating the thickness
of cover-glasses corresponding to the most perfect correction: six
glasses, having the exact thickness marked on each, 0·09 to 0·24 mm.,
cemented in succession on a slip, their lower surface silvered and
engraved with parallel lines, the contours of which form the test.
These being coarsely ruled are easily resolved by the lowest powers;
yet, from the extreme thinness of the silver, they form also a delicate
test for objectives of the highest power and widest aperture. The
test-plate in its original size is seen in Fig. 120, with one of the
circles enlarged.
[Illustration: Fig. 120.--Abbe’s Test-plate for estimating thickness of
glass-covers.]
To examine an objective of large aperture, the discs must be focussed
in succession, observing in each case the quality of the image in the
centre of the field, and the variation produced by using, alternately,
central and very oblique illumination.
When the objective is perfectly corrected for spherical aberration,
the outlines of the lines in the centre of the field will be perfectly
sharp by oblique illumination, and without any nebulous doubling or
indistinctness of the edges. If, after exactly adjusting the objective
for oblique light, central illumination is used, no alteration of the
focus should be necessary to show the outlines with equal sharpness.
If an objective fulfils these conditions with any one of the discs,
it is free from spherical aberration when used with cover-glasses
of that thickness. On the other hand, if every disc shows nebulous
doubling, or an indistinct appearance of the edges of the line with
oblique illumination, or, if the objective requires a different focal
adjustment to get equal sharpness with central as with oblique light,
the spherical correction of the objective is more or less imperfect.
Nebulous doubling with oblique illumination indicates over-correction
of the marginal zone; indistinctness of the edges without marked
nebulosity indicates under-correction of the zone; an alteration of
the focus for oblique and central illumination points to an absence of
concurrent action of the separate zones, which may be due to either an
average under or over correction, or to irregularity in the convergence
of the rays.
[Illustration: Fig. 121.--Zeiss’s Cover-glass Gauge.]
COVER-GLASS GAUGE.
Zeiss has gone a step further to lay the microscopist’s ghost of the
cover-glass. He invented a measurer (Fig. 121) whereby the precise
determination of thickness of glass-covers can be obtained. This
measurement is effected by a clip projecting from a circular box; the
reading is given by an indicator moving over a divided circle on the
lid of the box. The divisions seen cut round the circumference show
1/100ths of a millimeter. This ingenious gauge measures upwards of 5
mm.
This necessary and important digression has led me away from the
consideration of the achromatic objective, and to which I shall now
return.
English Immersion and Dry Objectives.
The homogeneous immersion system met with its earliest as well as its
staunchest advocates among English opticians. Among its more energetic
supporters were Messrs. Powell and Lealand, who were the first to
construct a 1/8-inch immersion objective on a formula of their own,
and which was found to resolve test-objects not before capable of
resolution by their dry objectives. This encouraged them to make a
1/16-inch, acquired by Dr. Woodward for the Army Medical Department,
Washington, and subsequently a 1/25-inch; neither of which surpassed
their 1/8-inch in aperture, and a new formula was tried in the
construction of their first oil-immersion objective. This had a duplex
front, and two double backs; but even this did not quite accomplish
what was expected of it, and another change was subsequently made;
the anterior front combination became greater than a hemisphere--a
balloon-lens. This at once gave an increase of aperture to a 1/12-inch
objective of 1·43 numerical aperture. After some few more trials a more
important change of the formula took place. The front lens was made of
flint-glass, and the combination took the form represented in diagram
(Fig. 122). This, on an enlarged scale, represents Powell’s 1/12-inch
numerical aperture 1·50. It is a homogeneous apochromatic immersion
of high quality and very flat field. It will be noticed that in this
combination the four curves of the lenses are very deep compared with
those of other opticians.
[Illustration: Fig. 122.--Powell and Lealand’s 1/12-in. Oil-immersion
Objective, drawn on a scale of 6-1.]
_Messrs. Ross_ have made many important improvements and changes in
the construction of their several series of achromatic objectives; the
calculations and formulæ for which were made exclusively for them by
Dr. Schrœder. The list is too long to quote, but most of these lenses
are of a high-class character, and work with admirable precision.
Among the best of their objectives, I can commend a 1-inch of 30°
and two oil-immersions, a 1/8-inch of 1·20 and a 1/12-inch of 1·25
numerical aperture, each of which bear the highest oculars equally
well; a good test, as I have always maintained, of excellence. Their
1/10-inch has a somewhat larger aperture, and therefore shows a fine
image of the podura scale. The finish of Ross’s several series of
objectives fully maintains the high character and reputation of this
old-established firm of opticians.
_Messrs. R. and J. Beck_ have bestowed great attention upon the
improvement of their dry-objective series, much in demand for
histological work, especially among the students of city hospitals,
who usually commence their pathological work with the cheaper forms of
objectives. In that case an inch objective of about 25° air angle, a
1/2-inch of not less than 40°, and a 1/4-inch or 1/5-inch magnifying
from 50 to 250 diameters, is quite sufficient for most of their work.
For bacteriological research, Messrs. Beck supply a 1/6-inch immersion
taken from a series, having a high aperture and a better finish at a
moderate price. Their 1/10-inch immersion has in my hands proved a
serviceable power for bacteriological research; it requires a good
sub-stage illuminating achromatic condenser to obtain the best results.
[Illustration: Fig. 123.--1/6-in. English Combination, largely used.]
_Messrs. Watson and Sons_ have much enhanced their reputation by
the marked improvement lately brought about in the manufacture of
their whole series of objectives. This probably is chiefly due to
the introduction of the _Jena_ glass into their manufacture, and
which has enabled them to give increase of aperture to one series in
particular, that of the para-chromatic, all of which in consequence are
of very high quality. It is difficult to particularise their several
objectives, the whole having special features in proportion to their
magnifying powers, while much care seems to have been bestowed on them
for the elimination of residual colour. A 1/8-inch with correction
collar is comprised of a single deep and rather thick front lens,
plano-concave flint, and double convex-crown for the middle and triple
combination for the back, the latter consisting of two crown lenses
cemented to a dense flint (Fig. 124) drawn to scale of 5-1, with lined
portions intended to represent the flint, and white the crown glass
lenses of the combination. The initial magnification of this objective
is 83 diameters, and the numerical aperture ·94. This superior
objective can be had for the small sum of £2. Another remarkably useful
and cheap objective, their 1-inch numerical aperture 0·21, consists of
two achromatic systems forming the front and back with the separation
between them of about half an inch, and may also be especially
recommended for students’ work.
In the accompanying diagram the lenses are drawn on too large a scale,
and therefore the distance between the two combinations should be much
greater.
Among the more useful of Watson’s series, the 1-inch, the 1/2-inch, and
the 1/6-inch, together with the 1/8-inch dry-objective, and a 1/9-inch,
will be found the most serviceable.
[Illustration: Fig. 124.--Watson’s 1/8-in. Objective Para-chromatic
Combination, scale 5-1.]
[Illustration: Fig. 125.--Watson’s 1-in. Achromatic Combination.]
_Messrs. Baker_ have their own series of objectives, most of which
are so very nearly allied to those of the continental opticians; and
what has been said of Zeiss’s and Leitz’s objectives may be taken to
apply also to Baker’s, who have an established reputation for their
histological series, all of which are well suited for students’ and
class-room work.
_Messrs. Swift and Son_ have a new series of objectives,
semi-apochromatic and pan-aplanatic, most of which are excellent
in quality and show increased flatness of field together with that
of achromatism; the index of refraction in each series having been
correctly determined together with exact radial focal distance,
thus affording more available aperture. I may select for special
commendation their 1/12-inch £5 5_s._ homogeneous immersion objective,
which is in every way suitable for bacteriological work; its
definition is very good, as is seen in a micro-photograph of podura
scale, given further on. Their dry 1/6-inch can be had for £1 16_s._--a
marvel of cheapness. Of their general series the most useful for
histological work are the 1/2-inch, the 1/3-inch at £1 12_s._, and
their 1/5-inch of numerical aperture 0·87 at £3.
_Mr. Pillischer_, of Bond Street, has manufactured many excellent
objectives. A fine homogeneous oil-immersion 1/12-inch numerical
aperture 1·25 is worthy of special notice; it will be found suitable
for bacteriological work; it has fine definition with a considerable
amount of penetration.
A more intelligent idea of the magnifying power of the objective
combined with the eye-piece will be gained by consulting the table
given below; precision in this respect has long been a desideratum with
microscopists.
Magnifying Powers of Eye-Pieces and Objectives.
A TYPICAL AND INITIAL SELECTION OF POWERS OF EYE-PIECES CALCULATED FOR
THE 10-INCH TUBE-LENGTH.
HUYGHENIAN EYE-PIECES.
NAME A B C D E F
OF MAKER. 0 or No. 1 2 3 4 5 6
Baker 6 8 12 15 -- -- Diameters.
Beck, R. & J. 4 8 15 20 25 not made. "
Leitz 5 6 7 8 10 12 " "
Powell & Lealand 5 7·5 10 20 40 " "
Reichert 2·5 3·5 4 5 6·5 " " [24]
Ross 3 8 12-1/2 20 25 40 " [25]
Swift & Son 6 9 12 15 18 21 "
Watson & Sons 4 6 8 10 12 15 "
Zeiss 3 4 5·5 7 9 not made. "
COMPENSATING EYE-PIECES FOR USE WITH APOCHROMATIC OBJECTIVES.
Zeiss 2 4 8 12 18 27 Diameters.
This may be taken as a typical set, further treated of among Eye-pieces.
INITIAL POWERS OF OBJECTIVES CALCULATED FOR THE 10-INCH TUBE-LENGTH.
This is ascertained by dividing the distance of distinct vision 10
inches by the focus of the objective, thus--
Focus-inches 4 3 2 1-1/2 1 2/3 1/2 4/10 1/4 1/5 1/6 1/8 1/12
Initial magnifying
power 2·5 3·3 5 7·5 10 15 20 25 40 50 60 80 120 diameters.
A reference to the above table will at once show that the nomenclature
of objectives expresses at once the initial magnifying powers, but as
makers have great difficulty in so calculating their formulæ so as to
obtain the _exact_ power, these figures must be taken as approximate.
Thus a 1/4-inch, which should magnify 40 diameters if true to its
description, might actually magnify a little more or less.
The magnifying powers of Zeiss’s and other apochromatic objectives
can be ascertained by dividing the focal length of the objective in
millimeters into 250 mm. (the distance of distinct vision), thus
Focus millimetres 24 16 12 4 3 2 1·5
Initial magnifying
power 10·5 15·5 21 63 83 125 167 diameters.
The total magnification, when any eye-piece is working in conjunction
with an objective, is ascertained by multiplying the initial power of
the objective by that of the eye-piece.
The above calculations are all for a 10-inch tube-length. Should,
however, a shorter or longer length of body be employed, the
magnification can at once be ascertained by a proportion sum. If the
magnification be 180 with 10-inch tube-length, what would it be with a
6-inch body--10 : 6 :: 180 = 108 diameters.
Abbe designed three different forms of eye-pieces: 1, the searcher
eye-piece; 2, the working eye-piece; and 3, the projecting eye-piece.
The _Searcher_ is a negative form of low power. The working is both
negative and positive, the positive form of which is constructed
on a newer principle; while the projection is chiefly intended
for microphotography, its field being small and its definition
superlatively sharp. These are severally explained among eye-pieces.
High-Power Objectives.
_Points of Importance for securing the best results with High-power
Objectives._--Always give to the body-tube of the microscope the
length for which the objective is corrected, 0·160 mm. for the short
continental tube, and 0·250 mm. for the English tube (10-inch). Employ
both dry and immersion objectives mounted for correction, commencing
with a numerical aperture of 0·75 (that is about 100° in air). If the
graduation is not given in thickness of cover-glass apply to the maker
to correct this omission.
With the homogeneous oil-immersion objective it is highly necessary to
utilise all marginal pencils of light, to optically unite the upper
lens of the condenser with the preparation as well as the front lens of
the objective by means of a liquid having the same index of refraction
or at least equal to that of the immersion. _Cedar Oil_ has been
generally adopted for the purpose mentioned, the better way of using
which is as follows: place a drop on the centre of the front objective,
or on the top of the cover-glass, and then lower the objective by means
of the coarse adjustment until it comes in contact with the oil, and
carefully bring into focus by the fine adjustment. If the slide is held
between the finger and thumb of one hand and moved from side to side,
while the other hand is working the fine adjustment, there can be no
danger of injuring either the objective or the specimen. Before putting
the microscope away, take a fine camel-hair brush dipped in ether,
alcohol, or methylated spirit, and carefully remove the oil from the
objective and the glass cover of the object; a soft chamois leather or
cambric pocket handkerchief will dry it off, or a piece of fine white
blotting paper answers equally well. Should the lens come accidentally
into contact with the Canada balsam, it must be very carefully removed
either by ether or alcohol. The former is by far the safest, as
alcohol, if not very carefully used, quickly dissolves out the balsam
and loosens the cover-glass of the object.
Achromatic Condensers.
_The Achromatic Condenser_ can no longer be classed among the
_accessories_ of the microscope, since it is an absolutely
indispensable part of its optical arrangements. Its value, then,
cannot be overrated, and the corrections of the lenses which enter
into the construction of the condenser should be made as perfect as
they can be made--in fact, as nearly approaching that of the objective
as it is possible to make them. It may therefore be of interest to
know something of the rise and progress of the achromatic condenser.
In my first chapter I have noticed the earlier attempts made by Dr.
Wollaston, whose experiments led him to fit to the underside of the
stage of his microscope a short tube, in which a plano-convex lens
of about three-quarters of an inch focal length was made to slide
up and down (afterwards moved up and down by two knobs); to improve
definition he placed a stop between the mirror and the lens. The stop
was found to act better when placed between the lens and the object.
From this improvement Dr. Wollaston enunciated that “the intensity of
illumination will depend upon the diameter of the illuminating lens and
the proportion of the image to the perforation, and may be regulated
according to the wish of the observer.” Dujardin in France and Tully
in England were at work in the same direction. The former a year or
two later on contrived an instrument, which he termed an _eclairage_,
to remedy the defects of Wollaston’s, and for illuminating objects
with achromatic light. This was submitted for approval to Sir David
Brewster, who, when the use of the achromatic condenser was first
broached, used these encouraging words:--“I have no hesitation in
saying that the apparatus for illumination requires to be as perfect
as the apparatus for vision, and on this account I would recommend
that the illuminating lens should be perfectly free from chromatic and
spherical aberration, and that the greatest care be taken to exclude
all extraneous light both from the object and eye of the observer.”
This far-seeing observer in optical science has borne good fruit,
and the outcome of his views is seen in the great development and
improvement of the achromatic condenser. In 1839 Andrew Ross made his
first useful form of condenser, and gave rules for the illumination
of objects in an article written for the “Penny Cyclopædia.” These,
epitomised, read as follows: 1. That the illuminating cone should equal
the aperture of the objective, and no more. 2. With daylight, a white
cloud being in focus, the object has to be placed nearly at the apex
of the cone. The object is seen better sometimes above and sometimes
below the apex of the cone. 3. With lamplight a bull’s-eye lens is
to be used, to parallelise the rays, so that they may be similar to
those coming from the white cloud. It has been seen that Mr. Lister
foreshadowed the sub-stage condenser.
The early form of Ross’s condenser consists of two small brass tubes
made to slide one in the other. To the outer one is attached a flat
brass plate which slides underneath the stage of the microscope, and
by means of a screw the adjustment of the axis of the illuminator is
effected. The upper portion of the apparatus carries the achromatic
combination, which by a rack and pinion movement is brought nearer to,
or removed further from the object on the stage. The several parts of
the illuminator unscrew, so that the lenses may be used either combined
for high powers, or separated for low powers.
[Illustration: Fig. 126.--Original form of Gillett’s Achromatic
Condenser.]
Messrs. Smith & Beck greatly improved upon Ross’s condenser by adding
another achromatic lens to the combination, three being employed when
used with high-power objectives and two or even one with the lower,
the adjustment and focussing being made by rack and pinion arrangement
beneath the stage. Some further changes for the better were made in the
condenser by Powell, and in 1850 an amateur microscopist, Mr. Gillett,
fully grasping the value of controlling the cone of rays passing into
the microscope, devised a new form of condenser, in connection with
which a revolving series of diaphragms of different values were made
to pass between the achromatic lenses and the source of light.
Andrew Ross constructed the first condenser on Gillett’s principle,
and this proved to be one of the most successful pieces of apparatus
contrived. _Gillett’s Condenser_ consists of an achromatic lens _c_,
about equal to an object-glass of one quarter of an inch focal length,
with an aperture of 80°. This lens is screwed into the top of a brass
tube, and intersecting which, at an angle of about 25°, is a circular
rotating brass plate _a b_, provided with a conical diaphragm, having
a series of circular apertures of different sizes _h g_, each of which
in succession, as the diaphragm is rotated, proportionally limits the
light transmitted through the illuminating lens. The circular plate
in which the conical diaphragm is fixed is provided with a spring and
catch _e f_, the latter indicating when an aperture is central with
the illuminating lens, also the number of the aperture as marked on
the graduated circular plate. Three of these apertures have central
discs for circularly oblique illumination, allowing only the passage
of a hollow cone of light to illuminate the object. The illuminator
above described is placed in the secondary stage _i i_, which is
situated below the general stage of the microscope, and consists
of a cylindrical tube having a rotatory motion, also a rectangular
adjustment, which is effected by means of two screws _l m_, one in
front, and the other on the left side of its frame. This tube receives
and supports all the various illuminating and polarising apparatus, and
other auxiliaries.
_Directions for using Gillett’s Condenser._--In the adjustment of the
compound body of the microscope for using with Gillett’s illuminator,
one or two important points should be observed--first, centricity; and
secondly, the fittest compensation of the light to be employed. With
regard to the first, place the illuminator in the cylindrical tube,
and press upwards the sliding bar _k_ in its place, until checked
by the stop; move the microscope body either vertically or inclined
for convenient use; and, with the rack and pinion which regulates
the sliding bar, bring the illuminating lens to a level with the
upper surface of the object-stage; then move the arm which holds the
microscope body to the right, until it meets the stop, whereby its
central position is attained; adjust the reflecting mirror so as to
throw light up the illuminator, and place upon the mirror a piece of
clean white paper to obtain a uniform disc of light. Then put on the
low eye-piece, and a low power (the half-inch), as more convenient
for the mere adjustment of the instrument; place a transparent
object on the stage, adjust the microscope-tube, until vision is
obtained of the object; then remove the object, and take off the cap
of the eye-piece, and in its place fix on the eye-glass called the
“centring eye-glass,”[26] which will be found greatly to facilitate
the adjustment now under consideration, namely, the centring of the
compound body of the microscope with the illuminating apparatus of
whatever description. The centring-glass, being thus affixed to
the top of the eye-piece, is adjusted by its sliding-tube (without
disturbing the microscope-tube) until the images of the diaphragms in
the object-glass and centring lens are distinctly seen. The illuminator
should now be moved by means of the left-hand screw on the secondary
stage while looking through the microscope, to enable the observer to
recognize the diaphragm belonging to the illuminator, and by means
of the two adjusting screws to place this diaphragm central with
the others: thus the first condition, that of centricity, will be
accomplished. Remove the white paper from the mirror, and also the
centring-glass, and replace the cap on the eye-piece, also the object
on the stage, of which distinct vision should then be obtained by
the rack and pinion, or fine screw adjustment, should it have become
deranged.
[Illustration: Fig. 127.--The Ross Improved Achromatic Condenser, with
diaphragm stops.]
The re-publication of the original directions is given with the view
of showing what a clear conception Gillett had of the value of his
invention. The careful directions given for centring must be regarded
with interest, although nearly superseded by the centring screw
arrangement in connection with the sub-stage. The best results, he
goes on to say, will be secured by using the plain mirror and focussing
the window-bar on the object, while a white-cloud illuminator will
afford as much light as may be required. It is a mistake to suppose
that direct light is more critical than indirect. As a rule, the
student is given to over-illuminate the object. These questions will,
however, be discussed further on.
Very many modifications of Gillett’s condenser have, since 1850, become
known to microscopists. Ross’s present improved form (Fig. 127) is
made to drop into the sub-stage of the microscope, and when adjusted,
is an extremely efficient instrument. The optical part is similar to
a 4/10-inch objective. It has two sets of revolving diaphragms, with
apertures and stops for showing surface markings in a perfect manner.
Abbe’s Condenser.
The essential feature of this condenser is its short focus, which
collects the light reflected by the mirror, so as to form a cone of
rays of very large aperture, having its focus in the plane of the
object.
[Illustration: Fig. 128.--The Iris Diaphragm, and carrier for Stops.]
The full aperture of the illuminating cone should only be used when
finely granular and deeply stained particles (protoplasm, bacteria,
&c.) are being examined with objectives of large aperture. In all
cases the cone must be suitably reduced, either by an iris, or other
form of diaphragm (_central illumination_). By placing the diaphragm
excentrically, by means of rack-work attached to the carrier, the
central rays are excluded and a certain extra-axial portion of the
illuminating pencil falls upon the object (_oblique illumination_).
When the diaphragm is thus excentrically placed, this oblique pencil
can be directed from all sides by rotating the carrier round the optic
axis. The central stop diaphragm shuts off all the axial and transmits
only the marginal rays, thus producing _dark-ground illumination_. The
iris diaphragm (Fig. 128) is so shaped that the edge of its smallest
opening closely approximates the object-slide on the stage.
[Illustration: Fig. 129.--The Abbe Condenser, detached from the
Sub-stage of the Microscope.]
The Abbe condenser is the most popular form in use, for all purposes.
Owing to the large aperture of the cone of light which it projects,
it can be employed with the highest powers; by removing the top lens
it can also be used with low powers. Dark ground illumination may be
obtained with it up to a 1/4-inch objective.
[Illustration: Fig. 130.--Optical Arrangement of Abbe Illuminator, 1·2
N.A.]
[Illustration: Fig. 131.--Optical Arrangement of Abbe Illuminator, 1·4
N.A.]
The condenser is made in two forms of 1·2 and 1·4 numerical aperture
by Messrs. Watson. The lenses are mounted in aluminium. Fig. 130 is
in more general use, but by workers with high powers Fig. 131 is
preferred, as it ensures the most oblique illumination with objectives
of largest aperture. It is preferred for photo-micrographic purposes.
[Illustration: Fig. 132.--The Optical Arrangement of Watson’s
Achromatic Condenser.]
_Watson’s Achromatic Condenser_ (Fig. 132), 1·0 numerical aperture,
shown in section, although originally designed for use with the
micro-spectroscope, is equally efficient for ordinary purposes. This
condenser transmits a larger aplanatic cone of light than Abbe’s.
It may therefore be employed with higher power objectives, and by
removing the top lens it is just as useful a condenser for lower
powers. Being constructed with lenses of an unusually large size, it is
well adapted for use with the micro-spectroscope. It is certainly one
of the best all-round condensers in use. The new Schott glass enters
into the construction of the lenses, and these are mounted in aluminium.
[Illustration: Fig. 133.--Powell’s Achromatic Condenser.]
Many microscopists consider on the whole that Powell’s sub-stage
apochromatic condenser with collar correction (Fig. 133) surpasses
that of Abbe. The mechanical arrangement of Powell’s is very simple:
the correction collar is similar to that of an ordinary objective, it
has a steeper spiral slot and only half a revolution of movement; a
long arc is fixed to the collar so that it may conveniently be reached
by the finger. It is so constructed as to turn easily and smoothly
at the slightest touch. The collar moves only the back lens of the
combination, leaving the mount rigid. The diaphragms are regulated by A
and B.
[Illustration: Fig. 134.]
[Illustration: Fig. 134_a_.]
[Illustration: Fig. 134_b_.--Powell’s Apochromatic Oil Immersion
Condenser, N.A. 1·40.]
The object of the correctional movement is to increase the maximum
aplanatic aperture of the condenser by separating the lenses. If
the back of a wide-angled objective be examined when an object is
illuminated by the full aperture of the condenser, the edge of the
flame being in focus, it will be noticed that the illuminated portion
of the back lens will be oval and pointed instead of circular. Also
that when the condenser is racked up, although the external shape of
the illuminated portion becomes more circular, two dark patches will
appear on either side of the centre, showing the operation of the
spherical aberration of the condenser. If under these circumstances
the lenses are separated by means of the collar adjustment, the black
spots will be closed up, and a circular and evenly-illuminated disc of
illumination of a larger size will result. The wheel of diaphragms,
or a series of graduated diaphragm discs to drop into a holder, is
intended for critical work; the diaphragm can always be recorded, and
the identical illuminating cone reproduced.
Hence we have a simple method of graduating apertures between any two
contiguous diaphragms; if, for example, we place the lever to the left,
so that the lens may be separated as far as possible, and use a No.
6 diaphragm, and if, on examining the object, it is thought that the
illuminating cone is not large enough, and if when No. 7 is turned
on it is found too much, we can go back to No. 6, and by turning the
lever 60° towards the right, closing the lenses and increasing the
power a little, we shall obtain an aperture somewhere between Nos. 6
and 7 diaphragm. Thus we can by means of the correction collar graduate
the aperture with the facility as with an iris, and we can record any
particular aperture with a degree of accuracy foreign to the iris. It
must be admitted, however, that the cone of light transmitted by the
condenser is a very small one.
Powell also supplies an apochromatic oil-immersion condenser, numerical
aperture 1·40, but without collar correction; Fig. 134 shows the
sliding tube lowered by arm A and cell B withdrawn for changing stops,
which can be done without altering the focus of the condenser. Fig.
134_a_ shows the cell B closed and raised by arm A close to the back
lens of optical combination. In Fig. 134_b_ six of the principal stops
are shown. Powell’s dry apochromatic condenser, of nearly 0·9 aplanatic
cone, is also very good; but the high price of all is a bar to their
more general use. The speciality of these is the conversion of axis
light into condensed oblique incident light by the refraction of the
condenser.
Messrs. R. & J. Beck have various forms of achromatic condensers, some
of which partake of a somewhat elaborate arrangement; others are simple
and inexpensive, to suit the students’ microscope; as when the light of
the concave mirror proves insufficient for any object requiring intense
transmitted light, an achromatic condenser must be adapted to even the
students’ form of microscope. The latest form of condenser (Fig. 135)
is fitted with revolving stops and iris diaphragm, and other appliances
for obtaining satisfactory results.
[Illustration: Fig. 135.--Beck’s newer form of Achromatic Condenser.]
_Beck’s Compound Illuminating Apparatus_ (Fig. 136).--It is useful
in working with the microscope to be enabled to rapidly change the
illumination, and for this reason this compound form of condenser has
been constructed. It consists of an upper portion A, a wide-angle
condenser, the aperture of which can be reduced at will by an iris
diaphragm, moved by the lever B. This can be used for all other
purposes. Below this diaphragm is a plate C, which can be swung back
out of position at will, as shown in outline. Into a cell in this
plate the stops D can be dropped, and the condenser can be used for
dark field illumination, or for high powers as an oblique illuminator.
A large-size polarising prism E, fastens to the plate C, and can be
removed when not required. In this way any of the various modes of
illumination may be separately or conjointly obtained.
[Illustration: Fig. 136.--Beck’s Compound Condenser.]
[Illustration: Fig. 137.--Beck’s Spherical Achromatic Condenser.]
Their condenser (Fig. 137) has a large aperture, and facilities for
rotating the series of diaphragms. It is available for either dry or
immersion objectives up to 1·3 numerical aperture on diatoms, and wet
or dry histological objects. The spherical form of the front is worked
by a milled-head that rotates a series of lenses and diaphragms. It
also avoids the inconvenience of having the connecting fluid drawn away
by capillary attraction, as would be the case if mounted on a flat
surface. It is also less in the way of the sub-stage movements.
[Illustration: Fig. 138.--Watson’s Parachromatic Condenser.]
_The Parachromatic Condenser_ of Messrs. Watson (Fig. 138) was made to
meet a demand for a condenser giving a large solid cone of illumination
free from colour. The optical part of this condenser consists of a
full hemispherical front lens, and the middle and back combinations of
such forms as to produce the necessary corrections. The Jena phosphate
crown and silicate flints are used in its manufacture, and to these
are due its special qualities. The total aperture of the condenser is
1·0, and it yields an aplanatic aperture of ·90 numerical aperture. The
magnifying power is 2/7ths of an inch. From this it will be seen that
it is especially suitable for use with high-power objectives.
It can also be employed without the front lens, when the magnifying
power is 4/10ths of an inch, and the numerical aperture ·35. It is
mounted in an exceedingly convenient manner, the iris diaphragm being
fitted in such a way as to be absolutely central with the optical
system.
The arc through which the handle controlling the iris travels is
divided, and indicates the aperture at which the condenser may be
working at any time. An important feature in this condenser is that it
is almost wholly free from colour. As a rule condensers of the same
form are found difficult to work with, because of the small diameter of
the field or back lens. This difficulty has been successfully overcome
by increasing the size of this lens, and the whole of which is fully
utilised.
Most London opticians have their own especial form of achromatic
condenser, designed for and fitted to their several stands and
objectives, varying from a small price to the more expensively-fitted
accessories.
[Illustration: Fig. 139.--Swift’s Illuminating Polarising Apparatus.]
[Illustration: Fig. 139_a_.--Swift’s Diaphragms and Central Stops.]
Messrs. Swift’s illuminating apparatus (Fig. 139) is conveniently
supplied with numerous useful appliances. The optical combination A is
computed to be used as an effective spot lens from a 3-inch objective
up to a sixth. C C are two small milled heads by means of which the
optical combination A is centred to the axis of the objective. The
revolving diaphragm E has four apertures for the purpose of receiving
central stops, oblique light discs, and selenite films. D is a frame
carrying two revolving cells, into one of which a mica film is placed,
which can be revolved with ease over either of the selenites below,
whereby changes of colour can be obtained in experimenting with
polarised light. The darts and P A’s indicate the position of the
positive axis of the mica and selenite films, and by this means results
can be recorded, etc. Either of the revolving cells can be thrown into
the centre of the condenser, and there stopped by means of a spring
catch; when so arranged the mica film, &c., may be revolved in its
place by turning the cell D, as both cells are geared together with
fine racked teeth. F is a polarising prism mounted on an eccentric
arm, rendered central when in use, or thrown out, as seen, when out of
use. G is the rack dove-tail slide for indicating and focussing the
condenser on the object. The advantages associated with this condenser
consist in having the polarising prism, selenite films, dark-ground,
and oblique light stops, so that they may be brought close under the
optical combination.
[Illustration: Fig. 140.--Baker’s Nelson Achromatic Condenser.]
Baker’s Nelson Condenser, shown in Fig. 140, is intended for use with
their medium instruments. It has, however, many pieces of apparatus
essential to those of a higher class. It is applicable, indeed, to all
instruments having sufficient depth beneath the stage to receive it. It
comprises an achromatic combination of 90° aperture, available with all
powers up to 1/8-inch tinted glass for neutralising the yellow rays of
artificial light, focussing adjustment, dark-ground illuminator, large
diaphragm with rotating tube to carry oblique light stops, small wheel
of apertures, polarising prism with two selenite films, clear aperture,
and oblique light-shutter for low powers.
Baker’s Students’ Condenser (Fig. 141) is designed to take the place of
Abbe’s, and costs much less. It transmits a larger aplanatic cone of
light, and can be used either with high or low powers by removing the
front lens. It is equally useful for photo-micrographic work.
Mr. J. Mayall’s semi-cylinder or prism for oblique illumination (Fig.
142) is a convenient form, as it permits of the semi-cylinder being
tilted and placed excentrically; in this manner, without immersion
contact, and by suitable adjustment, a dry object can be viewed
with any colour of monochromatic light. If placed in immersion
contact with the slide, the utmost obliquity of incident light can
be obtained. Objects in fluid may be placed on the plane-surface of
the semi-cylinder, and illuminated by ordinary transmitted light, or
rendered “self-luminous” in a dark field, as with the hemispherical
illuminator or Wenham’s immersion paraboloid. A concave mirror with a
double arm is quite sufficient to direct the illuminating pencil. This
semi-cylinder was originally made by Tolles, of Boston, for measuring
apertures, but, at Mr. Mayall’s suggestion, Messrs. Ross mounted it as
an illuminator.
[Illustration: Fig. 141.--Optical Arrangement of Baker’s Abbe
Condenser.]
The spiral slot should be fixed close beneath the larger lens of the
condenser, and when properly arranged will be found a convenient mode
of obtaining oblique light.
[Illustration: Fig. 142.--Mayall’s Semi-Cylinder Illuminator and Spiral
Diaphragms.]
_The Webster-Collins Universal Condenser_ (Fig. 143) is so well
known that it scarcely calls for any lengthy description. It is an
inexpensive form of condenser, designed in the first instance for use
with the students’ microscope. It is fitted into the sub-stage; has an
iris diaphragm as well as a series of revolving diaphragms moved by a
milled head screw arrangement.
[Illustration: Fig. 143.--The Webster-Collins Universal Condenser.]
Oblique Illumination.
_Wenham’s Parabolic Condenser._--Mr. Wenham’s many useful additions
to the microscope and its accessories demand especial notice. When
mention is made of the various immersion condensers (illuminators,
as he preferred to call them), his original right-angled prism, his
truncated hemispherical lens, his immersion paraboloid, and his reflex
illuminator, in which rays beyond the angle of total reflexion are
utilised by reflex action from cover-glass on to the surface of the
object, every one of these well-devised inventions will always be
spoken of in terms of praise. All in their turn conferred a great
service upon the microscope, and enabled the student to clear up
difficulties that stood in the way of developing structure when
achromatic lenses and dry-objectives were considered perfect. The
superior illumination of the object was wholly due to, and effected by,
_reflected_ rays from the object to the aperture of the objective, and
obviously, reflex action could only take place with dry-objectives.
This reflex action must be regarded as Mr. Wenham’s special discovery.
It must be observed, however, that it is not the same as the more
modern achromatic appliances used for throwing _direct_ rays upon the
object, and which proved the existence of apertures capable of direct
transmission up to 27° measured in the body of the front lens.
[Illustration: Fig. 144.--Wenham’s Parabolic Reflector.]
The most practical of Mr. Wenham’s inventions is probably the
hemispherical lens, since adopted by Messrs. Ross in connection with
their excellent Zentmayer stand, and which has proved eminently
serviceable. But the fact is that devices of the kind for obtaining
direct oblique light require a thin stage, and therefore most of those
who possess the earlier-made microscope stand would doubtless hail
the appearance of any appliance which will convert axial light into
oblique light; as by so doing the possessors of such instruments, in
which the stage is generally of considerable thickness, would enjoy the
pleasure of seeing the best resolution it is possible to get with their
dry-objectives.[27]
_Wenham’s Parabolic Reflector._--This will be better understood by
reference to Fig. 145, which represents it in section A B C, and shows
that the rays of light _r r′ r′′_, entering perpendicularly at its
surface C, and then reflected by its parabolic surface A B to a focus
at F, can form no part of the largest pencil of light admitted by the
object-glass and represented by G F H; but an object placed at F will
interrupt the rays and be strongly illuminated. A stop at S prevents
any light from passing through direct from the mirror.
In the microscope the _parabolic reflector_ fits into the cylindrical
fitting under the stage, and the adjustment of its focus upon the
object is made by giving it a spiral motion when fitted in--that is,
carefully pushing it up or down at the same time that it is turned
round by the milled edge B B. It must then be focussed by the rack
and pinion motion. As the rays of light must be parallel when they
enter it, a _flat mirror_, which in this case should be added to the
instrument, is generally used; daylight will then require only direct
reflection, but the rays from an artificial source will have to be made
parallel by placing a side condenser between the light and the mirror,
about 1-3/4 inch from the former and 4-1/2 inches from the latter.
Nearly the whole surface of the mirror should be equally illuminated;
this may be tested by temporarily placing upon it a card or piece
of white paper. Parallel rays can also be obtained from the concave
mirror, if the light is placed about 2-1/2 inches from it. Dark-ground
illumination is not suitable for very transparent objects--that is,
unless there is a considerable difference in their index of refraction,
or they are pervaded by air-cells.
[Illustration: Fig. 145.--Parabolic Reflector.]
One very remarkable example of this may be seen in the tracheal system
of insects. If any of the transparent larvæ of the various kinds of
gnat be mounted in gelatine and glycerine jelly, slightly warmed
but not enough to kill the insect outright, about the third day the
fluids circulating in the body will be absorbed and replaced by air.
Illuminated by the parabolic condenser, and viewed with a binocular
microscope, and a low power, the gnat-larva becomes a superb object.
The body of the insect is but faintly visible, and in its place is
displayed a marvellous tracheal skeleton, with the tubes standing out
in perspective, shining brilliantly, like a structure of burnished
silver. Unfortunately, such objects are not permanent, for when the
whole of the water dries up, the tracheal tubes either collapse or
become refilled with fluid.
As to the blackness of field, and luminosity of the object, this
depends upon excess of light from the paraboloid received beyond the
angle of aperture of the object-glass. It is found in practice that
more and more of the inner annulus of rays from the paraboloid has
to be stopped off, until at last, with high-angled objectives, it is
scarcely possible to obtain a black field.
The light, on the whole, most suitable for this method of illumination
is lamp, the rays of which should in all cases be rendered more
parallel by means of a large plano-convex lens, or condenser.
[Illustration: Fig. 146.--Wenham’s Hemispherical Lens.]
_Wenham’s Immersion Condenser._--Mr. Wenham, in the year 1856,
described various forms of oblique illuminators, one of which was an
immersion; a simple right-angled prism, connected by a fluid medium of
oil of cloves. This, however, was abandoned for a nearly hemispherical
lens connected with the slide, and although an improvement, did not
touch the point of excellence Mr. Wenham was looking for. Ultimately
he adopted a semi-circular disc of glass of the exact form and size
represented in the drawing, Fig. 146, having a quarter-inch radius,
with a well-polished rounded edge, the sides being grasped by a simple
kind of open clip attached to the sub-stage, the fluid medium used
for connecting the upper surface with the slide being either water,
glycerine, or oil; an increase of oblique illumination being obtained
by swinging the ordinary mirror sideways. By means of an illuminator of
the kind difficult objects mounted in balsam are resolved. This simple
piece of glass collects and concentrates light in a marvellous manner,
and is by no means a bad substitute for some of the more costly forms
of achromatic condenser. It can be used either in fluid contact with
the slide, or dry, as an ordinary condenser.
Mr. Wenham subsequently contrived a small truncated glass paraboloid,
for use in fluid contact with the slide; water, glycerine, oil, or
other substance being employed as a contact medium. The rays of light
in this illuminator, being internally reflected from a convex surface
of glass, impinge obliquely on the under surface of the slide, and
are transmitted by the fluid uniting medium, and internally reflected
from the upper surface of the cover-glass to the objective. To use the
reflex illuminator efficiently it must be racked up to a level with the
stage. The centre of rotation is then set true by a dot on the fitting,
seen with a low power, a drop of water is then placed on the top, and
upon this the slide is laid. Minute objects _on the slide_ must be
found either by the aid of a low power, by their greater brilliancy, or
by rotating the illuminator; the effect on the podura scale is superb,
the whole scale appearing dotted with bright blue spots in a zig-zag
direction. Objects for this illuminator should be especially selected
and mounted.
[Illustration: Fig. 147.--The Amici Prism.]
The Amici Prism, originally designed for oblique illumination, consists
of a flattened triangular glass prism, the two narrower sides of
which are slightly convex, while the third or broadest side forms the
reflecting surface. When properly used, it is capable of transmitting
a very oblique pencil of light. The prism is either mounted, as in
Fig. 147, for slipping into the fitting of the sub-stage, or on an
independent stand, as arranged for Powell’s microscope, page 85, Fig.
56.
Method of Employing the Achromatic Condenser to the Greatest Advantage.
_Its Illumination._--Good daylight is the best for general work. The
microscope should be placed near a window with a northern aspect.
Direct sunlight should never be utilised; the best light is that
reflected from a white cloud. A good paraffin lamp is the most
serviceable artificial source of light, and it is quite under control.
As an illuminant more often brought into requisition in the smoky
atmosphere of towns, the paraffin lamp is on the whole the handiest
and the most useful. If gas-light can be brought into use as suggested
for micro-photography, with the incandescent mantle, it will be found
to be the purest and best form of artificial illumination for the
microscope. Among paraffin lamps those constructed by Baker and Swift
are all that can be desired.[28]
[Illustration:
Sectional view of the Optical Arrangement of the Aplanatic Bull’s-eye
Lens, fitted in gymbal on the front of the lamp.]
[Illustration: Fig. 148.--Baker’s Microscope Lamp.]
As the chimneys of these lamps are made of metal, and blackened, no
reflected light disturbs the eye. Care must be taken to have the wick
evenly trimmed; the metal chimney has a glazed front, giving exit to
the rays of light, the flat of the flame being used with low powers,
and the image of the flame being reflected by a plane mirror to give
equal illumination of the whole field. In working with high powers,
the lamp is turned with the flame edge-wise, and at the same time the
mirror must be dispensed with. By working, as it is termed, directly
on the edge of the flame, the illumination is greatly increased, and a
band of light can be concentrated on any part of the preparation it is
desired to make a careful study of.
To obtain the best results, time and care must be given to the
illumination of the object. The lamp and microscope having been placed
in position, a low power is first used and the smallest diaphragm. On
looking through the microscope it will probably be observed that the
image of the diaphragm is not in the centre of the field; by moving
the centring screw of the condenser this may be adjusted. The low
power is then replaced by a high power, the largest diaphragm used,
and the bacteria or diatom brought into focus. The diaphragm must now
be replaced by one of medium size, and by racking the condenser up and
down, a point will be arrived at when the image of the edge of the
flame appears as an intensely bright band of light. If this is not
exactly in the centre of the field the centring screw of the condenser
must again be adjusted. With regard to the use of diaphragms, various
sizes should be tried while focussing with the fine adjustment, at
the same time using the correction colour; in this way we obtain
the sharpest possible image. When the condenser has been accurately
centred, it will still be necessary to focus it for each individual
specimen, so as to correct for difference in the thickness of slides
and the layers of mounting medium. Correction for different thickness
of cover-glasses must be made by the aid of the collar adjustment
in the following way: a high-power eye-piece is substituted for the
ordinary eye-piece, and the faults in the image will thereby be
intensified. By moving the collar completely round, first in one
direction and then in the other, while carefully observing the effect
of the image, it will be seen to become obviously worse whichever way
the collar is turned. The collar must then be turned through gradually
diminishing distances until an intermediate point is reached at which
the best image results with the high-power eye-piece, and on replacing
this by the low-power eye-piece the sharpest possible image will be
obtained.
_Effect of the Sub-stage Condenser._--The sub-stage condenser gives
the most powerful illumination when it has been racked up until it
almost touches the specimen. It produces a cone of rays of very short
focus, and the apex of the cone should correspond with the particular
bacterium or group of bacterias under observation. The effect of the
condenser without a diaphragm is to obliterate what Koch has termed a
_structure picture_. If the component parts of a tissue section were
colourless and of the same refractive power as the medium in which the
section is mounted, nothing would be visible under the microscope. As,
however, the cells and their nuclei and the tissues do not differ in
this respect, the rays which pass through them are diffracted, and an
image of lines and shadows is developed. If in such a tissue there were
minute coloured objects, and if it were possible to mount the tissue
in a medium of exactly the same refractive power, the tissue being
then invisible, the detection of the coloured objects would be much
facilitated. This is exactly what is required in dealing with bacteria
which has been stained with aniline dyes, and the desired result can be
obtained by the use of the sub-stage condenser.
If we use the full aperture of the condenser the greatly converged
rays play on the component parts of the tissue, light enters from
all sides, the shadows disappear, and the structure picture is lost.
If now a diaphragm is inserted, so that we are practically only
dealing with parallel rays, the structure picture reappears. As
the diaphragm is gradually increased in size the structure picture
gradually becomes less and less distinct, while the colour picture, the
image of the stained bacteria, becomes more and more intense. When,
therefore, bacteria in the living condition and unstained tissues are
examined, a diaphragm must be used, and when the attention is to be
concentrated upon the stained bacteria in a section or in a cover-glass
preparation the diaphragm must be removed and the field flooded with
light--(Crookshank).
The wide-angle condenser, it will be understood, consists of a
combination of lenses, which concentrate all the light entering them
to a small point, and the condenser must be so accurately focussed
that this brilliant cone of light, when it emerges from the upper lens
of the condenser, falls upon the object from all directions, forming
a wide-angle cone of light, at the apex of which the object must be
placed (see Fig. 149). That is to say, the object is illuminated by a
cone of rays passing through it in all directions.
[Illustration: Fig. 149.--Front Lens of Condenser.]
There are, however, objects which require a fully illuminated field,
when the lamp should be turned round and the Herschel lens condenser
(shown in section, Fig. 148) should be used to collect the light and
throw it upon the mirror. For moderate powers, as a four-tenth or
one-fifth, the condenser should be used a little below the focus to
give an even illumination over the whole field. Moreover, as to the use
of the condenser for defining general objects, it must be borne in mind
that to show different kinds of structure different apertures in the
iris diaphragm are necessary, and that whereas some objects show their
structure better with a large angle of light cut down in intensity
by the use of blue glass, others show better with a small pencil of
direct rays. For the resolution of diatoms it is often necessary to
use oblique light only, and for this purpose diaphragms with central
patches are used, the iris diaphragm being opened to its full extent.
An annular ring of oblique light emerges from the condenser upon the
object, and it is in this manner also that dark-ground illumination is
obtained with moderate and low powers.
THE DIAPHRAGM.
[Illustration: Fig. 150.--The Diaphragm.]
The early form of diaphragm in use was that shown in Fig. 150.
[Illustration: Fig. 151.--Shutter Diaphragm.]
It consists simply of a circular brass plate with a series of circular
openings of different sizes, arranged to revolve upon another plate by
a central pin or axis, the last being also provided with an opening
as large as the largest in the diaphragm-plate, and corresponding
in situation to the axis of the microscope body. The holes in the
diaphragm-plate are centred and retained in place by a bent spring in
the second plate, which rubs against the edge of the diaphragm-plate
and catches in a notch. The blank space shuts off the light from the
mirror when condensed light is about to be used. It is usually made
to fit in under the stage of the microscope. This has been almost
superseded by the iris diaphragm, originally designed by Wales, of
America. It was made by this optician for his working students’
microscope. An early form of the iris diaphragm is seen in Fig. 151.
By pressing upon the lever handle at the side the aperture gradually
closes up, and without for a moment losing sight of the object under
examination.
The Mirror.
The mode in which an object is illuminated is, in the words of the
late Andrew Ross, “second only in importance to the excellence of the
glass through which it is seen.” To ensure good illumination the mirror
should be in direct co-ordination with the objective and eye-piece;
it must be regarded as a part of the same system, and tending by a
combined series of acts to a perfect result. Illumination of the object
is recognised as of three kinds or qualities--reflected, transmitted,
and refracted light. For the illumination of transparent objects,
transmitted light is brought into use; for opaque objects, reflected
light is needed.
The mirror should be about 2-1/2 or 3 inches in diameter, and it must
not be fixed, but made to slide up and down the stem under the stage,
so that the rays of light emanating from it may be brought to a focus.
The utility of the mirror is so obvious that it is occasionally passed
over in silence by writers. To myself it appears to be an important
accessory of the microscope, and I shall therefore proceed to combine
theory with practice in what I have to say with regard to the mirror.
[Illustration: Fig. 152.--Principal Focus of Mirror.]
The microscope mirror should be the segment of a true sphere, and its
centre that of a true curvature. If the mirror has a true circular
boundary, the central point on line A (Fig. 152) of the reflecting
surface, is the pole of the same. The line A C is known as its
principal axis, and any other straight line through C, which meets the
mirror, is its secondary axis. When the incident axis is perfectly
parallel to the principal axis, the reflected rays converge to a point
F, its principal focus. So much for the theory of the mirror. Now we
come to its practical use.
Simple as the mirror of the microscope may appear to be, if the curve
of the surface is not perfect, it will yield a secondary reflection or
double pencil of rays. The plane mirror will occasionally be found to
emit more than one reflection of the lamp-flame; this we find may be
corrected by rotating the mirror in its cell. Many years ago I proposed
to meet a difficulty of the kind by arranging a rectangular prism on a
separate stand, shown in Fig. 153, consisting of a prism A B, mounted
in gimbal C, D, and E, secured to a brass tube G, fitted to the stem,
and thus made to take the place of the mirror.
The direct method of employing the mirror, that more generally resorted
to, is by reflecting rays from the concave surface; the plane surface
is preferred when the condenser is used. Whichever is employed, it
should not be forgotten that the _optic axis_ must be preserved
throughout, and so brought to the centre of the open tube of the
microscope. Another method is to interpose a bull’s-eye lens, and in
this way supply the mirror with a beam of parallel rays of light. The
plane side of the bull’s-eye lens should be turned towards the lamp,
so that lamp, bull’s-eye, sub-stage condenser, and objective, are
brought into an exact line, the bull’s-eye being set at right-angles
to the line. A piece of thin white paper held across the bottom of
the sub-stage will serve to show whether the rays of light are fairly
parallel. The next care is to focus the object on the stage, and then
the sub-stage condenser on the slide; further correction should be made
by means of the centring screws of the sub-stage, or by moving the
bull’s-eye lens or lamp slightly, thus perfecting the arrangements for
working with parallel rays of light.
[Illustration: Fig. 153.--Rectangular Prism.]
Accessories of the Microscope.
The accessories and appliances of the microscope have become so very
numerous, that any attempt to describe them and explain the uses to
which they are put would demand more space than I find myself in a
position to bestow upon them. I must therefore confine my remarks to
those accessories in more general use.
[Illustration: Fig. 154.--The Lieberkühn.]
Having described the method of employing transmitted light, I have a
few words to add with regard to the illumination of opaque objects by
reflected light. A very early and efficient form of opaque illumination
is the well-known _Lieberkühn_. This has not been entirely surpassed by
more recent inventions. The concave speculum termed a Lieberkühn, so
named after its celebrated inventor, directly reflects down upon the
object the light received either from the mirror or bull’s-eye lens.
It consists of a silver cap, which slides over the objective (Fig.
154), _a_ indicating the lower part of the compound body, and _b_ the
objective over which slides the Lieberkühn, _c_; the rays of light are
collected to a focus upon the object at _d_. The object may either
be mounted on a slip of glass, or held by the stage-forceps, _f_; if
very small, or transparent, it may be gummed to the dark well, _e_, or
mounted on a Beck’s opaque disc-revolver.
[Illustration: Fig. 155.--Stage Forceps, for holding objects while
under examination.]
[Illustration: Fig. 156.--Beck’s Disc-holder.]
This holder will be found useful for the examination of opaque or
other objects that cannot be conveniently held by the stage forceps,
the specimen being temporarily attached to it by gum or gold size. The
holder is intended to rotate, so that every portion of the object can
be brought into view. In this way it will be found useful in the study
of insects, foraminifera, &c.
With the Lieberkühn, however, the illumination of opaque objects must
be more or less one-sided, and therefore, the silver side-reflector
has superseded it for general use (Fig. 157). To ensure a more perfect
illumination of the object, the bull’s-eye lens should also be used.
Mr. Sorby devised a reflector to fit over the objective. It consists
of a semi-circular cap; is, in short, a modification of the parabolic
reflector. The light from the mirror can, by slightly varying its
inclination, be brought into use with this reflector.
The silver side-reflector is usually made with a ball-and-socket
joint, so that it can be turned in any direction. It is secured to the
stage of the microscope by the pin, which drops into a hole purposely
drilled to receive it, and facility given for turning up and down, or
in any position. If daylight is used the microscope should be placed
in such a position that the light from a white cloud falls upon the
speculum, but the light of the lamp is far more manageable for use with
the reflector.
[Illustration: Fig. 157.--Silver Side-reflector.]
The Lieberkühn is only intended to be used with low powers--a 2-inch,
1/2-inch and a 2/3-inch. Such objects as the elytra of the diamond and
other beetles are well suited for examination.
[Illustration: Fig. 158.--Sorby’s Modification of the Parabolic
Reflector.]
While experimenting with a parabolic reflector (Fig. 158), Mr. Sorby
saw the value of examining objects under every kind of illumination.
As on viewing specimens of iron and steel with this reflector he found
that, from the great obliquity of the illumination obtained, the more
brilliantly polished parts of the specimen reflected the light beyond
the aperture of the objective, and these could not be distinguished
from those parts which absorbed light, he thereupon proceeded to place
a small flat mirror in front of the objective, and cover half its
aperture, and at the same time stop off by means of a semi-cylindrical
tube the light from the parabolic reflector. This arrangement produced
the reverse appearance of that first employed, and it proved to be a
useful aid in determining structure.
The Bull’s-eye Condensing Lens.
This accessory is brought into constant use for the purpose of
converging rays from a lamp or mirror; or, for reducing the diverging
rays of the lamp to parallelism with the parabolic illuminator, or
silver side-reflector. The form in use is a plano-convex lens of about
three or four inches in focal length (Fig. 159). It is usually mounted
on a brass stand, so that it may be placed and turned in any direction,
and at any height. When used by daylight, its plane side should be
turned towards the object, and the same position maintained when used
for converging the rays of light from the lamp; but when used with the
side-reflector the plane side must be towards the lamp. Much attention
has been paid to this very necessary accessory, the bull’s-eye lens.
A doublet has been brought into use which has increased the value
of the bull’s-eye condenser in bacteriological research, and in
micro-photography generally.
[Illustration: Fig. 159.--Bull’s-eye Lens.]
“During a recent investigation of the spherical aberration in doublets,
it was believed to be impossible to construct a doublet of the form
known as ‘Herschel’s doublet’ free from aberration, although these
doublets figure in many books on optics. In a condenser made by Baker
the aberration is reduced to a minimum, 27 per cent. less than Sir John
Herschel’s. This doublet, it appears, differs from Herschel’s both in
the ratio of the radii of the meniscus, and also in the ratio of the
foci of the two lenses; indeed, the only point of similarity is in the
first lens, which is crossed. To test this, project the image of the
flat lamp-flame on a piece of white card with a plano-convex lens (the
field-lens of the Huyghenian eye-piece), use first the convex side and
then the plane side towards the card, the lamp being placed about 6
feet from the lens. Focus the lamp-flame as sharply as possible, and a
circular halo of misty light will be seen to surround the lamp-flame;
but when the plane side of the lens is made to face the card this halo
of misty light will be seen to be greatly reduced, and the brightness
of the image of the flame proportionately increased. If the lens, then,
were strictly aplanatic there should be no misty halo, all the light
being concentrated in the image of the lamp-flame, and the image of
maximum brightness. In short, the diameter of the halo or misty light
is the measure of the spherical aberration. If the condenser referred
to above, having the form of minimum aberration for two planes, be
compared in the same manner with an ordinary single bull’s-eye of the
same focus, the diameter of the misty halo will be found reduced to
a radius of about 1/5-inch, but, with this new condenser there is a
further reduction, so that the radius of the misty halo measures only
1/20-inch. These experiments are instructive, because the brightness,
or the mistiness of the microscopical image is an associated
phenomenon.”[29]
A sectional view of the optical arrangement of Baker’s aplanatic
bull’s-eye doublet is shown, together with lamp, in Fig. 148.
_The Microscope Lamp._--The introduction of paraffin into household
use has somewhat modified our views with regard to the most suitable
artificial source of illumination. Good paraffin burns with a whiter
and purer flame than colza oil, and consequently is less liable to
fatigue the eyes. The first cost of the lamp is trifling; for a
moderate sum a handy form of lamp can be had, mounted on an adjustable
sliding ring stand, and with a porcelain, metal or paper shade, to
protect the eyes from scattered rays of light. All opticians supply
accepted forms of lamps.
To give the increased effect of whiteness to the light (“white cloud
illumination” as it is termed), take a piece of tissue paper, dip
it into a hot bath of spermaceti, and, when nearly cold, cut out a
circular piece and secure it over the largest opening in the diaphragm
plate. This will be found to materially moderate and soften the light.
[Illustration: Fig. 160.--Beck’s Complete Lamp.]
_Beck’s Complete Lamp_ is constructed especially for delicate
microscopical work. It has a burner giving a flat flame; this can
be rotated to enable the edge or the flat of the flame to be used;
likewise a metal chimney with two apertures, in which 3 × 1 glass
slips slide; either white or coloured glasses may be used. A Herschel
aplanatic condenser is carried on a swinging arm, which rotates around
the lamp flame as a centre, and can be clamped in any position. The
whole lamp has a raising and lowering motion, with a spring clamp to
hold it in any position. The lamp is so designed that at its lowest
position the flame is only three inches from the table. Here the
microscopist is furnished with a lamp which will accomplish all he may
require with regard to illumination.
[Illustration: Fig. 161.--Watson’s Microscope Lamp.]
[Illustration: Fig. 162.--Glass Holder for carrying Coloured Glasses.]
Watson’s lamp (Fig. 161) has a metal chimney, and is somewhat simpler
in structure than those already referred to. For the student, the
simpler and cheaper form will answer every purpose. A glass holder for
carrying various tinted slips of coloured glass to act as a screen or
modifier of the light is much employed, and assists in determining fine
structures (Fig. 162).
Nose-pieces and Objective Changers.
A convenient appendage to the microscope is the rotating nose-piece,
invented by Mr. Charles Brooke, F.R.S., and intended to carry two or
more objectives, whereby a saving of time is effected, and the trouble
of repeatedly screwing and unscrewing is avoided. In the application of
the nose-piece attention should be given to centring. Messrs. Baker’s
objective changer is intended to facilitate the placing and replacing
the nose-piece in position. This adaptation consists of a milled head,
acting on three jaws, having a universal screw thread, a decided
improvement on the screw. Zeiss has adopted a tube-sliding objective
changer with centring adjustments. Messrs. Watson met the difficulty
of centring by making the nose-piece a part of the body-tube of their
microscopes (Fig. 163). This, when adapted to the shorter body of the
students’ microscope, fully compensates for want of length.
[Illustration: Fig. 163.--Watson’s Centring Nose-piece of Microscope.]
Their triple nose-piece is constructed with much care, and when in use
is found very effective. It is manufactured of that very light metal
aluminium, and which minimises the strain produced by the heavier brass
nose-piece.
_Finders._--The finder affords a necessary and useful means of
registering the position of any particular object, so that it may
be readily found again at any subsequent period. In the work of
examination the finder will save time when making a special research,
extending over a considerable surface.
[Illustration: Fig. 164.--Triple Nose-pieces.]
That the finder has been of use may be surmised from the number
invented and figured in the “Journal of the Royal Microscopical
Society.” By far the most useful form is that of graduating the plates
of the mechanical stage, dividing a certain portion into 100 parts.
Powell and Lealand have adopted this system in their No. 1 stands,
while Baker and Watson have added a graduated scale on silver to
1/100th mm. as a finder, and also a stage micrometer in 1/10th and
1/100th of a millimetre, together with a Maltwood finder for lodging
the position of any desired portion of a specimen under examination.
The _Maltwood_ finder (Fig. 165) can be used with any microscope, and
without a mechanical stage. This useful finder continues to occupy a
permanent place among the accessories of the microscope. It consists
of a glass slide, 3 × 1-1/4 inches, on which is photographed a scale
occupying a square inch; this is divided by horizontal and vertical
lines into 2,500 squares, each of which contains two numbers marking
its “latitude,” or place in the vertical series, and its “longitude,”
or place in the horizontal series. The scale is in each instance an
exact distance from the bottom and left-hand end of the glass slide;
and the slide, when in use, should rest upon the ledge of the stage of
the microscope, and be made to abut against a stop, a simple pin, about
an inch and a half from the centre of the stage.
[Illustration: Fig. 165.--Maltwood’s Finder.]
Dr. Pantacsek’s finder appears to have some advantage over Maltwood’s,
but it cannot be used with the same facility, and therefore will not
displace an old favourite. _The Amyot_ finder I have long had in use;
it is efficient and inexpensive--can indeed, if misplaced or lost, be
replaced by the aid of the square and compasses.
[Illustration: Fig. 166.--Amyot’s Object Finder.]
_The Okeden_ finder consists of two graduated scales, one _vertical_,
attached to the fixed stage-plate, the other _horizontal_, attached to
an arm carried by the intermediate plate; the first of these scales
enables the worker to “set” the vertically-sliding plate to any
determinate position in relation to the fixed plate, while the second
gives the power of setting the horizontally-sliding plate by that of
the intermediate.
_Micrometers._--It is of the utmost importance to have a means of
measuring with accuracy the objects, or part of objects, under
observation. The most efficient piece of apparatus for the purpose is
the micrometer eye-piece, the earlier form of which, Jackson’s, has
been described under the heading _Eye-pieces_ (p. 144). In the case of
micrometers, as in that of most other accessories, every optician has
his own adaptation and method of employing the same.
For the measurement of bacteria, a stage micrometer should be used with
a camera lucida. The stage micrometer consists of a slip of thin glass
ruled with a scale consisting of tenths and hundredths of a millimetre.
The image is projected on to a piece of paper placed on the table, and
the drawing made, and the object to be measured can be readily compared
with the scale.
[Illustration: Fig. 167.--The Ramsden Micrometer Eye-piece.]
In the Ramsden micrometer eye-piece, as previously explained, two fine
wires are stretched across the field of an eye-piece, one of which can
be moved by a micrometer screw. In the field there is also a scale with
teeth, and the interval between them corresponds to that of the threads
of the screw.
The circumference of the brass head is usually divided into one hundred
parts, and a screw with one hundred threads to the inch is used. The
bacterium to be measured is brought into a position in which an edge
appears to be in contact with the fixed wire, and the micrometer screw
is turned until the travelling wire appears to be in contact with
the other edge. The scale in the field and scale on the milled head,
together, give the number of complete turns of the screw and the value
of a fraction of a turn in separating the wires.
In the micrometer eye-piece constructed by Zeiss, the eye-piece with
a glass plate with crossed lines is carried across the field by means
of a micrometer screw. Each division on the edge of a drum corresponds
to ·01 mm. Complete revolutions of the drum are counted by means of a
figured scale in the visual field.
In the micrometer used with Zeiss’s _apochromatic_ objectives and
compensating eye-pieces the divisions are so computed, that, with a
tube-length of 160 mm., the value of one interval represents, with each
objective, just as many micra (·0001 mm.) as there are millimetres in
its focal length. A value of tables is therefore not required for these
eye-pieces, since the focus of the lenses indicates their micrometer
values within 5 per cent.
[Illustration: Fig. 168.--The Wollaston Camera Lucida.]
The _Camera Lucida_ will prove an extremely useful adjunct to the
micrometer, and a large number of contrivances have been devised for
its employment. There are those which project the image on to the
surface of a sheet of paper provided for the drawing, and those which
project the pencil and paper into the field of the microscope. The
former method is that usually adopted. To draw an object, with either
a Wollaston camera lucida or a neutral tint reflector, such as that
of Beale’s, both of which are made to slide on and take the place of
the cap of the eye-piece, as shown in Fig. 168, with its flat side
uppermost, the whole instrument must be raised until the edge of the
prism is exactly 10 inches from a piece of paper placed upon the
table; with the latter the instrument retains its vertical position,
and the image of the object is thrown on the paper placed in front of
the stand. The light must be so regulated that no more than is really
necessary is upon the object, whilst a full light should be thrown upon
the paper. Only one eye is to be used; and if one half of the pupil be
directed over the edge of the prism, the object will appear upon the
paper, and can be traced on it by a pencil, the point of which will
also be seen. Should any blueness be visible in the field, the prism is
pushed too far on, and should be drawn back till the colour disappears.
[Illustration: Fig. 169.--Microscope in position for drawing.]
[Illustration: Fig. 170.--Beale’s Neutral Tint Reflector.]
The position in which the microscope must be placed is shown in the
accompanying illustration (Fig. 169).
Beale’s neutral tint reflector (Fig. 170) is much in use, and its
advantages are utility, simplicity, and inexpensiveness.
[Illustration: Fig. 171.--The Abbe Model Camera Lucida.]
The Abbe model of camera lucida has been brought into use because the
projected image can be better illuminated, and is consequently so much
brighter. This form is now made in aluminium by Messrs. Watson & Sons.
In place of the image being traced by projection on paper, the reverse
is the case, both the paper and pencil are projected into the field of
view. The mirror reflects the paper on to the silvered surface of a
prism placed over the eye-lens of the eye-piece of the microscope, and
it is thereby conveyed to the eye. There is a central opening in the
silvering through which microscopic vision is obtained. It is fitted
in a new manner by means of a cloth-lined adapter, fitting over the
outside of the microscope tube; this saves all trouble in centring and
ensures concentricity. Where the instrument has capped eye-pieces, the
camera lucida must be adapted to the eye-piece, the cap being removed.
The apparatus can be disconnected from the fitting adapter by means of
a sliding pin, and readily replaced, or can be lifted over out of the
way, as shown in the drawing. Being made almost entirely in aluminium
it is very much lighter than other forms of apparatus, and does not
cause vibration. It can be used with the microscope _at any angle_, the
only necessity being that the paper on which the sketch is made should
be kept at the same angle as the instrument.
Micro-Photography.
Micro-photography or photo-micrography, as it is indifferently termed,
has, to a very considerable extent, superseded the use of the camera
lucida for the delineation of images seen under the microscope. I may
claim to be among the first workers with the microscope (1841) to prove
beyond a doubt that the camera could be made to render invaluable aid
to the microscopist, whereby a great saving of time might be effected,
and a drawing obtained with greater accuracy than that of the pencil of
the draughtsman.
It was about 1864-5 that Dr. Woodward’s earlier micro-photographs
were first seen in London. His skill in the manipulation of the
microscope had been long known. His first series of photographs of test
diatoms created, I remember, quite a sensation; they have probably
never been surpassed. These were taken by sun-light, magnesium, and
electric-light. I was the recipient of a series taken at a later date
(1870), and which, bound in quarto volume, are almost as perfect in
definition as any of a later date taken by oil-immersion objectives.
The objectives used by Dr. Woodward, throughout, were a 1/8-inch of
Wales’s (new series), and a 1/16-inch immersion, of Powell & Lealand’s,
especially produced for work with the camera. The magnification varied
from 800 to 3,000 diameters, a frustule of _Grammatophora Marina_
magnified 2,500, and a scale of podura, marked 3,000 in my collection,
are equal in definition to those taken by a high-angle 1/12-inch
oil-immersion. Pathological specimens taken with lower powers are
equally instructive, a section of epithelial cancer showing both nuclei
and cells with distinctness.
Dr. Maddox in 1864 was also experimentally engaged in the improvement
of the processes of photography for the purpose of promoting the work
of microscopists. His labours were attended with great success. To
him we are indebted for the gelatine dry-plate process, which gave
a remarkable impetus to photography in general. Dr. Maddox has, for
a period extending over forty years, diligently and successfully
cultivated and promoted micro-photography. Among other workers to whom
we are indebted for improvements in micro-photography I may mention
Wenham, Draper, Shadbolt, Highley, Koch, Sternberg, Pringle, Leitz, and
Pfeiffer.
Dr. Koch justly claims the credit of having extended the application
of micro-photography to the delineation of bacteria. A series of
instructive micro-photographs were published by him in 1877.
The importance of the camera has become more manifest as the work
of the bacteriologist has progressed. Koch strongly advocated
micro-photography on the ground that illustrations, especially of
bacteria, should be as true to nature as possible. Dr. Edgar Crookshank
holds the same opinion, and in support of his views we have numerous
illustrations of the bacteria given in his valuable “Text-book of
Bacteriology.” But he does not disguise the truth that there are
difficulties to be encountered, the first of which is owing to the
fact that the smallest and most interesting bacteria can only be made
visible in animal tissues by _staining_. This drawback has been very
nearly overcome by the use of eosin-collodion. With this medium, and
by shutting off portions of the spectrum by coloured glasses, Koch
succeeded in obtaining photographs of bacteria, which were stained
with blue and red aniline dyes. This method, however, introduced a
disturbing element of another kind. Owing to the longer exposure
required, the results were wanting in definition, attributable, it was
thought, to vibrations of the apparatus produced by passing traffic, or
by assistants moving about over the floor of the laboratory.
Koch nevertheless showed, at the great meeting of the International
Medical Association in London, 1881, a series of micro-photographs
of bacteria and tissue sections, which were the admiration of all
who saw them. To meet a difficulty occasioned by the aniline dyes,
Koch recommended that the preparations should be stained brown;
other experimenters found that preparations stained either yellow or
yellowish-brown gave good photographic representations; but it is by
no means an easy matter to find a good differential stain of bacteria
in the tissues, as even Bismarck brown is not entirely successful.
Other bacteriologists have encountered similar difficulties at the
outset. Hauser succeeded in showing the value of micro-photography
in the production of pictures of _impression_ preparations and
colonies of bacteria in nutrient-gelatine. But to give the general
effect, as well as faithfully reproduce the minute details in these
preparations of bacteria by the aid of the pencil, would in most
cases create insurmountable difficulties, except in the hand of the
most accomplished draughtsman. Hauser employed Gerlach’s apparatus,
and Schleusser’s dry-plates, and obtained his illumination by means
of a small incandescent lamp, which gave a strong white light. The
preparations so photographed were for the most part stained brown, and
mounted in the ordinary way in Canada balsam.
In 1884, Van Ermengen succeeded in photographing preparations of
comma-bacilli stained with fuchsine and methyl violet. These pictures
afforded the first practical illustration of the value of iso-chromatic
plates in micro-photography, and their introduction marks a distinct
era in the progress of micro-photography. The iso-chromatic, or more
properly the ortho-chromatic, dry-plate process was introduced because
in photography blue or violet comes out almost or quite white, while
other colours, yellow and red, are represented by a sombre shade or
even by black. This is due to the want of equality of strength between
the luminous and the actinic or chemical rays of light. In other
words, the violet and blue rays are more chemically active than any
other portion of the spectrum. It was found, then, that if plates
were coloured yellow with turmeric, the blue and violet rays were
intercepted, and their actinism proportionately reduced.
“In 1881, the so-called iso-chromatic plates were introduced. The
emulsion of bromide of silver and gelatine was stained with eosin, and
it was claimed that colours could be represented with their relative
intensity; chlorophyll and other stains have also been tried, and
by such methods the ordinary gelatine dry-plates can be so treated
that they will reproduce various colours, according to their relative
light intensity, and thus be rendered _iso_-, or what is now known as
ortho-chromatic.”
Apparatus and Material.
_Apparatus and Material_ used in micro-photography have, from time to
time, been greatly varied by different workers, some preferring to use
the microscope in the vertical position with the camera superimposed
or fitted on the eye-piece of the microscope tube; others, again,
prefer that both the microscope and the camera should be arrayed
horizontally. In another form the ordinary microscope is dispensed with
and the objective stage and mirror are adapted to the front of the
camera, together with a suitable arrangement for holding the object.
Lastly, the camera is lain aside, and an operating-room rendered
impervious to light, takes its place, and the image is projected and
focussed upon a ground glass screen held in its place by a separate
support. This method has been made practical since the introduction
into microscopy by Zeiss of the _projection_ eye-piece. It is well
known that micro-photographs can be produced by employing these
projection eye-pieces, as well as for screen illustrations in the
lecture-room.
[Illustration: Fig. 172.--Swift’s Horizontal Apparatus.]
With regard to the position of the microscope and camera, the
horizontal affords greater stability than the vertical, and is on this
account to be preferred. The simplest apparatus consists of a camera
fixed upon a base board, four or five feet in length, upon which
the microscope can be clamped, and which also carries the lamp and
bull’s-eye lens (Fig. 172). This arrangement I have found economical
and useful. No more elaborate arrangement is actually necessary.
Sunlight is no doubt the best, but a good paraffin lamp is a handy and
available illuminant.
With the former, and rapid plates, a short exposure of three or four
seconds, even when high powers are used, is found sufficient; whereas,
with the paraffin lamp it will vary from three to ten minutes.
Walmsley gives the following table for exposures with the lamp:--
1-1/2-inch objective 3 to 45 seconds.
2/3-inch " 7 to 90 "
4/10-inch " 1/2 to 3 minutes.
1/5-inch " 2 to 7 "
1/10-inch " 4 to 10 "
For micro-photography the following practical rules must be observed.
The sub-stage condenser may be dispensed with when low powers are used,
as well as the mirror, and the lamp so placed that the image of the
flat of the flame appears accurately adjusted in the centre of the
field of the microscope. The bull’s-eye lens is so interposed, that
the image of the flame disappears, and the whole field becomes equally
illuminated with high powers; the sub-stage achromatic condenser must
be used, and a greater intensity of illumination is obtained by placing
the lamp-flame edgeways. It is advisable to begin the practice of
micro-photography with low powers, and a trial experiment should be
made with some well-known object as the blow-fly’s tongue.
Dr. Crookshank is of opinion that, in the case of micro-organisms
when their biological characters are studied under low powers of the
microscope, photographs are preferable, because they give a more
faithful representation of the object. A micro-organism, even under
the highest powers of the microscope, is so minute an object, that to
represent it in a drawing requires a very delicate touch, and it is
only too easy to make a _picture_ which gives an erroneous impression
to those who have not seen the original. Photography enables the
scientific worker to record rapid changes, and it is quite possible as
the art advances we may find the film more sensitive than the human
retina, and that it will bring out details in bacteria which would be
otherwise unrecognised. The result, therefore, of experience is that in
research laboratories it will come into more general use as a faithful
and graphic method. I cannot better bring these observations to a close
than by giving a quotation from Dr. Piersoll’s practical method of
obtaining micro-photographs.
The three essential conditions to ensure success in micro-photography
are:--(1) Satisfactory apparatus; (2) good illumination; (3) suitable
preparations. With high amplifications (1,000 diameters and over),
the conditions are greatly changed by the approach to the limit both
of the shortness of the focus of the objective and of the length of
the camera which can be advantageously used; for the first experience
leads to the adoption of the 1/12-inch, for the second four feet is
the limit, since a given high amplification, say 2,000 diameters,
can be more satisfactorily and more conveniently obtained with a
superior 1/12-inch connection with suitable optical means to increase
the initial magnifying power of the objective, than with an unaided
1/25-inch lens, and the plate removed to a greater distance. Until
quite recently the various amplifiers offered the best means of
increasing the power of an objective, but the introduction of the
_projection-oculars_ of Zeiss is an accessory piece of apparatus,
far superior to any older device. These projection-oculars resemble
ordinary microscopical oculars or eye-pieces only in general form
and name, being optically a projection-objective in connection with
a collecting lens. The new oil-immersion apochromatic lenses, in
combination with these projection-oculars, form undoubtedly the more
efficient equipment for high-power work; it is as true for high-power
photography as for microscopical observation in general, that the best
results are obtained with fine and necessarily expensive, optical
appliances. If for the satisfactory study of the intimate structure of
a cell, or of a micro-organism, the most improved immersion lenses are
necessary, it is to be expected that, for the successful photography of
the same, tools at least as good are needed. Sunlight certainly affords
the most satisfactory illumination whereby good micro-photographs can
be obtained, as well as for recording microscopical images. That by
good lamp-light fair impressions of objects under extreme magnification
can be secured is encouraging, but the negatives produced by such
illumination seldom, if ever, possess the characteristics of a really
good sunlight negative, where the sharpest details are combined with an
exquisite softness and harmony of half-tones.
If the mirror of the microscope be of good size, it will only be
necessary to make an arm on which to support the removed mirror outside
some southerly exposed window, since it is desirable to have a greater
distance between the mirror and the stage than would be possible were
the mirror attached in its usual place. Where the microscope mirror
is too small to be satisfactorily used, a rectangular wood-framed
looking-glass is readily mounted, with the aid of a few strips of wood,
so as to turn about both axes.
The rays from the plane side of the mirror should pass through a
condensing lens (of 8-10-inch focus, if possible), so placed that
they are brought to a focus before reaching the plane of the object.
The exact position of the condensing lens is a matter of experience;
usually, however, the most favourable illumination is obtained at that
point where the field is brilliantly and _uniformly_ illuminated,
just before the rays form the image source of light; the nearer the
focus the less disturbance from diffraction rings. Ordinary objectives
will require the employment of monochromatic light--produced either
by a deep blue solution of ammonia-sulphate of copper, or by the
green glass screen--since the optical and actinic foci do not usually
perfectly coincide. Powers up to the 3/4-inch will require no further
condenser; with the 1/4 or 1/6-inch objectives, the low power (1 or
3/4-inch) serves with advantage as an achromatic condenser, when
attached to the sub-stage. The Abbe condenser, although so important
for fine microscopical investigation, is not adapted to photography
unless a very wide cone of light is desired, which, for the majority
of preparations, is some advantage; a low-power objective, used as a
condenser, is found to be more satisfactory than the Abbe with a small
diaphragm.[30]
The greatest delicacy in manipulation is necessary, as in working with
a 1/12-inch objective a turn too much of the fine adjustment will cause
the image to vanish. With fine preparations of bacteria it is not
easy to trace the image, and hence the advantage of commencing with
a well-marked object, as that of the fly’s tongue. The development
and fixation of the image must be proceeded with as in the ordinary
photographic process. In the text-books of photography full accounts
of failures will be found, their causes and prevention. Numerous
papers and suggestions for micro-photographic work will also be found
scattered throughout the “Journal of the Royal Microscopical Society.”
The _Projection Eye-piece_ has become an essential part of
micro-photography, and it is so arranged that it may be employed with
advantage with objectives of either the apochromatic or ordinary series
for photographic purposes, projecting an exquisitely sharp image of the
object on the plate. A diaphragm between the lenses limits the field,
and a sharp image of it should appear on the screen when the eye-piece
is adjusted. The adjustment may be effected by revolving the eye-piece
cap in a spiral slot, so that the eye or top lens is either brought
closer or removed farther away from the diaphragm, as may be required,
and divisions and a reader are usually provided for registering
positions. Such eye-pieces are made to fit any size microscope body.
Initial magnifying powers:--
English length of tube 10-in. 3 and 6.
Continental " " 6-in. 2 and 4.
[Illustration: Fig. 173.--Baker’s Pringle Vertical Micro-photographic
Apparatus.]
The microscope and camera (Fig. 173) are here seen to be part of
the same instrument. The bellows of the camera have an extension
varying from 6 in. to 30 in. The board on which the microscope and
limelight jet are fixed is made to turn out of the line of the camera
to facilitate adjusting the instrument and radiant, either limelight,
electric light or paraffin lamp; when this is done the board carrying
the same is turned back to a stop which brings the microscope into a
central position with the focussing screen. An adjustment is supplied
at the side of the camera, geared to the slow movement, for finely
focussing the object upon the screen. A light-excluding connection is
fitted to the front of the camera and microscope; immediately behind
this, in the bellows, is an exposing shutter which is manipulated
by means of a small milled head. Two focussing screens are usually
supplied, one grey, and one patent plate, together with a double dark
slide.
Mr. Andrew Pringle’s vertical micro-photographic apparatus is an
excellent form; it consists of a heavy base and brass support, carrying
a quarter-plate camera, grey and plain glass focussing screen, double
dark back, camera extending to 24 inches, and turning aside as shown in
Fig 173. It is light-tight in all its connections.
To secure uniform results in micro-photography, only thin preparations,
which lie as nearly as possible in one plane, can be relied upon for
good and perfect negatives.
[Illustration: Fig. 174.--Ross’s Arc Lamp.]
An electric arc lamp specially designed for micro-photographic work,
wherever the electric current is available, is that known as “the
Ross-Hepworth projection arc lamp.” The advantage gained by this
form of lamp is not only on account of the ease with which it may be
employed, but also on account of its superior power and quality. It is
of primary importance that the lamp employed to convert the electricity
into light should be of a good and reliable pattern. It is not
essential that it should be automatic in its working--many experienced
micro-photographers preferring a simple hand-feed lamp to the one of a
more complicated kind, being so much less difficult to keep in order. A
good hand-feed microscope-lamp has the advantage of greater simplicity
and portability.
The argand gas-light arranged for me many years ago for
micro-photography may be employed with advantage. It is clean, and
always ready for use when brought down to the table attached by a piece
of india-rubber tubing. The incandescent form of burner enhances its
value, since the light is thereby rendered whiter. The arrangement is
shown in the diagrammatic drawing, Fig. 175.
Over the argand burner B, is a pale-blue glass chimney, resting on a
wire gauze, stage A; this secures a uniform current of air. The colour
of the flame may be still more influenced by a disc of neutral tint,
or other coloured glass, inserted into the circular opening at E, in a
half-cylinder of metal, G, used to cut off all extraneous light; can
be rotated on the stage by the ivory nob at H, a metallic reflector I,
attached to the standard rod, on being brought parallel to F serves to
concentrate the light and send it on to the bull’s-eye, and through it
to the mirror, or directly to the photo-microscopic camera.
[Illustration: Fig. 175.--Table Incandescent Gas-lamp.]
By removing the shield G, and bringing the shade M over the burner, it
is at once converted into a useful microscopical lamp, for all ordinary
purposes. The screw R clamps the lamp-flame at any height, while the
support N carries a water-bath O, or a plate P, both of which will be
found useful in preparing and mounting objects.
A special incandescent gas-lamp is made by Messrs. R. & J. Beck.
Polarisation of Light.
Common light moves in two planes at right angles to each other, while
polarised light moves in one plane only. Common light may be turned
into polarised light either by transmission or reflection; in the
first instance, one of the planes of common light is got rid of by
reflection; in the other, by absorption. Huyghens was one of the first
physicists to notice that a ray of light has not the same properties in
every part of its circumference, and he compared it to a magnet or a
collection of magnets; and supposed that the minute particles of which
it was said to be composed had different poles, which, when acted on in
certain ways, arranged themselves in particular positions; and thence
the term _polarisation_, a term having neither reference to cause nor
effect. It is to Malus, however, who, in 1808, discovered polarisation
by reflection, that we are indebted for the series of splendid
phenomena which have since that period been developed; phenomena of
such surpassing beauty as to exceed most ordinary objects presented to
the eye under the microscope.
Certainly no more misleading name could well have been found to
describe the causation, in one particular direction, of small
displacements in the medium, through which the light waves are made to
pass.
The effect of “polarising” light is simply to alter the directions of
the vibrations of light, and allow of certain waves to pass which are
vibrating in one direction only, vertical, horizontal, or oblique,
as the case may be. The most efficient agent discovered for the
polarisation of light is that of Iceland spar, cut and mounted as a
“Nicol” prism.
By cutting crystals of Iceland spar into two parts, at a particular
angle, and cementing them together again in the reverse way, Nicol
succeeded in showing that one of the two polarising pencils could be
totally deflected to one side, while the other is directly transmitted
through the Nicol prism, and thereby the beam of light becomes at once
“polarised” in one plane only. No apparent difference can be seen in
the prism on holding it up to the light, except it be in a very slight
loss of brightness; but if another similarly heated crystal be held
before, and made to revolve around, a quarter of the circle just where
the two cross each other, total darkness results. This phenomenon
alternately recurs at every quadrature of the circle. A pair of Nicol
prisms, when appropriately mounted, constitute “a polarising apparatus”
for the microscope, one being fitted into the sub-stage, and the other
either immediately above the objective or eye-piece, where it can be
easily rotated, the object to be examined being placed on the stage of
the microscope, that is, between the polarising and analysing prisms.
[Illustration: POLARISCOPE OBJECTS.
Tuffen West, del. Edmund Evans.
PLATE VIII.]
The significance of polarised light centres in the fact that it affords
a wider insight into the structure of crystals, minerals, and a number
of other substances, and which could not otherwise be obtained without
its aid. Its usefulness is multifold, as even glass itself, when
not properly annealed, exhibits points of fracture, by a display of
Newton’s rings. The knowledge thus acquired is turned to account by
glass manufacturers.
_Double refraction._--When an incident ray of light is refracted into
a crystal of any other than the cubic system, or into compressed or
_unannealed glass_, it gives rise to two refracted rays which take
different paths; this phenomenon is termed _double refraction_.
Attention was called to this in 1670, by Bartolin, who first observed
it in Iceland spar; and the laws for this substance were accurately
determined by Huyghens.
Iceland spar or calc spar is a form of crystallized carbonate of
lime. It is composed of fifty-six parts of lime and forty-four parts
of carbonic acid, and is usually found in rhombohedral forms of
crystallization.
To observe the phenomenon of double refraction, a rhomb of Iceland
spar may be laid on a page of a printed book, when all the letters
seen through it will appear double; the depth of the blackness of the
letters is seen to be considerably less than that of the originals,
except where the two images overlap.
In order to state the laws of the phenomena with precision, it is
necessary to attend to the crystalline form of Iceland spar, which has
equal obtuse angles. If a line be drawn through one of these corners,
making equal angles with the three edges which meet there, it, or
any line parallel to it, is called the _axis_ of the crystal; the
axis being, properly speaking, not a definite _line_ but a definite
_direction_.
The angles of the crystals are the same in all specimens. If the
crystal is of such proportions that these three edges spoken of are
equal, as in the smaller crystal (Fig. 176), the axis is the direction
of one of its diagonals, as represented.
Any plane containing (or parallel to) the axis is called the _principal
plane_ of the crystal.
In the next diagram, Fig. 177, the line appears double, as _a b_ and
_c d_, or the dot, as _e_ and _f_. Or allow a ray of light, _g h_, to
fall thus on the crystal, it will in its passage through be separated
into two rays, _h f_, _h e_; and on coming to the opposite surface of
the crystal, will pass out at _e f_ in the direction of _i k_, parallel
to _g h_. The plane _l m n o_ is designated the principal section of
the crystal, and the line drawn from the solid angle _l_ to the angle
_o_ is where the axis of the crystal will be found; this is its optic
axis. Now when a ray of light passes along this axis, it is undivided,
and there is only one image; but in all other directions there are two
images.
[Illustration: Fig. 176.--Axis of Crystals of Iceland Spar.]
[Illustration: Fig. 177.--A Rhomb showing the passage of Rays of Light.]
Mr. Nicol, of Edinburgh first succeeded in making a rhomb of Iceland
spar into a _single-image prism_. His method of splitting up the
crystal into two equal parts was as follows:--
A rhomb of Iceland spar of one-fourth of an inch in length, and about
four-eighths of an inch in breadth and thickness, is divided into two
equal portions in a plane, passing through the acute lateral angle, and
nearly touching the obtuse side angle. The sectional plane of each of
these halves must be carefully polished, and the two portions cemented
firmly together with Canada balsam, so as to form a rhomb similar to
that before division; by this management the ordinary and extraordinary
rays are so separated that only one is transmitted: the cause of this
great divergence of the rays is considered to be owing to the action
of the Canada balsam, the refractive index of which (1·549) is that
between the ordinary (1·6543) and the extraordinary (1·4833) refraction
of calcareous spar, and which will change the direction of both rays
in an opposite manner before they enter the posterior half of the
combination. The direction of rays passing through such a prism is
indicated by the arrow, Fig. 178.
[Illustration: Fig. 178.]
Polarised light cannot be distinguished from common light, as
already said, by the naked eye; and for all experimental purposes in
polarisation, two pieces of apparatus must be employed, one to produce
polarisation, and the other to show or an analyse it. The former is
called the _polariser_, the latter the _analyser_; and every apparatus
that serves for one of these purposes will also serve for the other.
[Illustration: Fig. 179.--Polariser.]
[Illustration: Fig. 179_a_.--Analyser.]
Polarising Apparatus for Students’ Microscope.
In all cases there are two positions, differing by 180°, which give
a minimum of light, and the two positions intermediate between these
give a maximum of light. The extent of the changes thus observed is a
measure of the completeness of the polarisation of light.
The two prisms mounted as shown in Figs. 179 and 179_a_ constitute the
apparatus adapted to the microscope. The polariser slips into place
below the stage, and the analyser, with the prism fixed in a tube, is
screwed in above the objective.
The definition is considered by some experimenters as somewhat better
if the analyser be used above the eye-piece, and is certainly more
easily rotated.
[Illustration: Fig. 180.--Prism mounted as an Eye-piece.]
_Method of employing the Polarising Prism_ (Fig. 179).--After having
adapted it to slide into a groove on the under-surface of the stage,
where it is secured and kept in place by the small milled-head screw,
the other prism (Fig. 179_a_) is screwed on above the object-glass,
and thus passes directly into the body of the microscope. The light
from the mirror having been reflected through them the axes of the
two prisms must be made to coincide; this is done by regulating the
milled-head screw until, by revolving the _polarising_ prism, the field
of view is entirely darkened twice during its revolution. If very
minute salts or crystals are submitted for examination then it will be
found preferable to place the analyser above the eye-piece, as in Fig.
180. Thus the _polariscope_ is seen to consist of two parts; one for
_polarising_, the other for _analysing_ or testing the light. There is
no essential difference between the two parts, except what convenience
or economy may lead us to adopt; and either part, therefore, may be
used as polariser or analyser; but whichever is used as the polariser,
the other becomes the analyser.
[Illustration: Fig. 181.--More Modern Polariser and Analyser.]
Opticians have their own methods of adapting the polariser and analyser
to their several microscopes. Watson’s special form of apparatus is
represented in Fig. 181, the polariser being adapted to the sub-stage,
and the analyser to screw into the objective.
_Tourmaline._--A semi-transparent mineral, of a neutral or bluish tint,
called tourmaline, when cut into thin slices (about 1/20-inch thick)
with their faces parallel to their axes exhibit the same phenomena as
the Nicol prism. The only objection to which is that the transmitted
polarised beam is more or less coloured. The tourmaline to be preferred
stops the most light when its axis is at right-angles to that of the
polariser, and yet admits the most when in the same plane. Make choice
of a tourmaline as perfect as possible; size is of less importance when
intended for use with the microscope.
Transmission of rays through tourmaline is only one of several ways
in which light can be polarised. When a beam of light is reflected
from a polished surface of glass, wood, ivory, leather, or any other
non-metallic substance, at an angle of 50° to 60° with the normal, it
is more or less polarised, and in like manner a reflector composed of
any of these substances may be employed as an analyser. In so using it,
it should be rotated about an axis parallel to the incident rays which
are to be tested, and the observation consists in noting whether this
rotation produces changes in the amount of reflected light.
For every reflected substance there is a particular angle of incidence,
which gives a maximum of polarisation in reflected light. It is called
the _polarising angle_ for the substance, and its tangent is always
equal to the index of refraction of the substance; or, what amounts to
the same thing, it is that particular angle of incidence which is the
complement of the angle of refraction, so that the refracted rays are
at right angles. This important law was discovered experimentally by
Sir David Brewster.
Tourmaline, like Iceland spar, is a negative uniaxial crystal; and
its use as a polariser depends on the property which it possesses of
absorbing the ordinary much more rapidly than the extraordinary ray, so
that a thickness which is tolerably transparent to the latter is almost
completely opaque to the former. Its pale cobalt blue colour enhances
the beauty of certain crystal and mineral substances, but like Iceland
spar, the paler and more perfect crystals are becoming scarce.
_Selenite_ is another mineral of value in polarisation experiments. It
is a native crystalline hydrated sulphate of lime. A beautiful fibrous
variety called _satin-gypsum_ is found in Derbyshire. The form of the
crystal most frequently met with is that of an oblique rectangular
prism, with ten rhomboidal faces, two of which are much larger than the
rest. It is usually split up into thin laminæ parallel to their lateral
faces; each film should have a thickness of from one-twentieth to
one-sixtieth of an inch. In the two rectangular directions these films
allow perpendicular rays of polarised light to traverse them unchanged,
termed their _neutral axes_. In two other directions, however, which
form respectively angles of 45° with the neutral axes, these films
have the property of double refraction, a direction known as the
_depolarising axis_.
[Illustration: Fig. 182.--Darker’s Selenite Films and Stage.]
The thickness of the film of selenite determines the particular tint.
If, therefore, we use a film of irregular thickness, different colours
are presented by the different thicknesses. These facts admit of very
curious and beautiful illustration, when used under the object placed
on the stage of the microscope. The films employed should be mounted
between two glasses for protection. Some persons employ a large film,
mounted in this way between the plates of glass, with a raised edge,
to act as a stage for supporting the object, it is then called the
“selenite stage.” The best film for the microscope is that which
gives blue, and its complementary colour yellow. The late Mr. Darker
constructed a selenite stage for the purpose (Fig. 182). With this a
mixture of colours will be brought about, by superimposing three films,
one on the other. By slight variations in their positions, produced
by means of an endless-screw motion, all the colours of the spectrum
can be shown. When objects are thus exhibited, it should be borne in
mind that all negative tints, as they are termed, are diminished, and
all positive tints increased; the effect of which is to mask the true
character of the phenomena.
For a certain thickness of selenite the ellipse will become a circle,
and we have thus what is called _circularly-polarised_ light, which is
characterised by the property that rotation of the analyser produces
no change of intensity. Circularly-polarised light is not, however,
identical with ordinary light; for the interposition of an additional
thickness of selenite converts it into elliptically (or in a particular
case into plane) polarised light.
It is necessary, for the exhibition of colour in our experiments, that
the plate of selenite should be very thin, otherwise the retardation
of one component vibration as compared with the other will be greater
by several complete periods for violet than for red, so that the
ellipses will be identical for several different colours, and the total
non-suppressed light will be sensibly white in all positions of the
analyser.
Two thick plates may, however, be so combined as to produce the effect
of one thin plate. For example, two selenite plates of nearly equal
thickness may be laid one upon the other, so that the direction of
greatest elasticity in the one shall be parallel to that of least
elasticity in the other. The resultant effect in this case will be that
due to the difference of their thicknesses. Two plates so laid are said
to be _crossed_.
[Illustration: Fig. 183.--_Red_ is represented by perpendicular lines;
_Green_ by oblique.]
The following experiments will well serve to illustrate some of the
more striking phenomena of double refraction, and will also be a useful
introduction to its practical application. Take a plate of brass (Fig.
183) three inches by one, perforated with a series of holes from about
one-sixteenth to one-fourth of an inch in diameter; the size of the
smallest should be in accordance with the power of the objective, and
the separating power of the double refraction.
_Experiment_ 1.--Place the brass plate so that the smallest hole shall
be in the centre of the stage of the microscope; employ a low power
(1-1/2 or 2 inches) objective, and adjust the focus as for the ordinary
microscopic object; place the double image prism over the eye-piece,
and two distinct images will be seen; by revolving the prism, the
images will describe a circle, the circumference of which will cut the
centre of the field of view; one of which is the ordinary, the other
the extraordinary ray. By moving the slide from left to right the
larger orifices will appear in the field, the images seen will not be
completely separated, but will overlap, as represented in the figure.
_Experiment_ 2.--Insert the Nicol’s prism into its place under the
stage, still retaining the double image prism over the eye-piece;
then, by examining the object, there will appear in some positions two
images, in others only one image; it will be seen, that at 90° this ray
will be cut off, and that which was first observed will become visible;
at 180°, or one-half the circle, an alternate change will take place;
at 270°, another change; and at 360°, the completion of the circle, the
first image will reappear.
Before proceeding to make the next experiment, the position of the
Nicol’s prism should be adjusted, and its angles brought parallel with
the square of the stage. The true relative position of the selenite
should also be determined by noticing the natural flaws in the film,
which should run parallel with each other, and be adjusted at an angle
of about 46° with the square bars of the stage.
_Experiment_ 3.--If we now take the plate of selenite thus prepared,
and place it under the piece of brass on the stage, we shall see,
instead of the alternate black and white images, two coloured images
composed of the constituents of white light, which will alternately
change by revolving the eye-piece at every quarter of the circle; then,
by passing along the brass, the images will overlap; and at the point
at which they do so, white light will be produced. If, by accident, the
prism be placed at an angle of 45° from the square part of the stage,
no particular colour will be perceived, and it will then illustrate the
phenomena of the neutral axis of the selenite, because when placed in
the relative position no depolarisation takes place. The phenomena of
polarised light may be further illustrated by the addition of a second
double image prism, and a film of selenite adapted between the two.
The systems of coloured rings in crystals cut perpendicularly to the
principal axis of the crystal are best seen by employing the lowest
object-glass.
_Biaxial Crystals._--To show perfectly the beautiful series of _rings
and brushes_ which biaxial crystals exhibit, it becomes necessary
to convert the microscope, for the time being, into (so to speak) a
wide-angled telescope.
[Illustration: Huyghenian Eye-piece.
Inner draw-tube.
Objective in draw-tube.
Analysing Prism.
Objective.
Specimen under Examination.
Sub-stage Condenser.
Polarising Prism, fixed in sub-stage below.
Fig. 184.--Diagrammatic arrangement of the Polarising Microscope.
_In Sub-stage_: P, polarising prism; C, sub-stage condenser on stage;
M, mineral or crystal. On nose-piece: O^1, objective, 4/10-inch; A,
analysing prism.
_In Draw-tube_: O^2, 2 or 3 inch Objective; H, Huyghenian eye-piece.]
For the purpose, screw on a low-power objective to the end of the
draw-tube (Fig. 184).[31] As the light requires to be passed through
the crystals at a considerable angle, a wide-angled condenser should be
employed, but it need not be achromatic. The objective most suitable
is a 4/10-inch, of ·64 numerical aperture, but a 1/4-inch of ·71
numerical aperture, or a 1/3-inch of ·65 numerical aperture, will
answer the purpose equally well. As the whole of the back lens of the
objective should be visible through the analysing Nicol prism, the back
lens of the objective must not be too large; thus a 1/2-inch of ·65
numerical aperture will not be so effective. The analysing prism may be
placed either where it is in the drawing, below the stage, or above the
eye-piece. It works equally well above the objective, the position it
ordinarily occupies in the microscope.
For the draw-tube a 2-inch objective and a B Huyghenian eye-piece
answers very well. Before screwing the objective on to the end of the
draw-tube centre the light in the usual manner, the Nicol’s being
turned so as to give a light field, then screw the objective on to
the end of the aperture, and put the crystal on the stage, rack down
the body so that the objective on the nose-piece nearly touches the
crystal, then focus with the draw-tube only. The sub-stage condenser
should be racked up close to the underside of the crystal.
Opticians, however, have more recently furnished a special form of
microscope (_The Petrological Microscope_, Fig. 79, p. 112), for the
use of those students who may desire to prosecute so fascinating a
study, and determine the optic axial angles of crystals.
Fuess[32] lately introduced a new form of microscope for polarising and
viewing biaxial crystals, which he believes to be needed, as in the
ordinary microscope the opening of the polariser is scarcely a third of
that of the condenser; moreover, it is not absolutely necessary that
the polariser and analyser should be Nicol’s prisms. This fact was
discovered by myself many years ago. Fuess utilises a bundle of thin
glass plates, as in the older Nuremberg polariscope. The frame holding
plates can be readily adjusted at the proper polarising angle, the
analyser being the ordinary small Nicol, screwed above the objective.
The illuminator is an Abbe’s triple condenser, of numerical aperture
1·40, which can be adjusted in the ordinary way. The front lens of
this should have a diameter of 11·12 mm. and the lower lens of 30 mm.
This increase in the condenser fully compensates for the loss of light
by the bundle of glass plates, and also enables thick sections of
crystals to be examined in convergent polarised light. The ocular used
should have a large field; the A Huyghenian answers best. A suggestion
to return to the original Nuremberg polariser is very opportune, as
_Iceland spar is becoming scarce_.
Mr. A. Mickel accidentally discovered that an opalescent mirror can
be converted into an excellent and inexpensive substitute for the
Nicol-prism polariser.
Rotation of Plane of Polarisation.
When a plate of quartz (rock-crystal), even of considerable thickness,
cut perpendicular to the axis, is interposed between the polariser
and analyser, colour is exhibited, the tints changing as the analyser
is rotated; and similar effects of colour are produced by employing,
instead of quartz, a solution of sugar enclosed in a tube with plain
glass ends.
The action thus exerted by quartz and sugar is called _rotation of
the plane of polarisation_, a name which sufficiently expresses the
observed phenomena. In the case of ordinary quartz, and solutions of
sugar-candy, it is necessary to rotate the analyser in the direction
of watch-hands as seen by the observer, and the rotation of the plane
of polarisation is said to be _right-handed_. In the case of what is
called _left-handed_ quartz, and of solutions of non-crystallisable
sugar, the rotation of the plane of polarisation is in the opposite
direction, and the observer must rotate the analyser against
watch-hands.
_Quartz_ belongs to the uniaxial system of crystals, and accordingly
exhibits one series of rings only, and no perfect central black cross.
On revolving the tourmaline the colour gradually changes, and passes
through all the colours of the spectrum. It can be cut to exhibit
either right-handed polarisation or left-handed polarisation and also
to exhibit straight lines.
_Calc Spar._--A uniaxial crystal showing only one system of rings, and
a black cross, changing into a white cross on revolving the tourmaline.
_Topaz._--A biaxial crystal exhibiting only one system of rings with
one fringe, owing to the wide separation of the axes. The fringe and
colours change on revolving the tourmaline.
_Borax._--A biaxial crystal; the colours are seen to be more intense
than in topaz, but the rings not so complete--only one set of rings can
be seen, owing to their wide separation.
_Rochelle Salt._--A biaxial crystal; the colours are more widely spread
out than the former, and only one set of rings seen at the same time.
_Carbonate of Lead._--A biaxial crystal; axes not so far separated, and
both systems of rings are more widely spread than those of potassium
nitrate.
_Aragonite._--A biaxial crystal; axes widely separated, but both
systems of rings seen at the same time. A fine crystal for displaying
the biaxial system.
[Illustration: Fig. 185.--Crystal of Potassium Nitrate.]
It was long believed that all crystals had only one axis of double
refraction; but Brewster found that the greater number of crystals
which occur in the mineral kingdom have _two axes_ of double
refraction, or rather axes around which double refraction takes place;
in the axes themselves there is no double refraction.
Potassium nitrate crystallises in six-sided prisms with angles of
about 120°. It has two axes of double refraction. These axes are each
inclined about 2-1/2° to the axes of the prism, and 5° to each other.
If, therefore, a small piece be split off a prism of potassium nitrate
with a knife driven by a sharp blow of a hammer, and the two surfaces
polished perpendicular to the axes of the prism, so as to leave
the thickness of the sixth or eighth of an inch, and then a ray of
polarised light be transmitted along the axes of the prism, the double
system of rings will be clearly visible.
When the line connecting the two axes of the crystal is inclined 45°
to the plane of primitive polarisation, a cross is seen on revolving
the potassium nitrate; it gradually assumes the form of two hyperbolic
curves, as in Fig. 185. But if the tourmaline be again revolved
through half a quadrant, the black cross will be replaced by white
spaces, as in the second figure. These systems of rings have, generally
speaking, the same colours as those of thin plates, or as those of a
system of rings revolving around one axis. The orders of the colours
commence at the centres of each system; but at a certain distance,
which corresponds to the sixth ring, the rings, instead of returning
and encircling each pole, encircle the two poles as an ellipse does
its two foci. If the thickness of the plate of _nitre_ be diminished
or increased, the rings are diminished or increased according to the
thickness of the crystal.
Small specimens of various salts may be crystallised and mounted
in Canada balsam for viewing under the stage of the microscope; by
arresting crystallisation at certain stages, a greater variety of forms
and colours will be obtained: we may enumerate salicine, asparagine,
acetate of copper, phospho-borate of soda, sugar, carbonate of lime,
potassium chlorate, oxalic acid, and all the oxalates found in urine,
with the other salts from the same fluid, a few of which are shown in
Plate VIII.
The late Dr. Herapath described a salt of quinine, remarkable for
its polarising properties. The crystals of this salt, when examined
by reflected light, have a brilliant emerald-green colour, with
almost a metallic lustre; they appear like portions of the elytræ
of the cantharides beetle, and are also very similar to murexide in
appearance. When examined by transmitted light, they scarcely possess
any colour, there is only a slightly olive-green tinge; but if two
crystals, crossing at right-angles, be examined, the spot where they
intersect appears perfectly black, even if the crystals are not more
than one five-hundredth of an inch in thickness. If the light be in the
slightest degree polarised--as by reflection from a cloud, or by the
blue sky, or from the glass surface of the mirror of the microscope
placed at the polarising angle 65° 45′--these little prisms and films
assume complementary colours: one appears green, and the other pink,
and the part at which they cross is chocolate or deep chestnut-brown,
instead of black. Dr. Herapath succeeded in making artificial
tourmalines large enough to surmount the eye-piece of the microscope;
so that all experiments with those crystals upon polarised light may
be made without the tourmaline or Nicol’s prism. The finest rosette
crystals are made as follows:--To a moderately strong solution of
_Cinchonidine_ add a drop or two of Herapath’s test-fluid.[33] A few
drops of this is placed on the centre of a glass slide, and put aside
until the first crystals are observed to be formed near the margin.
The slide should now be placed upon the stage of the microscope, and
the progress of formation of the crystals closely watched. When these
are seen to be large enough, and it is deemed necessary to stop their
further development, the slide must be quickly transferred to the palm
of the hand, the warmth of which will be found sufficient to stop
further crystallisation. These crystals attract moisture, deliquesce,
and should therefore be kept in a perfectly dry place.
[Illustration:
Fig. 186.--In this figure heraldic lines are adopted to denote colour.
The dotted parts indicate _yellow_, the straight lines _red_, the
horizontal lines _blue_, and the diagonal, or oblique lines, _green_.
The arrows show the plane of the tourmaline, _a_, blue stage; _b_, red
stage of selenite employed.]
To render these crystals evident, it merely remains to bring the
glass-slide upon the field of the microscope, with the selenite stage
and single tourmaline, or Nicol’s prism, beneath it; instantly the
crystals assume the two complementary colours of the stage: red and
green, supposing that the pink stage is employed; or blue and yellow,
provided the blue selenite is made use of. All those crystals at
right angles to the plane of the tourmaline produce that tint which an
analysing-plate of tourmaline would produce when at right angles to the
polarising-plate; whilst those at 90° to these educe the complementary
tint, as the analysing-plate would also have done if revolved through
an arc of 90°.
This test is a delicate one for quinine (Fig. 186, _a_ and _b_); not
only do these peculiar crystals act in the way just related, but
they may be easily proved to possess the optical properties of that
remarkable salt, the sulphate of iodo-quinine.
[Illustration: Fig. 187.--Polarised Crystals of Quinidine.]
To test for quinidine, it is merely necessary to allow a drop of acid
solution to evaporate to dryness upon the slide, and to examine the
crystalline mass by two tourmalines, crossed at right angles, and
without the stage. Immediately little circular discs of white, with a
well-defined black cross, start into existence, should quinidine be
present even in very minute traces. These crystals are represented in
Fig. 187.
If the selenite stage be employed in the examination of this object,
one of the most gorgeous appearances in the whole domain of the
polarising microscope is displayed: the black cross disappears, and is
replaced by one consisting of two colours, and divided into a cross
having a red and green fringe, whilst the four intermediate sectors are
a gorgeous orange-yellow. These appearances alter on the revolution
of the analysing-plate of tourmaline; when the blue stage is employed,
the cross assumes a blue or yellow tint, varying according to the
position of the analysing plate. These phenomena are analogous to those
exhibited by certain circular crystals of boracic acid, and to circular
discs of salicine (prepared by fusion), the difference being that the
salts of quinidine have more intense depolarising powers than either of
the other substances; the mode of preparation, however, excludes these
from consideration. Quinine prepared in the same manner as quinidine
has a very different mode of crystallisation; but it occasionally
presents circular corneous plates, also exhibiting the black cross and
white sectors, but not with one-tenth part of the brilliancy, which of
course enables us readily to discriminate the two.
[Illustration: Fig. 188.--Urinary Salts, seen under Polarised Light.
_a_, Uric acid; _b_, Oxalate of lime, octahedral crystals of; _c_,
Oxalate of lime allowed to dry, forming a black cube; _d_, Oxalate of
lime as it occasionally appears, termed the dumb-bell crystal.]
Urinary salts are more readily seen under polarised light than by white
light. Ice doubly refracts, while water singly refracts. Ice takes the
rhomboidic form; and snow in its crystalline forms may be regarded as
the skeleton crystals of this system (Fig. 189). A sheet of clear ice,
of about one inch thick, and slowly formed in still weather, shows
circular rings with a cross by polarised light.
[Illustration: Fig. 189.--Snow Crystals.]
It is probable that the conditions of snow formation are more complex
than might be imagined, familiar as we are with the conditions relating
to the crystallisation of water on the earth’s surface. A great
variety of animal, vegetable, and other substances possess a doubly
refracting or depolarising structure, as: a quill cut and laid out flat
on glass; the cornea of a sheep’s eye; skin, hair, a thin section of a
finger-nail; sections of bone, teeth, horn, silk, cotton, whalebone;
stems of plants containing silica or flint; barley, wheat, &c. The
larger-grained starches form splendid objects; _tous-les-mois_, the
largest, may be taken as a type of all others. This presents a black
cross, the arms of which meet at the hilum (Fig. 190). On rotating the
analyser, the black cross disappears, and at 90° is replaced by a white
cross; another, but much fainter, black cross is seen between the arms
of the white cross, no colour being perceptible. But if a thin plate of
selenite be interposed between the starch-grains and the polariser, a
series of delicate colours appear, all of which change on revolving the
analyser, becoming complementary at every quadrant of the circle. West
and East India arrow-root, sago, tapioca, and many other starch-grains,
present a similar appearance; but in proportion as the grains are
smaller, so are their markings and colourings less distinct.
[Illustration: Fig. 190.--Potato Starch, under Polarised Light.]
Molecular Rotation.
For the purpose of studying the various interesting phenomena of
molecular rotation, a few necessary pieces of apparatus must be added
to the microscope. First, an ordinary iron three-armed retort stand,
to the lower arm of which must be attached either a polarising prism
or a bundle of glass plates inclined at the polarising angle; in the
upper an analysing prism. The fluid to be examined should be contained
in a narrow glass tube about eight inches in height, and this must be
attached to the middle arm. If the prisms be crossed before inserting
a fluid possessing rotatory power, the light passing through the
analyser will be coloured. If a solution of sugar be employed, and
the light which passes through the second prism is seen to be red,
but on rotating the analyser towards the right the colour changes
to yellow, and passes through green to violet, it may be concluded
that the rotation is right-handed. If, on the contrary, the analyser
requires to be turned towards the left hand, we conclude that the
polarisation is left-handed. These phenomena are wholly distinct from
those accompanying the action of doubly refracting substances upon
plane polarised light. It is not easy to explain in a limited space the
course to be followed in ascertaining the amount of rotation produced
by different substances. Monochromatic light should be used. If we are
about to examine a sugar solution with the prisms crossed, the index
attached to the analyser must first be made to point to zero. The sugar
is then introduced, when it will be necessary to rotate the analyser
23° to the right, in order that the light may be extinguished. This is
the amount of rotation for that particular fluid at a given density and
that height of column. As the arc varies with increase or decrease of
density and height of the fluid, it is needful to reduce it to a unit
of height and density. The following formula is that given by Biot:--P
= quantity of matter in a unit of solution; _d_ = sp. gr.; _l_ = length
of column; _a_ = arc of rotation; _m_ = molecular rotation.
Then _m_ = _a_/(_l p d_).
The application of the polarising apparatus to the microscope is of
much value in determining minute structure. It may also be defined as
an instrument of analysis; a test of difference in density between any
two or more parts of the same substance. All structures, therefore,
belonging either to the animal, vegetable, or mineral kingdom, in which
the power of unequal or double refraction is suspected to be present,
are those that should especially be re-investigated by polarised
light. Some of the most delicate of the elementary tissues of animal
structure, the ultimate fibrillæ of muscles, &c., are amongst the most
interesting subjects that might be studied with advantage under this
method of investigation. The chemist may perform the most dexterous
analysis; the crystallographer may examine crystals by the nicest
determination of their forms and cleavage; the anatomist or botanist
may use the dissecting knife and microscope with the most exquisite
skill; but there are still structures in the mineral, vegetable, and
animal kingdoms which will defy all such modes of examination, and will
yield only to the magical analysis of polarised light.
Formation and Polarisation of Crystals.
The inorganic kingdom will afford to the microscopist a never-ending
number of objects of unsurpassed beauty and interest. The phenomena
of crystallisation in its varied combinations can be made a useful
and instructive occupation. Although ignorant of the means whereby the
great majority of minerals and crystals have been formed in the vast
laboratory of Nature, we can, nevertheless, imitate in a small degree
Nature’s handiworks by crystallising out a large number of substances,
and watch their numerous transformations in the smallest appreciable
quantities, when aided by the microscope.
Among natural crystals we look for the material for the formation
of our lenses, while the varieties of granites present us with the
earliest crystallised condition of the earth’s crust as it cooled down,
the structure of which is beautifully exhibited under polarised light.
In Plate VIII. various crystalline and other bodies are displayed. In
No. 158 is a section of new red sandstone; 159 of quartz; and 160 of
granite. Special reference is made to others in the following list of
salts and other substances which form a beautiful series of objects for
study under polarised light:--
SALTS.
Alum.
Asparagine.
Aspartic Acid. Plate VIII. No. 168.
Bitartrate of Ammonia.
Boracic Acid.
Borax. No. 164.
Carbonate of Lime.
" Soda.
Chlorate of Potash.
Chloride of Barium.
" Cobalt.
" Copper and Ammonia.
" Sodium.
Cholesterine.
Chromate of Potash.
Cinchonine.
Cinchonidine.
Citric Acid.
Hippuric Acid.
Iodide of Mercury.
" Potassium.
" Quinine.
Iodo-disulphate of Quinine.
Kreatine. No. 166.
Murexide.
Nitrate of Bismuth.
" Barytes.
" Brucine.
" Copper.
" Potash.
" Strontian.
" Uranium.
Oxalate of Ammonia.
" Chromium.
" Chromium and Potash.
" Lime.
" Soda.
Indurated Sandstone, Howth.
Indurated Sandstone, Bromsgrove.
Gibraltar Rock.
Granite, various localities. No. 160.
Hornblend Schist.
Labrador Spar.
Norway Rock.
Quartz Rock, various. No. 159.
" in Bog Iron Ore.
Quartzite, Mont Blanc.
Sandstone. No. 158.
Satin Spar.
Selenites, various colours.
Tin Ore, with Tourmalin.
Oxalic Acid.
Oxalurate of Ammonia.
Permanganate of Potash.
Phosphate of Lead and Soda.
Platino-cyanide of Magnesia.
Plumose Quinidine.
Prussiate of Potash, red and yellow.
Quinidine.
Santonine.
Salicine.
Salignine. No. 162.
Sulphate of Cadmium.
" Copper. No. 161.
" Copper and Potash.
" Iron. No. 163.
" Iron and Cobalt. No. 165.
" Magnesia.
" Nickel and Potash.
" Soda.
" Zinc.
Sugar.
Tartaric Acid.
Thionurate of Ammonia.
Triple Phosphate.
Urate of Ammonia.
" Soda.
Urea, and most urinary deposits.
Uric Acid.
MINERALS.
Agates, various.
Asbestiform Serpentine.
Avanturine.
Carbonate of Lime.
Carrara Marble.
ANIMAL STRUCTURES.
Cat’s Tongue. No. 174.
Grayling Scale. No. 176.
Holothuria, Spicules of. Nos. 171-2.
Prawn Shell. No. 175.
VEGETABLE CRYSTALLINE SUBSTANCES.
CUTICLE of Leaf of Correa Cardinalis.
" " Deutzia scabra. No. 173.
" " Elæagnus.
" " Onosma taurica.
Equisetum. No. 170.
Fibro cells from orchid. No. 169.
" Oncidium bicallosum.
Scalariform Vessels from Fern.
Scyllium Caniculum. No. 177.
SILICIOUS CUTICLES, various.
Starches, various. No. 167.
The formation of artificial crystal may be readily effected, and the
process watched, under the microscope, by simply placing a drop of
saturated solution of any salt upon a previously warmed slip of glass.
Interesting results will be obtained by combining two or more chemical
salts in the following manner. To a nearly saturated solution of
the sulphate of copper and sulphate of magnesia add a drop on the
glass-slide, and dry quickly. To effect this, heat the slide so as to
fuse the salts in its water of crystallisation, and there remains an
amorphous film on the hot glass. Put the slide aside and allow it to
cool slowly; it will gradually absorb a certain amount of moisture
from the air, and begin to throw out crystals. If now placed under the
microscope, numerous points will be seen to start out here and there.
The starting points may be produced at pleasure by touching the film
with a fine needle point, so as to admit of a slight amount of moisture
being absorbed by the mass of salt. Development is at once suspended by
applying gentle heat; cover the specimen with balsam and thin glass.
The balsam should completely cover the edges of the thin glass circle,
otherwise moisture will probably insinuate itself, and destroy the form
of the crystals.
Mr. Thomas succeeded in crystallising “the salts of the magnetic
metals” at very high temperatures, with very curious results. In Plate
VIII. are seen crystals of sulphate of iron and cobalt, No. 163; and
of nickel and potash, No. 165, obtained in the following manner:--Add
to a concentrated solution of iron a small quantity of sugar, to
prevent oxidation. Put a drop of the solution on a glass slide, and
drive out the water of crystallisation as quickly as possible by the
aid of a spirit lamp; then with a Bunsen’s burner bring the plate to a
high temperature. Immediately a remarkable change is seen to take place
in the form of the crystal, and if properly managed the “foliation”
represented in the plate will be fairly exhibited. The slide must not
be allowed to cool down too rapidly or the crystals will probably
absorb moisture from the atmosphere, and in so doing the crystals alter
their forms. Immerse them in balsam, and cover in the usual way before
quite cold.
_Sublimation of Alkaloids._--The late Dr. Guy, F.R.S., directed the
attention of microscopists to the fact that the crystalline shape of
bodies belonging to the inorganic world might be of service in medical
jurisprudence. Subsequently, Dr. A. Helwig, of Mayence, investigated
this subject, and found the plan applicable not only to inorganic but
also to organic substances, and especially to poisonous alkaloids. By
using a white porcelain saucer Dr. Guy was able to watch the process
of crystallisation more minutely, and to regulate it more exactly. He
was, in fact, able to obtain characteristic crusts composed of crystals
of strychnine weighing not more than 1/3000th or 1/5000th of a grain.
Morphia affords equally characteristic results. For the examination
of these, Dr. Guy recommended the use of a binocular microscope with
an inch object-glass. But it is not to crystalline forms alone that
one need trust; the whole behaviour of a substance as it melts and
is converted into vapour is eminently characteristic, and when once
deposited on the microscopical slide, under the object-glass, the
application of re-agents may give still more satisfactory results.
The re-agents, however, which are here to be applied are not of the
kind ordinarily employed. Colour-tests under the microscope are,
comparatively speaking, useless; those that give rise to peculiar
crystalline forms are rather to be sought after. For instance, the
crystals produced by the action of carbozotic acid on morphia are by
themselves almost perfectly characteristic. These experiments should
not, however, be undertaken for medico-legal purposes by one unskilled
in their conduct, for the effects of the reagents themselves might be
mistaken by the uninitiated for the result of their action on the
substance under examination. For the special method of procedure, see
Dr. W. Guy, “On the Sublimation of the Alkaloids.”[34]
The Micro-spectroscope.
Spectrum analysis has, from its first introduction by Kirschoff in
1859, maintained its fascination over men of science throughout
the civilised world. Microscopists, astronomers, and chemists have
assigned to the spectroscope a highly important position amongst
scientific instruments of research. At quite an early period of its
history it appeared to ourselves to promise an extension of the work
of the microscope in pathology and microscopy, and second only to
that of astronomy and chemistry. The chief hindrances to the use of
the spectroscope were, in the early days, of a twofold nature; a
widespread, but quite erroneous view of the serious difficulties of
employing the instrument, and the want of a first aid to its use.
So valuable a means of research has this process of analysis proved to
be, that the discoveries made by the spectroscope appear marvellous.
The spectroscope was first made known as a refined instrument for the
analysis of light by two Germans, a physicist and a chemist, Kirschoff
and Bunsen. In 1860, the latter succeeded in detecting and separating
two new alkaline bodies from all other bodies from the waters obtained
from the Durkeim springs, less than 0·0002 part of a milligramme of
which can be detected by spectrum analysis. It is to the labours
of Huggins, Norman Lockyer and others that we are indebted for the
wonderful discoveries made in astronomy; and chiefly so to Brewster,
Herschel, and Talbot, for showing that certain metals give off light of
a high degree of refrangibility; that distinct bands are situated at a
distance beyond the last visible violet ray ten times as great as the
length of the whole visible spectrum from red to violet.
With regard to the discoveries made in connection with physiological
research, we are indebted to F. Hoppe, who in 1862 first described
the absorption bands of human blood. His results were confirmed by
the investigations of Professor Sir George Gabriel Stokes, who, by
adding certain reducing agents to the blood, found that he could change
scarlet blood into purple--“purple cruorine”--and in this way the
place occupied by the absorption band in the spectrum could be made to
change. He reduced the hæmoglobin by robbing the blood of its oxygen.
Thus, by Stokes’ and other methods, we have since arrived at extremely
valuable results, and the explanation of the difference in colour
between arterial and venous blood; and it has also enabled us to show
wherein the breathing power of the red corpuscles resides, and further
explains phenomena which before his investigations were inexplicable.
[Illustration: Fig. 191.--Fräunhofer’s Spectrum Lines.]
The spectroscope seems likely to be of almost as great use in medicine
as it has already proved to be in solar and terrestrial chemistry, if
we may form an opinion from the large amount of literature which has
appeared on the subject. The inception of this magical instrument arose
on the instance of a discovery made by Dr. Wollaston in 1802, who, on
making a slit in the shutter of his room, instead of a round hole, the
spectrum of sunlight, instead of being composed of a number of coloured
discs, was now a band of pure colours, each colour being free from
admixture with the next to it. Moreover, he found that this colour band
was not continuous, as Newton described it, but interrupted here and
there by _fine black lines_.
In 1814, Fräunhofer,[35] a German optician, discovered these lines
quite independently, and mapped out 576 of them, calling the more
prominent of them A, B, C, D, E, F, G, H, which lines he used as marks
of comparison. He also found that the distances of these lines from
each other may vary according to the nature of the substance composing
the prism; thus, their relative distances are not the same in prisms
of flint-glass, crown-glass, and bisulphide of carbon, but they always
occupy the same position relatively to the colours of the spectrum.
Kirschoff and Angström had mapped out in 1880 no less a number than
2,000 Fräunhofer lines, a portion of which are correctly shown in the
accompanying chart (Fig. 191).
In 1830, Simms, a London optician, made an improvement in the
construction of the spectroscope by placing a lens in front of the
prism, so arranged that the slit was in the focus of the lens. This
lens turns the light, after it has passed through the slit, into
a cylindrical beam before entering the prism. Another lens, also
introduced by him, receives the circular beam emerging from the prism,
and compels it to throw an image of the slit, which may be magnified
at pleasure for each ray. The lens between the prism and the slit is
termed the _collimating_ lens. Thus the following are the essential
parts of a chemical spectroscope:--(1) a slit, the edges of which are
two knife-edges of steel very truly ground, and exactly parallel to
each other, and in a direction parallel to the refracting edge of the
prism, to admit a pencil of rays. (2) A collimating lens; a convex
lens with the slit at its principal focus, which renders the rays
parallel before entering the prism. (3) A prism of dense glass, in
which the rays are refracted and dispersed. (4) An observing telescope
constructed like an astronomical refractor of small size, and placed so
that the rays shall traverse it after emerging from the prism. Such are
the essentials of a one-prism chemical spectroscope.
The form of instrument in use with the microscope is the “_direct
vision_” spectroscope, consisting of two prisms of flint-glass, placed
between three of crown-glass cemented together by Canada balsam; the
spectrum being viewed directly by the eye. The earliest constructed
form of micro-spectroscope is shown in Fig. 192, the Browning-Huggins.
It was, however, Mr. Sorby who suggested that the prism should be
made of dense flint-glass and of such a form that it could be used
in two different positions, and that in one it should give twice the
dispersion that it would in the other, but that the angle made by the
incident and emergent rays should be the same in both positions.
[Illustration: Fig. 192.--The Browning-Huggins Micro-spectroscope.]
[Illustration: Fig. 193.]
[Illustration: Fig. 193_a_.]
Figs. 193 and 193_a_ represent prisms of the kind arranged to use in
two different positions, i and i′ being the same angle as I and I′.
For most absorption-bands, particularly if faint, the prism should be
used in the first position, in which it gives the least dispersion;
when greater dispersion is required, so as to separate some
particular lines more widely, or to show the spectra of the metals,
or Fräunhofer’s lines in the solar spectrum, then the prism must be
used as in Fig. 193_a_. This answers well for liquids or transparent
objects, but it is, of course, not applicable to opaque objects.
To combine both purposes, some form of direct vision-prisms that maybe
applied to the body of the microscope is required. Fig. 194 represents
an arrangement of direct vision-prisms, invented by Herschel. The line
R R′ shows the path of a ray of light through the prisms, where it
would be seen that the emergent ray R′ is parallel and coincident with
the incident ray R.
[Illustration: Fig. 194.]
[Illustration: Fig. 194_a_.]
Another very compact combination is shown in Fig. 194_a_. Any number
of these prisms (P P P) may be used, according to the amount
of dispersion required. They are mounted in a similar way to a
Nicol’s prism, and are applied directly over the eye-piece of the
microscope. The slit S S is placed in the focus of the first glass
(F) if a negative, or below the second glass if a positive eye-piece
be employed. One edge of the slit is movable, and, in using the
instrument, the slit is first opened wide, so that a clear view of
the object is obtained. The part of the object of which the spectrum
is to be examined is then made to coincide with the fixed edge of the
slit, and the movable edge is screwed up, until a brilliant coloured
spectrum is produced. The absorption-bands will then be readily found
by slightly altering the focus. This contrivance answers perfectly
for opaque objects, without any preparation; and, when desirable, the
same prism can be placed below the stage, and a micrometer used in the
eye-piece of the microscope, thus avoiding a multiplicity of apparatus.
[Illustration: Fig. 195.--The Sorby-Browning Micro-spectroscopic
Eye-piece.]
A later and better form of instrument is the Sorby-Browning eye-piece
(Fig. 195), shown in section (Fig. 196) ready for inserting into the
body-tube of the microscope, the prism of which is contained in a
small tube, removable at pleasure. Below the prism is an achromatic
eye-piece, having an adjustable slit between the two lenses, the upper
lens being furnished with a screw motion to focus the slit. A side
slit, capable of adjustment, admits, when required, a second beam of
light from any object whose spectrum it is desired to compare with that
of the object placed on the stage of the microscope. This second beam
of light strikes against a very small prism, suitably placed inside the
apparatus, and is reflected up through the compound prism, forming a
spectrum in the same field with that obtained from the object on the
stage.
[Illustration: Fig. 196.--Sectional view of bright-line Spectroscope;
the letters also apply to the standard spectrum scale (Fig. 198).]
A is a brass tube, carrying the compound direct vision prism; B, a
milled head, with screw motion to adjust the focus of the achromatic
eye lens C, seen in the sectional view as a triple combination of
prisms. Another screw at right angles to C, which from its position
cannot be well shown in the figure, regulates the slit horizontally.
This screw has a larger head, and when once recognised cannot be
mistaken for the other. D D is a clip and ledge for holding a small
tube, so that the spectrum given by its contents may be compared with
one from an object on the stage. E is a round hole for a square-headed
screw, opening and shutting a slit, admitting the quantity of light
required to form the second spectrum. A light entering the round hole
near E strikes against the right-angled prism, which is placed inside
the apparatus, and is reflected up through the slit belonging to the
compound prism. If any incandescent object be placed in a suitable
position with reference to the round hole, its spectrum will be
obtained. F shows the position of the field lens of the eye-piece. The
tube is made to fit the microscope to which the instrument is applied.
To use this instrument insert F as an eye-piece in the microscope tube,
taking care that the slit at the top of the eye-piece is in the same
direction as the slit below the prism. Screw on to the microscope
the object-glass required, and place the object whose spectrum is to
be viewed on the stage. Illuminate with the stage mirror if it be
transparent; with mirror, Lieberkühn, and dark well, by side reflector,
or bull’s-eye condenser if opaque. Remove A, and open the slit by means
of the milled-head, not shown in figure, but which is at right angles
to D D. When the slit is sufficiently open the rest of the apparatus
acts as an ordinary eye-piece, and any object can be focussed in the
usual way. Having focussed the object, replace A, and gradually close
the slit till a good spectrum is obtained. The spectrum will be much
improved by throwing the object a little out of focus.
[Illustration:
Figs. 197 and 197_a_.--The Beck-Sorby Micro-spectroscope Eye-piece,
drawn on a scale of one half size.]
Every part of the spectrum differs a little from adjacent parts in
refrangibility, and delicate bands or lines can only be brought out by
accurately focussing that particular part of the spectrum. This can be
done by the milled-head B. Disappointment will occur in any attempt
at delicate investigation if the directions given be not carefully
followed out.
Opposite E a small mirror is attached. It is like the mirror below the
stage of a microscope, and is mounted in a similar manner. By means of
this mirror light may be reflected into the eye-piece, and in this way
two spectra may be procured from one lamp.
Method of using the Micro-Spectroscope.
A beginner with the micro-spectroscope should first make himself fully
acquainted with the spectroscope by holding it up to the sky and noting
the effects of opening and regulating the slit, by rotating the screw
C, Figs. 195 and 197. The lines will be well seen on closing down
the opening. This screw diminishes the length of the slit, when the
spectrum is seen as a narrow ribbon of prismatic colours. The screw
E regulates the admission of light through the aperture above D. The
better objects with which to commence the study of the absorption
bands are, aniline dye, much diluted, madder, permanganate of potash,
and blood. As each colour varies in refrangibility, the focus must
be adjusted by the screw E. When it is desired to view the spectrum
of a very minute object, the prisms should be removed by withdrawing
the tube containing them, the slit set open, and the object brought
into the centre of the field; the vertical and horizontal slits
must then be partially closed up, and the prisms replaced, when a
suitable objective is employed to examine the spectrum. For ordinary
observations a magnifying power of an inch and a half or two inches
will be suitable, but for small quantities of material a higher power
must be employed, when a single blood corpuscle can be made to show its
characteristic absorption band. After having obtained the best image of
any object on stage, throw it slightly out of focus, and substitute the
micro-spectroscopic eye-piece for the Huyghenian. Opaque objects should
be examined by reflected light, by means of the bull’s-eye condenser,
or side reflector. Mr. Sorby uses a binocular microscope, which enables
him to regulate the focussing and throwing out of focus of the object.
In examining crystals or other small objects, a small cardboard
diaphragm should be placed beneath them; and when examining the spectra
of liquids in cells, slip a small cap with a perforation of 1/10-inch
in diameter over the tube containing the 1/2-inch or 2-inch objective.
Substances which give absorption bands or lines in the red are best
seen by artificial light, while those which show bands in the violet
are better seen by daylight. By following rules of the kind we are less
likely to mix the bands of the absorption spectrum with the Fräunhofer
lines. For example, if the edge of a band happens to coincide with a
Fräunhofer line, the observer is apt to imagine that the band is better
defined and more abruptly shaded on one side than it really is.
[Illustration: Standard Spectrum Scale.
Cells for use with Spectroscope.
Fig. 198.]
_Cells and Tubes._--These are either supplied ready-made by the
optician, or can be formed out of small pieces of barometer tubing,
with the edges ground down and cemented on ordinary glass slips. In
Fig. 198 is seen the several kinds of cells and tubes usually employed,
while the little flat tubes commonly in use as bouquet holders will be
found of use, with the side stage reflecting spectrum as comparison
tubes; being of different diameters they allow of two or more depths of
colour in the fluid intended for examination.
In the case of many other fluids the sloping form of cell (Fig. 198)
will be useful, as different shades of fluids can be examined without
removal from the stage of the microscope. The deeper cells are cut from
a piece of barometer tubing of about half to an inch long, one end
being cemented to a piece of flatted glass, and the other covered over
temporarily or permanently with a thin piece of glass on the top, held
in its place by capillary attraction, thus admitting of the tube being
turned upside down.
_Re-agents required._--A diluted solution of ammonia, citric acid,
double tartrate of potash and soda (the last being used to prevent
the precipitation of oxide of iron), and the double sulphate of the
protoxide of iron and ammonia (employed to deoxidise blood, etc.). In
some special cases, dilute hydrochloric acid, purified boric acid, and
sulphate of soda are required.
The character of stains of blood varies with age and with the nature
of the substance with which it happens to be combined. This is
important to remember in connection with _Jurisprudence_, when the
micro-spectroscope is brought into use for the detection of blood
stains. The spectrum used in important cases of the kind should have
a compound prism, with enough, but not too great dispersive power,
otherwise the bands become, as it were, diluted, and less distinct.
If the blood stain is quite recent, the colouring matter will be
hæmoglobin only. This easily dissolves out in water, and when
sufficiently diluted gives the spectrum of oxy-hæmoglobin, which on
the addition of ammonia, together with a small quantity of the double
tartrate, a small piece of ferrous salt, and stirring carefully without
the admission of air, changes the spectrum of reduced hæmoglobin.
When stirred again, so as to expose the solution as much as possible
to air, the two bands reappear; on gradually adding citric acid in
small quantities the colour begins to change, and the bands are seen
to gradually fade away; if there should have been much blood present,
a band appears in the red; the further addition of ammonia makes all
clear again, but does not restore the original bands, because the
hæmoglobin has been permanently changed into hæmatin. This reaction
alone distinguishes blood from most other colouring matters, since
other substances after being changed by acids are restored by alkalies
to their original state. There are many other curious facts connected
with the spectroscopic analysis of blood, which are fully explained and
illustrated by Dr. Maemunn in his book on “The Use of the Spectroscope
in Medicine,” and also in Dr. Thudicum’s[36] reports and charts, which
are the most complete. Sir George Stokes, F.R.S., was one of the first
to show the essential value of the spectral phenomena of hematine,
and who proved, after Hoppe had first drawn attention to the fact,
that this colouring matter is capable of existing in two states of
oxidation, and that a very different spectrum is produced according
as the substance, which he termed _cruorine_, is in a more or less
oxidised condition. The chart appended to his paper[37] affords an
imperfect representation of the changes seen in the spectrum.
[Illustration: No. 1.--Arterial Blood, Scarlet Cruorine.
No. 2.--Venous Blood, Purple Cruorine.
No. 3.--Blood treated with Acetic Acid.
No. 4.--Solution of Hæmatin.
Fig. 199.--Sir George Stokes’ Chart of the Absorption Bands of Blood.]
Proto-sulphate of iron, or proto-chloride of tin, causes the reduction
of the colouring-matter, but, on exposure to air, oxygen is absorbed,
and the solution again exhibits the spectrum characteristic of the
more oxidised state. The different substances obtained from blood
colouring-matter produce different bands. Thus, _hæmatin_ gives rise
to a band in the red spectrum D; _hæmato-globulin_ produces two bands,
the second twice the breadth of the first in the yellow portion of the
spectrum between the lines D and E, No. 1. The absorption-bands differ
according to the strength of the solution employed, and the medium in
which the blood-salt is dissolved; but an exceedingly minute proportion
dissolved in water is sufficient to bring out very distinct bands. B
represents the red end of the spectrum and G the green as it approaches
the violet end.
_Mapping the Spectra._--In the sectional view given of the
micro-spectroscope (Fig. 196), the internal construction of the
instrument is shown, and the arrangement made for throwing a bright
point on to the surface of the upper prism is clearly seen. The mapping
out is accomplished by means of a photographic scale fixed as a
standard spectrum (Fig. 198), in the position of A A, illuminated by
the small mirror at R, and focussed by a small lens at C, so that on
looking into the instrument one can see the spectrum accurately divided
into one hundred equal parts, and scale readings can be made at once;
the only precaution needed is to be sure the D (or the sodium line,
if D cannot be got) always stands at the same number on the scale. To
map absorption spectra on this scale we have to lay down a line, as
many millimetres long as there are divisions in the scale, and mark
the position of the bands on this line. Mr. Browning supplies scales
printed off ready for use. But the mapping out of spectra, as Mr. Sorby
pointed out, requires some consideration; since the number of divisions
depends on the thickness of the interference-plate, it becomes
necessary to decide what number should be adopted. Ten it was thought
would be most suitable; but, on trial, it appeared to be too few for
practical work. Twenty is too many, since it then becomes extremely
difficult to count them. Twelve is as many as can well be counted; it
is a number easily remembered, is sufficiently accurate, and has other
practical advantages. With twelve divisions the sodium-line 0 comes
very accurately at 3-1/2; thus, by adjusting the plate so that a bright
sodium-light is brought into the centre of the band, when the Nicol’s
prisms are also crossed accurately at 3-1/2, parallelism is secured,
together with a wider field of observation. The general character of
the scale will be best understood from the following figure, in which
the bands are numbered, and given below the principal Fräunhofer lines.
The centre of the bands is black, and they are shaded off gradually at
each side, so that the shaded part is about equal to the intermediate
bright spaces. Taking, then, the centres of the black bands as 1, 2,
3, &c., the centres of the spaces are 1-1/2, 2-1/2, 3-1/2, &c., the
lower edges of each 3/4, 1-3/4, &c., and the upper 1-1/4, 2-1/4, &c.,
we can easily divide these quarters into eighths by the eye: and this
is as near as is required in the subject before us, and corresponds
as nearly as possible to 1/100th part of the whole spectrum, visible
under ordinary circumstances by gaslight and daylight. Absorption-bands
at the red end are best seen by lamp-light, and those at the blue end
by daylight.
[Illustration:
0 1 2 3 4 5 6 7 8 9 10 11 12
(Red end.) (Bue end.)
A B C D E _b_ F G
Fig. 200.--
]
On this scale the position of some of the principal lines of the solar
spectrum is about as follows:--
A 3/4
B 1-1/2
C 2-3/8
D 3-1/2
E 5-11/16
b 6-3/16
F 7-1/2
G 10-5/8
At first plates of selenite, which are easily prepared, were used,
because they can be split to nearly the requisite thickness with
parallel faces; but their depolarising power varied much with
temperature. Even the ordinary atmospheric changes alter the position
of the bands. However, quartz cut parallel to the principal axis of
the crystal is but slightly affected, and is not open to the same
objection; but this is prepared with some difficulty. The sides should
be perfectly parallel, the thickness about ·043-inch, and gradually
polished down with rouge until the sodium-line is seen in its proper
place. This must be done with care, since a difference of 1/10000-inch
in thickness would make it almost worthless.
The two Nicol’s prisms and the intervening plate are mounted in a tube,
and attached to a piece of brass in such a manner that the centre of
the aperture exactly corresponds to the centre of any of the cells used
in the experiments, and must be made to correspond with equal care, so
that any of them, or this apparatus in particular, may be placed on the
stage and in proper position without further adjustment, whereby both
time and trouble are saved.
Absorption Spectrum of Chromule.
In 1869 I published in the Journal of the Royal Microscopical
Society[38] a paper on results obtained by the spectrum analysis of
the colouring-matter of plants and flowers, some of which were of
considerable interest in many respects. My examinations extended to
several hundred different specimens, from which I was led to conclude
that the chromule of flowers is, for the most part, due to the chemical
action of the actinic rays of light over the protoplasm of the plant,
more so than to that of soil. But as certain roots of plants, as those
of the alkanet, yield their colouring-matter to oil, and in a much
smaller degree to spirit or water, it follows then that conclusions
of any kind can only be drawn after a long and careful study of the
question. Some of the results obtained were, however, of some interest
at the time, that, for example, seen in three different solutions of
the chlorophyll of _Cinchona succirubra_, one of three solutions in
alcohol, scarcely coloured, having in fact only a faint tinge of green
colour, and the spectrum of which much astonished me at the time. It
gave four well-marked absorption-bands; one deep sharp line _in the
red_; another, rather narrower, in the orange, coincident with D, or
the sodium-line; one in the green, about _b_, coincident with the
Thallium green band; and a fourth on the blue line F, nearly as broad
as that in the red. The ethereal solution gave different results. It
showed only three bands of absorption, nearly the same as in the last
case (though all of them fainter); but the fourth in the blue was not
apparent, the whole of that end of the spectrum being absorbed a little
beyond the green line _b_. This solution was _deep emerald-green_,
and even dilution did not alter the phenomena. The _acid_ alcoholic
solution was as deeply green as the last, but gave only the sharp broad
absorption-band in the red, and two very faint ghostly bands in the
position described above of the D and _b_ lines respectively.
Further additional researches on the chlorophyll of plants furnished
curious results, the chlorophyll being dissolved out by alcohol,
digested for some hours, and without heat; some plants being fresh,
and others dried. Five classes of phenomena exhibited themselves, but
_all_ agreed in having the red absorption-band broad, sharp, and well
defined, some having this one band only, the Lilac being of this type.
There are two classes in which two absorption-bands occur. One has the
red and the orange bands, of which the Fuchsia, Guelder-rose, and Tansy
are examples; another, in which the red and the green bands are alone
co-existent. Ivy is the type of the class, and it is immaterial whether
we take last year’s leaves or those of the early spring; the results
are the same.
The fourth class consists of the two former spectra superposed. Three
lines occur, the red, the orange, and the green bands, at C, D, and
_b_, as before. This is by far the largest class, and I have thirty or
forty examples of it. _Œnothera biennis_, Laurestinus, &c., are types
with the ethereal solution of the leaves of Red Bark.
The fifth class consists of those having properties similar to the
alcoholic solution of Red Bark described. But I only found eight of
these, and not all equal in colour power, namely: Berberry, Sloe,
Tea, Hyoscyamus, Digitalis, Senna, and Red Bark. The results obtained
appeared at the time to be well worth following up to a more practical
conclusion than that arrived at. It should be noted that in the
preparation of vegetable colouring matters for the micro-spectroscope,
care must be taken to employ only a small quantity of spirits of wine
to filter the solution, and evaporate it at once to dryness at a very
gentle heat, otherwise if we attempt to keep the colouring matters in
a fluid state they quickly decompose. It is necessary also to employ
various re-agents in developing characteristic spectra. The most
valuable re-agent is sulphite of soda. This admits of the division of
colours into groups.
It is better to use a dilute alcoholic solution for the extraction of
colour from plants, and to observe the spectrum in a column of about
three-quarters of an inch in height. By this means it is quite possible
to ascertain that the spectrum of chlorophyll presents seven distinct
absorption bands.
For further information on this interesting subject I must refer the
reader to Mr. Sorby’s paper “On a Definite Method of Qualitative
Analysis of Vegetable and Animal Colouring Matter by means of the
Spectrum Microscope,” “Proc. Roy. Soc.,” No. 92, 1867.
CHAPTER IV.
Practical Microscopy: Manipulation, and Mode of Using the Microscope.
In this chapter it will be my aim to discuss the best practical
methods of employing the microscope and its appliances to the greatest
advantage. First, the student should select a quiet room for working
in, with, if possible, a northern aspect, free from all tremor
occasioned by passing vehicles. The table selected for use should
be firm, and provided with drawers, in which his several appliances
can be kept ready to hand. The microscope must be placed at such an
inclination as will enable him to work in comfort, and without putting
strain on the muscles of the neck or fatiguing the eyes. The next
important point is that of light. Daylight, in some respects, is an
advantage; this should come from a white cloud on a bright day, but as
a rule more satisfactory results will be obtained by using a well-made
lamp, as this can be controlled with ease, and used at a proper height
and distance from the microscope. To have a good form of lamp is as
much to be desired for the student as for those engaged in the more
advanced work of microscopy.
Whatever the source of light we must on no account over-illuminate.
The object having been placed on the stage of the microscope, the
body should be racked down to within a quarter or half an inch of
the specimen, and then, while looking through the eye-piece, should
be slowly withdrawn until a sharp image comes into view. The fine
adjustment may now be used for the more delicate focussing of the
several parts of the field.
Accurate adjustment of focus is required when using a 1/4-inch
objective; details of the object, as striæ, being brought into view
when a stronger light is thrown obliquely upon them from the mirror.
If a 1-inch objective is used the light often proves to be in excess
of what is required, and this must be regulated by the aid of the
diaphragm.
The iris diaphragm, made to drop into the under-stage, is more
generally employed, as when racked up to the object it affords every
necessary graduation of illumination.
[Illustration: Fig. 201.--Bull’s-eye Lens.]
To illuminate opaque objects the light should be thrown upon them from
above by the bull’s-eye lens (Fig. 201). The focus of such a lens and
the lamp placed at four inches from it, is about three inches for
daylight, or two inches for artificial light. A large object may be
placed upon the stage of the microscope at once, but smaller objects
are either laid on a glass slide or held in the stage forceps.
When illuminating objects from above all light from the mirror, or
that which might enter the objective from below the stage, should be
carefully excluded. _Dark-field illumination_ is a means of seeing a
transparent object as an opaque one. The principle, however, is that
all the light shall be thrown from below the object, but so obliquely
that it cannot enter the object-glass unless interrupted by the object;
this is best accomplished by _Wenham’s Parabola_.
_Glass_ of any kind requires occasional cleaning; a piece of soft
washed chamois leather should be used for this purpose. The fronts of
the objectives may be carefully wiped, but not _unscrewed_ or tampered
with; a short thick-set camel’s hair brush may be passed down to
the back lens, and all dust removed without doing any harm. If the
objective is an _immersion_, carefully remove the fluid from the front
lens, as even distilled water will leave a stain behind. For removing
oil see special directions given at page 171.
When cleaning the _eye-pieces_, which should be done occasionally, the
cells containing the glasses must be unscrewed and replaced one at a
time, so that they may not be made to change places.
Any dirt upon the _eye-pieces_ may be detected by turning them round
whilst looking through the instrument; but if the _object-glasses_ are
not clean, or are injured, it will, for the most part, only be seen by
the object appearing misty.
The _object-glasses_, when in use but not on the microscope, should be
stood upon the table with the screw downwards, to prevent dust getting
into the lenses, and they should always be put into their brass cases
when done with. A large bell-glass shade will be found the most useful
cover for keeping dust from the instrument when not in use.
When looking through the eye-piece be sure to place the eye in close
proximation to the cap, otherwise the whole field will not be perfectly
visible; it should appear as an equally well-illuminated circular disc.
If the eyelashes are reflected from the eye-glass, the observer is
looking upon the eye-piece, and not through it.
_The Mirror._--The working focal distance of the mirror is that which
brings the images of the window-bars sharply out upon the object
resting upon the stage. In other words, the focus of the mirror is that
which brings parallel rays to a correct focus on the object-glass.
If employing artificial light, then the flame of the lamp should be
distinguishable; a slight change in the inclination of the mirror will
throw the image of the lamp-flame out of the field.
The strongest light is reflected from the concave side of the mirror,
that from the flat side is more diffuse and less intense. Oblique light
can be obtained by turning the mirror on one side and then adjusting
it so as to illuminate the field from that position. All the necessary
mechanism of the microscope is easily and quickly learned. The
object-glasses or objectives are, as previously explained, designated
according to the focal distance of a single lens of the same magnifying
power. Thus a 2-inch objective is understood to be a combination which
has the magnifying power of a single lens whose focal point is two
inches from the object, and so on with reference to other powers.
By the aid of different eye-pieces an extensive range of magnifying
power can be obtained; for example, the 2-inch objective with a deep
eye-piece will give the same amplification as the quarter objective
with the ordinary eye-piece. Indeed, for certain observations, the
combination of a wide-angled low-power objective, with a deep
eye-piece, or _compensating eye-piece_, is considered to have an
advantage.
It has been already explained that two objectives, one of much greater
power than the other, but both having only the same numerical aperture,
will show only the same amount of _detail_; the higher power on a
larger scale. That is, supposing with a 1/4-inch objective of 1·0
numerical aperture certain structure is resolved, then a 1/8-inch
substituted with exactly the same numerical aperture, but with double
the magnification, no more _resolving power_ will be found in the
latter objective than in the former. For this reason a doubt has been
expressed as to whether high-power objectives--especially the more
expensive oil-immersions, made to transmit large pencils of light
through their larger apertures--are so well adapted for ordinary
research as the best series of dry achromatic objectives, or even,
in some instances, the medium aperture lenses; undoubtedly, for
histological (physiological and pathological) work, the latter will be
found to meet the students’ requirements quite as well as the former.
The student or amateur will do well to commence with moderate or medium
powers, a 2-inch, a 1-inch, a 1/2-inch, a 4/10-inch, or 1/4-inch.
These, together with the A and B eye-pieces, will give a range of
magnification from 30 to 250 diameters.
_Penetration_ in the objective is a quality for consideration, as the
adjustment of high powers is a work of delicacy, and in some cases
their penetration is impaired by the arrangement made to obtain finer
definition. The value, however, of penetration in an objective is
always considered to be of more or less importance. It is a quality
whereby, under certain conditions, a more perfect insight into
structure is obtained. As a rule, the objective having the largest
working distance possesses the better penetration. Theoretically, the
penetration of an objective decreases as the square of the angular
aperture increases. For this reason the medical student will be
justified in choosing the objectives I have named, since these will
be better adapted to his work and pursuits. The penetration of the
objective is a relative quality assessed at a different value by
workers whose aims are widely different. But for the observation of
living organisms, the cyclosis within the cell of the closterium or
valisneria, for instance, preference will undoubtedly be in favour of
the objective with good penetration.
_Resolving Power._--This is a quality highly prized by the
bacteriologist. In the case of the high-angled apochromatic
oil-immersion, with its compensating eye-piece, its resolution is
found to be of very considerable advantage, because of its capacity
to receive and recombine all the diffraction spectra that lie beyond
the range of the older achromatic objective, with its smaller angular
aperture. The actual loss of resolving power consequent upon the
contraction of aperture from 180° to 128-1/2° is ten per cent., if
not more. Resolution depends, then, upon the quality and quantity
of the light admitted, the power of collecting the greatest number
of rays, and the perfection of centring. In other words, upon the
co-ordination of the illuminating system of the microscope--mirror,
achromatic condenser, objective and eye-piece. If diatoms are employed
as test-objects, it should not be forgotten that there are great
differences, even in the same species, in the distances their lines are
apart. For this reason ruled lines of known value, as Nobert’s lines,
are to be preferred. The following example will suffice to show the
value of a dry 1/8-inch objective of 120° in defining the rulings of a
19-band plate, which is equivalent to the 1/67000th of an inch. This
objective, with careful illumination, showed them all; but when cut
down by a diaphragm to 110°, the eighteenth line was not separable;
further cut down to 100° the seventeenth was the limit, to 80° the
fourteenth, and to 60° the tenth was barely reached.
_Flatness of Field._--This quality in the objective has, by the
introduction of the immersion system, lost much of the importance
formerly attached to it. Some writers assume it to be an “optical
impossibility.” The compensating eye-piece has had the effect of
contracting the visual field, consequently the peripheral imperfections
of the objective are of a less disturbing character. It has, however,
not been made perfectly clear whether the highest perfection of the two
primary qualities of a good objective, _defining power and resolving
power_, can be always obtained in one and the same combination of
lenses.
Doubtless, _defining_ power can be more satisfactorily determined by
the examination of a suitable object, and the perfection of the image
obtained; to assist in securing which, a solid axial cone of light
equal to about three-fourths of the aperture of the objective must be
employed.
To sum up, then, “the focal power of all objectives depends in their
perfect _definition_, a property on which their converging power
depends, and in turn their magnifying action is dependent; again, focal
power is the curvature imprinted by the lens on a plane wave, and is
reciprocal of the true focal length. It is appropriately expressed in
terms of the proper unit of focal curvature, the _dioptric_; a unit of
curvature.”[39]
[Illustration: Fig. 202.--Seiler’s Test Slide.]
It may be taken as an axiom with microscopists that “neither the
penetrating power nor the high-power defining objective is alone
sufficient for every kind of work. The larger the details of ultimate
structure, the narrower the aperture--and the converse; the minuter the
dimensions of elementary structure, the wider must be the aperture of
the objective.” Every worker with the microscope must have satisfied
himself of the truth of this statement, when engaged in the study of
the movements of living organisms, or defining the intimate structure
of the minuter diatoms, or of the podura scale.
_Test for Illumination._--Dr. C. Seiler recommends the human blood
corpuscle as the best test of good illumination. He prepares the object
in the following manner: Take for the purpose a clean glass slide of
the ordinary kind, and place near its extreme edge a drop of fresh
blood drawn by pricking the finger with a needle. Then take another
slide of the same size, with ground edges, and bring one end in contact
with the drop of blood, as shown in Fig. 202, at an angle of 45°; then
draw it evenly and quickly across the underslide, and the result will
be to spread out the corpuscles evenly throughout. Blood discs being
lenticular bodies, with depressed centres, act like so many little
glass-lenses, and show diffraction rings if the light is not properly
adjusted.[40]
_Errors of Interpretation._--To be in a position to draw an accurate
conclusion of the nature and properties of the object under
examination is a matter of great importance to the microscopist. The
viewing of objects by transmitted light is of quite an exceptional
character, rather calculated to mislead the judgment as well as the
eye. It requires, therefore, an unusual amount of care to avoid falling
into errors of interpretation. Among test objects the precise nature
of the structural elements of the Diatomaceæ have given rise to great
divergence of opinion. Then, again, the minute scales of the podura
Springtails, one of the Collembola, and their congeners _Lepisma
saccharina_, the structure of which is equally debatable. Mr. R. Beck,
in an instructive paper published in the “Transactions of the Royal
Microscopical Society,” says that the scales of the Lepisma can be made
to put on an appearance which bears little resemblance to their actual
structure.
[Illustration: Fig. 203.--Portions of Scales of Lepisma.]
In the more abundant kind of scales the prominent markings appear
as a series of double lines. These run parallel and at considerable
intervals from end to end of the scale, whilst other lines, generally
much fainter, radiate from the quill, and take the same direction as
the outline of the scale when near the fixed or quill end; but there
is, in addition, an interrupted appearance at the sides of the scale,
which is very different from the mere union, or “cross-hatchings,” of
the two sets of lines (Fig. 203, Nos. 1 and 2, the upper portions).
The scales themselves are formed of some truly transparent substance,
for water instantly and almost entirely obliterates their markings, but
they reappear unaltered as the moisture leaves them; therefore the fact
of their being visible at all, under any circumstances, is due to the
refraction of light by superficial irregularities, and the following
experiment establishes this fact, whilst it determines at the same
time the structure of each side of the scale, which it is otherwise
impossible to do from the appearance of the markings in their unaltered
state:--
“Remove some of the scales by pressing a clean and dry slide against
the body of the insect, and cover them with a piece of thin glass,
which may be prevented from moving by a little gum at each corner. No.
3 may then be taken as an exaggerated section of the various parts. A
B is the glass slide, with a scale, C, closely adherent to it, and D
the thin glass-cover. If a very small drop of water be placed at the
edge of the thin glass, it will run under by capillary attraction; but
when it reaches the scale, C, it will run first between it and the
glass slide, A B, because the attraction there will be greater, and
consequently the markings on that side of the scale which is in contact
with the slide will be obliterated, while those on the other side will,
for some time at least, remain unaltered: when such is the case, the
strongly marked vertical lines disappear, and the radiating ones become
continuous. (_See_ No. 1, the lower left-hand portion.) To try the same
experiment with the other, or inner surface of the scales, it is only
requisite to transfer them, by pressing the first piece of glass, by
which they were taken from the insect, upon another piece, and then
the same process as before may be repeated with the scales that have
adhered to the second slide, the radiating lines will now disappear,
and the vertical ones become continuous. (_See_ No. 2, left portion.)
These results, therefore, show that the interrupted appearance is
produced by two sets of uninterrupted lines on different surfaces, the
lines in each instance being caused by corrugations or folds on the
external surfaces of the scales. Nos. 1 and 2 are parts of a camera
lucida drawing of a scale which happened to have opposite surfaces
obliterated in different parts. No. 4 shows parts of a small scale in a
dry and natural state; at the upper part the interrupted appearance is
not much unlike that seen at the sides of the larger scales; but lower
down, where lines of equal strength cross nearly at right angles,
the lines are entirely lost in a series of dots, and exactly the same
appearance is shown in No. 5 to be produced by the two scales at a part
where they overlie each other, although each one separately shows only
parallel vertical lines.”
[Illustration:
Fig. 204.--Outer Membrane of Upper Plane of Red Beads thrown by each
alternate hole of grating; on lowering the focus white interspaces turn
into blue beads.]
[Illustration:
Fig. 204_a_.--Outer Membrane of Lower Plane of Beads thrown from
remaining holes of grating; on raising the focus white interspaces turn
into red beads.
Objective used, Zeiss’s apochromatic 1/12-inch oil-immersion, numerical
aperture 1·40, magnifying power 1,750 diameters.]
A well-known skilled observer of test objects[41] says: “Practically
the resolving power of our achromatic objectives on lined objects
reached their maximum in the late Dr. Woodward’s hands. _Amphipleura
pellucida_ was then, as now, the finest known regular structure
of the diatoms. There appeared then nothing more to be gained in
resolution when one of the apochromatic 1/12-inch objectives of
Zeiss, with its entire absence of colour, passed into my hands, and
I soon became convinced that it possessed the power of separating
the different layers of structure in the valve, beyond the grasp of
the dry-objective. The result of this increase of power enabled me
to split up, as it were, the one plate of silex forming the valve of
_Pleurosigma formosum_ into three layers, and which had never before
appeared to be possible; proving, in fact, that magnification without
corresponding aperture is of little or no account.”
“The intimate structure of these test objects,” says Mr. Smith, “is
built up on one plan, each being composed of two or more layers,
(1) a valve with two layers, as in _Pleurosigma balticum_; (2) two
layers with a grating and secondary markings placed diagonally, as in
_Pleurosigma formosum_; (3) with two layers of a net-like structure,
as in _Pleurosigma angulatum_, the fineness of the striæ or gratings
of which measure the 1/50000th of an inch. Five other diatoms
afford evidence of this compound structure. The presence of beads
or hemispheres in one of the focal planes, and depressions or pits
in another, are emphasised in the micro-photograph itself; reduced
portions of the valve are represented in Figs. 204 and 204_a_.”
A portion of a diatom valve, _Pleurosigma angulatum_,
micro-photographed on a higher scale of magnification, 4,500 diameters,
is given further on.
[Illustration: Fig. 205.--Sections of an old-fashioned Glass Tumbler,
from photographs by the late Mr. R. Beck.]
_Errors of interpretation_ arise either from the small cones of
illumination afforded by the dry-objective, or the oblique illumination
formerly resorted to for the resolution of these difficult test
objects, and several of the lights and shadows resulting from the
refractive power of the object itself. But the most common error is
that produced by the reversal of the lights and shadows resulting
from the refractive powers of the object itself. To make this
clear, I reproduce two reduced photographs of a small section of
an old-fashioned glass tumbler, covered externally with numerous
hemispheres, illuminated by transmitted light (Fig. 205).
This illustration well emphasises the difficulty there is in
determining structure under precisely similar conditions to those we
are accustomed to of examining valves of diatoms under the microscope.
If these photographs be held in front of a strong light, they at once
convey different impressions to the mind, the hemispheres appearing
depressions in the one, and raised beads in the other. Both are prints
from the same negative, but in mounting are reversed; and therefore the
apparent dissimilarity is due to a slight inequality of illumination,
which the mind accepts as light and shade.
Very similar appearances to those described will result if a thin plate
of glass were studded with minute, equal, and equi-distant plano-convex
lenses, the foci of which would very nearly lie in the same plane. If
the focal surface, or plane of vision, of the objective be made to
coincide with this plane, a series of bright points will result, from
the excess of light falling on each lens. If the plane of vision be
next made to coincide with the surfaces of the lenses, these points
would appear dark, in consequence of the rays being refracted towards
points _now_ out of focus. Lastly, if the plane of vision be made
to coincide with the plane _beneath_ the lenses that contain their
several foci, so that each lens may be, as it were, combined with the
object-glass, then a second series of bright points will result from
the accumulation of the rays transmitted at those points. Moreover,
as all rays capable of entering the objective are concerned in the
formation of the second series of bright focal points, the first series
being formed by the rays of a cone of light only, it is evident that
the circle of least confusion must be much less, and therefore the
bright points better defined in the first than in the last series.
There are no set of objects which have given rise to more discussion
as to their precise character than the scales of the podura
(_Lepidocyrtus cervicollis_), to the intimate structure of which Mr.
Smith turned his attention, and succeeded, I am inclined to think, in
his attempt to settle the structure of these very minute scales, and
which heretofore have been described as “notes of exclamation.” By
the aid of the same power as that employed in the examination of the
_pleurosigma formosum_, the old conventional markings have disappeared,
and well-defined “_featherlets_” have taken their place. By careful
focussing up and down, a series of whitish pin-like bodies is to be
seen, with an intervening secondary structure. A micro-photograph of
a portion of a scale taken by Mr. Smith shows that these pin-like
bodies are inserted in a fold of the basement membrane, which, in
his opinion, furnish unmistakable evidence of the fact that these
projecting bodies are real, and must no longer be looked upon as mere
_ghosts_. Quite recently, a micro-photograph of a portion of a podura
scale was placed in my hands, taken by Mr. J. W. Gifford with a Swift’s
1/12-inch apochromatic objective, of numerical aperture 1·40, and a
deep eye-piece, having a combined magnifying power of 3,827 diameters.
Fig. 206 shows a portion of the photograph which, it will be admitted,
supports Mr. Smith’s view of the structure of the podura scale.
[Illustration: Fig. 206.--Podura Scale, taken with a 1/12 Swift’s
Immersion × 3,827.]
Many other errors of interpretation are not unknown to the experienced
operator with the microscope, arising, for the most part, from an
influence exerted by peculiarities in the internal structure of certain
objects; for example, that offered by the human hair, and which, when
viewed by transmitted light, presents the appearance of a flattened-out
band, with a darkish centre, due to the refractive influence of the
rays of light transmitted through the hair. That it is a solid or
tubular structure is proved by making a transverse section of the
hair-shaft, when it is seen filled up by medullary matter, the centre
being somewhat darker than the outer part. It is, in fact, a spiral
outgrowth of the epithelial scales, overlapping each other, imparting
a striated appearance to the surface. A cylindrical thread of glass in
balsam appears as a flattened, band-like streak, of little brilliancy.
Another instance of fallacy arising from diversity in the refractive
power of the internal parts of an object is furnished by the mistakes
formerly made with regard to the true character of the _lacunæ_ and
_canaliculi_ of bone structure. These were long supposed to be solid
corpuscles, with radiating opaque filaments proceeding from a dense
centre; on the contrary, they are minute chambers, with diverging
passages--excavations in the solid osseous structure. That such is the
case is shown by the effects of Canada balsam, which infiltrates the
osseous substance.
Air bubbles are a perplexing source of trouble. The better way of
becoming accustomed to deceptive appearances of the kind is to compare
the aspect of globules of oil in water with bubbles of air in water, or
Canada balsam.
The molecular movements of finely divided particles, seen in nearly
all cases when certain objects are first suspended in water, or other
fluids, are a frequent cause of embarrassment to beginners. If a minute
portion of indigo or carmine be rubbed up with a little water, and a
drop placed on a glass slide under the microscope, it will at once
exhibit a peculiar _perpetual motion_ appearance. This movement was
first observed in the granular particles seen among pollen grains of
plants, known as _fovilla_, and which are set free when the pollen is
crushed. Important vital endowments were formerly attributed to these
particles, but Dr. Robert Brown showed that such granules were common
enough both in organic and inorganic substances, and were in no way
“indicative of life.”[42]
Professor Jevons succeeded in throwing light on these curious
movements. He showed that they were not due to evaporation, as
some observers contended, as they continue when all possibility of
evaporation is cut off, when the fluid is surrounded by a layer of oil,
and enclosed in an air-tight case: but as Professor Jevons pointed
out, these movements are greatly affected by the admixture of various
substances with water, being increased by a small quantity of gum,
and checked by a drop of sulphuric acid, or a few grains of some
saline substance, which increases the conducting power of water for
electricity. The Brownian movement, now termed _pedesis_, much depends
upon the size of the particles, their specific gravity, and the nature
of the liquid in which they are immersed.
The correct conclusions to be drawn by the microscopist regarding the
nature of an object will necessarily depend upon previous experience
in microscopic observations, a knowledge of the class of bodies
brought under observation, and the skill of the observer in the use
of the instrument--that is, in securing the best focus possible with
any objective brought into use. I am indebted to Messrs. Beck for the
following series of illustrations, showing the effect of under and over
correction of the objective.
DIRECTIONS FOR FINDING THE BEST FOCUS.
The method of finding and determining when the screw-collar adjustment
of the high-power objective has arrived at a point of perfect
definition and magnification is as follows:--
Select any dark speck of dust, or an opaque portion of the object,
and carefully focus this small particle by working the screw of the
fine adjustment, move the screw up and down until you are satisfied
the image is the sharpest and blackest that can be obtained, then once
more test the focus a little above and a little below while closely
scrutinising the effect on the image. It will now be seen that whereas
in focussing on one side of the best focus the object disappears in a
fog, by focussing on the other side it remains in view for a longer
period, but alters its appearance; it is now no longer a black dot, but
a bright dot of light surrounded by a black margin. The effects being
thus dissimilar on different sides of the best focus, show that the
objective is not perfectly adjusted for the cover-glass in use.
The next step is to find out whether the bright image is above or
below the best focus, as on this depends the direction in which the
adjustment-collar should be turned. To determine this it is only
necessary to ascertain which way the slow-motion milled head of the
microscope turns when moving the objective upwards.
In the case under consideration, the bright image will be _above_
the best focus, which shows that the cover-glass in use is _thicker_
than that for which the objective is adjusted, consequently the
adjustment-collar must be moved in the opposite direction.
[Illustration: Fig. 207.--Podura Scale Test.]
If the collar be turned too far in the opposite direction, it will
be found that the bright image is _below_ the best focus, and the
cover-glass is then _thinner_ than that for which the objective is
adjusted. The collar must then be turned back again _until the effect
on each side of the best focus is exactly similar_. This effect in the
case of a circular speck of dust will be that the object disappears
equally rapidly on either side, and does not instantly vanish into
fog, on either side presenting the bright spot appearance, though not
in so marked a degree on either side. When the object is in perfect
adjustment the expansion of the outline is exactly the same, both
within and without the focus.
A different indication, however, is afforded by such test-objects as
the finer diatoms, and the podura scale, in which we have to do with
a set of distinct dots and other markings. If the dots have a tendency
to run into lines when the object is _without_ the focus, the glasses
should be brought closer together; on the contrary, if the lines appear
when the object is _within_ the focal point, the glasses should be
farther separated.
The adjustment of the objective by the screw-collar in the case of
the podura scale should be carried out in the way described, when the
following effects will be observed to take place, usually in the order
of their arrangement.
Fig. 1 shows the appearance of a podura scale when the adjustment of
the object-glass is correct, and Fig. 2 shows the effect produced
on each side of the exact focus. Fig. 3 shows the way in which the
markings individually divide when all the adjustments are correct, and
when the focus is altered the least possible amount only each way.
Figs. 4 and 5 show the two appearances on one and the other side of
the best focus when the adjustment is incorrect, Fig. 6 showing the
appearance of the same at its best focus.
_The scales_ are magnified 1,300 diameters, and each square measures
·001 of an inch.
This method, however, of finding the best focus of an objective
can scarcely be accomplished without a sub-stage condenser. It may
therefore be of service to the student, and to those who are not
disposed to purchase expensive forms of condensers, to know that either
an inch or an inch and a half objective, or convex-lens mounted on a
simple wooden ring with a flange, can be arranged to slip in the place
of the diaphragm under the stage. This kind of condenser will prove to
be of considerable value with a 1/2-inch, a 4/10-inch, and a 1/4-inch;
while a still more excellent achromatic condenser can be made out
of a Steinheil’s _aplanatic-loup_ arranged to drop into the central
fitting of the sub-stage. As without a condenser of some kind it is
hardly possible to enter upon any course of histological or scientific
research.[43]
Working Accessories.
TROUGHS--LIVE-CAGES--COMPRESSORS.
_A glass plate_ with a ledge, and some pieces of _thin glass_, although
applicable for many purposes, are specially designed for objects in
fluid. Thus a drop of fluid containing the object sought for is placed
upon the slide and covered by a piece of thin glass; or, the object
being put upon the glass slide and the thin glass over it, the fluid is
applied near one side, and runs under by capillary attraction.
[Illustration: Fig. 208.--Varley’s Live-box.]
_Troughs and Live-box._--These are made of various materials, glass,
vulcanite, brass, &c., expressly for examining infusoria and live
animals. They should be so constructed as to admit of the use of a
medium power, a 1/2-inch at least, under the microscope. They should
also admit of being easily cleaned and repaired when broken; matters
rarely thought of by those who construct them. An early devised
_live-box_ (Varley’s, Fig. 208) consists of two circular pieces of
brass tubing, one sliding over the other carrying a disc of glass and
fitting over another glass with bevelled edges to prevent the fluid
flowing away.
[Illustration: Fig. 209.--Ross’s Compressorium.]
_The Compressorium_ is used for similar purposes. By a graduated
pressure the fluid is _thinned out_ and a higher power can be employed
for the examination of the object. Ross’s early compressorium consists
of a plate of brass about three inches long, having in its centre a
circle of glass like the bottom of the live-box. This piece of glass is
set in a frame, _B_, which slides in and out so that it can be removed
for the convenience of preparing any object upon it--under water if
desirable. The upper movable part, _D_, is attached to a screw-motion
at _C_; and at one end of the brass plate, _A_, which forms the bed of
the instrument, is an upright piece of brass grooved so as to receive
a vertical plate, to which a downward motion is given by a single fine
screw, surrounded by a spiral spring, which elevates the plate as soon
as the screw-pressure is removed.
[Illustration: Fig. 210.--Beck’s Parallel-plate Compressor.]
_Beck’s Parallel-plate Compressor_ (Fig. 210) affords a more exact
means of regulating the pressure, and can be used for a variety of
purposes. It is also easily cleaned.
[Illustration: Fig. 211.--Rousselet’s Compressorium.]
_Rousselet’s Compressorium_ (Fig. 211) is a very effective form for
general use. It is so arranged that the student has perfect control
over the pressure to which the specimen should be subjected. The
cover-glass is large in comparison with that beneath; being bevelled
causes evaporation to go on very slowly while the pressure between the
two glass surfaces is kept perfectly parallel.
[Illustration: Fig. 212.--Botterill’s Live-trough.]
_Botterill’s Live-trough_ (Fig. 212) consists of two brass plates
screwed together by binding screws, and holding between them two plates
of thin glass, which are maintained at a proper distance by inserting a
semicircular flat disc of india-rubber.
[Illustration: Fig. 213.--Glass Trough.]
Glass troughs for chara and polypes (a sectional view of one shown at
Fig. 213) are made of three pieces of glass, the bottom being a thick
strip, and the front (_a_) of thinner glass than the back (_b_); the
whole is cemented together with Jeffery’s marine-glue. The method
adopted for confining objects near the front glass varies according to
circumstances. The most convenient is to place in the trough a piece of
glass wide enough to stand across diagonally, as at _c_; then, if the
object be heavier than water, it will sink until stopped by the glass
plate. At other times, when used to view chara, the diagonal plate may
be made to press it close to the front by means of a wedge of glass or
cork. When using the trough the microscope should be placed in a nearly
horizontal position.
[Illustration: Fig. 214.--Weber’s Slip with Convex Cell for use as a
Live-trough.]
[Illustration: Fig. 215.--Current-slide Live-cell.]
Cells for viewing living objects, and watching their movements, take
many forms, usually determined by the makers for the purposes they
are required to serve. The smaller glass troughs (Figs. 216, 216_a_)
are made for examining the small infusoria, rotifers, &c., some of
which take special forms, as the double or divided trough (Fig. 217)
intended for viewing the circulation of the blood in the tail of a
small fish, and at the same time keep up a supply of water and air.
[Illustration: Fig. 216.]
[Illustration: Fig. 216_a_.]
The Frog-plate consists of a strip of plate-glass, or wood, pierced
with holes on either side, through which tapes are passed to secure
the frog in its place. At the extreme end is a shallow glass trough,
made to hold a sufficient quantity of water to keep the web of the foot
moist while under examination. In this way a continuous view of the
circulation of the blood of the animal is obtained.
[Illustration: Fig. 217.]
_Growing Cells_ have received more attention from those who devote
attention to the lower forms of life, the construction of which, for
the purpose of maintaining a continuous supply of fresh water to
objects under observation, and for sustaining their vital energy for
a long period, is of some importance. The employment of live-cells
is resorted to by microscopists, as doubtless there is much to be
discovered concerning the metamorphoses which some of the lower
micro-organisms, both of plant and animal life, pass through.
[Illustration: Fig. 218.--Frog-plate.]
Holman’s life slide consists of a 3 × 1 inch glass slide, with a deep
oval cavity in the middle to receive the specimen for observation. A
shallow oval is ground and polished around the deep cavity, forming a
bevel. From this bevel a fine cut extends, to furnish fresh air to the
living low forms of life which invariably seek the bevelled edge of the
cavity, thus bringing them within reach of the highest powers. He also
contrived a convenient form of “moist chamber,” or animalcule-cage
(Fig. 220), for the purpose of studying the growth of minute organisms,
without in any way disturbing them for a lengthened period. This is
also found useful as a dry chamber for holding minute insects.
[Illustration: Fig. 219.--Holman’s Life Slide. Full size.]
[Illustration: Fig. 220.--Holman’s Moist Chamber.]
_Zentmayer’s Holman Syphon Slide_ is used either as a hot or cold
water cell. It should be deep enough to hold a small fish or newt, and
retain it without any undue pressure. When in use it is only necessary
to place the animal into it (as shown in Fig. 221), with some water,
and secure it with a glass cover; then immerse the upper tube in a jar
of water, while another, at a lower level, maintains a current. When
the slide is on the stage of the microscope, one jar should stand on a
lower level than the other, the slide being made the highest part of
the syphon. The pressure of the atmosphere is sufficient to keep the
cover-glass in its place.
The examination of the various kinds of infusorial life--rotifers,
for instance--is facilitated by the addition of the smallest particle
of colouring matter, either carmine or indigo. A small quantity of
either of these colours should be rubbed up in a little water in a
watch-glass, and a portion taken up on the point of a brush, and the
brush run along the edge of the cover-glass; sufficient will be left
behind to barely tinge the water with the colour, and this gradually
distributes itself over the rotifers. Under the microscope this minute
quantity will be seen like a rising cloud of dust, and as it approaches
a rotifer it is whirled round in different curves, showing at once the
action of its wonderfully rapid cilia. This colouring matter appears to
be devoured, as it may be traced from the mouth to the digestive canal.
Monads may be detected by this means, and the smaller forms of _algæ_,
_Euglena viridis_ and _Protococcus pluvialis_.
[Illustration: Fig. 221.--Holman’s Syphon Slide.]
_Dipping-tubes._--In dealing with infusorial or monad life it is
convenient to keep a stock-bottle ready for their reception, and in
a light favourable to health. When a live specimen is required for
examination, the dipping-tube is brought into requisition. These tubes
are open at both ends, and vary in length and diameter. Their ends
should be nicely rounded off in the flame of a blow-pipe; in form
either straight, or bent and drawn out to a fine point, as represented
in Fig. 222. When any special specimen is required for examination,
then one of the tubes must be passed down into the water, the upper
orifice having been previously closed by the forefinger, and kept
tightly pressed, until its lower orifice comes in contact with the
object. On the finger being removed, the water rushes up and carries
the creature sought for with it. The finger is once more replaced at
the top of the tube; it is then lifted out, and the contents deposited
in one or other of the glass cells described. Tubes with india-rubber
covers can be had.
[Illustration: Fig. 222.--Dipping-tubes.]
[Illustration: Fig. 223.--Stock-bottle.]
_Moist and Warm Stages._--In addition to the moist cells and chambers
described it is often found necessary in working out the histories of
minute organisms to keep them for some time under observation, and
as far as possible in an undisturbed condition, and it is equally
necessary to prevent evaporation of the water in which they are
immersed. One of the best warm stages is that known as Maddox’s growing
stage; this can be had of any optician. More elaborate adaptions are
required for the study of special organisms, and for experimental
research.
[Illustration: Fig. 224.--Bartley’s Warm Stage.]
In that case _Bartley’s Warm Stage_ (Fig. 224) is recommended. There
are other forms of warm stages in use, many of an inexpensive kind
and readily adaptable to any stage. Bartley’s has proved useful; it
consists of a vessel, _E_, three parts filled with water and supported
on a ring stand. This may be kept at any temperature by the small
spirit-lamp, _C_; a syphon tube _d_ conveys the warm water along
_f_, and through the bent tubing which surrounds the object under
observation on the stage, _D_, and then passes off through the open
end, _C_, into the receptacle, _B_, placed to receive the overflow.
Steam can be used for heating, or iced water for observing the effects
of cold upon the organism.
A simple form of warm stage may be made of an oblong copper plate,
two inches long by one wide, from one side of which a rod of the
same material projects. The plate has a round aperture, the centre
half an inch in diameter, and is fastened to an ordinary slide
with sealing-wax. The drop or object to be examined is placed on a
large-sized cover-glass and covered over with a smaller one. Olive oil
or vaseline is painted round the edge of the smaller one to prevent
evaporation, and the preparation is placed over the aperture in the
plate. The slide bearing the copper plate is clamped to the stage
of the microscope. The flame of the spirit-lamp is applied to the
extremity of the rod, and the heat is conducted to the plate and
thence transmitted to the specimen. In order that the temperature of
the copper plate may be approximately that of the body, the lamp is so
adjusted that a fragment of cacao butter and wax placed close to the
preparation is melted.
Professors Stricker and Schäfer have constructed warm stages for
accurate observations, and which fully answer every purpose.
[Illustration: Fig. 225.--Stricker’s Warm Stage.]
_Stricker’s Stage_ (Fig. 225) consists of a rectangular box with a
central opening, _C_, permitting the passage of light through the
specimen under examination. The water makes its exit and entrance
at the side tubes _B B′_, and the temperature is indicated by a
thermometer in front. In this apparatus either warm or cold water can
be continuously used.
[Illustration: Fig. 226.--Schäfer’s Warm Stage.]
_Schäfer’s apparatus_ (Fig. 226) consists of a vessel filled with water
(seen near the stage) which has been first boiled to expel the air,
and then heated by means of a gas flame. The warm water ascends the
india-rubber tubing to the brass box on the stage. The box is pierced
by a tubular aperture to admit light to the object, and has an exit
tube by which the cooled water from the stage returns by another piece
of tubing to be reheated by the gas flame. There is a gas-regulator, by
means of which any temperature can be maintained.
Methods of Preparing, Hardening, Staining and Section Cutting.
Numerous methods are employed for the preparation, hardening, staining,
and section cutting of animal and vegetable tissues for the microscope,
the details of which are modified, or varied as may be found needful,
from time to time, by those whose intimate acquaintance with the
subject entitles them to make innovations and changes in this very
important department of microscopy. In the hands of the original
worker, formulæ and methods will only be regarded as finger-posts
pointing out a means of saving time in turning over pages to find this
or that special method of staining. For this particular reason I have
collected all the most accredited formulæ together in an Appendix
at the end of the book, and arranged them alphabetically for ready
reference.
As to section cutting, the student will do well to practise himself
in making dissections, thick and thin sections, of vegetable and
animal substances. The medical student will require no advice on this
point, as the use of the scalpel, and those instruments needed for
microscopical work, form an important part of his education. Of all
the instruments contrived for delicate dissections, none are more
serviceable than those which the student may make for himself out of
ordinary needles. These may be fixed in handles as represented in Fig.
229, in addition to which, a pair of scissors and forceps, and a few
small knives, such as those used in eye-operations, will prove most
suitable. The double-bladed scissors represented in Fig. 227, with
curved blades, are brought into use for cutting vegetable and other
soft structures, the disadvantage attendant upon the use of which is
owing to the curvature of the blades; when dealing with flat surfaces,
the middle of the section is left too thick to exhibit structure.
The double-bladed knife of Professor Valentin was formerly held in high
estimation by the microscopist, but this has been almost superseded
by the microtome, which has taken the place of all other instruments,
since by its aid uniform series of nearly all substances can be cut.
The standard unit of a perfect section cutter, of any kind, has been
fixed by the Royal Microscopical Society at the one-thousandth of a
millimetre.
[Illustration: Fig. 227.--Section Scissors and Forceps.]
The use of the razor for cutting sections has not been wholly
abandoned, the method of using which is as follows:--Take the tissue
between the thumb and finger of the left hand, hold the finger
horizontally, so that its upper surface may form a rest for the razor
to glide upon, take the razor firmly, and keep the handle in a line
with the blade, then draw it through the tissue from heel to point and
towards yourself. While cutting keep the razor well wetted with diluted
methylated spirit.
[Illustration: Fig. 228.--Dissecting Knives.]
Some preparation is required for cutting sections with the single
microtome. The substance to be cut must be embedded in some other
material, as carrot, turnip, potato, alder pith, paraffin, or thick
gum, with either of which the cylinder or well of the microtome must
be so nearly filled as to leave only an excavation in the centre for
the specimen to be operated upon to occupy. The various forms of
microtomes in use, and the selection of the most suitable, is therefore
a matter of some difficulty. I must content myself by particularising
two or three typical forms in general use. As all the substances
intended for cutting require preparation, it will be first necessary to
attend to the following directions given by one experienced in section
cutting, Mr. M. J. Cole[44]:--(1) Always use fresh tissues. (2) Cut the
organs into small pieces with a sharp knife. (3) Never wash a specimen
in water; when it is necessary to remove any matter, allow some weak
salt solution to flow over the surface of the tissue, or wash it in
some hardening re-agent. (4) All specimens should be hardened in a
large quantity of the re-agent; too many pieces should not be put into
the same bottle, and keep them in a cool place. (5) In all cases the
hardening process must be completed in spirits. (6) Label the bottles,
stating the contents, the hardening fluid used, and when changed.
Attention to details is necessary, as if hardening is neglected, good
sections cannot be made.
_Embedding in Paraffin Wax or Lard._--Melt together, by the aid of
gentle heat, four parts of solid paraffin and one part of lard. A
quantity of this may be made and kept ready for use. Melt the paraffin
mass over a water bath, take the specimen, and dry it between the folds
of a cloth to remove the spirit, so that the paraffin may adhere to its
surface, place it in a small chip-box, in the desired position, and
pour in enough melted paraffin to cover it, then set aside to solidify;
when quite cold break away the box, and cut sections from the embedded
mass with a sharp razor.
To infiltrate a tissue with paraffin, place the specimen in absolute
alcohol or chloroform for an hour or two, then transfer to a bath of
melted paraffin, at its melting point (about 110° F.), and keep it at
this temperature for several hours, so that the paraffin may penetrate
to the middle of the tissue. Then remove the specimen from the paraffin
and put it into a small chip-box, pour in enough paraffin to cover it,
and set aside to cool. When quite cold, make sections as before, with a
razor, or fix it into a microtome, with a little melted paraffin. The
sections when cut must be placed in turpentine to remove the paraffin,
and then into absolute alcohol to remove the turpentine, and finally
in distilled water to remove the alcohol, when they may be forthwith
stained. It is often found better to stain the tissue in bulk before
embedding. In this case the sections will only require the turpentine
to dissolve away the paraffin, and may then be mounted in Canada balsam.
_Hardening and Preparing Animal Tissues_ for section cutting and
microscopical examination.--Fresh tissues are not well suited for
microscopical examination, but it is sometimes advisable to observe
the appearances of a fresh specimen, especially if it is suspected
to contain amaloid bodies or parasites. It will then be necessary to
_tease_ out a small portion of the tissue immersed in a weak solution
of salt and water by the aid of a pair of fine needles (Fig. 229) and
the dissecting microscope (Fig. 230).
[Illustration: Fig. 229.--Needles for teasing out Sections.]
[Illustration: Fig. 230.--Dissecting Microscope.]
The most important point in connection with an instrument of this kind
is, that it affords firm and convenient rests for the hands, and should
not be raised too high from the table.
The stage should either be made of glass, or provided with a glass dish
for dissecting under water, or preservative fluid. A pair of aplanatic
lenses, mounted on a focussing bar as shown in Fig. 230, will be found
the most convenient to work with.
Investigations of this nature should be always carried out in the
manner described, but preparations of the kind cannot be preserved
any length of time, unless properly hardened in spirit or Formalin
solution. The method of teasing out under the light of a condensing
lens is shown in Fig. 231.
[Illustration: Fig. 231.--Method of teasing out Muscular Fibre, &c., in
a fluid medium under Condensed Light.]
It may be as well to state at the outset that physiological and
pathological tissues can be hardened by immersion in methylated spirit
alone, or a saturated solution of picric acid in methylated spirit in
about a week, and it is said to yield satisfactory results, even some
of the tissues being ready in twenty-four hours. The only drawback is
that sections thus quickly hardened must be stained with picro-carmine.
But, whatever method of hardening adopted, the tissue should be washed
by means of a stream of water for half an hour, to remove all traces of
the hardening agent, and on its removal pressed between folds of cotton
cloth or fine Swedish filtering paper.
The principal hardening re-agents usually kept in bulk ready for use
are the following:--
_Absolute Alcohol._--This is suitable for the internal organs of
animals, glands, &c. These organs must be perfectly fresh, and should
be cut into small pieces, so that the alcohol may penetrate them as
quickly as possible. The hardening is usually complete in a short
time.[45]
_Chromic Acid and Spirit._--Chromic acid one-sixth per cent., water
solution two parts, and methylated spirit one part. This reagent
hardens in about ten days. Then transfer to methylated spirit, which
should be changed every day until all colour is discharged from the
tissue. This is a suitable reagent for the preparation of cartilage,
nerve trunks, heart, lips, blood vessels, trachea, lungs, tongue,
intestines, and gullet.
_Potassium Bichromate._---Make a two per cent. water solution of this
salt. This will harden specimens in about three weeks. Then transfer
the preparation to methylated spirit, and change it every day until
all colour is discharged. This is suitable for spinal cord, medulla,
cerebellum, and cerebrum.
_Müller’s Fluid._--Bichromate of potash 30 grains, sulphate of soda 15
grains, distilled water 3-1/2 ounces. This hardens in from three to six
weeks. Then transfer, as before, to methylated spirits, and change it
every day until colour ceases to appear. Most suitable for lymphatic
glands, eye-ball and its internal structures, as well as for tendons,
and thymus gland.
_Methylated Spirit_ may be generally employed, but it has a tendency to
shrink some tissues too much; it hardens in about ten days. It is usual
to change the spirit daily, for the first three days at least. Skin,
mammary gland, supra-renal glands, tonsils, and all injected organs may
be hardened in it. (See note on the adulteration of methylated spirit
with rack-oil, which utterly spoils it for use.)
_Decalcifying solution_ for bones and teeth. Take one-sixth per cent.
watery solution of chromic acid, and to every measured ounce add five
drops of nitric acid. This reagent will soften the femur of any small
animal in about three weeks; larger require a longer time. Change the
fluid several times, and test its action by running a needle through
the thickest part of the bone. Should it not pass through easily,
then continue the process until it does. When soft enough transfer to
water, let it soak for an hour or two, then pour off the water and
add ten per cent. solution of carbonate of soda, and soak for twelve
hours to remove all trace of acid. Wash again in water, and transfer to
methylated spirit until required. Teeth require a large quantity of the
decalcifying solution for softening.
_Microtomes._--The simplest form of “hand-cutting machine” is that
worked by a screw, which raises the preparation, and at the same time
regulates the fineness of the section. When a number of sections are
required, or when a complete series of sections of an organ is desired,
Cole’s simple microtome (Fig. 233) is in every way adapted.
[Illustration: Fig. 232.--Hand Section Cutter.]
[Illustration: Fig. 233.--Cole’s Section Cutting Microtome.]
_The method of using it_ is as follows:--Screw the microtome firmly
to the table, and with the brass tube supplied with the microtome,
punch out a cylinder of carrot to fit into the well. Cut this in half
longitudinally, and scrape out enough space in one half of the carrot
to take the specimen; then place the other half of carrot in position,
and make sure that the specimen is held firmly between them, but it
must not be crushed. Now put the cylinder of carrot and specimen into
the well of the microtome and commence cutting the section. A good
razor will do, but it is better to use the knife which Messrs. Watson
supply with the microtome. While cutting keep the knife and plate
of the microtome well wetted with dilute methylated spirit, and as
sections are cut place them in a saucer of dilute spirit. A number of
sections may be cut and preserved in methylated spirit until required
for examination or mounting.
When a specimen has a very irregular outline, it cannot be very
successfully embedded in carrot; paraffin will then be found to be more
suitable. Place the tissue in the well of the microtome in the proper
position, pour in enough melted paraffin to cover it, and put it by to
get cold and hard before attempting to cut sections.
[Illustration: Fig. 234.--The Cambridge Rocking Microtome.]
_Cambridge Rocking Microtome._--This new pattern Cambridge Rocking
Microtome (Fig. 234) possesses advantages over other instruments in use
for cutting flat sections, and not parts of a cylindrical surface. The
tube containing the paraffin is 30 millimetres in internal diameter
instead of 20 millimetres, as in the earlier forms. The forward
movement is also increased, so that an object 12 millimetres long can
be cut throughout its whole length. It is provided with a dividing
arc for reading off the thickness of the section in thousandths of a
millimetre. The razor may be fixed either with its edge at right angles
to the direction of motion of the object, or diagonally, for giving a
slicing cut. The object can also be raised and fixed in position clear
of the razor.
This microtome has both steadiness and stiffness in its geometrical
arrangement and bearings, while the simplicity and efficiency of its
mechanism for advancing the section between each stroke of the razor is
remarkable. Although it may appear more complicated at first sight, it
is found not to be so when brought into use.
[Illustration: Fig. 235.--Cathcart’s Microtome.]
[Illustration: Fig. 235_a_.--Section Cutting Holder for Microtome.]
_Cathcart’s Freezing Microtome._--This is a convenient and useful
microtome for freezing purposes. Since its first introduction it has
been much improved. The clamping arrangements give steadiness, and the
principal screw is more effective; the freezing-plate is circular,
and the arrangements made for preventing the ether from reaching the
upper plate secures the object in view. This instrument can now be used
for embedding as well as freezing. The directions for freezing are as
follows:--
1. Place a few drops of mucilage (one part gum to three parts water) on
the zinc plate.
2. Take a piece of the tissue to be cut, of about a quarter of an inch
in thickness, and press it into the gum.
3. Fill the ether bottle with anhydrous methylated ether, and push the
spray points into their socket. All spirit must of course have been
previously removed by soaking for a night in water. The tissue should
afterwards be soaked in gum for a like time before being cut.
Work the spray bellows briskly until the gum begins to freeze; after
this work more gently. Be always careful to brush off the frozen vapour
which, in a moist atmosphere, may collect below the zinc plate. If the
ether should tend to collect in drops below the plate, work the bellows
slower.
5. Raise the tissue by turning the milled head, and cut by sliding the
knife along the glass plates.
6. After use, be careful to wipe the whole instrument clean.
7. Should the ether point become choked, clear by means of the fine
wire provided for the purpose.
8. The instrument is intended for use with methylated sulphuric ether.
9. In clamping the instrument to a table, or other support, care should
be taken that the zinc plate is in a horizontal position. If the plate
be not horizontal, the gum will tend to run to one side.
The arrangement made for cutting and embedding sections consists of a
cylindrical tube (Fig. 235_a_) fitting into the principal well of the
microtome, within which is a hinged plate, upon which the screw acts,
as in an ordinary vice. To bring this into use the freezing apparatus
must be first removed, and the embedding tube placed in the well, and
firmly pressed into place.
Staining Animal Structures.
Specific stains are chiefly employed to assist the eye in
distinguishing one elementary tissue from another. It is therefore
necessary to stain all structures, as certain parts are seen to have
a special affinity for one colouring agent rather than another,
whereby they become more deeply stained, and consequently more clearly
differentiated. For staining animal structures, borax, carmine, and
hæmatoxylin are more frequently employed than others. The formulæ for
each will be found in the Appendix “Formulæ and Methods.”
_Staining Process._--Place the section in distilled water to wash
away the alcohol; place a little of the carmine in a watch glass, and
immerse the section in it for four or five minutes; then remove it
to a solution composed of methylated spirit five parts, hydrochloric
acid one part; shake well together. This solution should be kept ready
for use. Immerse the section in this solution and leave it to soak
for about five or ten minutes if over-stained, until the desired tint
has been obtained. Sections of skin and fibrous tissue may be left
until nearly all colour is removed, the glands and hair follicles will
then be brought out more clearly. The section must be transferred to
methylated spirit to remove all traces of acid, then to oil of cloves
contained in a watch glass, lift the section from the methylated spirit
by one of the _lifters_ (Fig. 250), and carefully float it on the
oil, in which it should be allowed to remain for about five minutes.
This is the clearing process, the object of which is to remove the
spirit and prepare the section for mounting in Canada balsam. First,
however, place the section in filtered turpentine to wash away the oil
of cloves; this is found to answer better than another plan adopted,
that of removing the section from the oil of cloves and mounting it in
balsam direct. The oil, however, has a tendency to darken the balsam.
_Logwood or Hæmatoxylin Stains_ (see Appendix for the several formulæ).
Staining by this agent is effected as follows:--
After the specimen has been hardened in any of the chromic acid
solutions in use, transfer it to a seven per cent. watery solution of
bicarbonate of soda for about five minutes, then wash well in distilled
water. Spirit prepared preparations do not require to be transferred
to the soda solution, but all sections must be washed before they are
transferred to the logwood stain. To a watch glass nearly full of
distilled water add ten or twenty drops of the logwood stain, in which
it should remain for twenty or thirty minutes. Wash well with the
ordinary tap water, which will fix the dye and cause it to become blue.
Dehydrate in methylated spirit, clear in clove oil, and mount in dammar
or Canada balsam.
_Double-staining with Hæmatoxylin and Rosin._--Stain the section as
directed above, then place it in an alcoholic solution of rosin, about
one gramme of rosin to an ounce of methylated spirit, and let it soak
for a few minutes; wash well in methylated spirit, clear in oil of
cloves, and mount in balsam.
_Canada balsam_ should be prepared for use as follows:--One ounce of
dried balsam to one fluid ounce of pure benzole; dissolve, and keep
in an _outside_ stoppered bottle. Clear the section in clove oil, and
place it in turpentine, clean a cover-glass and slide, place a few
drops of balsam on the centre of the latter, take the section from the
turpentine on a _lifter_, allow the excess of turpentine to drain away,
and with a needle-point lift the section on to the balsam slide. Now
take up the cover-glass with a pair of forceps (Fig. 236), and bring
its edge in contact with the balsam, ease it down carefully as shown
in Fig. 237, so that no air bubbles are enclosed, and with the needle
point press the surface of the cover until the section lies quite
smoothly and flat, and the excess of balsam is pressed out. The slide
should now be transferred to the _warm-chamber_, and there allowed to
remain for a day or two, or until set and hardened.
[Illustration: Fig. 236.--Forceps for Mounting.]
Any exuded balsam may be washed away with benzole and a soft camel’s
hair brush; then dry the slide with an old piece of linen cloth, and
apply a ring of cement or Japanner’s gold size. Other methods for
staining and mounting will be found to answer quite as well--that of
Beneke’s is a useful one for staining connective tissue.
[Illustration: Fig. 237.--Mode of placing Glass Cover on Object.]
For staining connective tissue a modification of Weigert’s method of
staining fibrine is resorted to. Portions of tissue that have been
fixed in alcohol having been embedded in paraffin and cut, the sections
are detached and placed on glass slides, and stained for ten or twenty
minutes with gentian violet, ten parts, well shaken with water 100
parts; filter, and add five to ten parts of a concentrated alcoholic
gentian violet solution. Afterwards treat for one minute with lugol
solution, of a port wine tint, dry with filter paper and decolourise
with aniline xylol (aniline oil two parts and xylol three parts).
Decolourisation having been stopped at the right point (judged from
experience) mount the sections in xylol balsam. The fibres of the
connective tissue should appear stained of various shades of violet.
_Double Staining_ nucleated blood corpuscles. Two kinds of staining
agents are required. Stain A: dissolve five grammes of rosin in half an
ounce of distilled water, and add half an ounce of rectified alcohol.
Stain B: dissolve five grammes of methyl green in an ounce of distilled
water. Place a drop of frog’s blood on a glass slide, and with the edge
of another slide spread it evenly over the centre of the slip, and put
it away to dry; when quite dry flood the slide with stain A for three
minutes, and wash with water, now flood the slide with Stain B for five
minutes, wash again with water, and allow the slide to dry. Apply a
drop of the prepared Canada balsam and a cover-glass.
[Illustration: Fig. 238.--Shadbolt’s Turn-table.]
The blood of such mammals as are non-nucleated should be treated in a
slightly different way. Spread a drop or two of blood on a slide and
dry it quickly; then put the slide on Shadbolt’s turn-table (Fig. 238)
and run a ring of cement around it; allow this time to dry, and then
apply a second coating, and before this becomes quite dry place on it
a clean glass cover, and press it down gently with one of the fine
needles (Fig. 229), until firmly adherent.
_Epithelium_.--Remove from the mouth of a frog by scraping some
_squamous_ epithelium; the columnar must be taken from the stomach;
place it in glycerine, or Farrant’s solution on the slide; apply a
cover-glass, and with the point of the needle press it down until the
epithelium cells are separated and spread evenly over the slide. Set
this aside for a day or two, then wash away any of the medium which
may have escaped; dry the slide, and run a ring of cement around the
edges, on the turn-table. Portions of the intestine of a rabbit or
other animal may be treated in the same way. If it is wished to make
permanent specimens of such structures, the intestine must be hardened
in a two per cent. solution of bichromate of potash for a couple of
days, then washed until all colour is discharged, and removed to a
solution of picro-carmine for twenty-four hours, after which allow the
stain to drain away, when it will be ready for mounting.
By the aid of the handy little spring clip (Fig. 239), objects of
delicacy when mounted may be left to dry and harden for any length of
time.
[Illustration: Fig. 239.--Spring-clip for Mounting.]
_Striped muscular fibre_, taken from the pig, must be teased out in
a two per cent. solution of bichromate of potash, in which it should
remain for two or three weeks, when it may be transferred to methylated
spirit, and allowed to remain until required for mounting. Soak a piece
in water to remove the spirit, place a small fragment on a slide in a
few drops of water, and with a couple of needles tease the tissue up,
so as to separate the fibres. Drain away the water, and apply a drop or
two of Farrant’s medium and a cover-glass, which cement down as before
directed.
_Fibrous tissue_ may be served in the same way. _Yellow elastic tissue_
must be first placed in a solution of chromic acid and spirit for ten
days, and then treated as directed for muscular fibre.
_Non-striated Muscle._--A piece of the intestine of a rabbit should be
steeped in chromic acid and spirit for ten days, then washed in water;
strip off a thin layer of the muscular coat, and stain in hæmatoxylin
solution. Well wash in ordinary water until the colour changes to
blue, when it will be fit for mounting. Place a fragment on a slide
and a drop of water, and carefully separate the fibres with a pair of
needles. Drain off the water, as it is now ready for mounting, place
on slide, and add a drop or two of Farrant’s medium, and place on the
cover-glass.
_Nerve Tissue._--Dissect out the large sciatic nerve from a frog’s
thigh, and stretch it on a small piece of wood, to which pin both ends
of the nerve, and transfer it to a one per cent. solution of osmic acid
for an hour or two. Wash in distilled water; tease up a small fragment
on a slide (as shown in Fig. 240), and apply a drop or two of Farrant’s
solution and cover-glass.
Tissues containing air should be soaked in water that has been boiled
for ten minutes; this will displace the air. (For Farrant’s medium, see
Appendix.)
_Glycerine Jelly._--Dissolve one ounce of French gelatine in six ounces
of distilled water, and melt together in a hot-water bath. When quite
dissolved, add four ounces of glycerine, and a few drops of creosote
or carbolic acid. Filter through white filtering paper while warm, and
keep in a capped bottle. This may be used instead of Farrant’s solution.
[Illustration: Fig. 240.--Method of Teasing out Tissue.]
_Nitrate of silver_ darkens by exposure; it is used in a half per
cent. watery solution. Specimens to be acted upon should be washed in
distilled water, to remove every trace of sodium chloride, and then
steeped in the silver solution for some two or three minutes, after
which they should be again washed until they cease to turn milky;
then place them in glycerine and expose them to the action of light
until they assume a dark brown colour, when they should be mounted in
glycerine or glycerine jelly.
By means of this stain the endothelial cells of the lymphatics, blood
vessels, &c., and the nodes of Ranvier, constrictions in medullary
nerves, are rendered visible. Sections of any of these may subsequently
be stained by logwood or carmine.
Several methods have been adopted for staining with gold chloride. Dr.
Klein’s and Professor Schäfer’s are among the best.
Dr. Klein’s method of showing the nerves of the cornea is as
follows:--Remove the cornea within fifteen minutes of death; place it
in a half per cent. chloride of gold solution for half an hour, or
an hour; wash in distilled water, and expose to the light for a few
days; in the meantime occasionally change the water. Then immerse it in
glycerine and distilled water, in the proportion of one to two; lastly,
place it in water, and brush gently with a sable pencil to remove any
precipitate, when it will be fit for mounting in glycerine. The colour
of the cornea should be grey-violet.
Schäfer adopts another method--a double chloride of gold and potassium
solution.
Osmic acid, first used by Schultze, is useful for the demonstration of
fatty matters, all of which it colours black; it is also valuable for
certain nerve preparations. Specimens should be allowed to remain in
a one or two per cent. aqueous solution of the acid from a quarter to
twenty-four hours, when the staining will be completed; but if it is
desired to harden specimens at the same time, they should remain in it
for some few days. Osmic acid does not penetrate very deeply, therefore
small portions should be selected for immersion. This is a useful stain
for infusorial animals.
Chloride of palladium, another of Schultze’s staining fluids, is used
to stain and harden the retina, crystalline lens, and other tissues of
the eye, the cornified fat and connective tissues remaining uncoloured.
The solution should be used very weak:--Chloride of palladium, one
part; distilled water, 1,000 parts. Specimens should be mounted in
glycerine at once, or further stained with carmine.
Dr. Schäfer employs a silver nitrate and gelatine solution for
demonstrating lung epithelium; this is made as follows:--Take of
gelatine ten grammes, soak in cold water, dissolve, and add warm water
to 100 cc. Dissolve a decigramme of nitrate of silver in a little
distilled water, and add to the gelatine solution. Inject this with
a glass syringe into the lung until distension is pretty complete.
Leave it to rest in a cool place until the gelatine has set; then cut
sections as thin as possible, place them on a slide with glycerine, and
expose to light till ready for mounting.
Of the double stains Mr. Groves prefers only those where the double
colour is produced by a single process--or stains in which one colour
is first employed, and then another. Single stains are picro-carmine,
carmine and indigo carmine, aniline blue and aniline red.
Picro-carmine is specially useful for staining sections hardened in
picric acid. It is prepared in several ways:--
1. Add to a saturated solution of picric acid in water a strong
solution of carmine in ammonia to saturation.
2. Evaporate the mixture to one-fifth its bulk over a water bath,
allow it to cool, filter from deposit, and evaporate to dryness, when
picro-carmine is left as a crystalline powder of red-ochre colour.
Sections can be stained in a one per cent. aqueous solution, requiring
only ten minutes for the process; wash well in distilled water, and
transfer them to methylated alcohol, then to absolute alcohol, after
which they are rendered transparent by immersing in oil of cloves or
benzole, before mounting in balsam or dammar.
To summarise Mr. Groves’ recommendations:--
1. Let the material be quite fresh.
2. (_a_) Take care that the hardening or softening fluid is not too
strong. (_b_) Use a large bulk of fluid in proportion to the material.
(_c_) Change the fluid frequently. (_d_) If freezing be employed, take
care that the specimen is thoroughly frozen.
3. (_a_) Always use a sharp razor. (_b_) Take it with one diagonal
sweep through the material. (_c_) Make the sections as thin as
possible; and (_d_) Remove each one as soon as cut, for if sections
accumulate on the knife or razor they are sure to get torn.
4. (_a_) Do not be in a hurry to stain, but (_b_) Remember that a weak
colouring solution permeates the section better, and produces the best
results; and (_c_) That the thinner the section the better it will take
the stains.
5. (_a_) Always use glass slips and covers free from scratches and
bubbles, and chemically clean. (_b_) Never use any but extra thin
circular covers, so that the specimens may be used with high powers.
(_c_) Always use cold preservatives, except in the case of glycerine
jelly, and never use warmth to hasten the drying of balsam or dammar,
but run a ring of cement round the cover.
6. Label specimens correctly; keep them in a flat tray, and in the
dark.
Double and Treble Staining.
Dr. W. Stirling[46] furnishes a brief but useful account of the methods
he has employed with much success.
_Osmic Acid and Picro-carmine._--Mix on a glass slide a drop of the
blood of newt or frog and a drop of a one per cent. aqueous solution
of osmic acid, and allow the slide to stand by. This will fix the
corpuscles without altering their shape. At the end of five minutes
remove any excess of acid with blotting-paper, add a drop of a solution
of picro-carmine, and a trace of glycerine to prevent evaporation, and
set aside for three or four hours to see that no overstaining takes
place. At the end of this time the nucleus will be found to be stained
red, and the perinuclear part yellow.
_Picric Acid and Picro-carmine._--Place a drop of the blood of a frog
or newt on a glass slide, and add a drop of a saturated solution
of picric acid: put the slide aside and allow it to remain for
five minutes; at the end of that time, when the acid has fixed the
corpuscles (that is, coagulated their contents), any excess of acid
should be removed as before. A drop of solution of picro-carmine
should now be added, and a trace of glycerine, and the preparation set
aside for an hour. At the end of that time remove the picro-carmine
solution by means of a narrow slip of blotting-paper, and add a drop of
Farrant’s solution of glycerine and apply glass-cover. The perinuclear
part of the corpuscles will be seen to be highly granular and of a
deep orange colour, whilst the nucleus is stained red. Some of the
corpuscles will appear of a delicate yellow colour, and threads are
seen extending from the nucleus to the envelopes. The preparation
should be preserved and mounted in glycerine.
_Picro-carmine and Aniline Dye._--For glandular tissue, none of the
aniline dyes answer so well as iodine green, used in the form of a one
per cent. watery solution. Stain the tissue in picro-carmine, wash
it in distilled water acidulated with acetic acid, and stain it in a
solution of iodine green. As it acts rapidly, care must be taken not to
overstain. Wash the section in water, and then transfer it to alcohol;
finally clear with oil of cloves. The washing should be done rapidly,
as the spirit dissolves out the green dye. All preparations stained
with iodine green must be mounted in dammar.
_Picro-carmine and Iodine Green._--Stain a section of the cancellated
head of a very young bone (fœtal bone) in picro-carmine, wash it in
distilled water, and stain it with iodine green, and mount in dammar.
All newly-formed bone is stained red; that in the centre of the osseous
trabeculæ, the residue of the calcified cartilage in which the bone
is deposited, is stained green. Many of the bone corpuscles are also
stained green.
Ossifying cartilage, the back part of the tongue, Peyer’s Patches,
solitary-glands, trachea, and bronchus, may all be treated in the
same way. In preparing the skin, take a vertical section from the
sole of the foot of a fœtus. The cuticle and superficial layers of
the epithelium are dyed yellow, the rete Malpighii green; and the
continuation of these cells can be traced into the ducts of the
sweat-glands, which are green, and form a marked contrast to the red
stained connective tissue of the cutis vera, through which they have to
ascend to reach the surface. The outer layer of the grey matter of the
cerebellum with Purkinge’s cells is, when double stained, red, while
the inner or granular layer is green. Logwood and iodine green stains
the mucous glands of the tongue green, and the serous glands, lilac
logwood stain.
_Eosin and Iodine Green._--Eosin is used as the ground colour. Stain
the tissue in an alcoholic solution of eosin, which will colour it
very rapidly, usually in a few seconds. Wash the section thoroughly
in water acidulated with acetic or hydrochloric acid, a one per cent.
solution, and stain with iodine green. This will double stain bone and
cerebellum; but if logwood is substituted for the latter, the cerebrum
and general substance become stained by the eosin, while the logwood
colours the nerve-cells a lilac.
_Gold Chloride and Aniline Dyes._--The tissue must be impregnated with
chloride of gold, and then stained with either aniline blue, iodine
green, or rosin. The tail of a young rat, containing as it does so many
different structures, is an excellent material for experimenting upon.
Remove the skin from the tail, and place pieces half an inch long into
the juice of a fresh lemon for five minutes, wash it to get rid of the
acid. The fine tendons swell up under the action of the lemon acid, and
permit of the more ready action of the chloride of gold solution. Place
the piece for an hour or more in a one per cent. solution of gold,
remove it and wash it thoroughly, and then place it in a twenty-five
per cent. solution of formic acid for twenty-four hours. This reduces
the gold. During the process of reduction the preparation must be
kept in the dark. The osseous portion has then to be decalcified in
the ordinary way, with a mixture of chromic and nitric acid. After
decalcification preserve the whole in alcohol. Transverse sections of
the decalcified tail are made, and may be stained with a red dye, as
rosin, and afterwards with a watery solution of iodine green. Mount in
dammar.
Injecting Small Animal Bodies.
[Illustration: Fig. 241.--Injecting Syringe.]
[Illustration: Fig. 242.--Water Bath and Melting Vessels.]
The injection of animal bodies practised by the older anatomists, to
render the vascular system more apparent, has not been superseded by
the more modern methods of staining. The method of injecting even
small bodies requires some skill, and a few pieces of apparatus made
expressly for the purpose. First, a special form of brass syringe of
such a size that it may be grasped with the right hand, the thumb at
the same time covering the button at the top of the piston-rod when
drawn out to the full. In Fig. 241 the piston rod is seen withdrawn,
_a_ is the body, with a screw at the top for firmly screwing down the
cover, _b_, after the piston, _c_, is replaced; _e_ is a stop-cock,
to the end of which either of the smaller cannulæ, _g_, is affixed.
The transverse wires are for securing them tightly with thread to the
vessels into which they are to be inserted. In addition to the syringe,
two or three tinned vessels are required to contain size, injecting
fluid, and hot water.
The size must be kept hot by the aid of a water bath; if a naked
fire be used there is danger of burning it. A convenient form of
apparatus for melting the size, and afterwards keeping it at a proper
temperature, is Fig. 242.
[Illustration: Fig. 243.--Artery Needle.]
A pair of strong forceps for seizing the vessel, and a small needle
(Fig. 243) is also necessary for passing the thread round the vessel
into which the injection pipe has been inserted. These complete the
list of apparatus. To prepare the material for opaque injections,
take one pound of the finest and most transparent glue, break it into
small pieces, put it into an earthen pot, and pour on it three pints
of cold water; let it stand twenty-four hours, stirring it now and
then with a stick; set it over a slow fire for half an hour, or until
all the pieces are perfectly dissolved, skim off the froth from the
surface, and strain through a flannel for use. Isinglass and cuttings
of parchment make an excellent size, and are preferable for particular
injections. If gelatine be employed an ounce to a pint of water will be
sufficiently strong, but in very hot weather it is necessary to add a
little more gelatine. It must be first soaked in part of the cold water
until it swells up and becomes soft, when the rest of the water, made
hot, is to be added. The size thus prepared may be fixed with finely
levigated vermilion, chrome-yellow, blue salts, or flake white.
To prepare the subject, the principal points to be attained are: to
dissolve the fluids and completely empty the vessels; relax the solids;
and prevent the injection from coagulating too soon. For this purpose
it is necessary to place the animal, or part to be injected, in warm
water, as hot as the operator’s hand will bear. This should be kept at
nearly the same temperature for some time by occasionally adding hot
water. The length of time required is in proportion to the size of the
part and the amount of its rigidity.
_Injecting the systems of Vessels with different colours: Carmine and
Gelatine Injection._--Carmine 30 grains, strong liquid ammonia 60
drops, glacial acetic acid 43 drops, gelatine solution (one ounce in
six ounces of water) two ounces, water one ounce: dissolve the carmine
in the ammonia and water in a test tube, and mix it with one half of
the warm gelatine, add the acid to the remaining half of gelatine, and
drop it little by little into the carmine mixture, stirring it well
with a glass rod during the mixing; filter through flannel, and add a
few drops of carbolic acid to make it keep. It is very important that
the stain should be quite _neutral_, the test of which is the colour
and smell of the fluid. It should be a bright red, and all trace of
smell of ammonia must be removed.
_Prussian or Berlin Blue and Gelatine._--Take 1-1/2 ounces of gelatine,
place it in a vessel and cover it with water; allow it to stand until
all the water is absorbed and the gelatine is quite soft, then dissolve
in hot water. Dissolve one drachm (60 grains) of Prussian or Berlin
blue in six ounces of water, and gradually mix it with the gelatine
solution, stirring well with a glass rod during the mixing; then filter
as before.
_Watery Solution of Berlin Blue._--Dissolve 2-1/2 drachms of the blue
in 18 ounces of distilled water, and filter. This staining fluid is
used for injecting the lymphatic system.
_Directions for Injecting._--The animal to be injected must be first
killed by chloroform, and injected while still warm; to secure this
place the body in a water bath, at a temperature of 104° Fahrenheit.
Expose the main artery of the parts to be injected, clear a small
portion of it from the surrounding tissues, and place a ligature of
thin tissue or silk round it, by means of the small artery needle (Fig.
243). With a pair of sharp-pointed scissors make an oblique slit in the
wall of the vessel, insert the cannula, and tie the ligature firmly
over the artery behind the point of the cannula, into which put the
stop-cock. Fill the syringe with injection fluid, which must not be too
warm, and take care not to draw up any air-bubbles; insert the nozzle
of the syringe into the stop-cock and force in a little fluid; remove
the syringe so that the air may escape, re-insert the syringe, repeat
the process until no air-bubbles escape, and then proceed slowly with
the injection. Half an hour will be required to complete the process
in an animal the size of a rabbit. To judge of the completeness of
the injection, examine the vascular parts of the lips, tongues and
eyes; if satisfactory, tie the ligature round the artery and withdraw
the syringe; place the animal in cold water for an hour to consolidate
the injection fluid. When cold dissect out the organs, cut them up,
and place them in methylated spirit to harden. Change the spirit every
twenty-four hours for the first three days. The hardening process will
be complete in ten days.
To inject lymphatics by the puncture process, a small-sized
subcutaneous syringe should be used, filled with a watery solution of
the prepared stains. Thrust the nozzle into the pad of the foot, (or
tongue), and then rub the limb to cause the injection fluid to flow
along the lymphatic vessels into the glands.
When the blue stain is used add a few drops of acetic acid to the
spirit while the hardening process is going on.
_Of Injecting Different Systems of Vessels with Different Colours._--It
is often desirable to inject different systems of vessels distributed
to a part with different colours, in order to ascertain the arrangement
of each set of vessels and their relation to each other. A portion of
the gall-bladder in which the veins have been injected with white lead,
and the arteries with vermilion, forms an attractive preparation. Each
artery, even to its smallest branches, is seen to be accompanied by
two small veins, one lying on either side of it. By this method four
different sets of tubes have been injected--the artery with vermilion,
the portal vein with white lead, the duct with Prussian blue, and the
hepatic vein with lake. There are also opaque colouring matters which
may be employed for double injections.
_Injecting the Lower Animals._--The vessels of fishes are exceedingly
tender, and require great caution in filling them. It is often
difficult or quite impossible to tie the pipe in the vessel of a fish,
and it will generally be found a much easier process to cut off the
tail of the fish, and put the pipe into the divided vessel which lies
immediately beneath the spinal column. In this simple manner beautiful
injections of fish may be made.
_Mollusca_ (slug, snail, oyster, &c.).--The tenuity of the vessels of
the mollusc often renders it impossible to tie the pipe in the usual
manner. The capillaries are, however, usually very large, so that the
injection runs very readily. In different parts of the bodies of these
animals are numerous lacunæ or spaces, which communicate directly
with the vessels. Now, if an opening be made through the integument of
the muscular foot of the animal, a pipe may be inserted, and thus the
vessels may be injected from these lacunæ with comparative facility.
_Insects._--Injections of insects may be made by forcing the injection
into the general abdominal cavity, when it passes into the dorsal
vessel and is afterwards distributed throughout the system. The
superfluous injection is then washed away, and such parts of the body
as may be required removed for examination.
Natural injection of Medusæ may be effected without injuring the
vessels, with an opening at the side remote from it. The medusa must
be placed in a glass vessel, with the bell downwards, and a bell-jar
ending in a narrow tube above is placed over it and made air-tight;
the medusa is then covered with the injection-mass, the air in the
glass is exhausted, and as the sea-water runs out by slits in the lower
side of the annular canal, the coloured fluid runs in. In the case
of leeches and large species of earthworms, the natural injection is
made from the ventral sinus. In all cases a glass tube is used, with a
finely drawn-out point. The injection is complete when the injection
issues from the counter-opening. Besides the animals mentioned, large
caterpillars, beetles, and larvæ of various kinds are favourable
objects for injection; the glass cannula being introduced into the
posterior end of the dorsal vessel, and the counter-opening made in the
ventral vessel, and _vice versâ_.
_Staining Living Protoplasm with Bismarck Brown._--Henneguy having
treated _Paramœcium aurelia_ with an aqueous solution of aniline
brown (known as “Bismarck Brown”), found that they assumed an intense
yellow-brown colour. The colour first appears in the vacuoles of the
protoplasm, and then in the protoplasm itself, the nucleus generally
remaining colourless, and becoming more visible than in the normal
state. If a yellow-tinted paramœcium be compressed so as to cause a
small quantity of the protoplasm to exude, it is seen that it really is
the protoplasmic substance which becomes coloured. All the Infusoria
may be stained with Bismarck brown, but no other aniline colour
employed exhibits the same property--they merely stain the Infusoria
after death, and are in fact poisonous. Living protoplasm does not as
a rule absorb colouring matters, and as Infusoria are chiefly composed
of protoplasm, attempts have been made to ascertain whether protoplasm
in general, of animal or vegetable origin, behaved in the same way in
the presence of aniline brown. A tolerably strong solution of Bismarck
brown was therefore injected under the skin of the back of several
frogs. After some hours the tissues became uniformly tinted a deep
yellow; the muscular substance especially had a very marked yellow
tint. The frogs did not appear in the least incommoded. Small fry of
trout placed in a solution stained rapidly and continued to swim about.
Finally, a guinea-pig, under whose skin some powder of Bismarck brown
had been introduced, soon presented a yellow staining of the buccal and
anal mucous membranes and of the skin. Seeds of cress sown on cotton
soaked with a concentrated solution of the Bismarck brown sprouted,
and the young plants were strongly stained brown; but on crushing the
tissues and examining them under the microscope, it was ascertained
that the protoplasm of the cells was very feebly coloured: the vessels,
on the contrary, showed a deep brown stain up to their termination of
the leaf. The mycelium of a mould developed in a solution of Bismarck
brown was clearly stained after having been washed in water, whilst
it is known that the mycelium, which frequently forms in coloured
solutions, picro-carmine, hæmatoxylin, &c., remained perfectly
colourless. Other aniline colours injected under the skin of frogs
stained the connective tissue as deeply as did the Bismarck brown; but
the striæ of muscle remained colourless. We may conclude, then, that
Bismarck brown possesses the quality of colouring living protoplasm
both in plants and in animals.
Cutting, Grinding, and Mounting Hard Structures.
Take the femur of cat, or rabbit, remove as much of the muscle as
possible and macerate it in water until quite clean; on removal hang it
up to dry. With a fine saw make transverse and longitudinal sections.
File the section down until flat, and smooth. Take some Canada balsam,
place a piece on a square of glass and warm gently over a lamp until
the balsam is plastic enough to allow of the section being pressed
into it, and set it aside to consolidate. Take a hone (“Water-of-Ayr”
stone), moisten it with water, and rub one side of the section upon it
until quite smooth, then place the glass slip, with the section still
attached, into methylated spirit, and in a very short time the section
will be separated; wash it and remount it on the reverse side, and
proceed to rub it down on the hone until it appears to be thin enough
for mounting. Polish both sides on a polishing strop with Tripoli
powder, and mount in Canada balsam.
[Illustration: Fig. 244.--Small Lathe for cutting and polishing
Sections of Teeth.]
_Teeth._--The enamel of the teeth is a much harder structure than
that of bone, consequently it is found necessary to have recourse
to a cutting machine. Hand machines have been introduced for this
purpose, but the small lathe described in the earlier editions of my
book has in no way been superseded by later cutting machines. Fig. 244
represents the small lathe used for cutting and polishing every kind of
hard substance. With regard to the teeth, two sections should be made
perpendicular to one another through the middle of the crown and fang
of the tooth from before backwards, and from right to left, which will
show the peculiar structure of the enamel. The section must be cemented
to the carrier of the stock of the lathe, or to the metal plate _a_,
and kept in position by the steel holder _b_; the wheel being set in
motion by the first treadle. The embedding materials in use are either
gum-shellac or Canada balsam. The former is more generally employed by
the lapidary and grinder of lenses than the latter. As the enamel is
liable to fracture under the saw, it will be necessary to lessen the
friction by dripping water on the saw as it is made to revolve. Thick
sections can be quickly ground down against the corrondum wheel. The
final polishing of the section may be done on the lathe, or by rubbing
the flattened surface with water upon a “Water-of-Ayr” stone, and
ultimately set up in Canada balsam, which must not be too fluid, or it
will penetrate the _lacunæ_ and _canaliculi_, fill up the interspaces,
and cause them to become quite invisible. As the flatness of the
polishing surfaces is a matter of importance, the stones themselves
should be tested from time to time, and when found to present an uneven
surface must be rubbed down on a granite stone with fine sand, or on
a lead plate with emery powder. If it is decided to use Canada balsam
as the embedding material, this must be prepared in the following
manner:--The section of tooth or bone must be attached to a slip
of well-annealed glass by hardened Canada balsam, and its adhesion
effectually secured by placing the slide on the cover of a water
bath, or in the hot-chamber (Fig. 256), when the balsam, a thick drop
of which should be used, will spread out by liquefaction. The slide
should then be removed and allowed to cool in order that the hardness
of the balsam may be tested. If too soft, as indicated by its readily
yielding to the pressure of the thumb-nail, the heating process must be
repeated, care being taken not to cause it to boil and form bubbles;
if too hard, which will be shown by its chipping, it must be remelted
and diluted with fluid balsam, and then set aside as before. When it
is found to be of the right consistence, the section must be laid upon
its surface with the polished side downwards; the slip of glass is
next to be gradually warmed until the balsam is softened, care being
taken to avoid the formation of bubbles, then press the section gently
down with a needle upon the liquefied balsam, the pressure being just
applied on one side rather than over the whole surface, so as to drive
the superfluous balsam towards the opposite side; finally, an equable
pressure over the whole will secure a perfect attachment of the section
without air bubbles. If, however, these should present themselves in
drying, and they cannot otherwise be expelled by pressure, it will
be found better to take the section off and relay it as before. The
thickness of the layer of balsam may be reduced by rubbing it down
before applying the glass-cover.
_Rock Sections._--Small pieces of rock may be ground down by the aid
of the lathe, or on a zinc plate, with emery powder and water, until
one side is rendered smooth and flat. Then fasten the polished side
of the section to a square of glass on the metal holder of the lathe,
with dried Canada balsam, as directed for bone, and allow it time to
become consolidated. When moderately thin take a piece of plate-glass
and some fine emery or putty-powder and rub the section down as thin as
possible. When found to be thin enough wash it well in water, and put
it aside to dry, or warm it over a spirit-lamp, and with a needle push
the section off the glass into a watch-glass of benzole or turpentine,
and allow it to soak until all the balsam is dissolved out. Wash
again in turpentine, and mount in Canada balsam, with or without a
cover-glass. Sections of echinus spines, shells, stones of fruits, &c.,
are prepared in the same way as those of bones and teeth; but when the
grinding is finished, the sections must be passed through alcohol into
oil of cloves, after which they should be mounted in Canada balsam. If
tolerably thin, sections of these substances can be cut in the lathe;
in the first instance, there will be no actual occasion to attach them
to glass at all, except for the purpose of obtaining a hold upon the
specimen for polishing, but the surface thus attached must afterwards
be completely removed in order to bring into view a stratum which the
Canada balsam may not have penetrated.
With regard to smaller bodies, these can scarcely be treated in any
other way than by attaching a number of them to slips of glass at once,
and in such a way as to make them mutually support each other. Thus in
making horizontal and vertical sections of _foraminifera_, it would be
impossible to slice them through unless they were laid close together
in a bed of hardened Canada balsam, and first grinding away one side
and then turning and rubbing down the other. My friend, Dr. Wallich,
many years ago communicated to me the ingenious plan adopted by himself
when mounting and turning a number of these minute objects together.
The specimens being cemented with Canada balsam, in the first instance,
to a thin film of mica, and then attached to a glass slide by the same
means, when ground down to the thinness desired, the slide must be
gradually heated just sufficiently to allow of the detachment of the
mica-film and the specimen it carries; a clean slide with a thin layer
of hardened balsam having been prepared, the mica-film is transferred
to it with the ground surface downward. Its adhesion by drying having
been complete, the grinding and polishing should be proceeded with;
and as the mica-film will yield to the stone without any difficulty,
the specimen now reversed in position may be further reduced to the
requisite thickness for mounting as a permanent object.
_Staining and Mounting Vegetable Tissues._--Bacteria I propose to
treat of in a separate section. Vegetable tissues generally will first
receive attention, and their differentiation is based on the employment
of delicate gradations of colour stains. The more striking results are
obtained by _Multiple Staining_, while the cell contents are rendered
more palpable. On this account colouring media have been divided into
_nuclear_, _plasmic_, and _specific_. The first are chiefly valued in
proportion as they prove to have a selective affinity for the nuclei of
cells, and leaving the protoplasm comparatively unstained. Such stains
are needful for fresh and young tissues. On the other hand, _plasmic_
stains colour tissue uniformly, and are used to give a ground colour
by way of contrast; and _specific_ stains are chiefly employed to
distinguish certain elementary structures from the mass of cellulose
which forms the basis of vegetable tissue, and which is also met with
to a slight extent in animal membranes.
Cellulose, as it occurs in plant life, presents a variety of physical
properties: sometimes it is soft, as in young plants, and again quite
dense in older structures. This fact accounts for the varying results
obtained when cellulose is subjected to the action of staining fluids,
and whether the cellulose occurs in a nearly pure form, as in cotton
fibre, or in the modified form of lignine or woody-fibre. Stains
which readily attack young tissue have little or no effect upon it in
its maturer form. It is of much importance, then, in the staining of
fibres, as well as sections for the microscope, that the cellulose
should take the stain uniformly.
The staining of tissues may be effected in four ways. First, when the
stain has sufficient affinity for the tissue to be retained by it
without the intervention of any outside agent. Second, when the stain
and mordant are mixed and applied to the tissue in one solution. These
two are the simplest and easiest methods of staining. Third, when the
tissue is first immersed in the staining liquid and then transferred to
some other liquid which shall fix the colour upon the tissue. Fourth,
when the tissue is first impregnated with the mordant, or fixing agent,
and then immersed in the stain. The last method is the one usually
followed in commercial works, and it is to be recommended in the
staining of microscopical preparations which do not readily take the
stain.
_Nuclear Stains._--As in both vegetable and animal sections it is
generally the nuclei which form the landmarks of the structure, so the
most important class of reagents which are used in any of the branches
of microscopical work are the “nuclear stains.” There are several of
these stains, the most important of which is the hæmatoxylin, and
when proper solutions are used the results are very satisfactory.
Many formulæ have been given, but there are three only reliable,
Delafield’s, Kleinenberg’s, and Ehrlich’s, in all of which alum is
present as an ingredient; the idea being that the alumina forms with
the colouring matter an insoluble lake, and so acts as a mordant.
In _Delafield’s_ solutions a large proportion of alum to hæmatoxylin
is used, and methylic alcohol (wood-spirit in the place of rectified
spirit).
For _Kleinenberg’s_ solution many different formulæ exist. Squire’s
improved formulæ for both stains is given in the Appendix, “_Formulæ
and Methods_.”
Hæmatoxylin solutions stain the nuclei violet, and in order to change
this into blue, the sections should be transferred to water taken from
the house supply, not distilled water; but as the alkalinity of the
water varies in different localities, a better and more uniform result
is obtained by using a weak solution of bicarbonate of sodium (half a
grain to the ounce).
_Carmine_ is also much in vogue as a nuclear stain, and the two
solutions more generally employed are Greenacher’s alcoholic borax
carmine, and Orth’s lithium carmine. Under ordinary circumstances they
act as general stains, affecting the ground tissue as well as the
nuclei. By subsequent treatment with acidulated alcohol or acidulated
glycerine the colour is discharged from the ground tissue without
seriously affecting the nuclei. Used in this way, carmine becomes a
good nuclear stain. It should be remembered that the sections must
not be washed in pure water, as the colour will to a great extent
be discharged; nor in acidulated water, as the carmine will be
precipitated.
Alum carmine and alum cochineal are useful nuclear stains, not
requiring after-treatment.
_Picro-carmines_ are also largely used. The following formulæ will be
found the most useful:--
_Ammonia Picro-carmine._--Carmine, one gramme; strong solution of
ammonia, three cc.; distilled water, five cc. Dissolve the carmine in
the ammonia and water with a gentle heat, then add saturated aqueous
solution of picric acid, 200 cc.; heat to boiling, and filter.
_Picro-Lithium Carmine._--The following is generally preferred for
use--Lithium carmine solution, 100 cc.; saturated solution of picric
acid, 270 cc.
There are several aniline dyes which are used for nuclear staining:
methylene blue, methyl green, safranine, gentian violet, vesuvine,
fuchsine, and Hoffmann’s blue.
The usual process is to stain in 1/4 or 1/2 per cent. aqueous solutions
and wash in methylated spirit. Methylene blue and methyl green have the
reputation of being so readily washed out in the methylated spirit as
to be worthless. This is obviated by washing the sections (when removed
from the stain) in distilled water, previous to the differentiation in
methylated spirit. Treated in this manner, the nuclear staining is very
beautiful. This also applies to Hoffmann’s blue and partly to vesuvine;
with the latter, however, it is not a necessity. Safranine and gentian
violet worked better by transferring the sections directly from the
stain into 90 per cent. alcohol.
_Contrast Stains._--Very frequently other stains are used to dye the
ground a colour which is in contrast to that employed for the nuclei.
Brown, orange, or pink are used after nuclear blue or green. Carmine is
generally counterstained yellow or indigo-blue; and fuchsine red, as in
tubercle bacilli, is counterstained with nuclear blue. It is important
that the ground stain should be made weaker than the principal
stain, so that the whole tissue may be shown without detracting from
the nuclei. The following colours are used as counterstains for
animal sections, but they prove less useful for vegetable sections:
benzo-purpurine, eosin, erythrosine, orange, acid rubin, and picric
acid.
Examples of _specific_ stains are fuchsine, methylene blue, and gentian
violet for bacteria; osmic acid for fatty elements; victoria blue
and rose bengale, for demonstrating elastic tissue; methyl violet,
iodine, and safranine, for amyloid degeneration. Methylene blue is one
of the most useful of aniline dyes, and one of the most variable in
composition.
_Iodine green_, or methyl green, has long been in use as a reagent for
amyloid, starchy matters, in ignorance of the fact that the reaction
is due to the methyl violet, contained as an impurity in the iodine
green. It is exceedingly difficult to obtain a green quite free from
violet. As nuclear stains they are identical, and the amyloid reaction,
being dependent wholly upon the contained violet, varies, not with the
formula of the green, but with the extent to which it has been purified.
_Cellulose reactions._--After the nuclear stains, the most important
reagents to the botanist are those which affect cellulose and its
several modifications. Pure cellulose is coloured yellow by iodine,
the colour being changed to a blue on the addition of slightly dilute
sulphuric acid, or a strong solution of zinc. Solutions containing
iodine, iodide of potassium, and chloride of zinc, give a violet
reaction with unaltered cellulose, and yellow with lignine.
Schulze’s zinc re-agent must be used with a certain amount of caution,
as the chloride of zinc and potassium undergo decomposition. The
formula now in use is as follows: Take of zinc chloride solution (sp.
gr. 1·85) 70 cc., potassium iodide 10 grammes, iodine 0·1 gramme; but
this solution can only be employed as a re-agent and not as a dye, and
structures stained with it cannot be mounted in any of the ordinary
media, and the only fluid for ringing them down is caoutchouc cement.
Cellulose can be stained permanently by carmine, hæmatoxylin,
nigrosine, methylene blue, safranine, and fuchsine. The aniline
dyes are used in dilute aqueous solutions containing one-eighth or
one-fourth per cent. of dye. When the cellulose undergoes the change
known as lignification its reactions are altered. It is coloured yellow
by chloro-zinc iodine, red by phloroglucin, yellow by aniline chloride.
The two latter are much assisted by hydrochloric acid. The results of
these reactions also cannot be preserved in the usual mounting media.
Sections containing mixed tissue, partly unaltered cellulose and partly
lignified, give striking results with aniline dyes, and with this
additional advantage can be preserved for years.
_Double Staining._--When a section is passed through methyl green
solution and afterwards one of carmine, the lignified portion is
coloured green and the unlignified red. Acid green may be used in the
place of methyl green, with a similar result. When picric acid is used
with carmine, ingrosine, or Hoffmann’s blue, the picric acid dyes the
ligneous portion and the others colour the unlignified structure, red,
black, and blue respectively.
_Eosin stain_ is the most useful for sieve-tubes and plates. Make a
strong solution of eosin in equal parts of water and alcohol, and stain
the section for five or ten minutes. Wash well in methylated spirit,
dehydrate, clean in oil of cloves, and mount in Canada balsam.
_Bleaching Process._--The bleaching and clearing of vegetable
structures before staining is a very necessary process, especially
so if starch be present in any quantity. Clearing agents are of
two kinds--those which act by virtue of their property of strongly
refracting light, and those which disintegrate and dissolve the amyloid
cell contents. To the first class belong the essential oils, as oil
of cloves, Canada balsam, glycerine, and other similar bodies; to the
second class, solutions of potash, phenol, and chloral hydrate. The
actual value of some of these agents is questionable. The process
usually preferred is as follows: Place the sections in a fresh clear
solution of chlorinated lime, allowing them to remain until quite
bleached, say from two to four or five minutes; then gently warm in
a test-tube for a few seconds, and quickly replace the solution with
distilled water and boil for two or three minutes; repeat the treatment
with boiling water three times; wash with a one per cent. solution of
acetic acid, and finally with distilled water. The sections are then
quite ready for staining operations.
When the stem is hard and brown, a solution of chloride of lime should
be used--a quarter of an ounce of chloride dissolved in a pint of
water, well shaken and stood by to settle down, then pour off the clear
fluid for use. For hard tissues this solution answers well, but it is
not suitable for leaves, as they require not only bleaching, but the
cell contents should be dissolved out to render them transparent. A
solution of chlorinated soda answers well for both stems and leaves. It
is prepared as follows:--
To one pint of water add two ounces of fresh chloride of lime, shake
or stir it well two or three times, then allow it to stand till the
lime has settled. Prepare meanwhile a saturated solution of carbonate
of soda--common washing soda. Now pour off the clear supernatant
fluid from the chloride of lime, and add to it, by degrees, the soda
solution, when a precipitate of carbonate of lime will be thrown down;
continue to add the soda solution till no further precipitate is
formed. Filter the solution, and keep it in a well-stoppered bottle in
the dark, otherwise it speedily spoils.
Sections bleached in chlorinated soda must, when white enough, be
washed in distilled water, and allowed to remain in it for twenty-four
hours, changing the water four or five times, and adding a few drops
of nitric acid, or at the rate of eight or ten drops to the half-pint,
to the water employed before the final washing takes place. From water
transfer them to alcohol, in which they must remain for an hour or more.
Although alkaline glycerine has been recommended for several purposes
in micro-technique, it is not so well known as it should be how
serviceable it is as an extempore mounting solution in vegetable
histology. The best mixture for general use is composed of glycerine 2
ozs., distilled water 1-1/2 oz., solution of potash, B.P., 1/2 oz. This
combines the refringent property of the glycerine with the clearing
action of the caustic potash, while the swelling action of the potash
is considerably diminished.
_Cutting Sections of Hard Woods._--The lathe and circular saw will be
found as useful for cutting sections of the harder kinds of woods,
as for bone structure. It may be necessary to subject the older and
consequently harder pieces of wood to the action of steam for a few
hours to soften them, and afterwards transfer them to methylated
spirit, before making an attempt to cut sections. But the more open
woods, of one, two, or three years’ growth, will show all that may
be required, and these can be cut by hand, or with the microtome, as
already described.
With a little practice the finest and thinnest possible slices may be
cut by hand. It is usual to take off the first slice to give a smooth
and even surface to the specimen. Then turn the screw to raise it a
little, sprinkle the surface with spirit and water, and cut with a
light hand. Remove the cut sections with a fine camel’s-hair brush
or a section lifter (Fig. 250) to a small vessel containing water,
when the thinnest will float on the surface, and remove to methylated
spirit and water, where they should remain until they can be mounted.
Sections of hard woods, and those containing gum-resins, or other
insoluble material, must first be kept in methylated spirit or alcohol,
and finally transferred to oil of cloves, to render them sufficiently
transparent for mounting in Canada balsam.
If the structure of an exogenous wood is required to be examined,
the sections should be made in at least three different ways: the
transverse, the longitudinal, and the oblique, or, as they are
sometimes called, the horizontal, vertical, and tangential, each of
which will exhibit different appearances, as seen in Fig. 245: _b_ is a
vertical section through the pith of a coniferous plant, and exhibits
the medullary rays known to the cabinet-maker as the silver grain;
_e_ is a magnified view of a part of the same; the woody fibres are
seen with their dots _l_, and the horizontal lines _k_ indicating the
medullary rays cut lengthwise; _c_ is a tangential section, and _f_ a
portion of the same; the medullary rays _m m_, and the woody fibres
with vertical slices of the dots, are shown. Instructive preparations
will be secured by cutting oblique sections of the stem. The sections
seen are made from the pine. All exogenous stems, however, exhibit
three different appearances, according to the direction in which the
section is made.
[Illustration: Fig. 245.--Sections of Wood.]
Bacteria Cultivation, Sterilising, and Preparing for Microscopical
Examination.
That branch of mycology which is now looked upon as a separate
department of science, termed bacteriology, took shape in the years
1875-9, when its founder, the veteran botanist Cohn, who recognised
that the protoplasm of plants corresponded to the animal sarcode,
published his exact mode of studying bacteria. But it was a pupil
of his, Dr. Koch, who a year later discovered that a specific cattle
disease, anthrax, was due to a bacillus, and it was he also who gave
us the useful modification of gelatine as a medium in which to grow
bacteria; he hit upon the method of pouring melted gelatine containing
distributed germs on to plates, and thus isolating the colonies and
ensuring the further isolation of the spores, and so facilitate the
preparation of pure cultures on a large scale, and with great saving of
time.
The difficulty of isolating a bacterium and tracing its life history
under the microscope must at first sight appear great. A further
objection that such work is slow and difficult has no more weight here
than in any other department of science, as will be seen on proceeding
to follow out the directions I am about to furnish for the use of the
student.
Apparatus, Material, and Reagents employed in Bacteriological
Investigations.
A good microscope with a wide-angled sub-stage condenser, and
objectives of an inch, 1/4-inch, or 1/6-inch, and a 1/12-inch
homogeneous oil-immersion.
A large bell-glass for covering the same when fuming acids are in use
in the laboratory.
About a square foot of blackened plate-glass.
A white porcelain slab, or a shallow photographic dish of some size.
Glass bottles with ground stoppers for alcoholic solutions and aniline
dyes.
Glass bottles with funnels for filtering solutions of stains, with
pipettes.
A specialised form of pipette for the micro-chemical filtration of
solutions (Fig. 246).
A small stoppered bottle of cedar oil (Fig. 247).
Set of small glass dishes or watch-glasses for section staining.
Stock of glass slides sterilised, together with round thin
glass-covers, in boxes (Fig. 248).
Needle holders and platinum needles, with a packet of ordinary sewing
needles (Fig. 249).
Platinum, or plated copper section-lifters (Fig. 250).
Glass rods, drawn out to a fine point, for manipulating sections when
acids are employed.
[Illustration: Fig. 247.--Bottle and Dipper for Cedar Oil.]
[Illustration: Fig. 246.--Pipette for Micro-chemical Filtration.]
[Illustration: Fig. 248.--Box for keeping Glass-covers.]
[Illustration: Fig. 249.--Needle Holders, fine Lifter and Hook for
Manipulating Structure.]
[Illustration: Fig. 250.--Section Lifters.]
[Illustration: Fig. 251.--Spring Flat Forceps.]
[Illustration: Fig. 251_a_.--Forceps with fine Points.]
A pair of small spring steel platinum-pointed forceps for holding
glass-covers (Fig. 251).
One or two pairs of fine-pointed forceps (Fig. 251_a_).
Collapsible tubes for containing Canada balsam and dammar.
Soft rags or old pocket handkerchiefs for removing cedar oil from
lenses and cover-glasses. Chamois leather for wiping lenses and
removing dust.
_Reagents_, _alcohol_, bergamot oil, _celloidin_, dissolved in equal
parts of ether and alcohol.
_Ebner’s_ solution. (See Appendix.)
_Formalin_, glycerine, gelatine, _Klebs’_ and _Kleinenberg’s_
solutions. (See Appendix.) The latter consisting of a watery solution
of picric acid 100 parts; strong sulphuric acid two parts; filter, and
add distilled water 300 parts.
_Muller_ fluid. (See Appendix.)
_Osmic acid_, a five per cent. solution.
_Paraffin_, _spermaceti_ and _xylol_, _acetic acid_, _hydrochloric
acid_, a one per cent. solution with _alcohol_.
_Ammonia_ liquid, _ether_, _picro-lithium carmine_, _potash solution_.
_Safranine_, concentrated alcoholic solution of, and a watery solution.
_Turpentine_, _vesuvin_, water distilled and sterilised.
Aqueous solutions of the several dyes may be kept in bottles ready for
use.
To both aqueous and alcoholic solutions a few drops of phenol, or a
crystal of thymol, should be added as a preservative. For the rapid
staining of cover-glass preparations, it is convenient to have the
most frequently used stains--fuchsine, methyl-violet, &c.--in bottles
provided with pipette stoppers.
_Clearing Agents._--Oils of cedar wood, cloves, origanum, aniline,
terebene, toluol and xylol, benzol and spirits of turpentine.
_Mounting Media._--Acetate of potash solution concentrated, benzole,
balsam, glycerine jelly, Fanant’s medium, dammar and mastic, Canada
balsam in xylol, Hollis’s glue, zinc white.
Cement for fixing small specimens temporarily to a glass slide. Remove
all traces of moisture, place upon it a drop or two of a medium
prepared as follows:--Dissolve over a water bath 15 grammes of white
lac in 100 grammes of absolute alcohol, decant off the clear liquid,
and stand it by for a while.
As the alcohol evaporates from the warmed surface of the glass slide a
hard transparent coating is left. This may be slightly softened at any
time by means of a drop of oil of lavender. After arranging the objects
the heat of a spirit-lamp will cause the oil to evaporate, leaving
them firmly attached. Objects may be mounted on cover-glasses in a
similar way. A resinous mounting medium may then be employed in the
usual manner. If glycerine or glycerine jelly is the mounting medium
employed, collodion diluted with two or three times its volume of oil
of lavender may be found preferable as the fixing agent. The section
should be placed in position before the preparation dries and the oil
is evaporated.
Methylated spirit is often so largely adulterated with rock-oil as
to render it unsuitable for technical purposes. Even to varnishes it
imparts a fluorescent appearance as it dries off.
[Illustration: Fig. 252.--Iron Box for holding Sterilised Instruments
and Glass Plates.]
The needles and instruments used must not be passed through a Bunsen
burner flame, which is most destructive, but enclosed in a sheet-iron
box made for the purpose (Fig. 252), and placed in the hot-air
steriliser for an hour at 150°C. The box can be opened at the side,
and each instrument withdrawn with a pair of sterilised forceps when
required for use.
_Glass plates_ are sterilised in the same iron box, and the _platinum
needles_ for inoculating nutrient media, examining cultivations, &c.,
are served in the same manner before being used. The needles consist
of two or three inches of platinum wire fixed to the end of a glass
rod. Several of these needles should be made by fixing pieces of wire
into a glass rod about six inches long. The glass rod must be heated at
the extreme end in the flame of a Bunsen burner, or blow-pipe, and the
platinum wire held near one extremity with forceps, and fused into the
end of the glass rod. Some of these rods should be straight, and some
bent, and others provided with a loop, and kept especially ready for
inoculating test-tubes of nutrient jelly.
[Illustration: Fig. 253.--Damp Chamber for Plate-cultivations.]
_Glass Dishes._--Several shallow glass dishes are required for
preparing damp chamber cultivations, the upper covers fitting over
the under (as in Fig. 253), in the centre of which culture-plates are
stacked one above the other, and when necessary placed in the incubator.
Apparatus for Incubation and Cultivations in Liquid Media.
_Lister’s Flasks._--Lister devised a globe-shaped flask with two
necks, a vertical and a lateral one, the lateral being a bent spout,
tapering towards the extremity. When the vessel is restored to the
erect position after pouring out some of its contents, a drop of
liquid remains behind in the end of the nozzle, and thus prevents the
regurgitation of air through the spout. A cap of cotton-wool is tied
over the orifice, and the residue left in the flask for future use. The
vertical neck of the flask is plugged with sterilised cotton-wool in
the ordinary way.
[Illustration: Fig. 254.--Pasteur’s Bulb Pipette.]
[Illustration: Fig. 255.--Storing Cultivation Tube.]
Sternberg advocates the use of a glass bulb, provided with a slender
neck drawn out to a fine point and hermetically sealed. Special forms
of tubes, bulbs, and pipettes were devised by Pasteur, and are still
in use at the Bacteriological Institute, Paris, and known as the
Pasteur’s bulb pipette (Fig. 254).
Others are provided with lateral or with curved arms, one of which
is drawn out to a fine point, and the slender neck plugged with
cotton-wool, as in Fig. 255.
THE WARM CHAMBER, STERILISER, AND INCUBATOR.
[Illustration: Fig. 256.--Pfeiffer’s Warm Chamber.]
_The Warm Chamber._--This is an accessory of importance in
bacteriological work. For the continuous heating of specimens during
cultivation it is an absolute necessity. Pfeiffer’s warm chamber (Fig.
256) is suitable for microscopical work generally. It consists of a
hard-wood box, made air-tight, with doors and glass windows to allow
of the specimen being moved from time to time, and kept under constant
observation. The box is mounted on a metal plate tripod stand, and is
heated from below by a small gas burner, with a thermo-regulator. A
paraffin lamp will do as well, so long as it maintains a temperature of
from 25° to 45°C., and without danger of injury to the stand and lenses
of the microscope. A thermometer is placed in the air space to mark the
temperature.
[Illustration: Fig. 257.--Crookshank’s Incubator.]
_Hot-air Incubators and Sterilisers_ are usually made of sheet-iron,
in the form of a cubical chest, with double walls, supported on four
legs, as that of Dr. Crookshank’s (Fig. 257). They are heated by gas or
a lamp from below, while the temperature is indicated by a thermometer
inserted through a hole in the top, as in that of the Hearson’s
incubator. Test-tubes, flasks, funnels, cotton-wool, &c., must be
sterilised by exposure to a temperature of 150°C. for an hour or more.
_Wire Cages_ or crates are used for containing test-tubes, especially
when they are to be sterilised in the hot-air steriliser, or for
lowering tubes of nutrient jelly into the steam steriliser. All
instruments, needles, scalpels, &c., before using must be carefully
sterilised.
[Illustration: Fig. 258.--Dr. Koch’s Steam Steriliser.]
_Steam Sterilisers_ are made either of iron or tin, jacketed with thick
felt, and provided with a conical cap or lid perforated at the apex to
receive a thermometer (Fig. 258). Inside the vessel is an iron grating
or diaphragm about two-thirds of the way down, which divides the
interior into two chambers, the upper or steam chamber, and the lower
or water chamber. A gauge outside marks the level of the water in the
lower chamber; this should be kept about two-thirds full. The apparatus
stands upon three legs, and is heated from below with a Bunsen burner
or a lamp. It is employed for sterilising nutrient media in tubes or
flasks, for cooking potatoes or hastening the filtration of agar-agar.
When the thermometer indicates 100° C. the lid is removed, and
test-tubes are lowered in a wire-basket by means of a hook and string,
and the lid quickly replaced. Potatoes or small flasks are lowered into
the cylinder in a tin receiver with a perforated bottom, which rests
upon the grating, and admits of the contents being exposed to the steam
generated.
One of the most efficient forms of incubators introduced into the
bacteriological laboratory is that known as Hearson’s (Fig. 259).
This consists of a chamber surrounded by a water-jacket, with water
space below, to afford room for the pipe, L, which conveys the heated
products from the flame of the lamp, T, through the water and back
again to the lantern. A is the water-jacket surrounding the chamber
containing the cultures; O, the pipe through which the water supply is
admitted; N, the tap for employing the same; M, the overflow pipe; S,
the capsule in a case attached by a tube to the lower plate outside;
D, a lever pivoted on the left, carrying at its free end a damper, F,
which, when resting on the chimney, V, effectually closes it; P, a
screw for adjusting the damper when starting the apparatus; H, a lead
weight for bringing more pressure on the capsule; K, a thermometer, the
bulb of which is inside and the scale outside the chamber.
[Illustration: Fig. 259.--The Baird-Hearson Biological Incubator.]
The treated products of combustion move in the direction indicated
until the water and chamber are sufficiently heated to distend the
capsule. When this point is reached the wire between S and P is pushed
up by the capsule, and the lever causes the damper to rise more or
less off the chimney, V, and on examining the thermometer the inside
of the chamber is at length found to remain steadily at the required
temperature.
When the thermometer registers the desired temperature, the lead
weight must be damped to the lever by means of the milled-head screw
which goes through it. After having been once adjusted the heat in
the interior will remain constant, notwithstanding the utmost changes
of temperature occurring in these latitudes, nor will very great
alterations in the size of the lamp-flame seriously interfere with the
results. The milled-head screw, P, must be turned, after the first
adjustment, during the whole time that the incubator is in use. Observe
the temperature before opening the door; observations taken afterwards
are worthless.
Preparation of Nutrient Media--Separation, and Cultivation of Bacteria.
[Illustration: Fig. 260.--Plate Cultivation Showing Colonies.]
To cultivate micro-organisms artificially they must be supplied
with the proper nutrient material, perfectly free from pre-existing
organisms. The secret of Koch’s methods greatly depends upon the
possibility, in the case of starting with a mixture of micro-organisms,
of being able to isolate them completely one from another, and to
obtain an absolutely pure growth of each cultivable species. When
sterile nutrient gelatine has been liquefied in a test-tube and
inoculated with a mixture of bacteria in such a way that the individual
micro-organisms are distributed throughout it, and the liquid is poured
out on a glass plate and allowed to solidify, the individual bacteria,
instead of moving about freely as in a liquid medium, are fixed to
one spot, where they develop their own species. In this way colonies
are formed, each possessing its own biological characteristics and
morphological appearances (Fig. 260).
To maintain individuals isolated from each other during growth,
and free from contamination, it is only necessary to thin out the
cultivation to protect the plates from the air, and to have facilities
for examining them from time to time, and observing the characteristic
microscopical appearances. The colonies on nutrient gelatine examined
with a low power (Fig. 260), if micro-organisms such as _Bacillus
anthraces_ and _Proteus mirabilis_, the naked eye appearances in
test-tubes of the growth of the bacilli of anthrax and tubercle, and
the brilliant growth of micro-coccus prodigiosus, may be given as
examples in which the appearances are often very striking and sometimes
quite characteristic. I must, however, first direct attention to a
well-recognised fact, that bacteriology only touches animal pathology
at a few points, and that so far from bacteria being synonymous with
_disease germs_, the majority of these remarkable organisms appear
to be beneficent rather than inimical to man. This is of immense
importance to science, as I shall attempt to show further on; although
even a brief description of all the useful ferments due to bacteria and
brought into use would occupy a volume to themselves, and call for a
school of bacteriology quite apart from that involved in the medical
aspect of the question, for the purpose of fully investigating problems
raised by the agriculturist, the forester, the gardener, the dairyman,
brewer, dyer, tanner, and other industries, which open up vistas of
practical application, and to some extent are already being taken
advantage of in commerce.
_The Preparation of Nutrient Gelatine and Agar-agar._--Take half a
kilogramme (one pound) of beef as free as possible from fat, chop
finely, transfer to a flask or cylindrical vessel, and shake up well
with a litre of distilled water. Place the vessel in an ice-pail, or
ice-cupboard, or in winter in a cold cellar, and leave for the night.
Next morning commence with the preparation of all requisite apparatus.
Thoroughly wash and rinse with alcohol about 100 test-tubes, and allow
them to dry. Plug the mouths of the test-tubes with cotton-wool, place
them in their wire cages in the hot-air steriliser, to be heated for
an hour at a temperature of 150°C. In the same manner cleanse and
sterilise several flasks, and a small glass funnel. In the meantime,
the meat infusion must be well shaken, and the liquid portion separated
by filtering and squeezing through a linen cloth or a meat press. The
red juice thus obtained must be brought up to a litre by transferring
it to a large measuring glass and adding distilled water. It is then
poured into a sufficiently large and strong beaker, and set aside after
the addition of ten grammes of peptone, five grammes of common salt,
and 100 grammes of best gelatine.
In about half an hour the gelatine is sufficiently softened, and
subsequent heating in a water bath causes it to be completely dissolved.
The next process requires the greatest care and attention. Some
micro-organisms grow best in a slightly acid, others in a slightly
alkaline, medium. For example, for the growth and characteristic
appearances of the _comma bacillus_ of Asiatic cholera a faintly
alkaline soil is absolutely essential. This slightly alkaline medium
will be found to answer best for most micro-organisms, and may be
obtained as follows:--With a clean glass rod dipped in the mixture, the
reaction upon litmus-paper may be obtained, and a concentrated solution
of carbonate of soda must be added drop by drop until red litmus-paper
becomes faintly blue. If it is too alkaline, it can be neutralised by
the addition of lactic acid.
Finally, the mixture is heated for an hour in the water-bath. Ten
minutes before the boiling is completed the white of an egg beaten up
with the shell is added, and the liquid is then filtered while hot.
During filtration the funnel should be covered over with a plate of
glass, and the process of filtering must be repeated, if necessary,
until a pale straw-coloured, perfectly transparent filtrate results.
The sterilised test-tubes are filled to about a third of their depth
by pouring in the gelatine carefully and steadily. The object of this
care is to prevent the mixture touching the part of the tube with which
the plug comes into contact; otherwise, when the gelatine sets, the
cotton-wool adheres to the tubes and becomes a source of embarrassment
to subsequent procedures. As the tubes are filled they are placed
in a basket, and then sterilised. They are either lowered into the
steam steriliser, when the thermometer indicates 100 cc., for twelve
minutes, for four or five successive days, or they may be transferred
to the test-tube water-bath, and heated for an hour or two for three
successive days.
If the gelatine shows any turbidity after, it must be poured back into
a flask, boiled for ten minutes, and filtered again, and the process of
sterilisation repeated.
_Nutrient Agar-agar_ is a substance prepared from seaweed which grows
on the coasts of Japan and India, and is supplied in long crinkled
strips. It boils at 90° C., and remains solid up to a temperature
of about 45° C. It is therefore substituted for gelatine in the
preparation of a jelly for the cultivation of those bacteria which
will grow best in the incubator at the temperature of the blood, and
also at ordinary temperature for bacteria which lignify gelatine. The
preparation is conducted on much the same principles as those already
described. Instead, however, of 100 grammes of gelatine, only about
twenty grammes of agar-agar (1·5 to 2 per cent.), and to facilitate the
solution it must be allowed to soak in salt water overnight. Flannel
is substituted for filter paper. The hot-water apparatus is invariably
employed. The final results, when solid, should be colourless and
clear; but if slightly milky, it may still be employed.
_Wort-gelatine_ is used in studying the bacteria of fermentation. It is
made by adding from five to ten per cent. of gelatine to beer-wort.
_Glycerine Agar-agar._--This is made by adding five per cent. of
glycerine to nutrient agar-agar, after the boiling and before the
filtration.
[Illustration:
1 2 3 4 5 6
Yellow. Yellow. Red. Orange. Green. Brown.
Fig. 261.--Pure Cultivation in Tubes (Crookshank).]
_Test-tube Cultivations._--To inoculate test-tubes containing nutrient
jelly, the cotton-wool plug is removed. A sterilised needle, charged,
for example, with blood or pus containing bacteria, is thrust once in
the middle line into the nutrient jelly, and steadily withdrawn. The
tube should be held horizontally or with its mouth downwards, and the
plug replaced as quickly as possible, and an india-rubber cap fitted
over the mouth of the tube.
The appearance produced by the growths in the test-tubes can be in
most cases sufficiently examined with the naked eye (Fig. 261). In
some cases the jelly is partially liquefied, while in others it
remains solid. The growths may be abundant or scanty, coloured or
colourless. When liquefaction slowly takes place in the needle tracts,
the appearances which result are often very delicate and in some very
characteristic. The appearance of a simple white thread with branching
lateral filaments, of a cloudiness, or of a string of beads in the
track of the needle, may be given as examples. In some cases much may
be learnt by means of a magnifying-glass.
Beneke recommends that gelatine culture tubes should be inoculated by
making a puncture quite at the side of the medium, close to the glass.
The advantage of this method over the plan of inoculating the mass in
the middle is that the growing culture can be microscopically examined
from the outside, and various details made out, such as the nature of
the growth, the comparative appearance of colonies near the surface and
those situated more deeply, and the presence of one or more distinct
organisms. If the tubes used have the opposite sides flat and parallel,
such examinations will be still further facilitated.
_Plate Cultivations._--By this method a mixture of bacteria, whether
in fluids, excreta, or in cultivations on solid media, can be so
treated that the different species are isolated one from the other, and
perfectly pure cultivations of each of the cultivable bacteria in the
original mixture established in various nutrient media. We are enabled
also to examine under a low power of the microscope the individual
colonies of bacteria. The same process, with slight modification, is
also employed in the examination of air, soil, and water.
In order to spread out the liquid jelly evenly on the surface of a
glass plate, and to hasten its solidification, it is necessary to
place the plate upon a level and cool surface. The glass plates are
sterilised in an iron box placed in the hot-air steriliser, at 150° C.,
from one to two hours.
The damp chambers for the reception of the inoculated plates are
prepared by cleansing and washing out with one in twenty carbolic
acid the shallow glass dish and bell-cover (Fig. 253). A piece of
filter-paper should cover the bottom of dish, moistened with the same
solution.
“In a glass-beaker with pad of cotton-wool at bottom place tube
containing cultivation, the three tubes to be inoculated, three glass
rods which have to be sterilised, and a thermometer. Liquefy the
gelatine in the three tubes by placing them in a beaker containing
water 30° C. Keep the tubes, both before and after the inoculation, in
the warm water to maintain the gelatine in a state of liquefaction.
Remove the plug from the culture and also the plug of test-tube with
liquefied jelly. With the needle take up a droplet of the cultivation
and stir it round in the liquefied jelly. Replace both plugs, and
set aside the cultivation. Hold the freshly-inoculated tube almost
horizontally, then raise it to the vertical, so that the liquid
gelatine gently flows back. By repeating this motion, and rolling the
tube, the micro-organisms which have been introduced are distributed
throughout the gelatine. Any violent shaking, and consequent formation
of bubbles, must be carefully avoided. Inoculate the second tube, and
also third, in the same way, but with three droplets from a sterilised
needle. The next process consists in pouring out the gelatine on glass
plates and allowing it to solidify.
“Remove cover of box containing sterilised plates, withdraw a plate
with sterilised forceps, and rapidly transfer it to the filter-paper
under the bell-glass and quickly replace cover of box. Remove plug from
the test-tube which was first inoculated, and the contents are poured
out on the plate. With a glass rod the gelatine must be then rapidly
spread out in an even layer within about half an inch of the margin of
the plate, the bell-glass is replaced, and the gelatine is allowed to
set. Meanwhile a glass bench is placed in damp chamber, upon which the
plate is placed when the gelatine is quite solid; precisely the same
process is repeated with the other tubes.
“The colonies will be found to develop in the course of a day or two,
the time varying with the temperature of the room. The lower plate will
contain a countless number of colonies, which, if the micro-organisms
liquefy gelatine, speedily commingle, and produce in a very short time
a complete liquefaction of the whole gelatine. On the middle plate
the colonies will also be very numerous, but retain their isolated
positions for a longer time; while on the uppermost plate the colonies
are completely isolated from one another, with an appreciable surface
of gelatine intervening.
“The microscopical appearances of the colonies are best studied by
placing the plate on a slab of blackened glass, or on a porcelain
slab if the colonies are coloured. A small diaphragm is used, and
the appearances studied principally with a low power. A much simpler
method of plate-cultivation is to pour the liquefied jelly into shallow
flat dishes; they take up much less room, and in many ways are more
convenient.
“Nutrient agar-agar can also be employed for the preparation
of plate-cultivations, but it is much more difficult to obtain
satisfactory results.”
Microscopical Examination of Bacteria.
_Bacteria in Liquids, Cultures, and Fresh Tissues._--In conducting
bacteriological researches, the importance of absolute cleanliness
cannot be too strongly insisted upon. All instruments, glass vessels,
slides, and cover-glasses should be thoroughly cleansed before use. The
same applies to the preparation and employment of culture media; any
laxity in the processes of sterilisation, or insufficient attention to
minute technical details, will be followed with disappointing results
by contamination of the cultures, resulting in the loss of much time.
For the preparation of microscopical specimens it will be found
convenient to use a platinum inoculating needle, sterilised, as
before directed, in the sheet-iron box; in a few moments it will be
cool enough not to destroy the bacteria with which it is brought into
contact.
_Unstained Bacteria._--The bacteria in liquids, such as blood and
culture-fluids, can be investigated in the unstained condition by
transferring a drop with a looped platinum needle, or a capillary
pipette, to a slide, covering it with a clean cover-glass, and
examining without further treatment. If it is desirable to keep the
specimen under prolonged observation, a drop of sterilised water or
salt solution must be run in at the margin of the cover-glass to
counteract the tendency to dry.
Cultures on the solid media can be examined by transferring a small
portion with a sterilised needle to a drop of sterilised water on
a slide, thinning it out, and covering with cover-glass as already
described. Tissues in the fresh state may be teased out with needles
(Fig. 249) in sterilised salt solution, and pressed out into a
sufficiently thin layer between the slide and cover-glass. Glycerine
may in many cases be substituted for salt solution, especially for such
as actinomyces and mould fungi.
Very small bacilli and micro-cocci are distinguished from granular
matter or fat-crystals, or _vice versâ_, by the fact that the latter
are altered or dispersed by the addition of acetic acid, and changed
by solution of potash; ether dissolves out fatty particles, while
micro-organisms remain unaffected. Baumgarten demonstrated tubercle
bacilli in sections by treating them with potash, which clarified
the tissues and brought the bacilli clearly into view. In examining
unstained bacteria the iris-diaphragm should be used, and the sub-stage
condenser carefully centred and focussed.
His’s Method of Staining.--A slide is prepared as for bacteria in the
fresh state; the reagents are then applied by placing them with a
pipette drop by drop at a margin of the cover-glass, and causing them
to flow through the preparation by means of a strip of filter-paper
placed at the opposite margin.
Babès’ Method is as follows: A little of the growth spread out on a
cover-glass into as thin a film as possible; when almost dry, apply a
drop or two of a weak aqueous solution of methyl-violet from a pipette
to the film; any excess of the stain must be removed by gentle pressure
with a strip of filter-paper.
_Cover-glass Preparations._--A cover-glass is smeared with the
substance to be examined spread out into a sufficiently thin layer; in
the case of cultures on solid media, diffuse the bacteria in a little
sterilised water. By means of another cover-glass the juice or fluid is
squeezed out from between them into a thin layer, and on sliding them
apart each cover-glass bears on it a thin film of the material. The
cover-glass is then placed with its film side upwards and allowed to
dry. After a few minutes it is passed from above downwards through the
flame of a Bunsen burner three times. Apply two or three drops of an
aqueous solution of fuchsine or methyl-violet to cover the film, wash
away any surplus stain after a few minutes with distilled water. The
cover-glass is then allowed to dry, when the preparation may be mounted
in Canada balsam, or while still wet, turned over on a slide, and the
excess of water removed with filter-paper.
If necessary to apply stain for a much larger period, pour staining
solution into a watch glass and allow cover-glass to swim on surface
with prepared side downwards.
Crookshank, instead of watery solutions of aniline dyes, prefers to use
stronger solutions, and to reduce the staining by a momentary immersion
in alcohol. The method is as follows: cover-glass preparations are
stained with carbolised fuchsine (_Neelsen’s solution_) for about two
minutes, rinsed in alcohol for a few seconds, and quickly washed in
water. This method is specially valuable for sarcinæ and streptococci.
_Gram’s Method._--The whole film is first stained violet with
gentian-violet, fixed by a solution of iodine, in iodide of potassium
in the bacilli, but not in any débris, pus cells, or tissue elements
present. Transfer cover-glass to alcohol, the bacilli alone remain
stained, the violet colour being changed to blue. By employing a
contrast colour, such as eosin, a double staining is obtained.
For staining preparations with gentian-violet Crookshank employs the
following useful method:--Place four or five drops of pure aniline in a
test-tube, add distilled water to three-quarters full, close mouth with
thumb, shake thoroughly. Filter the emulsion twice, pour filtrate into
watch-glass. To the perfectly clear aniline water thus obtained, add,
drop by drop, a concentrated alcoholic solution of gentian-violet till
precipitation commences. Cover-glasses must be left in this solution
ten minutes, transferred to iodine-potassic-iodide until the film
becomes uniformly brown, then rinsed in alcohol. The decolourisation
may be hastened by dipping the cover-glass in clove oil and returning
to alcohol. Again immerse cover-glass in clove oil, dry by gently
pressing between two layers of filter-paper, and mount in Canada balsam.
_Double-staining_ of cover-glass preparations.--They can be treated
by Ehrlich’s method for staining tubercular sputum, or by Neelsen’s
modification, or by staining with eosin after treatment by the method
of Gram.
Ehrlich’s Method is as follows: Five parts of aniline oil are shaken
up with one hundred parts of distilled water, and the emulsion filtered
through moistened filter-paper. A saturated alcoholic solution of
fuchsine, methyl-violet, or gentian-violet, is added to filtrate in
watch-glass, drop by drop, until precipitation commences. Cover-glass
preparations are floated in this mixture for fifteen minutes to half an
hour, then washed for a few seconds in dilute nitric acid (one part of
nitric acid to two of water), then rinsed in distilled water.
Neelsen’s Solution and Methylene Blue.--Ziehl suggested the use of
carbolic acid as a substitute for aniline blue. Neelsen recommended
a solution of carbolic acid, absolute alcohol and fuchsine. (See
Appendix.)
_Gram’s Solution and Eosin._--After using Gram’s method as above and
decolourising in alcohol, the cover-glass is placed in a weak solution
of eosin for two or three minutes, washed in alcohol, immersed in clove
oil, dried, and mounted in balsam.
_Staining of Spores._--The cover-glass preparation must be heated to
210° C. for half an hour, or passed about twelve times through the
flame of a Bunsen burner, or exposed to the action of strong sulphuric
acid for several seconds, then a few drops of a watery solution of
aniline dye applied in the usual way. To double-stain spore-bearing
bacilli the cover-glass preparation must be floated from twenty minutes
to an hour on Ehrlich’s fuchsine-aniline-water, or on the Ziehl-Neelsen
solution. The stain must be heated until steam arises.
Staining of Flagella.
Koch first stained flagella by floating the cover-glass on a watery
solution of hæmatoxylin, transferring them to a five per cent. solution
of chromic acid, or to Müller’s fluid, by which they obtained a
brownish-black coloration.
_Löffler’s Method._--Add together aqueous solutions of ferrous-sulphate
and tannin (twenty per cent.) until the mixture turns a violet-black
colour, then add three or four cc. of a one-in-eight aqueous
solution of logwood; a few drops of carbolic acid may be added
before transferring to a stoppered bottle; that is the mordant. The
dye consists of 1 cc. of a one per cent. solution of caustic soda,
added to 100 cc. of aniline water, in which four or five grammes of
either methyl-violet, methylene blue, or fuchsine, are dissolved. A
cover-glass preparation is made in the usual way, then the film is
covered with mordant, and cover-glass held over flame until steam
rises, the mordant is then washed off with distilled water. The stain
is filtered and a few drops allowed to fall on film, after a few
minutes the cover-glass is again warmed until steam rises. The stain is
then washed off with distilled water, and the preparation is ready to
be mounted for examination.
As Löffler’s process is somewhat complicated, a modification has been
said to afford more satisfactory results. A specimen is taken from
a recent gelatine culture and diluted with water. A little of the
fluid is then transferred to a warm cover-glass by means of a pipette
and allowed to dry, after which a drop of the following mordant is
applied:--Aqueous solution of tannin (twenty per cent.), ten cc.; cold
saturated solution of ferrous sulphate, five cc.; saturated solution of
fuchsine in absolute alcohol, one cc. The cover is next heated gently
for a short time until vapours are given off, then washed carefully.
This process is repeated two or three times, and the specimen washed
after each application. Subsequently, staining is effected by means
of Ziehl’s fuchsine solution, the cover is afterwards warmed once or
twice for about fifteen seconds, then washed, and the specimen examined
in water to ascertain if the colour is sufficiently intense. If
satisfactory, the preparation may then be dried and finally mounted in
Canada balsam or dammar.
_Preservation of Preparations._--After examining a cover-glass
preparation with an oil-immersion objective the cedar oil must be
carefully wiped off, and the slide set aside for the Canada balsam to
set. At a convenient time these preparations should be sealed with a
ring of Hollis’s glue.
Bacteria in Sections of Tissues.
_Method of Hardening and Decalcifying Tissues._--To harden small
organs, such as the viscera of a mouse, they should be placed on a
piece of filter-paper at the bottom of a small wide-mouthed glass jar,
and covered with about twenty times their volume of absolute alcohol.
Larger organs are treated in the same way, but must be cut up into
small pieces. Müller’s fluid, methylated spirit, or formalin may be
used.
Teeth, or osseous structures, must first be placed in a decalcifying
solution, as Kleinenberg’s. When sufficiently softened, soak in water,
to wash out picric acid, and transfer to weak spirit. Ebner’s solution
gives good results.
Methods of embedding, fixing, and cutting.--Crookshank finds that
after hardening, the pieces of tissue are embedded in a mixture of
ether and alcohol for an hour or more, then transferred to a solution
of celloidin in equal parts of ether and alcohol, and left there for
several hours.
The piece of tissue is then placed in a glass capsule, and some of the
celloidin solution poured over it. The capsule can be placed bodily in
60 to 80 per cent. alcohol, and left there until the following morning.
The celloidin should be of the consistency of wax. The piece of tissue
is next cut out, and after trimming is put into water until it sinks,
then transferred to gum, and cut with the freezing microtome.
Sections of fresh tissues are to be floated in ·8 per cent. salt
solution, and then carefully transferred by a platinum lifter to a
watch-glass containing absolute alcohol.
_Staining Bacteria in Tissue Sections._--Weigert’s method is as
follows:--Place sections for from six to eighteen hours in a one per
cent. watery solution of any of the basic aniline dyes. To hasten,
place the capsule containing solution in the incubator, or heat it to
45° C., or a stronger solution may be used. In the latter case the
sections must be treated with a half-saturated solution of carbonate of
potash, as they are easily over-stained. In either case the sections
are next washed with distilled water, passed through sixty per cent.
alcohol into absolute alcohol. When almost decolourised, spread
out on a platinum lifter and transfer to clove oil, or stain with
picro-carmine solution (Weigert’s) for half an hour, wash in water,
alcohol, and treat with clove oil, and transfer to clean glass slide.
_Gram’s Method._--Sections are stained for ten minutes in a capsule
containing aniline-gentian-violet solution, then placed in the iodine
and iodide solution until uniformly brown, then placed in absolute
alcohol, and washed by carefully moving sections in the liquid with a
glass rod. When completely decolourised, they are transferred to clove
oil and then to a slide.
Double-staining is obtained by transferring the sections after
decolourisation to eosin, Bismarck brown, or vesuvin (Crookshank).
_Formalin_ is an excellent preservative fluid; one part to 20,000 is
sufficient to prevent fermentation. For the preservation of vegetable
sections, a one per cent. solution is required; even the fresh
appearance of vegetable structures is preserved for some time when
immersed in it. In the nutrient gelatine for biological specimens, if
used early, will arrest the liquefaction of the gelatine by bacteria.
For hardening it saves time, and is even better than alcohol, chromic
acid, pot. bich., and many others. It does not cause shrinkage of
the cells. Tissue 1/2 to 3/4 inch thick hardens in twenty-four hours
in pure formalin; five to ten per cent. is best for loose tissue. In
another method, by which time can be saved, instead of placing the
specimen in the _formalin_ and afterwards in mucilage, prior to cutting
sections, make the mucilage with two per cent. (or stronger) formalin
water, and it will then answer both purposes at the same time.
Preparing, Mounting, Cementing and Collecting Objects.
Various materials are required for preparing and mounting microscopic
objects, as slips of glass, patent flatted plate measuring 3 × 1 inch,
thin glass covers, glass cells, preservative media, varnishes, cements,
a glazier’s diamond, and a Shadbolt’s turn-table.
The glass slides and covers, although sent out packed ready for use,
should be immersed in an alkaline solution to ensure perfect freedom
from any greasiness derived from touching by the fingers. Dr. Seller
recommends a particular solution for this purpose. (See _Formulæ_,
Appendix.)
Varnishes and cements must be selected with care, as these are not only
expected to adhere firmly to the glass slide, but also to resist the
action of the preservative fluid in which the specimen may be mounted.
Among the numerous preparations employed, I may enumerate Canada
balsam, gum dammar, Venice turpentine, Japanners’ gold size, used for
closing up cells, asphalte varnish, Brunswick black, shellac, glue and
honey, Hollis’ liquid glue, and marine glue. To give a finish to the
mounted specimen, coloured varnishes are sometimes resorted to. A red
varnish of sealing-wax is made by digesting powdered sealing-wax in
strong alcohol. Filter, and place the solution in a dish, and evaporate
by means of a sand bath to reduce it to a proper consistency. This
is said to resist the action of cedar oil. For white, zinc, cement
is the best. This is made of benzole, gum dammar, oxide of zinc, and
turpentine. Cole gives another formula, but either of these may be
obtained of Squire, who supplies every kind of staining and mounting
material.
[Illustration: Fig. 262.--Walmsley’s Cell-making Turn-table.]
_Cells for Mounting._--The minuter forms of life should be mounted in
thin cells, which may be readily made with Japanners’ gold size, dammar
or asphalte, and a Shadbolt or Walmsley’s turntable. The glass slide
being placed under the metal springs in such a manner that its two
ends shall be equi-distant from the centre (a guide to the position is
afforded by the circles traced out on the brass), take a camel’s hair
pencil and dip it into the Japanner’s gold-size, holding it firmly
between the finger and thumb, and set the wheel in motion, when a
perfect circle will be formed; put it aside to dry, or place it in the
warm chamber to harden. To cut cover-glasses place a sheet of thin
glass under the brass springs, and substitute for the pencil a cutting
diamond. A cutting diamond is not only useful to the microscopist for
the above purpose, but also for writing the names of mounted objects on
one end of the slide.
It will be found convenient to make a number of such cells, and keep
a stock ready for use. There are many objects whose structure is very
transparent. These should be mounted dry. Scales from the wings of
butterflies and moths, of the podura and lepisma, and some of the
diatomaceæ are of this class. All that is necessary in preparing
objects for dry mounting is to take care that they are free from
extraneous matter, and fix them permanently in the position in which
their structure will show to the best advantage.
For mounting specimens of greater thickness it is desirable to use
deeper cells. It will then be found convenient to make a second or a
third application of the gold-size, allowing sufficient time between
applications for the varnish to dry. Cells of a still deeper kind
are made up by cementing rings of glass or metal to the glass-slides
with marine glue or Brunswick black. The latter will be rendered more
durable by mixing in a small quantity of indiarubber varnish (made
by dissolving small strips of caoutchouc in gas-tar). The process
of mounting in glass-cells is similar to that employed in making
varnish-cells, except that a somewhat larger quantity of cementing
medium is required. Objects mounted in this way should be kept for a
time in the horizontal position, and a little fresh varnish must be
applied if the cement shows a tendency to crack. In mounting objects
in balsam, care must be taken to have the specimen _quite dry_ before
transferring it to turpentine. Objects mounted in cells should become
_perfectly saturated_ with the mounting fluid before being finally
cemented down.
[Illustration: Fig. 263.--Glass-cells for Mounting.]
It is preferable to mount and preserve specimens of animal tissues
in shallow cells, to avoid undue pressure on the preparation. Cells
intended to contain preparations immersed in fluid must be made of
a substance impervious to the fluid used, such as here represented
(Fig. 263). The surface of the fixed glass-circle should be slightly
roughened before applying the cement.
Different modes of mounting may be employed with advantage; for
instance, entomological specimens, as legs, wings, spiracles, tracheæ,
ovipositors, stings, tongues, palates, corneæ, should be mounted in
balsam; the trachea of the house-cricket, however, should be mounted
dry. Sections of bone may either be mounted dry or in a fluid. Other
objects, as sections of wood and stones of fruit, exhibit their
structure best in Canada balsam.
In mounting entomological specimens, the first thing, of course,
is the dissection of the insect. This is best accomplished by the
aid of a dissecting microscope, a pair of small brass forceps, and
finely-pointed scissors; the parts to be prepared and mounted should
first be carefully detached from the insect with the scissors, then
immersed in a solution of caustic alkali (_liquor potassæ_) for a few
days, to soften and dissolve out the fat and soft parts. The length
of time necessary for their immersion can only be determined by
experience, but, as a general rule, the objects assume a certain amount
of transparency when they have been long enough in the alkali; when
this is ascertained, the object must be placed in a proper receptacle
and put by to soak for two or three hours in soft or distilled water.
It should then be placed between two slips of glass, and gently pressed
till the softer parts are removed. Should any adhere to the edge of
the object, it will be necessary to wash the specimen carefully in
water, a process that will be much assisted by the delicate touches
of a camel’s-hair brush. Place the object now and then under the
microscope to see that all extraneous matter is removed, and when this
is accomplished take the specimen up carefully with the camel’s-hair
brush, or a lifter, and place it on a piece of very smooth paper (thick
ivory note is the best for the purpose), arrange it carefully with the
brush and a finely pointed needle, place a second piece of paper over
it, and press it flat between two slips of glass, and compress it by a
small spring clip (Fig. 264). A dozen clips may be had for a few pence.
When _thoroughly_ dry (which it will probably be in about twenty-four
hours, if in a warm room), separate the glasses, and gently unfold the
paper; then, with a little careful manipulation, the object may be
readily detached, and placed in a little spirit of turpentine, where
it should be allowed to remain until rendered transparent and fit for
mounting. The time during which it should remain in this liquid will
depend on the structure; some objects, such as wings of flies, will be
quickly permeated, while horny and dense objects require an immersion
of a fortnight or even longer. A pomatum pot with a _concave_ bottom
and well-fitting lid will answer admirably for conducting the soaking
process in; and it is well, in preparing several specimens at a time,
to have two pots, one for large and medium, the other for very small
objects, otherwise the smaller will adhere to the larger.
[Illustration: Fig. 264.--Spring Clip for Mounting.]
In mounting objects in fluid, the glass cover should come nearly, but
_not quite_, to the edge of the cell, a slight margin being left for
the cement, which should project slightly over the edge of the cover,
in order to secure it to the cell.
_Media for Preserving Algæ._--The most useful preservative media for
algæ are chrome-alum, formalin, and camphor water. The solution should
consist of one per cent. of chrome-alum and one per cent. of formalin;
this will render the gelatinous sheath and matrix form clear, while it
will retain the colour of the algæ in most cases. The Chlorophyceæ do
well in any of these media; but other species, as _Ulva Lactuca_, are
rendered somewhat brittle. For such use formalin alone. The Phæophyceæ
should be placed while fresh in the formalin; the larger forms are
better fixed by placing them for an hour or two in chrome-alum
solution. The Florideæ do well in any of the three solutions, but the
more delicate species, _Griffithsia_, require a two per cent. formalin
solution in sea-water; the plant preserves its natural appearance in
this medium.
To preserve and mount diatomaceæ in as nearly as possible a natural
condition, they should be first well washed in distilled water and
mounted in a medium composed of one part of spirits of wine to seven
parts of distilled water. The siliceous coverings of the diatoms,
however, which show various beautiful forms under the higher powers
of the microscope, require more care in preparation. The guano, or
infusorial earth containing them, should first be washed several times
in water till the water is colourless, allowing sufficient time for
precipitation between each washing. The deposit must then be put into
a test tube and nitro-hydrochloric acid (equal parts of nitric and
hydrochloric acids) added to it, when a violent effervescence will
take place. When this has subsided, the whole should be subjected to
heat, brought nearly to the boiling point for six or eight hours. The
acid must now be carefully poured off, and the precipitate washed
in a _large_ quantity of water, allowing some three or four hours
between each washing, for the subsidence of some of the lighter forms.
The sediment must be examined under the microscope with an inch
object-glass, and the siliceous valves of the diatoms picked out with a
coarse hair or bristle.
Dr. Rezner’s Mechanical Finger (Fig. 265) for selecting and arranging
diatoms, adaptable to any microscope, is made to slip on to the
objective far enough to have a firm bearing, and so that the bristle
point can be brought into focus when depressed to its limit. It is
clamped in its place by a small thumb-screw. The bristle holder slides
into its place, and is carefully adjusted to the centre of the field.
When using the finger, the bristle is first raised by means of the
micrometer screw till so far within focus as to be nearly or quite
invisible, then the objective is focussed on to the slide, and the
desired object sought for and brought into the centre of the field;
the bristle point is then lowered by the screw until it reaches the
object, which usually adheres to it at once, and can then be examined
by rotating the bristle wire by means of the milled head.
[Illustration: Fig. 265.--Rezner’s Mechanical Finger.]
The medium used for mounting diatomaceæ is of considerable importance,
inasmuch as their visibility is either diminished or much increased
thereby. Professor Abbe, experimenting with the more minute test
objects, diatoms, &c., found monobromide of naphthaline gave increased
definition to most of them. This liquid is colourless, somewhat of an
oleaginous nature, and is soluble in alcohol. Its density is 1·555, and
refractive index 1·6. Its index of visibility is about twice that of
Canada balsam.
Taking the refractive index of air as 1·0, and diatomaceous silex as
1·43, the visibility may be expressed by the _difference_ ·43.
The following table may be constructed :--
Refractive indices Visibility of silex
(taken approximately). (Refr. index = 1·43).
Water = 1·33 10
Canada balsam = 1·54 11
Bisulphide of carbon = 1·68 25
Sol. of sulphur in bisulph. = 1·75 32
" phosphorus " = 2·11 67
These data relating to visibility must be taken in connection with the
numerical aperture of the objectives and of the illuminating pencil.
The effect produced on diatoms is very remarkable, the markings on
their siliceous frustules being visible under much lower powers.
So that the visibility of the diatom mounted in phosphorus as compared
with balsam is as sixty-seven to eleven; in other words, the image is
six times more visible. Mr. Stephenson’s phosphorus medium is composed
of a solution of solid or stick phosphorous dissolved in bisulphide of
carbon. Great care is required in preparing the solution owing to the
very inflammable nature of the materials. So small a quantity of the
bisulphide of carbon is required to dissolve the phosphorus that the
diatom may be said to be mounted in nearly pure phosphorus. Remarkable
enough, this medium has the reverse effect upon such test-objects as
podura and lepisma scales. These lose their characteristic markings.
For mounting minute objects, carbolic acid solution will be found a
useful medium--the purest crystals of carbolic acid dissolved in just
sufficient water to render them fluid. No more should be dissolved than
may be wanted for the time being, as if left standing exposed to the
light it changes colour. Small crustacean foraminifera, the palates of
moluscs, after boiling a short time in liquid potash and well washing
to remove all traces of alkali, may be preserved in carbolic acid
solution. Should the specimens appear cloudy gently warm the slide over
a spirit lamp.
_Preserving and Killing Rotatoria with cilia in situ._--Mr. C.
Rousselet’s method of preserving and mounting the Rotatoria[47] has
been attended with so much success that the old difficulty attendant
upon the preservation of these various beautiful forms of infusorial
life has been practically overcome. The process resorted to consists
of four stages, namely, narcotising, killing, fixing, and preserving.
In dealing with rotifers hitherto, the difficulty has been that of
successfully killing them with their rotating organs fully extended.
It has been found needful to have recourse in the first instance to
a narcotising agent, and one that acts slowly. The most suitable is
a weak solution of the hydrochlorate of cocaine, a one per cent.
solution, or even weaker. This was first proposed by Mr. Weber for
keeping these active little bodies quiet while under observation. Mr.
Rousselet carries this agent further; he applied it to narcotise them
prior to killing, and this it does most effectually. The rotifers are
seen to sink to the bottom of the live-cell, and the cilia gradually
to slacken in motion, and the time for killing has arrived. This is
effected by Flemming’s chromo-aceto-osmic acid. A rather weak solution
must be employed--consisting of 1 per cent. solution of chromic acid,
15 parts; 2 per cent. osmic acid, 4 parts; glacial acetic acid, 1
part--which is at the same time a killing and fixing medium. The word
“fixing” must not be taken to imply simply fixing, as it includes
rapidly _killing_ and _hardening_ and preventing further change in
the tissues of the rotifers by subsequent treatment, as mounting. The
animal, therefore, must remain quietly for a few minutes, and then
taken out and washed in five or six changes of distilled water, and
hence transferred to the preservative fluid. All this must be effected
with great care. The best preservative fluid is simply distilled water,
rendered antiseptic by a trace of the fixing solution (about eight
drops to an ounce of water) giving the slightest tinge of yellow to
the solution. This slight tinge of colour is imparted to the rotifers,
otherwise they remain transparent and unchanged, while the nervous
tissue throughout the body is brought out to perfection.
Some slight difference in treatment is required by certain species, as
that of _Asplanchna priodonta_; after the application of the cocaine
solution, which should be added slowly, that is, by letting a few
drops trickle down the side of the live-trough; this, being heavier
than water, sinks to the bottom, thus narcotising the rotifers, and
assisting to kill them with the cilia fully expanded. They should be
left quietly for fifteen minutes, then thoroughly washed with distilled
water. On further experimenting, Mr. Rousselet found that a weaker
solution of osmic acid alone, 1/4 per cent., answers quite as well
as, if not better than, Flemming’s fluid; even this must be allowed
to act for only a very short time--a minute at most; the rotifers
then remain white and transparent, excepting the ova, in which a
fat-like substance, _lecithene_, is secreted. If they become too much
stained, they may be decolourised by passing them through peroxide of
hydrogen. For narcotising the following solution has been found most
useful:--Take a 2 per cent. solution of cocaine hydrochlorate, 3 parts;
methylated spirit of wood naphtha, 1 part; and distilled water, 6
parts. This must be added as before directed, drop by drop, watching
the effect upon the rotifers under the microscope.
All the rotatoria may be killed and preserved in the same way. For
mounting, Mr. Rousselet prefers a slightly _hollowed-out_ glass cell,
the advantage of which is that the rotifers are kept to the centre,
and cannot move to the edge. A little difficulty at first presents
itself to exclude air-bubbles, but this, with a little care, can be
overcome by placing a drop of a two or three per cent. solution of
formalin, just sufficient to fill the cell. Then transfer the rotifers
with a dipping pipette to the cell, and lower the cover-glass down very
gently, removing any excess of fluid by blotting-paper. The best cement
for the cover-glass is gold-size.
_Method of Cementing._--After many years’ experience, I have arrived
at the conclusion that for cementing down the cover-glass there is
nothing better than either gold size or gum dammar varnish. The latter,
for some preparations, will be improved by the addition of a small
proportion of indiarubber dissolved in naphtha. (See Appendix.)
Should glycerine be preferred, carefully wash away any surplus quantity
by gently syringing; then apply a ring of waterproof cement round the
cover-glass. An inexpensive one can be made by dissolving ten grains of
gum-ammoniac in an ounce of acetic acid, and adding to this solution
two drachms of Cox’s gelatine. This liquid flows easily from the brush
and is waterproof, rendered more so if subsequently brushed over with
a solution of ten grains of bichromate of potash in an ounce of water.
An especial recommendation to this cement is its adhesiveness to glass,
even should there be a little glycerine left behind on the cover. After
the gelatine ring is thoroughly dry any kind of cement may be employed.
A useful cement for fixing minute objects, diatoms, &c., temporarily to
thin glass covers, before permanently mounting them in Canada balsam,
is made as follows:--Dissolve, without heat, two or three grains of gum
arabic in one ounce of distilled water, then add glacial acetic acid,
three minims, and the least trace of sugar. Filter carefully through
filter paper, and repeat this in the course of three or four weeks.
This cement will be unaffected by the balsam.
_Mounting Chara._--It is often found difficult to preserve and mount
the fruit of chara, but this can be successfully accomplished in
glycerine jelly, by taking the following precautions. After cleaning
the specimen place it in 92 per cent. of alcohol for several hours,
then transfer it to a mixture of equal parts of spirit and glycerine
for several hours longer, pour off nearly all the mixture, and add pure
glycerine at intervals till the glycerine becomes concentrated. The
specimen is then mounted in glycerine jelly in a cell just deep enough
to take it without pressure.
There are some objects much more difficult to prepare than others, and
which tax the patience of the beginner in a manner which can hardly
be imagined by any one who has never made the attempt. The structure
of many creatures is so delicate as to require the very greatest care
to prevent mutilation, and consequent spoliation, of the specimen.
The beginner, therefore, must not be discouraged by a few failures in
commencing, but should persevere in his attempts, and constant practice
will soon teach him the best way of managing intricate and difficult
objects. The room in which he operates should be free from dust, smoke,
and intrusion, and everything used should be kept scrupulously clean,
since a very small speck of dirt, which may be almost invisible to the
naked eye, will assume unpleasant proportions under the microscope, and
not only mar, but possibly spoil a fine and delicate preparation.
Few students on commencing to work with the microscope will fully
realise the fact that under medium or high powers the natural
appearance of almost all objects is changed by the refractive nature of
the fluid medium in which they are immersed and which enters more or
less into their composition. The remarkable changes effected by the law
of diffusion, when alkaloid substances enter into their composition,
show the necessity of taking every precaution in the employment of
preservative fluids. Glycerine affords an example of the chemical
change induced, should the preparation have been passed through an
alkaline solution.
_Air Bubbles_ are a constant source of annoyance both in preparing and
mounting. These may be removed from the specimen by gently warming the
under part of the slide over a spirit lamp, or placing the slide in
the warm chamber, when the bubbles will move towards the edge of the
cover-glass and ultimately disappear. The air-pump is preferred by many
microscopists.
Collection of Objects.
_Infusorial Life_, with all its fascinations, was fully unveiled to
naturalists by the celebrated Ehrenberg. It was he who termed it
infusorial, because he first met with the more interesting forms
of minute life in infusions of hay and other vegetable substances.
Since his day it is a well-known experience of those who take up the
microscope that the most interesting objects to commence with are
infusorial living creatures of sufficient dimensions to be easily
understood and seen with moderate magnifying powers. Moreover,
infusoria are more readily found in almost any pool or running stream
of water, either near the surface or clinging to the under surfaces
of aquatic plants. At one time all the small shallow pools in the
neighbourhood of London--Hampstead Heath, Clapham, Wandsworth, and
other commons--abounded in the most interesting forms of life, were
famous hunting grounds for the marvellous volvox, the charming dismid
and diatom, the wonderful budding and self-dividing hydra. A few hours’
ramble furnished the microscopist with a bountiful supply of these and
many other forms of life. Now all is changed; our commons have been
devoted to other purposes, and with the general _levelling_ up all the
little pools have disappeared, and the microscopist has been warned
off and driven further afield, or seeks the good offices of a country
friend for an occasional peep into pond life.[48]
A teaspoonful, however, judiciously taken from a well-chosen locality
will often be found to contain a variety of living forms, every one of
which will deserve a careful and patient study.
Of the microscopic organisms, the collection of which requires no
other methods than those ordinarily pursued by the naturalist, most
of them must be sought for in pools or running waters, basking in the
sunshine, clinging to leaves and rootlets of all aquatic plants; some
freely moving about, others clinging to stones or pieces of wood at the
bottom. Dismids congregate in shallow waters or rise to the surface
in a quiet nook, while the diatomaceæ are seen covering the bottom of
clear water, to which they give a yellowish-brown tinge of colour.
Infusorial animal life, as vorticellæ, stentors, rotifers, and
various polyzoa, cling, as also do hydra, in colonies to vallisneria,
duck-weed, frogbit, or small branches dipping down under water; and
if some of the water-weed is brought home the little creatures will
live and thrive for several weeks. No waters, however, are so full of
minute animal life as the sphagnum bog. A number of species of diatoms,
as well as protozoids and the smaller molluscs, will be found in all
peat bogs. It is remarkable, too, that the same species, everywhere,
are associated with this kind of moss. Lord Sidney Godolphin Osborn
supplied his friends with moss growing in a damp part of the garden
walk of his rectory; this always furnished the same species of
rotifers. These proved to be most interesting objects to my friends,
and in an early communication I described them as _indestructible_,
since they will bear any amount of desiccation; nevertheless, they were
revived when a drop of water was introduced into the glass-cell.
[Illustration: Fig. 266.--Collecting Stick, Bottle, Hook, and Net.]
The Thames mud always furnishes a number of beautiful forms of
triceratum. Lower down the river, as brackish water is reached, greater
varieties of diatoms appear. But to secure them the collector must be
provided with a collecting stick. A convenient form is furnished by
Messrs. Baker (Fig. 266). This consists of an ordinary walking-stick,
together with a lengthening rod, a cutting hook to clear away weeds,
ringed bottles with screw tops, and a net with a glass tube attached.
Their uses are too obvious to need further description.
The siliceous skeletons of diatoms are met with in the fossil state.
Among the first discovered of the infusorial strata were the polishing
slates of Bilin and Tripoli, the berg-mehl or mountain meal, the entire
mass of which is composed of the siliceous skeletons of different
species of diatoms. Richmond, Virginia, is rich in the same organisms,
while the great mass of our chalk cliffs are composed of foraminiferous
shells, xanthidiæ, &c. One remarkable fact in connection with fossil
infusoria is that most of the forms are still found in the recent
state. The beautiful engine-turned discs, _Coscinodisci_, so abundant
in the Richmond earth, may be met with in our own seas, and in great
profusion in the deposits of guano on the African and American coasts,
and in the stomachs of the oyster, scallop, and other salt-water
molluscous animals common to our shores.
A great number of infusorial earths may be mounted as dry objects,
while others require careful washing and digesting in appropriate
media. The finer portions of the sediments will be found to contain the
better and more perfect siliceous shells.
Preparing and Mounting Apparatus.
[Illustration: Fig. 267.--Mounting Apparatus.
1.--Ross’s instrument for cutting thin covering-glass for objects. This
apparatus consists of a bent arm supporting the cutting portion of this
apparatus, which consists of a vertical rod with a soft cork at one
end. A brass arm at right angles carries the diamond parallel with and
close to the main rod.
2.--Covering-glass measurer. To measure the thickness of
covering-glass, place it between the brass plate and the steel bearing;
the long end of the lever will then indicate the thickness on the
scale, to 1/50-th, 1/100-th, or 1/1000-th inch.
3.--Brass table on folding legs, with lamp for mounting objects.
4.--Whirling table with eccentric adjustment for making cells and
finishing off slides.
5.--Air-pump with glass receiver, 3-1/2-inch brass plate for mounting
objects and withdrawing air-bubbles.
6.--Improved table with knife for cutting soft sections. This consists
of an absolutely flat brass table, with a square hole to receive the
wood, or other matter, on a movable screw, which adjusts the thickness
of the section.
7.--Smith’s holder with spring and screw for adjusting pressure when
mounting objects.
8.--Cutting diamonds for cell-making and cutting slips of glass.
9.--Writing diamonds for cutting thin covering-glass and naming objects.
10.--Page’s wooden forceps, for holding glass slips or objects when
heated, during mounting.]
PART II.
CHAPTER I.
Microscopic Forms of Life--Thallophytes--Pteridophyta,
Phanerogamæ--Structure and Properties of the Cell.
The time has long since passed by since the value of the microscope
as an instrument of scientific research might have been called in
question. By its aid the foundation of mycology has been securely laid,
and cryptogamic botany in particular has, during the last quarter of
a century, made surprising progress in the hands of those devoted to
pursuits which confer benefits upon mankind.
Little more than thirty years ago practically nothing was known of
the life history of a fungus, nothing of parasitism, of infectious
diseases, or even of fermentation. Our knowledge of the physiology of
nutrition was in its infancy; even the significance of starches and
sugars in the green plant was as yet not understood, while a number
of the most important facts relating to plants and the physiology
of animals were unknown and undiscovered. When we reflect on these
matters, and remember that bacteria were regarded merely as curious
animalculæ, that rusts and smuts were supposed to be emanations of
diseased states, and that spontaneous generation still-survived among
us, some idea may be formed of the condition of cryptogamic botany and
the lower forms of animal life some eight or ten years after my book on
the microscope made its first appearance (1854).
Indeed, long prior to this time, dating from that of even the earliest
workers with the microscope, it was known that the water of pools and
ditches, and especially infusions of plants and animals of all kinds,
teem with living organisms, but it was not recognised definitely that
vast numbers of these microscopic living beings (and even actively
moving ones) are plants, growing on and in the various solid and liquid
matters examined, and as truly as visible and accepted plants grow on
soil and in the air and water. Perhaps the most important discovery
in the history of cryptogamic botany was initiated here. The change,
then, that has come over our knowledge of microscopic plant life during
this last busy quarter of a century has been almost entirely due to the
initiation and improvement, first in methods of growing them, and in
the methods of “_Microscopic Gardening_”; and secondly, to the greater
knowledge gained in the use of the microscope.
“If we look at the great groups of plants from a broad point of view,
it is remarkable that the fungi and the phanerogams occupy attention
on quite other grounds than do the algæ, mosses, and ferns. Algæ are
especially a physiologist’s group, employed in questions on nutrition,
reproduction, and cell division and growth; the Bryophyta and
Pteridophyta are, on the other hand, the domain of the morphologist.
Fungi and Phanerogams, while equally or even more employed by
specialists in morphology and physiology, appeal widely to general
interest on the ground of utility.
“It is very significant that a group like the fungi should have
attracted so much scientific attention, and aroused so general an
interest at the same time. But the fact that fungi affect our lives
directly has been driven home; and whether as poisons or foods,
destructive moulds or fermentation agents, parasitic mildews or
disease germs, they occupy more interest than all other cryptogams put
together, the flowering plants alone rivalling them in this respect.
A marked feature of the period in which we live will be the great
advances made in our knowledge of the uses of plants, for, of course,
this development of economic botany has gone hand in hand with the
progress of geological botany, the extension of our planting, and the
useful applications of botany to the processes of home industries.”[49]
The intimate organic structure of the vegetable world is seen to
consist of a variety of different materials indeterminable by
unassisted vision, and for the most part requiring high magnification
for their discrimination. Chemical analysis had, however, shown
that vegetables are composed of a few simple substances, water,
carbonic acid gas, oxygen, nitric acid, and a small portion of
inorganic salts. Out of these simple elements the whole of the
immense variety of substances produced by the vegetable kingdom are
constructed. No part of the plant contains fewer than three of these
universally distributed elements, hence the greater uniformity in
their chemical constituents. It will be seen, then, that the methods
of plant chemistry are of supreme interest both to the chemist and the
physiologist, or biologist. Plants, while they borrow materials from
the inorganic, and powers from the physical world, whereby they are
enabled to pass through the several stages of germination, growth, and
reproduction, could not accomplish these transformations without the
all-important aid of light and heat, the combined functions of which
are indispensable to the perfect development of the vegetable world.
Light, then, enables plants to decompose, change into living matter,
and consolidate, the inorganic elements of carbonic acid gas, water,
and ammonia, which are absorbed by the leaves and roots from the
atmosphere and earth; the quantity of carbon consolidated being exactly
in proportion to the intensity of the light. Nevertheless, light in
its chemical character is a deoxidising agent, by which the numerous
neutral compounds common to vegetables are formed. It is the principal
agent in preparing the food of plants, and it is during the chemical
changes spoken of that the specific heat of plants is slowly evolved,
which, though generally feeble, is sometimes very sensibly evolved,
especially so when flowers and fruits are forming, on account of the
increase of chemical energy at this period.
The action of heat is measurable throughout the whole course
of vegetable life, although its manifestations take on various
forms--those suited to the period and circumstances of growth. Upon
the heat generated depends the formation of protein and nitrogenous
substances, which abound more directly in the seed buds, the points of
the roots, and in all those organs of plants which are in the greatest
state of activity. The whole chemistry of plant life, in fact, is
manifest in this production of energy for drawing material from its
surroundings; therefore the organising power of plants bears a direct
ratio to the amount of light and heat acting upon them.
The living medium, then, which possesses the marvellous property of
being primarily aroused into life and energy, and which either forms
the whole or the greater portion of every plant, is in its earliest and
simplest form nothing more than a microscopic cell, consisting of one
or two colourless particles of matter, in closest contact, and wholly
immersed in a transparent substance somewhat resembling _albumen_
(white of egg), termed _protoplasm_, but differing essentially in its
character and properties. This nearly colourless organisable matter
is the life-blood of the cell. It is sufficiently viscid to maintain
its globular form, and under high powers is seen to have a slightly
consolidated film enclosing semi-transparent particles, together with
vacuoles which are of a highly refractive nature. These small bodies
are termed nuclei, and they appear to be furnished with an extremely
delicate enveloping film. In a short time the nuclei increase in
number and split up the parent body. The protoplasmic mass, however,
is undoubtedly the true formative material, and is rightly regarded as
“the physical basis of life” of both the vegetable and animal kingdoms.
There are, however, certain members of the vegetable kingdom which
somewhat resemble animals in their dependence upon receiving organic
compounds already formed for them, being themselves unable to effect
the fixation of the carbon needed to effect the first stage in their
after chemical transformations. Such is the case with a large class
of flowering plants, among Phanerogams, and the leafless parasites
which draw their support chiefly from the tissues of their hosts. It
is likewise the case with regard to the whole group of fungi; the
lower cryptogams, which derive the greater portion of their nutritive
materials from organic matter undergoing some form of histolysis;
while others belonging to this group have the power of originating
decomposition by a fermentative (_zymotic_) action peculiarly their
own. There are many other protophytes which live by absorption, and
which appear to take in no solid matter, but draw nourishment from the
atmosphere or the water in which they exist.
With regard to motion, this was at one time considered the distinctive
attribute of animal life, but many protophytes possess a spontaneity of
power and motion, while others are furnished with curious motile organs
termed _cilia_, or whip-like appendages, _flagella_, by which their
bodies are propelled with considerable force through the water in which
they live.
Henceforth this protoplasmic substance was destined to take an
important position in the physiological world. It is, then, desirable
to enter somewhat more fully into the life history of so remarkable
a body. It has a limiting membrane, composed of a substance somewhat
allied to starch, termed _cellulose_, one of the group of compounds
known as carbo-hydrates. The mode of formation and growth of this
cell wall is not yet definitely determined; nevertheless, it is the
universal framework or skeleton of the vegetable world, although it
appears to play no special part in their vital functions. It merely
serves the purpose of a protecting membrane to the globular body called
the “_primordial cell_,” which permanently constitutes the living
principle upon which the whole fundamental phenomena of growth and
reproduction depend.
Sometimes this protoplasmic material is seen to constitute the whole
plant; and so with regard to the simplest known forms of animal
life--the amœba, for example. That so simple and minute an organism
should be capable of independent motion is indeed surprising. Dujardin,
a French physiologist, termed this animated matter _sarcode_. On a
closer study of the numerous forms of animal life it was found that
all were alike composed of this sarcode substance, some apparently not
having a cell wall. The same seemed to hold good of certain higher
forms of cells, the colourless blood corpuscles for instance, which
under high powers of the microscope are seen to change their shape,
moving about by the streaming out of this sarcode. At length the truth
dawned on histologists that the cell contents, rather than the closing
wall, must be the essential structure. On further investigation it
became apparent that a far closer similarity existed between vegetables
and animals than was before supposed. Ultimately it was made clear that
the vegetable protoplasm and the animal sarcode were one and the same
structure. Max Schultz found this to be the case, and to all intents
and purposes they are identical.
We have now to retrace our steps and look somewhat more closely
into the discovery of that important body, the _cell-nucleus_. It
was an English botanist, Dr. Robert Brown, who, in 1833, during his
microscopical studies of the epidermis of orchids, discovered in their
cells “an opaque spot,” to which soon afterwards he gave the name
of _nucleus_. Schleiden and Schwann’s later researches led them to
the conclusion that the nucleus is the most characteristic formative
element in all vegetable and animal tissues in the incipient phase
of existence. It then began to be taught that there is one universal
principle of development for the elementary parts of all organisms,
however different, and that is the formation of cells. Thus was
enunciated a doctrine which was for all practical purposes absolutely
new, and which opened out a wide field of further investigation for the
physiologist, and led up to a fuller knowledge of the cell contents. In
fact, it became a question as to whether the cell contents rather than
the enclosing wall should not be considered the basis of life, since
the cell at this time had by no means lost its importance, although it
no longer signified the minute cavity it did when originally discovered
by Schwann. It now implied, as Schultz defined it, “a small mass of
viscid matter, protoplasm, endowed with the attributes of life.” The
nucleus was once more restored to its original importance, and with
even greater significance. In place of being a structure generated _de
novo_ from non-cellular substance, and disappearing as soon as its
function of cell formation is accomplished, the nucleus is now known as
the central permanent feature of every cell, and indestructible while
the cell lives, and the parent, by division of its substance, of other
generations of nuclei and cells. The word _cell_ has at the same time
received its final definition as “a small mass of protoplasm supplied
with a nucleus.” In short, all the activities of plant and animal life
are really the product of energy liberated solely through _histolysis_,
or destructive processes, amounting to the combustion that takes place
in the ultimate cells of the organisms.
But there are other points of especial interest involved in the
question of cell formation beside those already mentioned.
The cell and its contents collectively are termed the _endoplasm_,
or when coloured, as in algæ, _endochrome_. With regard to the outer
layer of the cell and its growth nothing satisfactory has been clearly
determined and finally accepted.
The cell as a whole is a protoplasmic mass, and not an emulsion, as
some observers would have us suppose. It is, in fact, a reticulated
tissue of the most delicate structure, made up of canaliculate spiral
fibrils with hyaline walls capable of expansion and contraction. These
fibrils are probably composed of still finer spirals. The visible
granulated portion of the protoplasm, the only part that takes a stain
under ordinary circumstances, is simply the contents of these canals.
It is the chromatin of Flemming, and is capable of motion within the
canals. The nucleus, then, is probably nothing more than a granule of
the extra-cellular net, and is formed by the junction of the several
bands of wall-threads which traverse it in different directions. The
cell wall of plants possesses the same structure as protoplasm, and is
probably protoplasm impregnated by cellulose.
It is this portion of the protoplasmic mass that is now recognised
under the term _octoplasm_, or primordial utricle, and is of so fine
and delicate a nature that it is only brought into view when separated
from the cell wall either by further developmental changes, or by
reagents and certain stains or dyes. It was, in fact, discovered to be
a slightly condensed portion of the protoplasmic layer corresponding
to the _octosare_ of the lower forms of animal life. The octoplasm
and cell wall can only be distinguished from each other by chemical
tests. Both nucleus and nucleoli are only rendered visible in the same
way, that is, by staining for several hours in a carmine solution, and
washing in a weak acetic acid solution.
With the enlargement of the cell by the imbibition of water, clear
spaces, termed vacuoles, are seen to occupy a small portion of the
cell, while the nucleus and nucleoli lie close to the parietal layer.
The interesting phenomenon of cyclosis, to which I shall have occasion
to refer further on, is now believed to be due to the contractility
of certain wall-threads stretching from the nucleus to the outermost
layers of the cell. An intimate relationship is thereby established
between the nucleus, the nucleolus, and the parietal layer. This much
has been made clear by the more scientific methods of investigation
pursued in the use of the microscope. Nevertheless a large and
important class of cells, forming a kind of borderland between the
vegetable and animal kingdoms, still remains to be dealt with, in which
the cell contents are only imperfectly differentiated, while numerous
other unicellular organisms, owing to their extreme minuteness,
tenuity, and want of all colour, are apparently devoid of any nucleus,
and when present can only be differentiated by a resort to a specially
conducted method of preparation and staining. There is, however, a
remarkable feature in connection with many micro-organisms--that
certain of these protophytes possess motive organs, cilia or flagella,
bodies at one time supposed to be characteristic of, and belonging to,
the protozoa.
This being the case, the methods of plant chemistry are of supreme
interest, the more so because physiologists are in a position to
isolate a single bacterial cell, grow it in certain media, and thus
devote special attention to it, and keep it for some time under
observation. In this way it has become possible to further grasp facts
in connection with cell nutrition and the nature of its waste products.
We have, then, arrived at a stage when the history of the chemical
changes brought about by bacteria can be more definitely determined, as
we have here to do with the vegetable cell in its simplest form. The
chemical work performed by these micro-organisms has as yet occupied
only a few years; nevertheless, the results have been of the most
remarkable and encouraging character.
At an earlier period an interesting discovery in connection with the
pathogenic action of these bodies was, by the labours of Schöenlein,
Robin, and others, brought to the notice of the medical profession,
viz., that certain diseases affecting the human body were due to
vegetable parasites. In 1856 an opportunity offered itself for a
thorough investigation, and the microscopical part of the work fell
into my hands, with the result that I was able to add considerably to
Schöenlein’s list of parasitic skin diseases. My observations were
in the first instance communicated to the medical journals. But the
generalisation arrived at was that “If there be any exceptions to
the law that parasites select for their sustenance the subjects of
debility and decay, such exceptions are rarely to be found among the
vegetations belonging to fungi, which invariably derive nutrition from
matter in a state of lowered vitality, passing into degeneration, or
wherein decomposition has already taken place to a certain extent....
It scarcely admits of a doubt that all diseases observed of late years
among plants have been due to parasites of the same class favoured by
want of vigour of growth and atmospheric conditions, and that the
cause of the various murrains of which so much has been heard is also
due to similar causes.”[50]
Herein, then, is to be found the solution of a difficulty that so
long surrounded the question, but which subsequently culminated in
the specialisation and scientific development of bacteriology, due to
the unceasing labours of Pasteur, whose solid genius enabled him to
overcome the prejudices of those who were at work on other lines, and
who had no conception of the functions that parasitic organisms fulfil
in nature.
Going back to my earlier experimental researches to determine the part
taken by saccharomycetes and saprophytes in fermentation, I find,
from correspondence in my possession, that in 1859 I demonstrated to
the satisfaction of Dr. Bell, F.R.S., the then head of the chemical
laboratory of Somerset House, that a very small portion of putrefactive
matter taken from an animal body, a parasitic fungus (_Achorion
Schöenleinii_), a mould (_Aspergillus_ or _Penicillium_), and a yeast
(_Torula cerevisiæ_) would in a short time, and indifferently, set up
a ferment in sweet-wort and transform its saccharine elements into
alcohol, differing only in degree (quantitative), and not in kind
or quality. This, then, was the first step in the direction towards
proving symbiotic action between these several parasitic organisms. The
only apparent difference observed during the fermentative processes was
that putrefactive (saprophytic) action commenced at a somewhat earlier
stage, and that the percentage of alcohol was also somewhat less.[51]
In 1856, also, the ærobic bacteria attracted my attention, and,
together with the late Rev. Lord Sidney Godolphin Osborne, I exposed
plates of glass (microscopical slides), covered with glycerine and
grape sugar, in every conceivable place where we thought it possible to
arrest micro-organisms. The result is known, viz., that fungoid bodies
(moulds and bacterial) were taken in great numbers, and varying with
the seasons. The air of the hospital and sick-room likewise engaged
attention, each of which proved especially rich in parasitic bodies.
During the cholera visitation of 1858 the air was rich in ærobic and
anærobic bacteria, while a _blue mist_ which prevailed throughout
the epidemic yielded a far greater number than at any former period
(represented in Plate I., No. 13). This blue mist attracted the
especial attention of meteorologists. At a somewhat later period a more
remarkable fungoid disease, the fungus foot of India, _mycetoma_, came
under my observation, a detailed description of which I contributed to
the medical journals, and also, with further details, to the “Monthly
Microscopical Journal” of 1871. Interlacing mycelia, ending in hyphæ,
in this destructive form of parasitic disease were seen to pervade the
whole of the tissues of the foot, the bony structures being involved,
and it was only possible to stay the action of the parasite by
amputation.
So far, then, the study of parasitic organisms had at an early period
shared largely in my microscopical work, extending over several years,
and with the result that these micro-organisms were found to exhibit on
occasions great diversity of character, and that different members of
the bacteria in particular flourish under great diversity of action,
and often under entirely opposite conditions; that they feed upon
wholly different materials, and perform an immense variety of chemical
work in the media in which they live.
The study of the chemistry (_chemotaxis_) of bacteria has, however,
greatly enlarged our conception of the chemical value and power of
the vegetable cell, while it is obvious that no more appropriate
or remunerative field of study could engage the attention of the
microscopist, as well as the chemist, than that of bacterial life, and
which is so well calculated to enlarge our views of created organisms,
whether belonging to the vegetable or animal kingdom.
Pathogenic Fungi and Moulds.
It is scarcely necessary to go back to the history of the parasitic
fungi to which diseases of various kinds were early attributable.
The rude microscopes of two and a half centuries ago revealed the
simple fact that all decomposable substances swarmed with countless
multitudes of organisms, invisible to ordinary vision. Leuwenhoek, the
father of microscopy, and whose researches were generally known and
accepted in 1675, tells of his discovery of extremely minute organisms
in rain-water, in vegetable infusions, in saliva, and in scrapings
from the teeth; further, he differentiated these living organisms
by their size and form, and illustrated them by means of woodcuts;
and there can be no doubt that his figures are intended to represent
leptothrix filaments, vibrios, and spirilla. In other of his writings
attempts are made to give an idea of the size of these “animalcules”;
he described them as _a thousand times smaller than a grain of sand_.
From his investigations a belief sprung up that malaria was produced
by “animalcules,” and that the plague which visited Toulon and
Marseilles in 1721 arose from a similar cause. Somewhat later on the
natural history of micro-organisms was more diligently studied, and
with increasing interest. Müller, in 1786, pointed out that they had
been too much given to occupy themselves in finding new organisms, he
therefore devoted himself to the study of their forms and biological
characters, and it was on such data he based a classification. Thus the
scientific knowledge gained of these minute bodies was considerably
advanced, and the subject now entered upon a new phase: the origin
of micro-organisms. It further resolved itself into two rival
theories--spontaneous generation, and development from pre-existing
germs--the discussion over which lasted more than a century. Indeed,
it only ended in 1871, when the originator of the Abiogenesis theory
withdrew from the contest, and the more scientific investigations of
Pasteur (1861) found general acceptance. This indefatigable worker
had been investigating fermentation, and studying the so-called
diseases of wines and a contagious disease which was committing ravages
among silkworms. Pasteur in time was able to confirm the belief that
the “muscadine disease” of silkworms was due to the presence of
micro-organisms, discernible only by the microscope. The oval, shining
bodies in the moth, worm, and eggs had been previously observed and
described by Nägeli and others, but it was reserved for Pasteur to show
that when a silkworm whose body contained these organisms was pounded
up in a mortar with water, and painted over the leaves of the tree upon
which healthy worms were fed, all took the disease and died.
[Illustration: PLATE IX.
AFTER D^R CROOKSHANK _J. T. Balcomb. del._
TYPICAL FORMS OF BACTERIA, SCHIZOMYCETES, OR FISSION-FUNGI.]
As the contagious particles were transmitted to the eggs, the method
adopted for preventing the spread of the disease was as follows:--Each
female moth was kept separate from the others, and allowed to deposit
her eggs, and after death her body was crushed up in a mortar as
before, and a drop of the fluid examined under the microscope. When
any trace of muscadine was found present, the whole of the eggs and
body were burnt. In this way the disease was combated, and ultimately
stamped out.
Pasteur also pointed out that one form or cause of disease must not
be confounded with another. For example, muscadine, a true fungus
(_Botrytis bassiana_), should not be confounded with another disease
known to attack silkworms, termed _pebrin_, this being caused by a
bacterium, and, according to the more recent researches of Balbiani, by
a Psorospermia. Botrytis is a true mould, belonging to the Oomycetes,
and allied to the potato fungus, Peronospora. It is propagated by
spores, which, falling on a silkworm, germinate and penetrate its body.
A mycelium is then developed, which spreads throughout the body. Hyphæ
appear through the skin, and bear white chalky-looking spores; these
become detached, and float in the air as an impalpable dust-like smoke.
Damp further develops the fungus.
Insects suffer much from the ravages of fungi. The house-fly sticking
to the window-pane is seen to be surrounded by the mycelia of
_Penicillium racemosum_ (_Sporendonema muscæ_, or _Saprolegnia feræ_).
In other cases Cordiceps attacks certain caterpillars belonging to the
genera Cossus and Hepialus when they are buried in the sand and before
their metamorphosis into chrysalides; they are killed by the rapid
development of hyphæ and mycelium in their tissues.
_Sphæria miletaris_, a parasite of _Bombyx pilyocarpa_, the caterpillar
of which is found on pine-trees, is one of the few fungi which may
be regarded as beneficial to man, since it aids in the destruction
of multitudes of these caterpillars, which otherwise would devour
the young shoots and pine needles. Giard specialises other parasites
of insects, which he terms Entomophoreæ. Others, _E. rimosa_, attack
grasshoppers and the diptera, enveloping them in a dense coating of
mycelium and spores, which speedily kills the victim.
The study, then, of the life-history of germs, microbes,
micro-organisms, or bacteria (as they are indifferently termed), opened
up a new science, that of Bacteriology. By the more recent advances
in this science we are enabled to understand the very important part
these minute organisms fill in the great scheme of Nature, for almost
exclusively by their agency the soil is supplied with the requisite
nutritive material for plant life. And, as already pointed out,
wherever organic matter is present--that is, the dead and useless
substances which are the refuse of life--such material is promptly
seized upon by micro-organisms, by means of which histolysis is rapidly
accomplished.
Bacteria require a power of from 600 to 1,000 diameters or more for
the determination of the species to which they belong. The number of
species has been so much increased of late that a bulky volume is
found to be insufficient for their enumeration. I am, however, by the
courtesy of Professor Crookshank, enabled to present my readers with
the typical forms of thirty-nine species of Bacteria, Schizomycetes, or
fission-fungi, a selection, it will be seen, chiefly taken from among
pathogenic organisms--those believed to originate disease. But many of
the supposed _Saprophytic_ forms often described as originating disease
are merely accidental associates, that is, living in companionship for
a time.
_Size._--In ordinary terms of measurement, bacteria are on an average
from 1/25000th to about 1/5000th of an inch long. These measurements
do not convey a definite impression to the mind. It is calculated that
a thousand million of them could be contained in a space of 1/25th of
an inch. The best impression of the size of the bacteria is, perhaps,
obtained when it is stated that a 1/25-inch immersion objective gives a
magnification of nearly 2,200 diameters, and that under this power the
bacteria appear to be about the size of very small print. The standard
of measurement accepted by bacteriologists is the micro-millimeter.
One millimeter is equal to about 1/25000th an English inch. The
number of micrococci in a milligramme of a culture of _Staphylococcus
pyogenes aurens_ has been estimated by Bujwid by counting at eight
thousand millions. Not only do various species differ in dimensions,
but considerable differences may be noted in a pure culture of the
same species. On the other hand, there are numerous species which so
closely resemble each other in size and shape that they cannot be
differentiated by microscopic examination alone, and we have to look
to other characteristics, as colour, growth in various culture media,
pathogenic power, chemical products, &c., in order to decide the
question of identity.
_Reproduction._--The reproduction of bacteria takes place for the
most part by fission and by spore formation. _Fission_ is a process
of splitting up or division, whereby an organism divides into two or
more parts, each of which lives and divides in its turn. If certain
organisms are watched under the microscope, a coccus or bacillus will
be seen to elongate and at the same time become narrower, until its
two halves become free, the two individual organisms again dividing
and subdividing in their turn. This kind of reproduction is more
readily seen in a higher class of unicellular organisms, the desmids.
If, however, the new organisms do not break away from each other, but
remain connected in groups or clusters, they are termed Staphylococci;
if they remain connected in the form of a chain, or like a string of
beads, they are termed Streptococci. If the division takes place in
one plane, Diplococci are formed; if in two directions Tetracocci, or
Tablet-cocci, are formed. On account of this multiplication by fission,
the generic name of Schizomycetes, or fission-fungi, has been given to
bacteria.
_Spores._--A second method by which bacteria propagate is by spores.
These bodies are distinguished by their remarkable power of resistance
to the influence of temperature and the action of chemical reagents.
Some of them will resist their immersion in strong acid solutions for
many hours; also freezing and very high temperatures. Spore formation
may take place in two ways: firstly, by “endogenous spores” (internal
spores); secondly, by “arthrospores.”
_Endogenous Spores._--When the formation of the spores takes place in
the mother-cell, the protoplasm is seen to contract, giving rise to one
or more highly refractive bodies, which are the spores. The enclosing
membrane of the organism then breaks away, leaving the spores free.
_Arthrospores._--When the spore is not formed in the parent bacillus,
but when entire cells (owing to lack of favourable conditions of
growth) become converted into spores, the formation is known as
“arthrogenous,” the single individual being called an arthrospore. When
the conditions are again favourable, spores germinate, giving rise to
new bacilli. The germinating spore becomes elongated, and loses its
bright appearance, the outer membrane becomes ruptured, and the young
bacillus is set free. Certain conditions, such as the presence of
oxygen in the case of the anthrax bacillus, give rise to the formation
of spores; while various kinds of bacteria secure continuous existence
by developing spores when there is lack of proper food material.
With reference to the incredible rapidity with which the bacteria
multiply under conditions favourable to the growth and development,
Cohn writes as follows:--“Let us assume that a microbe divides into
two within an hour, then again into eight in the third hour, and so
on. The number of microbes thus produced in twenty-four hours would
exceed sixteen and a half millions; in two days they would increase to
forty-seven trillions; and in a week the number expressing them would
be made up of fifty-one figures. At the end of twenty-four hours the
microbes descended from a single individual would occupy 1/40th of a
hollow cube, with edges 1/25th of an inch long, but at the end of the
following day would fill a space of twenty-seven cubic inches, and in
less than five days their volume would equal that of the entire ocean.”
Again, Cohn estimated that a single bacillus weighs about
0·000,000,000,024,243,672 of a grain; forty thousand millions, 1 grain;
289 billions, 1 pound. After twenty-four hours the descendants from a
single bacillus would weigh 1/2666th of a grain; after two days, over
a pound; after three days, sixteen and a half million pounds, or 7,366
tons. It is quite unneccessary to state that these figures are purely
theoretical, and could only be realised if there were no impediment to
such rapid increase.
Fortunately, however, various checks, such as lack of food and
unfavourable physical conditions, intervene to prevent unmanageable
multiplication of these bodies.
These figures show, however, what a tremendous vital activity
micro-organisms do possess, and it will be seen later at what great
speed they increase in water, milk, broth, and other suitable media.
The following bacilli, among others, have numerous flagella distributed
over the whole of the organism: the bacillus of blue milk (_Bacillus
cyanogenus_)[52]; the bacillus of malignant œdema; the hay bacillus
(_Bacillus subtilis_); _Proteus vulgaris_, &c.
The following have only one or two flagella at the poles: the _Bacillus
pyocyaneus_, the _Spirillum finkleri_, the _Spirillum choleræ
Asiaticæ_, &c.
The _Spirillum undala_, _Spirillum rubrum_, _Spirillum concentricum_,
and _Sarcinæ_, pocket-cocci, have several flagella.
_Micrococcus agilis_ have also several flagella; these possibly arise
from one point. As I have already pointed out, the _classification_
of the bacteria is one of great difficulty, since new kinds are
being constantly discovered, and at present any attempt made in this
direction can only be considered as quite of a provisional nature.
The difficulties which stand in the way may be surmised from the fact
that _Sarcinæ_, pocket-cocci, were originally believed to be a single
species, described by me, under the name of _Sarcina ventriculi_, in
the fourth edition of my book, “as remarkable bodies invading the human
and animal stomach, and seriously interfering with its functions.”
[Illustration: Fig. 268.--Sarcinæ.]
The original woodcut of these curious parasites is reproduced in
Fig. 268, also in Plate IX., No. 7, and which evidently belong to a
different species, numbering thirty-nine altogether. Quite recently
Mr. G. H. Broadbent, M.R.C.S., Manchester, sent me a supply of these
interesting bodies lately discovered by him in an infusion of cow
manure. On examining a drop with a power of 1500 diameters they were
discovered moving over the field of the microscope with a gyrating
motion by the aid of flagella projecting from each corner of the
pocket. After some days, having attained their full growth of four,
eight or sixteen in a pocket, they break up, and recommence the
formative process. Sarcinæ are certainly pathogenic in their nature.
Cocci in groups, or asso-cocci, are similarly associated. These several
forms of spiro-bacteria are enclosed in a transparent cell-wall, and
are sometimes described as zooglæa.
Of bacteria the most characteristic groups are bacillus, bacterium, and
a species of clostridium, a bottle-shaped bacillus. It is, however,
difficult to draw a sharp line between so-called _species_.
_Spiro-bacteria_, or _spirilla_, possess short or long filaments,
rigid or flexible, and their movements are accordingly rotatory, or in
the long axis of the filaments. These bodies are again divided into
comma bacilli, or vibrios--a name invented by the older microscopists
who first described them--some species of which have a flagellate
appendage, to which their movements are due.
=Anthrax=, =Splenic Fever=, has been long known to be prevalent among
cattle at certain seasons of the year, and is believed to originate
from peculiar conditions of climate and soil. This view of splenic
fever on microscopical examination proved an entire fallacy. Bollinger
in 1872 discovered that the blood of the affected animal was still
virulent after death, owing to the presence of the _spores_ of the
bacillus, and that the soil also became infected and impregnated by
the disease germs wherever the fever first broke out. In 1877 Dr. Koch
made a more careful investigation into the source of the disease, and
was able to give a complete demonstration of the life-history of the
splenic fever bacillus, and to offer definite proofs of its pathogenic
properties. He pointed out that the rods grew in the blood and tissues
by lengthening and by cross division. Further, that they not only grew
into long leptothrix filaments but they produced enormous numbers of
seeds or spores. He watched the fusion of the rods to the formation
of spores and the sprouting of fresh rods. He furthermore inoculated
a mouse, watched the effect through several generations, and fully
demonstrated that in the blood and swollen spleen of the animal the
same rods were always present. Pasteur and Paul Bret pursued the same
course of investigations, which were always followed with precisely
similar results. It was, however, principally due to the researches of
Koch that the doctrine of _contagium vivum_ was placed on a scientific
basis.
Subsequently Koch formulated methods of cultivation, and dictated the
microscopical apparatus needful. Furthermore, he furnished postulates
for proving beyond doubt the existence of specific pathogenic
micro-organisms.
“The chain of evidence regarded by Dr. Koch as essential for
proving the existence of a pathogenic organism is as follows:--1.
The micro-organism must be found in the blood, lymph, or diseased
tissue of man or animal suffering from, or dead of the disease. 2.
The micro-organism must be isolated from the blood or tissue, and
cultivated in suitable media--_i.e._, outside the animal body. These
pure cultivations must be carried on through successive generations of
the organism. 3. Pure cultivation thus obtained must, when introduced
into the body of a healthy animal, produce the disease in question. 4.
In the inoculated animal the same micro-organism must again be found.
The chain of evidence will be still more complete if, from artificial
culture, a chemical substance is obtained capable of producing the
disease quite independently of the living organism. It is not enough to
merely detect, or even artificially cultivate, a bacterium associated
with disease. An endeavour must be made to establish the exact
relationship of the bacteria to disease processes. In many instances
disease bacteria regarded as the actual contagia have been found, on
a further searching inquiry, to be entirely misleading. It is almost
needless to remind the enthusiast that the actual contagion of the
disease must be fully demonstrated.”
[Illustration: Fig. 269.--Micro-Photograph of Typhoid Fever Bacteria.
Magnified 1000 ×. Taken by Leitz’s oil immersion 1/12-inch ocular No.
4, and sunlight exposure of one minute.]
_Typhoid Bacillus_ (Fig. 269).--Rods 1 to 3µ in length, and ·5 to ·8µ
in breadth, and threads. Spore-formation has not been observed, but
the protoplasm may be broken up, producing appearances which may be
mistaken for spores. Actively motile, provided, some with a single and
others with very numerous flagella, which are from three to five times
as long as the bacillus itself. They stain readily in aqueous solutions
of aniline dyes; and grow rapidly at a temperature of about 60° Fahr.
In plate cultivations minute colonies are visible in thirty-six to
forty-eight hours; they are circular or oval, with an irregular margin.
On agar they form a whitish transparent layer, and they flourish in
milk.
[Illustration: Fig. 270.--Plague Bacillus, Bombay, 1897. Magnified 1200
×.]
_The Plague_ (_Pestis Bacillus_).--The Bombay plague of 1897-98 will
ever be remembered as one of the most appalling visitations ever
known. The number of deaths will never be accurately determined,
as the native population, among whom the disease chiefly prevailed
and became so fatal, concealed their dead or carried them away by
night. The outbreak from the first proved to be most infectious, its
incubation lasting from a few hours to a week only. It prevailed in all
the over-crowded native quarters of the city. The rats and mice that
infested the dwellings of the poor were found to be equally susceptible
with human beings, and these vermin also died by hundreds. Those that
survived left their holes and made off, in this way helping to spread
the infective virus. On examining the bodies of dead rats, they were
found to have swollen legs, the blood being filled by bacilli and
curious monads, with whip-like appendages. The bacillus of plague
was discovered by Kitasato in 1894; it is characterised by short
rods with rounded ends, and a clear space in the middle. The bacilli
stain readily with aniline dyes, and when cultivated on agar, white
transparent colonies are formed which present an iridescent appearance
when examined by reflected light. In addition to the bubonic swellings,
the neighbouring lymphatic glands were also swollen and blocked by
bacilli.
[Illustration: Fig. 271.--Monads in Rat’s Blood, 1,200 ×. (Crookshank.)
_a_. Monad threading its way among the blood-corpuscles; _b_. Another
with pendulum movement attached to a corpuscle; _c_. Angular forms;
_d_. Encysted forms; _e_ and _f_. The same seen edgeways.]
My illustration (Fig. 270) is from a micro-photograph taken in 1897,
when the death rate stood very high. The general distribution of the
bacilli, together with phagocytes and the contents of swollen lymphatic
glands, magnified 1,200 ×, is from a preparation made in hospital. The
monads from the rat’s blood, 1200 ×, seen threading their way among the
blood corpuscles of a rat, and represented in Fig. 271, are somewhat
larger than those found in the Bombay rats, but the flagella in the
latter were quite as marked, while the encysted forms were wholly
absent and the blood corpuscles less crenated. The white bodies (Fig.
270) were in some preparations, together with the lymphatic bodies,
more numerous and more swollen.
With regard to the conditions of life of the bacteria, they may
be divided broadly into two classes. When the organisms draw
their nourishment from some living body or “host,” they are known
as “parasites.” These are further termed “obligate” parasites if
they exclusively live on their “host.” If the bacteria draw their
nourishment from dead organic matter, they are called “saprophytes.”
These are also divided into “obligate” and “facultative” saprophytes.
Thus it will be apparent that a parasite under certain circumstances
may readily become a saprophyte.
Some of the more important saprophytes are those organisms which
play an important and useful part in our every-day life, such as,
for instance, in the phenomena associated with fermentation, and
putrefaction agents which transform dead and decomposing organic matter
into their simpler elements, thus completing the great life cycle,
and rendering the dead and effete matter again ready for the vital
processes.
Among other life manifestations of certain bacteria may be mentioned
those which have the property of generating colouring matter,
though not chlorophyll. The bacteria themselves are colourless and
transparent, and the pigment is merely formed as a product of their
metabolism, especially under the influence of light. Many of the
bacteria give rise to various gases and odours, particularly the
anærobic organisms, which originate those foul putrefactive gases
(ammonia, sulphuretted hydrogen, &c.). The blood-rain, _Micrococcus
prodigiosus_, gives off an odour resembling trimethylamin.
Micro-organisms have the property of producing various changes in the
medium on which they are grown. In many cases albuminous bodies are
peptonized and gelatine is liquefied. Many bacteria have the faculty of
resolving organic bodies into their simplest elements; others, again,
have the property of converting ammonia into nitric and nitrous acid.
Certain microbes have the property of becoming phosphorescent in the
dark. These phosphorescent bacteria are often seen on decaying plants
and wood; sometimes in tropical climates the sea becomes luminous owing
to the presence of countless numbers of these organisms. Again, they
are frequently seen on the surface of dead fish, particularly mackerel,
which often become so bright as to strongly illuminate the cupboard in
which they lie.
The particular class of fungi that produce disease in man and the
higher animals are generally known as “pathogenic.” These pathogenic
organisms may exert their pernicious power in several ways. They
may be injurious on account of their abstracting nourishment from
the blood or tissues, or for the purely mechanical reason of their
stopping up the minute capillaries and blood-vessels by their excessive
multiplication. But the poisonous action of most of the pathogenic
bacteria is due to the chemical products secreted by the organisms,
and it is to the circulation and absorption within the body of these
poisons that the disturbances of the animal system, which characterise
disease, decay, and dissolution of every organism, must be traced.
Parasitic Diseases of Plants.
The subject of fungoid diseases and fungus epidemics are of worldwide
interest, if only because of the annual losses to agriculturists from
parasitic diseases of plants, amounting to millions of pounds sterling.
The history of wheat-rust, and that of oats and rye, each equally
susceptible to the ravages of the same Rufus, can be traced back to
Genesis. A description of it was given in 1805 by Sir Joseph Banks. He
suggested that the germs entered the stomata, and he warned farmers
against the use of _rusted_ litter, and made important experiments on
the sowing of rusted wheat-grains. A great discussion on the barberry
question followed, Fries particularly insisting on the difference
between _Æcidium berberidis_ and _Puccinia graminis_. Tulasne confirmed
the statement made by Henslow that the uredo and puccinia stages belong
to the same fungus, and are not mixed species. De Bary’s investigations
in 1860-64 proved that the _sporidia_ of some Uredinieæ (_e.g._,
_Coleosporium_) will not infect the plant which bears the spores, and
that the æcidia of certain other forms are stages in the life-history
of species of Uromyces and Puccinia. Furthermore, De Bary in 1864
attacked the question of wheat rust, and by means of numerous sowings
of the telentospores on barberry proved that they bring about the
infection.
This led to the discovery of the phenomenon of _Heterœcism_
(colonisation), introducing a new idea, and clearing up many
difficulties. In 1890 the rust question entered on a new phase: it was
taken up by men of science all over the world, and active inquiries
were set on foot. The result has been the confirmation of De Bary’s
results, but with the further discovery that our four common cereals
are attacked by no less than ten different forms of rust belonging to
five separate species or “form species,” and with several physiological
varieties, capable of turning the table upon the barberry by infecting
it. Some of these are found to be strictly confined to one or other of
the four common cereals, infecting two or more of them, while others
can infect various kinds of our common wild grasses.
[Illustration: Fig. 272.--_Puccinia_, displaying _uredospores_ and
_telentospores_.
_a._ _Aregma speciosum_; _b._ _Xenodochus paradoxus_; _c._ _P.
Amorphæ_; _d._ _Triphœmium dubens_; _e._ Younger spores; _f._ _P.
lateripes_; magnified 450 diameters.]
The fact is, that what has usually gone by the name of _Puccinia
graminis_ is an aggregate of several species, and that varietal forms
of this exist so especially adapted to the host, that, although no
morphical differences can be detected between them, they cannot be
transferred from one cereal to another, pointing to physiological
variations of a kind met with among bacteria and yeasts, but hitherto
unsuspected in these higher parasitic fungi. It now appears we must be
prepared for similar specialisation of varietal forms among Ustilagineæ
as well as among Uredineæ.
Moreover, it has been found that different sorts of wheat, oats,
barley, and rye are susceptible to their particular rusts in different
degrees, at the bottom of which, it is suggested, there must be some
complex physiological causes. De Bary gave proof, in 1886, that Peziza
(Plate I., Nos. 1, 4, 5, 6) succeeds in becoming parasitic only after
_saprophytic_ culture to a strong mycelium, and that its form is
altered thereby--probably by the excretion of a poison. Professor
Marshall Ward showed that similar results took place in the case of the
lily disease. Reinhardt, in 1892, showed that the apical growth of a
peziza is disturbed and interrupted if the culture solution is employed
concentrated; and Büsgen, in 1893, showed that _Botrytis cinerea_
excretes poison at the tips of the hyphæ, thus confirming Professor
Ward’s results with the lily disease in 1888, and of later years, that
a similar excretion occurs in rust-fungus. He further found that the
water contents of the infected plant exercises an influence, as in the
case of _Botrytis_ attacking chrysanthemums and other plants in the
autumn, and that cold increases the germinating capacity of the spores.
Pfeiffer, in his work on “_Chemotaxis_,” shows that bacteria will
congregate in the neighbourhood of an algal cell evolving oxygen. He
also found that many motile antherozoids, zoospores, bacteria, &c.,
when free to move in a liquid, are attracted towards a point whence
a given chemical substance is diffusing. He was concerning himself
less with the evolution of oxygen or movements of bacteria than with
a fundamental question of stimulation to movement in general. He
found the attractive power of different chemical substances vary with
the organism, and that various other bodies beside oxygen attract
bacteria--peptone, dextrose, potassium salts, &c.; that swarm spores
of the fungus _Saprolegnia_ are powerfully attracted towards the
muscles of a fly’s leg placed in the water in which they are swimming
about; also, that in many cases where the hyphæ of fungi suddenly and
sharply bend out of their original course to enter the body of a plant
or animal, the cause of the bending lies in a powerful chemotropic
action, due to the attraction of some substance escaping from the body.
Professor Ward has seen zoospores of a _Pythium_ suddenly dart out on
to the cut surface of a bean-stem, and there fix themselves.
This will be better understood by referring to the course pursued by
these bodies generally. When the spore of a parasitic fungus settles
on a plant, it frequently behaves as follows:--The spore germinates
and forms a slender tube of delicate consistency, blunt at the end,
and containing colourless protoplasm, as shown, highly magnified in
Fig. 272, and in Figs. 273 and 274 much less magnified. De Bary long
ago showed that such a tube--the germinal-hypha--only grows for a
short time along the surface of the organ, and its tip soon bends down
and enters the plant, either through one of the stomata or by boring
its way directly through the cell-walls. Professor Ward says these
phenomena suggested to himself that the end of the tube is attracted in
some way, and by some force which brings its tip out of the previous
direction, and De Bary has suggested that this attraction is due to
some chemical substance excreted by the host plant. It is remarkable
with what ease the tube penetrates the cell-walls, and which Ward
believes to be due to the solvent action of an enzyme, capable of
dissolving cellulose.
“Miyoshi carried these observations a step further when, in 1894,
he showed that if a leaf is injected with a substance such as
ammonium-chloride, dextrine, or cane-sugar (all substances capable
of exerting chemotropic attraction on fungus-hyphæ), and spores of a
fungus which is _not parasitic_ are then sown on it, the hyphæ of the
fungus penetrate the stomata and behave exactly as if the fungus were a
true parasite.
“So surprising a result lets in a flood of light on many known cases
of fungi, which are, as a rule, _non_-parasitic, becoming so, in fact,
only when the host plant is in an abnormal condition, _e.g._, the entry
of species of Botrytis into living tissues when the weather is cold
and damp and the light dull; the entry of Mucor into various fruits,
tomatoes, apples, pears, &c., when the hyphæ meet with a slight crack
or wound, through which the juices are exposed. It is exceedingly
probable that the rapid infection of potato leaves in damp weather in
July is traceable not merely to the favouring effect of the moisture on
the fungus, but that the state of super-saturation of the cell-walls
of the potato leaf--the tissues of which are now unduly filled with
water and dissolved sugars, &c., owing to the dull light and diminished
transpiration--is the primary factor which determines the easy victory
of the parasite, and, as Professor Ward suggested some time ago, that
the suppressed life of Ustilagineæ in the stems of grasses is due to
the want of particular carbo-hydrates in the vegetative tissues, but
which are present in the grain. A year later Miyoshi carried proof to
demonstration, and showed that a fungus-hypha is actually so attracted
by substances on the other side of a membrane, and that its tip pierces
the latter; for the hyphæ were made to grow through films of artificial
cellulose, of collodion, of cellulose impregnated with paraffin, of
parchment paper, and even the chitinous coat of an insect, simply by
placing the intact films on gelatine impregnated with the attracting
substance, and laying the spores on the opposite side of the membrane.
“Now this is obviously a point of the highest importance in the theory
of parasitism and parasitic diseases, because it suggests at once
that in the varying conditions of the cells, the contents of which
are separated only by membranous walls from the fungus-hyphæ, whose
entrance means ruin and destruction, there may be found circumstances
which sometimes favour and sometimes disfavour the entrance of the
hyphæ; and it is, at least, a remarkable fact that some of the
substances which experiments prove to be highly attractive to such
hyphæ--_e.g._, sugars, the sap of plums, phosphates, nitrates, &c.--are
just the substances found in plants; and the discovery that the action
depends upon the nature of the substance as well as on the kind of
fungus, and is affected by its concentration, the temperature, and
other circumstances, only confirms us in this idea.”
Moreover, there is one other fact which it is important to notice,
viz., that there are substances which repel instead of attract the
hyphæ. Is it not, then, asks Professor Ward, natural to conclude
that the differences in behaviour of different parasites towards
different host-plants, and towards the same host-plant under different
conditions, probably depend on the chemotropic irritability of the
hyphæ towards the substance formed in the cells on the other side of
the membranous cell-walls? And when, as often happens, the effusion of
substances, such as the cells contain, to the exterior is facilitated
by over-distension and super-saturation, or by actual wounds, we cannot
be surprised at the consequences when a fungus, hitherto unable to
enter the plant, suddenly does so. To this proposition my answer is
emphatically in the affirmative, since in my investigations into the
“fungus-foot disease” (“_Mycetoma_”), 1871, of India, the entry of the
fungus was in almost every case shown to be through an abrasion of
the skin or a direct open wound; the majority of the cases reported
were among the agricultural classes. When, then, as often happens, the
effusion of substances, such as the cells contain, to the exterior
is facilitated by over-distension and super-saturation, or by actual
wounds, we cannot be surprised at the consequences when a fungus,
hitherto unable to enter the plant, suddenly does so. Nevertheless,
it must be admitted that the knowledge gained of parasites does not
satisfactorily account for epidemic visitations over large areas.
Habitat of Fungi and Moulds.
[Illustration: Fig. 273.--Fungi and Moulds.
Description of Figures.--_a._ Fungi Spores, taken in a sick chamber;
_b._ _Aspergillus glaucus_; _c._ Yeast, recent state; _d._ Exhausted
yeast, budding; _e._ Penicillium spores more highly magnified; _g._
Aerobic spores and mould mycelium; _h._ Aspergillus spore, grown on
melon.]
Habitat, Specialised Forms of Parasites.
_Habitat._--The habitat of vegetable parasitic fungi is extremely
variable. Fungi are found everywhere, living and flourishing on all
the families of the vegetable and animal kingdoms. They attack our
houses, foods, clothes, utensils of every kind, wall papers and
books, the paste of which, to my astonishment, affords a sufficient
supply of nourishment. Members of the parasitic tribe of bacteria, by
a combined effort of countless myriads, have given rise to a sense
of supernatural agency. _Bacillus prodigiosus_, described also as
_Palmella mirifica_ and _Zoogalactina imetropia_, from its attacking
milk and other alimentary substances, the spores of which are often of
a deep red colour, have been found to cover whole tracts of country
in a single night with what is called a “_gory dew_,” changing in
daylight to a deep green colour. This was at one time regarded with
superstitious awe as a miracle, as it has been known to attack bread
and even the sacred wafer, and which in mediæval ages was described as
the “bleeding-host.” This parasitic plant belongs to anærobic bacteria,
and is only developed in the dark. The nitrogen required for nutrition
must be derived from the air. An algal form gives rise to the red scum
seen in ponds and reservoirs in the autumn. The discharge from wounds
is coloured blue by _Bacterium pyocyanine_. There are many other forms,
some of which have an orange colour, and the genus is recognised as
“_chromogenic microbes_.”
[Illustration: Fig. 274.--Fungi and Moulds.
Description of Figures.--_d._ _Puccinia graminis_ on wheat; _c._
Polycystis spore of rye-smut; _f._ Alder fungus spores, _Microspheria
penicellula_; _g._ _Dactylium roseum_, rose-coloured mould; _h._
_Verticillium distans_, whorled mould found on herbaceous plants; _i._
_Botrytis_, vine and lily fungus; _j, j′._ _Peronospora infestans_,
potato fungus; _k._ _P. gangliformis_, mould of herbaceous plants; _l._
Various _Penicillium_ and other spores taken in a bean-field.]
A cryptogam belonging to anærobic bacteria, described as _Protococcus
invalis_, on being set aside in a bottle, and a little rain water
added, was seen to set up spontaneous fermentation, and in a very short
time exhibited remarkable activity. The colour of the infusion changed,
it assumed a delicate pink hue in direct light, which deepened to a
red in reflected light. The fluid contents were now observed to be
dichoric, and the spectroscopic appearance subsequently presented was
one of much interest. The spectrum was a well-marked one, and might be
taken to determine the presence of a nitrogenous element or of glucose.
Among all the various plants known to suffer from the attacks of
parasites, the vine has been the greatest sufferer. The oïdium, or
_Erysiphe Tuckeri_, so called from the name of the discoverer by whom
it was first described, has been longest known to the vine grower.
This really belongs to the group Ascomycetes, and appears to have been
brought from America in 1845, whence it was passed on to France, where
it soon threatened to entirely destroy the vineyards. This was followed
by another parasite, belonging in this instance to the animal kingdom,
_Phylloxera vastatrix_. This oïdium appears on the grape in the form of
greyish filaments, terminating in an enlarged head, which contains an
agglomeration of spores, not free or in a chaplet, as in Aspergillus
(Fig. 273). These spores when ripe burst from the capsule as fine dust,
and are diffused by the air in all directions, thus spreading the
disease far and away. Another of the parasitic moulds, _Peronospora
viticola_, is a kind of mildew, differing from oïdium. The hyphæ
penetrate more deeply than that of oïdium. On the upper surface of the
leaf brown patches appear; these branch out and ramify as seen in the
potato-fungus, _P. infestans_ (Fig. 274). The parasite destroys the
tissue of the leaf, and it withers and dies. There are other well-known
parasites, the black-rot, _Phomauvicola_, belonging to the Ascomycetes.
This appears in early shoots in the form of round black spots, and
gradually spreads over leaves and young fruit. This same rot, one year,
devastated the American vineyards.
[Illustration: Fig. 275.--Fungi, Moulds.
_a._ Clustered Spores, _Gonatobotrys simplex_; _b._ Spore of _Puccinia
coronata_, the mildew of grapes; _c._ Barley smut; _d._ _Puccinia
althæa_; _e._ _Penicillium glaucum_; _m._ _Ixodes farinæ_, found in
damaged flour together with smut.]
Cereals, wheats and grasses, suffer from other well-known forms of
microscopic fungi termed _rusts_ and _smuts_, which cover the blades
or infect the full ear of the fruit. The name given indicates their
colour, and these belong, for the most part, to the genus Uredo and
the family of the Basidiomycetes. They have no endogenous spores but
as many as four forms of exogenous. This is also the case with wheat
and barley, whereby they are distinguished as _Uredo_ or _Puccinia
graminis_ (see Figs. 273 and 274, and Plate I., Nos. 19 and 22,
_Æcidium berberidis_). For a long time it was believed that _Uredo
linearis_ and _Puccinia graminis_ were so many distinct species, but
it is now known that there are only three successive phases of the
developmental stages of a single species--that, as a matter of fact,
puccinia presents the phenomenon of alternation of generations, that
is, that the complete development of the fungus is only effected by
its transference from one plant to another. Other uredines, Ustilago
and Tilletia smuts, are more apt to affect the ears of wheat, rye, and
other grasses than puccinia. Bread made from wheat affected by smut
has an acrid and bitter taste, while that made from rye flour often
produces a serious form of disease. The propagation of either, then,
should be stopped as quickly as possible by destroying all barberry
bushes growing near or within the vicinity of corn fields, and by other
means. The ergot of rye is due to distinct species of fungi having
endogenous spores enclosed in a sac or _ascus_, hence the name of the
family, Ascomycetes or _Tuberaceæ_, which are reproduced by the spores
contained in these asci. Truffles belong to this family. But other
members of the same family have several forms of spores, and these
again present us with the phenomenon of alternation of generations.
[Illustration: Fig. 276.--Fungi, Moulds.
_p._ Spores of _Tilletia caries_; _q._ Spores of _Tilletia caries_,
when germinating, produce a fœtid olive-coloured spore in cereal
grains; _r._ Telentospores of _Puccinia graminis_; _s._ _Crystopus
candidus_, spores growing in chains; _t._ _Petronospora infestans_,
mildew of turnips, &c.; _u._ A transverse section of ergot of rye,
showing spores in masses; _v._ _Claviceps purpuræ_, associated with
ergoted rye.]
Ergot of rye is used in medicine, but if not used with care it will
produce a dangerous disease. This parasitic fungi consists of minute
microscopic masses of spores, which cover the young flower of the
rye with a white flocculent mass, formerly termed _sphacelium_. The
mycelium formed spreads over the ear of corn in thick felt-like masses,
termed _sclerotis_. The sphacelium changes its form in the following
spring. Other changes are brought about, and it seems to pass through a
cycle of alternations of generations.
Bread made from rye so infested is known to produce grave consequences,
soon to become fatal if not detected in time. The disease is termed
ergotism, and gangrene of the extremities takes place among people
of the north of France and Russia, who consume bread made from rye
flour. Ergot of maize will also cause similar diseases. Fowls and
other animals fed upon this cereal become in a short time poisoned,
and the cause of death is not rightly suspected. There is another
fungus belonging to the same group of Ascomycetes, known as _Eurotium
repens_, which appears upon leather when left in a damp place, and also
upon vegetable or animal substances if badly preserved, and gradually
destroys it. This mould is of a darkish green colour.
The minute spores display themselves as rows of beads when fully ripe
on the erect mycelium. _Aspergillus glaucus_ represents the white
exogenous spores of the sphacelium of the ergot of rye; and those
subsequently produced in the yellow balls correspond with the asci
developed in sclerotis, the endogenous species. Many of the parasitic
species belonging to the genera _Erysiphe_, _Sphæria_, _Sordaria_,
_Penicillium_, &c., have a similar mode of propagation, and affect a
large number of plants.
Parasitic Fungi of Men and Animals.
In the microscopical examinations especially given to the elucidation
of parasitic diseases of the skin, previously referred to, I discovered
more varieties of spores and filaments of certain cryptogamic plants
associated with a larger number of specific forms of fungi than any
previous observer. I did not, however, feel justified in concluding,
with Küchenmeister, Schœnlein, and Robin, that these fungoid growths
were the primary cause of the diseases referred to. Indeed, the
foremost dermatologists of the period utterly refused to entertain
the specific germ theory of the German investigators. Nevertheless, I
contended, “the universality of their distribution is in itself a fact
of very considerable importance, and one pointing to the belief that
they are scavengers ever ready to fasten on decaying matter, and, on
finding a suitable soil, spread out their invisible filaments in every
direction in so persistent a manner as to arrest growth and overwhelm
the plant in destruction.”[53]
Special forms of fungi are given in Plate I., Nos. 10-14, and those of
the ascomycetes in Nos. 17-21.
[Illustration: Fig. 277.--Healthy fresh Yeast, from a large Brewery, in
an active stage of formation, × 400.]
_Oïdium albicans_ affects both animals and plants. It often attacks
the mucous membrane of the mouths of young children. The spores become
elongated and converted into hyphæ, and ramify about in all directions,
producing a troublesome form of disease. This parasitic fungus is
better known under another name, _Saccharomyces mycoderma_. Oïdium
resemble algæ in their mode of life, as they are mostly found in a
liquid media. The structure of all ferments is very simple: each plant
is composed of a single cell, either of a spherical, elliptical, or
cylindrical form, varying in size, and filled with protoplasmic and
nucleated matter. This grows, and is seen to bud out and divide into
two or more parts, all resembling the mother cell.
Fig. 277 represents the healthy cells of yeast, _Saccharomyces
cerevisiæ_, freshly taken from a brewer’s vat, and in an active stage
of growth. The mode of multiplication continues as long as the plant
remains in a liquid favourable to its nutrition.
The changes from one stage to another are rapid, as will be noticed on
reference to the consecutive formative processes the cells are known to
pass through, Fig. 278 (1859).
If the development of the plant is arrested by want of a saccharine
or nitrogenous substance, and the liquid dries up, the protoplasm
contained in the cell contracts, and the spores, or endogenous
reproductive organs, of the plant will remain in a state of rest,
become perfectly dry, and yet retain life. They are not easily killed,
even when subjected to a very high or low temperature, they do not lose
the power of germination when favourable conditions present themselves,
and at once take on a new birth.
There are, however, many other ferments besides that of beer-yeasts,
such as alcoholic and wine ferments, the commonest of which, according
to Pasteur, is _Saccharomyces ellipsoideus_.
[Illustration: Fig. 278.--Development of Yeast Cells.
1. When first taken; 2. One hour after introducing a few cells into
sweet-wort; 3. Three hours after; 4. Eight hours; 5. Forty-eight hours,
when the cells become elongated.]
But yeast-fungi and mould-fungi, like bacteria or fission-fungi, are
micro-organisms, belonging to two specific orders, the Saccharomycetes
and the Hyphomycetes, which are intimately related to each other, but
quite distinct from bacteria. Their germs occur widely distributed in
air, soil, and water. Many species are of hygienic, while others are
of pathological interest and importance in being either accidentally
associated with, or the cause of, disease processes, while others
are fermentations of very essential service in various industrial
processes. The making of beers, wines, and spirits, as we understand
them, constitutes but a small part of the province of fermentation.
The life activities of ferments open out a study of vast importance
to mankind, and while they have only been regarded in their worst
aspect--that of a bane--they are, nevertheless, a boon to mankind.
The first clear view we obtained of this was that of Reess, who in
1870 showed there were several species or forms of the yeast-fungus.
Hansen followed up this discovery in 1883, and, taking advantage of the
strict methods of culture introduced by bacteriologists, found that by
cultivating yeast on a solid media from a single spore it was quite
possible to obtain constant types of pure yeasts, each possessing its
own peculiar properties. One consequence of Hansen’s labours was that
it now became possible for every brewer to work with a yeast of uniform
type instead of with haphazard mixtures, in which serious disease
forms might predominate and injure the beer. Among other things made
clear was that a true yeast may have a mycelial stage of development.
Furthermore, there is the influence exercised by the nucleus of
the yeast cell. Many other points of interest arose out of these
investigations; one was, that many higher fungi can assume a yeast-like
stage of development if submerged in fluids, as, for instance, various
species of Mucor, Ustilago, Exoascus, and numerous others. Ascomycetes,
and Basidiomycetes as well, are known to form budding cells, and it was
thought that the yeasts of alcoholic fermentation are merely reduced
forms of these higher fungi, which have become habituated to the
budding condition--a conclusion supported by Hansen’s discovery that a
true Saccharomyces can develop a feeble, but a true, mycelium.
[Illustration: Fig. 279.--Saccharomyces and Moulds.
1. Section from a tomato, showing spores growing from cuticle; 2.
Portion detached to show budding-out process; 3. Lateral view of spore
sac with oospores issuing forth; 4. Apiculated ferment spores; 6 and 7.
_Mycoderma cerivisiæ_ in different stages of growth, as seen on wine
bottles; 8 and 9. _Torulæ diabeticæ_, torulæ and fission spores.]
“This view has been entirely confirmed by an inquiry into the mode of
brewing _saké_ by the Japanese, by the aid of the Aspergillus fungus.
Further researches established the fact that other forms of fungi,
_e.g._, those on the surface of fruits, developed endogenous spores,
which cause alcoholic fermentation. More recently, and by further
experimental inquiry, partly by pure cultures of separate forms, and
partly by well-devised cultures on ripening fruits still attached to
the plant but imprisoned in sterilised glass vessels, it has been found
that yeast and moulds are separate forms, not genetically connected,
but merely associated in nature, as are so many other forms of yeasts,
bacteria, and moulds. Further, Hansen has discovered that several
yeasts furnish quite distinct races or varieties in different breweries
in various parts of the world, so that we cannot avoid the conclusion
that their race characteristics have been impressed on the cells by the
continued action of the conditions of culture to which they have so
long been exposed--they are, in fact, domesticated races.”
The environments of yeasts are peculiar. Sauer found that a given
variety of yeast, whose activity is normally inhibited when the
alcohol attains a certain degree of concentration in the liquid, can
be induced to go on fermenting until a higher degree is attained by
the addition of a certain lactic acid bacterium. The latter, indeed,
appears to prepare the way for the yeast. It has been shown, also, that
damage may be done to beers and wines by allowing plant germs to gain
access with the yeast; there are, too, several forms of yeast that are
inimical to the action of the required fermentation. Other researches
show that associated yeasts may ferment better than any single yeast,
and such symbiotic action of two yeasts of high fermenting power has
given better results than either alone. English ginger-beer furnishes a
curious symbiotic association of two organisms--a true yeast and a true
bacterium--so closely united that the yeast cells become imprisoned
in the gelatinous meshes of the bacterium; and it is a curious fact
that this symbiotic union of yeast and bacterium ferments is far more
energetic than either when used alone, and the product is different,
large quantities of lactic and carbonic acids being formed, and little
or no alcohol.
Many years ago I gave an account of similar curious symbiotic results
obtained by introducing into a wort-infusion a small proportion of
_German yeast_, an artificial product composed of honey, malt, and a
certain proportion of spontaneously-fermented wheat flour. This, to
my astonishment, produced ten per cent. more alcohol than any of its
congeners, and did not so soon exhaust itself as brewer’s yeast.[54]
In the hephir used in Europe for fermenting milk, another symbiotic
association of yeast and a bacterium, it is seen that in this process
no less than four distinct organisms are concerned. I have already
instanced the fermentation of rice to produce saké, which is first
acted upon by an Aspergillus that converts the starch into sugar and
an associated yeast, and this is also shown to be a distinct fungus,
symbiotically associated in the conversion. “Starting, then, from
the fact that the constitution of the medium profoundly affects the
physiological action of the fungus, there can be nothing surprising
in the discovery that the fungus is more active in a medium which
has been favourably altered by an associated organism, whether the
latter aids the fungus by directly altering the medium, or by ridding
it of products of excretion, or by adding gaseous or other body. It
is not difficult to see, then, that natural selection will aid in
the perpetuation of the symbiosis, and in cases like that of the
ginger-beer plant it is extremely difficult to get the two organisms
apart, a difficulty similar to that in the case of the soredia of
lichens.”
Buchner discovered that by means of extreme pressure a something
can be extracted from yeast which at once decomposes sugar into
alcohol and carbon-dioxide. This something is regarded as a kind of
incomplete protoplasm--a body, as we have already seen, composed of
proteid--and in a structural condition somewhere between that of true
soluble enzymes like invertin and a complete living protoplasm. This
reminds me of an older experiment of mine, the immediate conversion
of cane-sugar into grape-sugar. If we take two parts of white sugar
and rub it up in a mortar with one part of a perfectly dry solid, the
German yeast before spoken of, it is immediately transformed as if by
magic into a flowing liquid mass--a syrup. This process of forming
“invert sugar” can be watched under the microscope; the liberation of
carbonic acid gas in large bubbles is seen to go on simultaneously with
the assimilation of the dextrose, and the breaking up of the crystals
of sugar; the cell at the same time increasing in size as well as in
refractive power; a curious state of activity appears to be going on in
the small mass, which is very interesting to watch throughout.
However, the enzymes of Buchner are probably bits off the protoplasm,
as it were, and so the essentials of the theory of fermentation remain,
the immediate agent being not that of protoplasm itself, but of
something made by or broken off from it. Enzymes, or similar bodies,
are known to be very common in plants, and the suspicion that fungi do
much work with their aid is abundantly confirmed. It seems, indeed,
that there are a whole series of these bodies which have the power of
carrying over oxygen to other bodies, and so bringing about oxidations
of a peculiar character. These curious enzymes were first observed
owing to studies on the changes which wine and plant juice undergo when
exposed to the action of the oxygen of the air.
The browning of cut apples is known to be due to the action of an
oxydase, that is, an oxygen carrying ferment, and the same is claimed
for the deep colouring of certain lacs obtained from the juice of
plants, such as Anacardiaceæ, which are pale and transparent when
fresh drawn, but which gradually darken in colour on exposure to the
air. Oxydases have been isolated from beets, dahlia, potato-tubers,
and several other plants. This fact explains a phenomenon known to
botanists, and partly explained by Schönbein as far back as 1868, that
if certain fungi (_e.g._, _Boletus beridies_) are broken or bruised,
the yellow or white flesh at once turns blue; this action is now traced
to the presence in the cell sap of an oxydase.
It is the diastatic activity of Aspergillus which is utilised in the
making of saké from rice, and in the preparation of soy from the soja
bean in Japan. Katz has recently tested the diastatic activity of
Aspergillus, of Penicillium, and of _Bacterium megatherium_, in the
presence of large and small quantities of sugar, and found all are able
to produce not only diastase, but also other enzymes; as the sugar
accumulates the diastase formed diminishes, whereas the accumulation of
other carbo-hydrates produces no such effect. Harting’s investigation
on the destruction of timber by fungi derives new interest from the
discovery of an emulsion-like enzyme in many such wood-destroying
forms, which splits up glucosides, amygdalin, and other substances into
sugar, and that hyphæ feed on other carbo-hydrates. The fact, also,
that Aspergillus can form inverts of the sucrase and maltase types,
as well as emulsin, inulate, and diastase, according to circumstances
of nutrition, will explain why this fungus can grow on almost any
organic substance it may happen to alight upon. The secretion of
special enzymes by fungi has a further interest just now, for recent
investigations promise to bring us much nearer to an understanding of
the phenomena of parasitism than it was possible when I was at work
upon them some forty or fifty years ago.
It was De Bary who impelled botanists to abandon older methods, and he
who laid the foundation of modern mycology. Later on he pointed out
that when the infecting germinal tube of a fungus enters a plant-cell,
two phenomena must be taken into account, the penetration of the
cell-walls and tissues, and the attraction which causes the tips of
the growing hypha to face and penetrate these obstacles, instead of
gliding over them in the lines of apparent least resistance. The
further development of these two factors shows that in the successful
attack of a parasitic plant on its victim or host these fungi can
excrete cellulose-dissolving enzymes, and that they have the power of
destroying lignine. Zopf has also furnished examples of fungi which
can consume fats. There is, however, one other connection in which
these observations on enzymes in the plant-cell promise to be of
considerable importance, viz., the remarkable action of certain rays of
the solar light on bacteria. It has been known for some time past that
if bacteria in a nutrient liquid are exposed to sunlight they quickly
die. The further researches of Professor Marshall Ward and other
workers in the same direction have brought out the fact that it is
really the light rays, and not high temperatures, that it is especially
the blue-violet and ultra-violet rays, which exert the most effective
bactericidal action. This proof depended upon the production of actual
photographs in bacteria of the spectrum itself. Apart from this, the
Professor demonstrated that just such spores as those of anthrax, at
the same time pathogenic and highly resistant to heat, succumb soonest
to the action of these cold light-rays, and that under conditions
which preclude their being poisoned by a liquid bath. It is in all
probability the action of these rays of light upon the enzymes, which
abound in the bacterial cells, that bring about their death.
The sun, then, is seen to be our most powerful scavenger, and this
apparently receives confirmation in connection with Martinaud’s
observations, that the yeasts necessary for wine-making are deficient
in numbers and power on grapes exposed to intense light, and to this is
due that better results are obtained in central France as contrasted
with those in the south. “When we reflect, then, that the nature of
parasitic fungi, the actual demonstration of infection by a fungus
spore, the transmission of germs by water and air, the meaning and
significance of polymorphism, heteræcism, symbiosis, had already been
rendered clear in the case of fungi, and that it was by these studies
in fermentation, and in the life-history of the fungus Saccharomyces,
that the way was prepared for the ætiology of bacterial diseases in
animals, there should be no doubt as to the mutual bearings of these
matters.”
Industrial uses of Fungi and Saccharomycetes.
There are many industrial processes which are more or less
dependent for success on bacterial fermentations. The subject is
young, but the results already obtained are seen to be of immense
importance from a scientific point of view, and to open up vistas of
practical application already being taken advantage of in commerce,
while problems are continually being raised by the forester, the
agriculturist, the gardener, the dairyman, the brewer, dyer, tanner,
and with regard to various industries, which will eventually confer
great advantages in their economic application.
The remarkable discovery made by Alvarez of the bacillus, which
converts a sterilised decoction of the indigo plant into indigo sugar
and indigo white, the latter then oxidising to form the valuable
blue dye, whereas the sterile decoction itself, even in presence of
oxygen, forms no indigo, plainly proves how these minute organisms
may be turned to a good account. There are, however, important points
to be determined as to the action of the fermentation brought about
by these enzymes, and the appearance of certain mysterious diseases
in the indigo vats. Again, certain stages in the preparation of tea
and tobacco leaves are found to depend upon very carefully regulated
fermentations, which must be stopped at the right moment, or the
product will be spoilt. Regarding the possible _rôle_ of bacteria,
the West Indian tobacco has a special bacterium, which has been
isolated and found to play a very important part in its flavour.
Every botanist knows that flax and hemp are the best fibres of Linum
and Cannabis respectively, separated by steeping in water until the
middle lamella is destroyed and the fibres isolated; but it is not
so well known that _not every water_ is suitable for this “retting”
or steeping process; and for a long time this was as much a mystery
as why some waters are so much better than others for brewing. Quite
recently Fribes has succeeded in isolating the bacillus upon which the
dissolution of the middle lamella depends. This investigation brought
out other interesting details as to the reaction produced by living
micro-organisms, and which can be utilised in deciding questions of
plant chemistry too subtile for testing with ordinary re-agents. One
other important fact connected with these researches is that botanists
have now discarded the view that the middle lamella of the plants
referred to is composed of cellulose, and know that it consists of
pectin compounds. Fribes’ anærobic bacillus is found to dissolve and
destroy pectins and pectinates, but does not touch cellulose or gum.
It is well known that the steeping of skins in water in preparation
for tanning involves bacterial action, owing to which the hair and
epidermal coverings are removed, but it also appears that in the
process of swelling the limed skins, the gases evolved in the substance
of the tissues, and the evolution of which causes the swelling and
loosens the fibre so that the tanning solutions may penetrate, are due
to a particular fermentation caused by a bacterium, which, according
to some investigators, is identical with a lactic ferment introduced
by the pine bark, and which is responsible for the advantageous
acidification of the tanning solutions.
Hay is made in different ways, and in those where a “spontaneous”
heating process is resorted to the fermentation is no doubt dependent
upon the presence of thermogenic bacteria. But probably no other
subject has attained to so much importance as the bacteriology of the
dairy: the study of the bacteria found in milk, butter, and cheese in
their various forms.