CHAPTER VI.

The Mineral and Geological Kingdoms.

The structure of rocks and the formation of crystals will be found to

furnish an endless supply of instructive material for the microscope.

In sciences of pure observation, as those of mineralogy and geology,

the facts to be observed are of several different kinds, and where

so many observers are at work all over the world, constant progress

will necessarily be made, as well as continued correction required

from change and improvement in the methods of observation. It would

be impossible to give even a slight sketch of what has been done in

the two departments of nature referred to during the past few years.

Mineralogical and geological research have derived very great advantage

from having been assigned to professional teaching. But, as Professor

Bonney reminds us, the progress made in geological work in particular,

has been directly due to the revelations of the microscope. It called

forth an instrument of special construction for the purpose, the

petrological microscope (Fig. 79), well equipped with Nicol’s prisms,

and numerous other appliances demanded for the important investigations.

“Upon the history of the two main groups of rocks the microscope has

thrown much light. For the igneous rocks it has simplified their

classification and determined their mutual relations; while for the

rudimentary group, it has shown the true nature of their constituents,

and pointed out the sources from which they were derived. But it is in

helping to elucidate the problem of the metamorphic rocks, of which

much less was known, that the microscope has been of the most service.

It has likewise greatly assisted in the attempt to determine the

history and mutual relation of these rocks. One of the most important

results within the last few years has been the demonstration that

without exception these crystallin schists are very old, all probably

older than the first rocks in which traces of life have been found. The

conclusion arrived at, is that “the environment necessary for changing

an ordinary sediment into a crystalline schist existed generally only

in the earliest ages, and but very rarely and locally, if ever, since

palæozoic time began.”

The crystalline schists then are the relics still preserved to us

of the early days of the earth’s history, when the temperature near

the surface was still high. Since that time the zone for marked

mineralogical changes has been continually sinking, until at the

present day it has reached a depth practically unattainable. “The

subterranean laboratory still exists, but the way to it was virtually

closed at a comparatively early period in the earth’s history.”

Greater progress has been made since the microscope was pressed into

the service of geology, and inspires the hope that we shall yet learn

something more of the earliest ages, when the mystery of life began.

“It may be regarded as one of the most remarkable results of geological

science, that an acquaintance with organic forms is at least as

necessary for a geologist as a knowledge of minerals, and that a

correct knowledge of organic remains (portions of fossil plants and

animals) should prove a more certain and unerring guide in unravelling

the structure of complicated districts of countries, than the most

wide and general acquaintance with inorganic substances. The cause of

this, however, is obvious, as the mineral substances produced at any

one period of a vast succession of ages, do not appear to have had

any essential difference from those formed under like circumstances

at another. The animals and plants, however, living at one period of

the earth’s history were widely different from those living at other

periods. There has been a continuous succession of different races of

living beings on the earth following each other in a certain regular

and ascertainable order, and when that order has been determined, it

is equally certain that we can at once assign to its proper period of

production, and therefore to its proper place in the series of rocks,

any portion of earthy matter we may meet with containing any one, or

even any recognisable fragment of one, of these once living beings.”

The method of preparing sections of minerals and rocks for microscopic

examination will be found at pp. 241, 307-309. The sections, it is

almost needless to say, must be prepared thin enough to permit the use

of transmitted light, as well as for that of polarised light: that is

to say, they should range from about 1/100th to 1/1000th of an inch.

Almost any lapidary will cut sections of any choice specimen.[88]

The formation of crystals, and the method of preparing them for

examination, has also been fully explained in the chapter on polarised

light, pp. 219 et seq., and illustrated on Plate VIII. It is well known

in micro-chemistry that “almost every substance, simple or compound,

capable of existing in the solid state, assumes, under favourable

conditions, a distinct geometrical figure, usually bounded by plane

surfaces and having angles of constant value.

Much useful information may be gained upon micro-crystallography, as

well as on almost everything having any relation to the _technique_ of

the microscope, in the “Journal of the Royal Microscopical Society.” To

the June number (1898) Mr. T. Charters White contributes an article on

crystals, and reminds us that the presence of much or little moisture

will modify and alter forms, as much and as often as varying degrees of

temperature. At the same time he offers a few useful suggestions for

the purpose of securing better results, for which purpose he employs

hippuric acid, hydroquinine, and picric acid alone or in combination

with hippuric acid, and an aqueous solution of bichromate of potassium,

crystallised in a tolerably thick emulsion of gum arabic. This is the

only aqueous solution; the other solvents have been methylated spirit,

acetone, and absolute alcohol, taking these three solvents as types

of the greatest volatility, because in making certain crystals it is

necessary that the solvent should evaporate quickly, otherwise the

crystals will assume their original forms. It is further desirable

to make saturated, or even super-saturated solutions of the three

chemicals named, as the colours produced under polarised light are

of a deeper and richer character than they are if made from weaker

solutions. Of the three chemicals named he prefers hippuric acid, for

reasons stated, that it is the most manageable, and allows of more time

being taken in modifying the formation of the crystals. It is also

advisable to slightly warm the glass slide before the drop of fluid is

applied. On the whole, picric acid appears to furnish a greater variety

of crystals when used in combination with bichromate of potassium and a

solution of gum arabic.

APPENDICES AND TABLES USEFUL TO THE MICROSCOPIST.

APPENDIX A.

ILLUMINATION ARRANGEMENTS OF THE MICROSCOPE.

A doubt has of late been expressed among practical microscopists

as to the value of the illumination arrangements of the lamp and

the microscope, so as to secure the more perfect definition of the

flagellate organ of the monas and other minute forms of infusorial

life. We have been told that better results will be obtained by

turning the mirror aside, and so disposing the microscope and lamp

in the horizontal position, that the central rays of light from the

mirror-edge of the lamp-flame shall pass through the optical axis of

the achromatic condenser, the focus of which must be accurately brought

upon the field of view by means of the substage centring screws and

rack-work, and in such a manner, that by employing a 1-inch objective,

a sharply-defined image of the lamp-flame, edge-on, is projected on

to the centre of the field in association with the specimen under

examination. If the 1-inch objective be now replaced by a 1-12th or

1-16th inch immersion and once again focussed into place, and a slight

re-adjustment of the centring made, it will be found that the field is

brilliantly illuminated, and the most minute portions of infusorial

life are well defined, and with a sharpness otherwise unattainable. At

the same time the graduating or iris diaphragm must be brought into use.

Dr. Clifford Mercer, the President of the American Microscopical

Society, who has quite recently reinvestigated the question of

illumination, utterly condemns the narrow cone, as well as that of

oblique light in all such investigations, and considers the 3-4ths axil

cone as the most suitable method for microscopical illumination, and

he bases his resolving limit accordingly. Some important experiments

are brought forward by Dr. Mercer, which at the same time demonstrate

the correctness of Lord Rayleigh’s limit of resolution (referred to in

a previous chapter, p. 44), for circular apertures as contrasted with

that calculated by the late Sir George Airy.

With regard to the Abbé Theory, Dr. Mercer says: “Resolution in the

Abbé Theory may be said to increase by bounds. So long as the central

image of the source of light alone is to be seen at the back of the

objective, resolution is not present. The aperture may be increased

without change in the contraction of the diffraction pattern, and in

accompanying resolution, so long as the central image alone is to

be seen at the back of the objective; but the moment the increase

in aperture is sufficient to uncover or admit one flanking spectrum

image, resolution is present. With greater increase in aperture, no

improvement in the picture as to the contraction of the diffraction

pattern is to be seen until another spectrum image is uncovered or

admitted. Dr. Mercer gives his reasons for considering that the

advantageous reduction in a cone of light between an object and the

objective should not exceed, in the case of first-class objectives,

one-fourth to one-third (never more than one-half) of the diameter of

the cone. On the other hand, with full cone illumination, resolution

increases continuously, and not by jumps or by periodic accessions.

With regard to the use of oblique light, he says his Photos 2, 3, and

4[89] are a pictorial warning for a second time against the use of

oblique illumination in ordinary work us a means of increasing, or of

attempting to exhaust the resolving power of the microscope. At the

same time it becomes evident that every substage should be provided

with a means by which its condenser may be accurately centred, and that

every student using the microscope should be familiar with a method of

centring his substage condenser.

Dr. Mercer summarises the results of his experiments thus:--

1. “Diffraction rays on leaving an object may be considered in the same

category with other rays changed in direction by an object.

2. “The diffraction phenomena seen in a projected image are essentially

the effect of changes in light _above_ the objective, due to a

function of aperture, and not to changes _below_ the objective, due to

diffraction of light in the plane of the object.

3. “Diffraction in the plane of the object does, under some

circumstances, furnish light to certain parts of an aperture from

which primary rays are absent, and this enables aperture to more fully

determine the character of the projected image, resulting in a more

nearly truthful image, or, on the other hand, in false appearances.

This is the gist of the Abbé phenomena of microscopic vision.

4. “But such phenomena are not peculiar to microscopic vision,

notwithstanding Professor Abbé’s claim to the contrary.

5. “With any positive lens similar and more brilliant results may be

got by utilising corresponding pencils of primary rays, instead of

isolated pencils of diffracted rays.

6. “Still more trustworthy results may be got by using primary rays in

place of the isolated pencils of primary rays.

7. “An advantage peculiar to using narrow cone illumination with

an objective of wide aperture (the only illumination admissible in

the Abbé theory), consists in giving, under suitable conditions,

approximately the acme of resolving power simultaneously in each

several diameters. Thus a circular aperture is approximately squared

or made rectangular as to resolving power in several of its diameters

simultaneously.

8. “Special attention is called to the fact that the Abbé theory

deals with complex objects; for only such objects are subject to

resolution. Single particles and uniform areas are outside its domain.

These latter, however, are microscopic objects, and all objects are

essentially different shaped aggregations of points. An isolated

point-like particle, no matter what its minuteness, may be seen if it

present sufficient contrast with the surrounding microscopic field.

The size of the disc image is no less than a limit determined finally

by aperture. That limit in size varying inversely with aperture,

determines the limit of resolving power. This is the gist of the theory

of microscopic vision which harmonises with our experimental study of

aperture.”

APPENDIX B.

MICRO-PHOTOGRAPHY.

Owing in some measure to the more complete knowledge of the subject

gained by the experience of years, and the extreme value of

micro-photography in the delineation of bacteria, and perhaps in a

measure to the advent of the perfected dry-plate process, photography

is being rapidly pressed forward in conjunction with the microscope. In

the course of the year [1898] no less than six, more or less, new forms

of micro-photographic apparatus have appeared; two are simple, one

for daylight, one for lamp, one for electric, and one for lime-light

illumination. Passing over the simpler forms, for a notice of which I

am unable to find room, there is one piece of new apparatus, that of

Mr. E. B. Stringer, which is not only new, but is in every way adapted

to the work of micro-photography. It is in fact a well-arranged camera,

fitted with a powerful condensing arrangement, each portion of which

is capable of being independently centred and controlled. Indeed, the

specially interesting feature of the apparatus is the control of the

gas and the beautiful and uniformally illuminating disc of zircon,

about a quarter of an inch in diameter.

[Illustration: Fig. 446.--Mr. E. B. Stringer’s Improved

Micro-photography Apparatus.

B. Oxyhydrogen jet with zirconium cylinder, covered by the cowl A when

working.

C. Doublet parallelising condenser, with centering screws.

D. Iris diaphragm.

E. Holder for trough and light-filtering media.

F. Plano-convex lens, 4-1/4ins. diameter, with centering screws G

H. Plano-concave lens, with iris diaphragm T.

K. Connecting pulleys between focussing rod of camera and fine adjustment of

Microscope.

L. Triangular frame in which Microscope feet are placed. M. Flap shutter.

N. Door through which image is observed on card screen, etc.

O. Solid block of mahogany on which camera body is fixed and supported.

P. Dark slide.]

This efficient photo-micrographic apparatus (Fig. 446) is made by

Messrs. W. Watson & Sons, under the instructions of Mr. E. B. Stringer.

The illuminating condensing system is mounted on a square brass bar,

the illuminant being oxygen-hydrogen light burning on zirconium.

Immediately in front of this is a condenser, c, four and a half

inches diameter, with an iris diaphragm, D, immediately in front of

it. The holder, E, carries the light filtering media through which the

beam passes and enters the condenser, F. It then goes through a tank of

water contained in the cone, F to H, and emerges a practically parallel

beam of great intensity through a plano-concave lens, h, of such a

diameter as to exactly fill the back lens of the substage condenser.

There is an iris diaphragm, T, for cutting off stray light.

The whole of the apparatus is fitted with centring screws and clamps,

and after having been once adjusted it is ready for use at any moment

without preparation. By means of this apparatus, instantaneous pictures

can be taken of living rotifers, so brilliant is the illumination,

while photographs of such fine objects as the flagella of bacteria

cannot be secured with the same amount of certainty by any other

microphotographic apparatus with which I have made myself acquainted.

APPENDIX C.

FORMULÆ AND METHODS:--CEMENTING, CLEARING, HARDENING AND MOUNTING.[90]

CLEARING AGENTS.

The object of employing a clearing agent is to replace the alcohol in

the dehydrated section by a liquid which has a refractive index about

the same as the balsam into which it is to be placed, and which will

readily mix with it.

OIL OF BERGAMOT will clear quickly from 90 per cent. of alcohol.

Clove oil clears more rapidly, but it dissolves out aniline colours

to a considerable extent. Xylol is without action on aniline colours.

This strength of alcohol is chosen because of its being that of the

methylated spirit sold in London, and which is much used in washing and

dehydrating on account of its cheapness.

OIL OF CEDAR WOOD, although an essential oil, resembles xylol, but

evaporates slowly. It has very little solvent action on the aniline

colours. It clears rapidly from absolute alcohol, but not well from

90 per cent. Sections can be left in it for several days. It is a

convenient medium in which to examine tissues before mounting them

permanently. It clears celloidin without dissolving it; and as a

connecting fluid between the object and objective nothing better has

been discovered.

Other clearing agents have been tried, but as they dissolve out the

aniline colours, are no longer used.

CEMENTS.

GROVE’S MASTIC AND BISMUTH.--Dissolve gum mastic in chloroform, and

thicken with nitrate of bismuth. The solution of mastic should be

nearly saturated.

GROVE’S OXIDE OF ZINC, DAMMAR, AND DRYING OIL.--Rub up well-ground

oxide of zinc, 2 ozs., with drying oil, to the consistence of

thick paint. Then add an equal quantity of gum dammar, previously

dissolved in benzoline, and of the thickness of syrup. Strain through

close-meshed muslin. Keep in well-corked bottle, and, if necessary,

thin with benzoline.

ISINGLASS CEMENT.--Heat the isinglass in a covered vessel on the

water-bath with a little glacial acetic acid, until it is thoroughly

softened and forms a stiff mass, then gradually add more acid until it

produces a thick solution which is of uniform consistence, and just

fluid while hot. Then run into wide-mouth bottles and close with good

corks.

KITTON’S CEMENT of white lead and red lead in powder, and litharge

powder in equal parts. Grind together with a little turpentine, until

thoroughly incorporated, and mix with gold size. The mixture should be

thin enough to use with a brush; in using, one coat should be allowed

to dry before applying another. No more cement should be mixed with the

gold size than is required for immediate use, as it sets quickly, and

becomes unworkable.

KRÖNIG’S CEMENT.--Gradually add ordinary resin, 7 to 9 parts, to melted

beeswax, 2 parts, then steam and cool.

SHELLAC CEMENT.--Dissolve shellac in an equal weight of methylated

spirit, then pour off the clear portion and add a few drops of balsam

and castor oil.

MARINE GLUE.--Dissolve indiarubber in mineral naphtha, and add twice

the quantity of powdered shellac; or make chloroform the solvent,

and use mastic instead of shellac. For casting battery trays, use a

composition of 4 parts resin and 1 of gutta percha, with a little

boiled oil.

SELIER (_Cleaning Glass Slides_).--New slides or cover-glasses must be

placed for a few hours in a mixture of 1 part of potassium bichromate,

1 of sulphuric acid, and 25 of water. Subsequently wash with water and

wipe dry with a linen rag, after draining off the excess of moisture.

Covers that have been used should be previously immersed for a few

days in a mixture of equal parts of alcohol and hydrochloric acid.

Scrape old slides free of mounting medium before immersing them in the

bichromate solution.

ELSCHING’S CELLOIDIN SOLUTION.--Allow the celloidin shavings to swell

up for 24 hours in the necessary quantity of absolute alcohol, then add

the proper amount of ether.

KOCH’S COPAL.--Stain small pieces of material in bulk, and dehydrate

with alcohol, then immerse in a thin solution of copal in chloroform.

Evaporate with a gentle heat until the solution is so far concentrated

as to draw out into threads that are brittle on cooling. Then remove

the objects and leave on a tile for a few days to dry. Sections may

then be cut by means of a fine saw. If objects are imbedded unstained,

remove copal from sections by soaking in chloroform, decalcify if

necessary, and stain.

EULENSTEIN’S CEMENT.--Mix equal parts of Brunswick black and gold size

with a very little Canada balsam.

DECALCIFYING AND BLEACHING.

In the case of bony structures, or tissues so impregnated with

calcium salts, the material should be decalcified by an acid capable

of dissolving out the mineral matter. Hydrochloric acid with alcohol

is in more general use. The older the bone the stronger will be the

acid required, nitric with alcohol and chromic acid. Picric acid is

preferred for fœtal bone.

ANDEER, J. J., finds an aqueous solution of phloroglucin acts as a

powerful decalcifying agent on the bones of animals, but is without

action on the most delicate organic tissue. If treatment with

hydrochloric acid be employed as well, the residual “ossein” will be

without a trace of either calcium phosphate or carbonate.

EBNER’S FLUIDS.--(1) Mix 100 C.c. of cold saturated aqueous solution of

sodium chloride, 100 C.c. of water, and 4 C.c. of hydrochloric acid.

Preparations are placed in the fluid, and 1 to 2 C.c. of hydrochloric

acid added daily until they are soft. (2) Mix 2·5 parts of hydrochloric

acid (sp. gr. 1·16) with 500 of alcohol (90 per cent.), 100 of water,

and 2·5 of sodium chloride.

FOL’S LIQUID.--Mix 70 volumes of 1 per cent. chromic acid, 3 of nitric

acid, and 200 of water.

MAYER’S DESILIFICATION PROCESS.--Place the objects in alcohol

contained in a glass vessel coated internally with paraffin, then

add hydrofluoric acid drop by drop until desilification is complete,

avoiding the fumes meanwhile.

MARSH’S CHLORINE METHOD.--Chlorine is generated in a small bottle by

treating crystals of potassium chlorate with strong HCl., and the gas

is led through a piece of glass tubing, bent twice at right angles, to

the bottom of a bottle containing the sections immersed in water.

RANVIER’S FLUID.--Use 50 per cent. hydrochloric acid with the addition

of sodium chloride to counteract its swelling action.

SQUIRE’S FLUID.--(1) Mix 95 parts of glycerine with 5 parts of

hydrochloric acid; used for softening teeth. (2) Use a 4 per cent.

aqueous solution of arsenic acid at a temperature of 30° to 40° C.

After softening tissues in this solution, keep them in alcohol.

WALDEYER.--To a 0·1 per cent. solution of palladium chloride, add

one-tenth its volume of hydrochloric acid.

HARDENING, FREEZING, AND EMBEDDING.

ALTMANN (_Fixing Solution_).--A mixture of equal parts of 5 per cent.

potassium bichromate solution and 2 per cent. osmic acid.

ALCOHOL.--Strengths of alcoholic solutions, as given by Squire, will be

found of practical value. Absolute alcohol (sp. gr. O·797) containing

about 98 per cent. of ethylic alcohol is taken as the basis in most

instances. Alcohol of 90 per cent. (sp. gr. 0·823) is prepared by

mixing 14 volumes of absolute alcohol and 1 volume of distilled water;

84 per cent. alcohol (sp. gr. 0·838) is rectified spirit B.P.; 70

per cent. alcohol (sp. gr. 0·872) may be obtained by adding 1 volume

of distilled water to 3 volumes of absolute alcohol, 6 volumes of

rectified spirit, or 4 volumes of methylated spirit; 50 per cent.

alcohol (sp. gr. 0·918) is prepared by adding 4 volumes of distilled

water to 5 volumes of absolute alcohol, 3 volumes of water to 5 volumes

of rectified spirit, or 3·5 volumes of water to 5 volumes of methylated

spirit. Absolute alcohol, 75 C.c., mixed with acetic acid, 25 C.c.,

serves as an excellent fixing agent for nuclei. Immerse tissues in

it for 6 to 12 hours, then transfer to 90 per cent. alcohol until

hardened, afterwards preserving in 70 per cent. alcohol till wanted.

BETZ’S HARDENING FLUID.--A mixture of equal parts of sulphuric ether

and alcohol. This is used for hardening the brain of insects prior to

cutting sections.

COLE’S FREEZING PROCESS.--Dissolve picked gum acacia, 4 ozs., in

distilled water, 6 ozs., and to each 5 parts of the resulting

mucilage add 3 parts of syrup made by dissolving loaf sugar, 1 lb.,

in distilled water, 1 pint. To each ounce of the medium add 5 grains

of pure carbolic acid, and soak the tissues in it prior to freezing.

For tissues liable to come to pieces, mix 4 parts of syrup with 5 of

mucilage.

FLEMMING’S FIXING SOLUTION.--Osmic acid (1 per cent. solution), 80

C.c.; chromic acid (10 per cent. solution), 15 C.c.; glacial acetic

acid, 10 C.c.; distilled water, 95 C.c.

FOL’S FIXING--Osmic acid (1 per cent. solution), 4 C.c.; chromic

acid (10 per cent. solution), 5 C.c.; glacial acetic acid, 10 C.c.;

distilled water, 181 C.c.

FISCHER’S IMBEDDING MASS.--Dissolve 15 parts of transparent soap in

17·5 parts of 96 per cent. alcohol.

KLEIN’S HARDENING.--Mix 1 C.c. of 10 per cent. chromic acid solution

with 60 C.c. of water, and add 30 C.c. of 90 per cent. alcohol.

MÜLLER’S FLUID FORMULA, see page 288.--This solution is sometimes mixed

with one-third its volume of 90 per cent. alcohol, its hardening action

being then much more rapid.

RABL’S HARDENING FLUID.--Chromic acid solution (10 per cent.), 7 C.c.;

water, 200 C.c.; formic acid (sp. gr. 1·2), 5 drops.

ROLLETT’S FREEZING PROCESS.--Small portions of tissue placed on the

stage of microtome, after immersion in the white of an egg, then frozen

and cut with a very cold knife.

RYDER (_Double Embedding_).--After the celloidin bath, soak objects

in chloroform, then remove into a mixture of chloroform and paraffin,

heated to not more than 40° C., and finally into a bath of pure

paraffin.

STRICKER (_Imbedding Mass_).--Prepare the objects in alcohol and imbed

in a concentrated solution in gum arabic in a paper case, then throw

the whole into alcohol and cut after 2 or 3 days.

WEBB (_Dextrin Freezing_).--A thick solution of dextrin (1:40)

in aqueous solution of carbolic acid is used for imbedding, and

subsequently frozen.

MOUNTING MEDIA.

Sections are usually mounted in balsam, dammar, glycerine, &c., but it

is not a necessity that the cover-glass should be fixed or cemented

down. Some cements (caoutchouc by preference) should be employed when

glycerine or aqueous (Farrant’s) media are used.

ALLEGER’S GELATINE PROCESS.--Add a few drops of formalin to each gramme

of 0·5 to 1 per cent. gelatine solution. After mounting the section in

this, apply heat to the slide until the paraffin is softened, and allow

the superfluous gelatine to drain from the edge of the slide.

APÁTHY’S MOUNTING MEDIUM.--Picked gum arabic, 50 Gm.; cane-sugar, 50

Gm.; distilled water, 50 Gm.; dissolve over a warm bath and add 0·05

Gm. of thymol. This medium sets very hard, and combined with a paper

cell it may be used for ringing glycerine mounts.

COLE’S SLOW OR EXPOSURE METHOD OF MOUNTING.--Dissolve dried Canada

balsam, 3 ozs., in benzole, 3 fl. ozs., and filter. Apply a clean

cover-glass to a slide that has been moistened by breathing on it,

and place a few drops of the balsam solution on the cover-glass. Then

remove a section from turpentine, and put it into the balsam. Put

aside for 12 hours to allow the benzole to evaporate, and having

warmed a slide and added a drop of fresh balsam solution to that on the

cover-glass, bring the fluid balsam in contact with the warmed slide.

Press the cover down carefully to avoid the inclusion of air bubbles,

and when the excess of balsam is squeezed out, put the slide aside to

cool, after which it may be cleaned with a camel-hair brush or soft rag

moistened with methylated spirit.

FARRANT’S SOLUTION.--Take of gum arabic 5 parts; water 5 parts; when

the gum is fairly dissolved add 10 parts of a 5 per cent. solution of

carbolic acid.

FLEMMING’S GLYCERINE PRESERVATIVE.--Mix equal parts of alcohol,

glycerine, and water. Lee recommends the addition of 0·5 to 0·75 per

cent. of acetic acid.

LEE’S TURPENTINE COLOPHONIUM MOUNTING MEDIUM.--This is highly

recommended for general work, and is prepared by adding small pieces

of colophonium to rectified oil of turpentine, heating in a stove, and

when the solution is sufficiently thick filtering twice in the stove.

SEAMAN (_Glycerine Jelly_).--Dissolve isinglass in water so as to make

a jelly that remains stiff at the ordinary temperature of the room, and

add one-tenth part of glycerine, together with a little solution of

borax, carbolic acid, or camphor water. Filter through muslin whilst

warm and add a little alcohol.

SEILER (_Alcohol Balsam_).--Heat Canada balsam until it becomes brittle

when cold, then dissolve in warm absolute alcohol and filter through

absorbent cotton-wool. This is chiefly useful as a mounting medium for

objects stained with carmine.

SQUIRE (_Farrant’s Medium_).--Dissolve in 200 C.c. of distilled water 1

Gm. of arsenious acid and 130 Gm. of gum arabic, then add 100 C.c. of

glycerine. Filter through fine Swedish filter paper upon which has been

deposited a thin layer of talc.

SQUIRE (_Glycerine and Gum_).--Dissolve 130 Gm. of gum arabic in 200

C.c. of chloroform water (1 in 200), then add 100 C.c. of glycerine and

filter.

SQUIRE (_Glycerine Jelly_).--Soak 100 Gm. of French gelatine in

chloroform water, drain when soft, and dissolve with heat in 750 Gm.

of glycerine. Add 400 Gm. of chloroform water, with which has been

incorporated about 50 Gm. of fresh egg albumen, mix thoroughly, and

heat to boiling point for about 5 minutes. Make up the total weight to

1550 Gm. with chloroform water and filter in a warm chamber.

SQUIRE (_Canada Balsam_).--Dry the balsam over a water bath until

brittle when cooled, then to each 200 Gm. add 100 C.c. of benzole or

rather less xylol.

SQUIRE (_Dammar Solution_).--(1) Dissolve 100 Gm. of dammar in 100 C.c.

of benzole. (2) Dissolve 100 Gm. of dammar in 200 C.c. of turpentine

oil, and add 50 Gm. of mastic dissolved in 200 C.c. of chloroform.

SQUIRE (_Potassium Acetate Solution_).--Dissolve 250 Gm. of potassium

acetate in 100 C.c. of water, by the aid of gentle heat, and filter.

This is used as a mounting medium.

SQUIRE (_Treatment of Sections_).--Imbed tissues to be cut in paraffin

melting between 45° and 50° C., according to the temperature of the

room and the nature of the material. Afterwards preserve the sections,

prior to staining and mounting, in 50 per cent. alcohol, or in a

mixture of equal volumes of glycerine and thymol water (1 in 1500).

Sections may be conveniently washed in alcohol, dehydrated, and

cleared, in small wide-mouthed bottles.

TOPPING’S SOLUTION.--Mix 1 part of absolute alcohol with 5 parts of

water, or 4 parts of water and 1 part of aluminium acetate. Add an

equal volume of glycerine before use.

STAINS AND STAINING METHODS.

APÁTHY’S HÆMATOXYLIN STAIN.--After staining in 1 per cent. solution

of hæmatoxylin in 70 or 80 per cent. alcohol, wash out in 1 per cent.

solution of potassium bichromate in alcohol of the same strength. The

bichromate solution should be freshly made by mixing 1 part of a 5 per

cent. aqueous solution with about 4 parts of 80 to 90 per cent. alcohol.

ALFEROW (_Silver Staining_).--An acid solution of silver picrate,

lactate, acetate, or citrate, is prepared by adding to 800 C.c. of the

solution 10 to 15 drops of a concentrated solution of the acid of the

salt taken.

BETHE’S STAIN FOR CHITIN.--Place series of mounted sections on slides

in a freshly prepared 10 per cent. solution of aniline hydrochloride,

containing 1 drop of hydrochloric acid for each 10 C.c., for 3 or 4

minutes, then rinse in water, and put the slide with sections downwards

in a 10 per cent. solution of potassium bichromate. The process may be

repeated if the stain is not sufficiently intense, but the sections

must be well rinsed with water after each immersion.

BEALE’S AMMONIA CARMINE.--Carmine, 10 grs.; strong solution of ammonia,

30 mins.; distilled water, 2 ozs.; alcohol, 0·5 oz.; glycerine, 2 ozs.

Dissolve the carmine in the ammonia by the aid of heat, boil for a few

seconds, and let the solution cool. Then allow the excess of ammonia

to evaporate, add the other ingredients, and filter. If any carmine

should deposit on keeping add one or two drops of ammonia solution to

redissolve it.

BENDA’S COPPER HÆMATOXYLIN.--Harden the material with chromic acid or

Flemming’s solution and leave sections for 24 hours in a 5 per cent.

solution of neutral copper acetate at a temperature of about 40°

C., wash out well with distilled water, and stain to a dark grey or

blackish tint in a saturated aqueous hæmatoxylin solution. Decolourise

the sections in 0·2 per cent. hydrochloric acid until light yellow, put

back into the copper solution until they turn bluish-grey, then wash,

dehydrate, clear, and mount in balsam.

BISMARCK BROWN.--Vesuvine 0·5 Gm., rectified spirit 2, and distilled

water 80 C.c.; or a concentrated alcoholic solution may be kept ready

for dilution.

BOCHMER’S HÆMATOXYLIN.--Dissolve (_a_) crystallised hæmatoxylin, 1

Gm., in absolute alcohol, 10 C.c., and (_b_) alum ammonia, 10 Gm., in

distilled water, 200 C.c. Mix the two solutions, and allow to ripen

for some days before use. Filter after standing a week. Wash out with

aqueous solution of alum (0·5 per cent.) or with acids.

CALBERLA’S INDULIN STAIN.--Dilute a concentrated aqueous solution with

6 volumes of water and stain sections for 5 to 20 minutes. Afterwards

wash in water or alcohol, and examine in glycerine or clove oil.

CALBERLA’S MACERATING MIXTURE (for nerve and muscle of

embryos).--Dissolve potassium chloride, 0·4 Gm., sodium chloride, 0·3

Gm., sodium phosphate, 0·2 Gm., and calcium chloride, 0·2 Gm., in

water, 100 Gm., saturated with carbon dioxide just before using. Mix

one volume of this solution with half a volume of Müller’s solution

and one volume of water. The Müller’s solution may be replaced by a

2·5 per cent. solution of ammonium chromate. Tissues macerated in this

mixture are isolated by teasing and shaking, and mount specimens in

concentrated potassium acetate solution.

CANOY’S SALT SOLUTION.--Add a trace of osmic acid to a 0·75 per cent.

solution of sodium chloride in water.

CHENZINSKY’S METHYLENE BLUE AND EOSINE.--Mix saturated aqueous solution

of methylene blue, 40 parts, with 0·5 per cent. solution of eosine in

70 per cent. alcohol, 20 parts, and distilled water or glycerine, 40

parts.

COHNHEIM’S GOLD METHOD.--Place pieces of tissue in 0·5 per cent. gold

chloride solution until quite yellow, then expose to light in water

acidulated with acetic acid until the gold is thoroughly reduced. Mount

specimens in acidulated glycerine.

CROOKSHANK’S METHOD OF STAINING FLAGELLA.--Cover-glass preparations

are stained with a drop of concentrated alcoholic solution of gentian

violet, then rinsed in water, allowed to dry, and mounted in balsam.

CZOKER’S ALUM COCHINEAL.--Dissolve alum 1 Gm. in distilled water, 100

C.c., add powdered cochineal, 1 Gm., and boil; evaporate down to half

of its original bulk, filter, and add 1/2 C.c. of liquid carbolic acid.

DELAFIELD’S HÆMATOXYLIN.--Dissolve hæmatoxylin, 4 Gm., in absolute

alcohol, 25 C.c., and add the solution to 400 C.c. of a saturated

aqueous solution of ammonia alum. Expose the mixture to light and air

for 3 or 4 days, then filter and add glycerine, 100 C.c., and methylic

alcohol, 100 C.c. Again expose the solution to light until it becomes

dark-coloured, then filter and preserve in a stoppered bottle.

EHRLICH’S ACID HÆMATOXYLIN.--Dissolve hæmatoxylin, 2 Gm., in absolute

alcohol, 100 C.c., and add glycerine, 100 C.c., distilled water, 100

C.c., ammonia alum, 2 Gm., glacial acetic acid, 10 C.c. Expose to

daylight for at least a month before use, removing the stopper at

intervals.

EHRLICH’S HÆMATOXYLIN (AMMONIATED).--Dissolve ammonium carbonate, 0·4

Gm., and hæmatoxylin, 2 Gm., in proof spirit, 40 C.c., and expose to

the air in a shallow dish for 24 hours. Then make up the volume to

40 C.c. with proof spirit (warming if necessary to re-dissolve any

separate crystals), and add ammonia alum, 2 Gm., dissolved in distilled

water, 80 C.c., together with glycerine, 100 C.c., rectified spirit, 80

C.c., and glacial acetic acid, 10 C.c.

EHRLICH-BIONDI MIXTURE (or Ehrlich-Biondi-Heidenheim

mixture).--Dissolve (_a_) methyl green, 0·5 Gm., in distilled water,

100 C.c.; (_b_) acid fuchsine, 0·5 Gm., in distilled water, 40 C.c.;

(_c_) orange, 2 Gm., in distilled water, 200 C.c. Mix the three

solutions and filter before use. Stain sections for 12 hours, then

wash, dehydrate, clear, and mount.

EHRLICH-WEIGERT-KOCH’S GENTIAN-VIOLET-ANILINE-WATER.--Aniline water,

100 C.c., concentrated alcoholic solution of gentian violet, 11 C.c.;

absolute alcohol, 10 C.c.

EVERARD, DEMOOR, AND MASSART’S HÆMATOXYLIN-EOSINE.--Dissolve alum, 20

Gm., in water, 200 Gm., by the aid of heat, then filter, and after 24

hours add a solution of hæmatoxylin, 1 Gm., in alcohol, 10 Gm. Let the

solution stand for 8 days, again filter, and mix with an equal volume

of the following solution:--Eosine, 1 Gm., alcohol, 25 Gm., water, 75

Gm., glycerine, 50 Gm.

FLEMMING’S GENTIAN VIOLET METHOD.--Use a concentrated alcoholic

solution of Gentian Violet diluted with about one half its bulk of

water. Differentiate the stained objects in alcohol acidulated with

about 0·5 per cent. of hydrochloric acid, followed by pure alcohol and

clove oil.

FLEMMING’S ORANGE METHOD.--Stain for days or weeks in strong alcoholic

safranine solution diluted with half its bulk of aniline water

(saturated); then rinse in distilled water, differentiate in absolute

alcohol containing 0·1 per cent. of hydrochloric acid, stain for 1

to 3 hours in strong aqueous gentian violet solution, again wash in

distilled water, and finally treat with concentrated aqueous solution

of Orange. After a few minutes transfer sections to absolute alcohol,

then clear in clove or bergamot oil, and mount in dammar or balsam.

FOL’S FERRIC CHLORIDE FIXING AND STAINING PROCESS.--Preparations are

treated with tincture of ferric chloride diluted with 5 to 10 times its

bulk of 70 per cent. alcohol, and then transfer for 24 hours to alcohol

containing a trace of gallic acid.

FREY’S FUCHSINE SOLUTION.--A solution of 0·01 Gm. of crystallised

fuchsine, 20 to 25 drops absolute alcohol, and 15 C.c. of water.

FRIEDLAENDER’S STAINING METHODS.--Cover-glass preparations are treated

for 3 minutes with a 1 per cent. solution of acetic acid, and allowed

to dry after removal of excess of liquid by filter paper. Next place

them in gentian violet aniline water (aniline water, 100 C.c.,

concentrated alcoholic solution of gentian violet, 11 C.c.; absolute

alcohol, 10 C.c.) for half a minute, wash in water, mount and dry

in balsam. Sections are kept for 24 hours in a warm place, in the

following solution:--Concentrated alcoholic solution of gentian violet,

50 C.c.; distilled water, 100 C.c.; glacial acetic acid, 10 C.c. Then

treat for 1 or 2 minutes with 0·1 per cent. acetic acid, dehydrate,

clear, and mount in balsam.

GAFFKY’S STAINING METHODS.--Sections of material hardened in alcohol

are left for 20 to 24 hours in a deep blue opaque solution, freshly

made by adding saturated alcoholic solution of methylene blue to

distilled water. Then wash in distilled water, dehydrate in absolute

alcohol, clear in turpentine oil, and mount in balsam.

GIACOMI’S STAINING METHOD.--Stain cover-glass preparations for a few

minutes in a hot solution of fuchsine, then place in water containing

a few drops of ferric chloride solution, and afterwards decolourise in

strong ferric chloride solution. If any precipitate be formed with the

iron solution, complete the decolourisation in alcohol. Counterstain

with vesuvine.

GIBBES’ DOUBLE STAINING METHOD.--Well mix magenta, 2 Gm., and methylene

blue, 1 Gm., then add slowly aniline oil, 3 C.c., dissolve in rectified

spirit, 15 C.c. Subsequently add 15 C.c. of distilled water and keep

the stain in a stoppered bottle. Cover-glass preparations are placed

for 4 minutes in the slightly heated stain and sections left for some

hours in the stain at the ordinary temperature. Afterwards, wash in

methylated spirit until no more colour comes away, then dehydrate,

clear in cedar oil, and mount in balsam.

GIBBES’ MAGENTA STAIN.--Mix magenta, 2 Gm.; aniline oil, 3 Gm.;

rectified spirit, 20 C.c.; and distilled water, 20 C.c.

GOLGI’S SUBLIMATED METHOD.--Small cubes of tissue are hardened for

15 to 30 days in Müller’s fluid, which should be frequently changed.

Then transfer for 8 to 10 days to 0·25 to 1 per cent. aqueous mercuric

chloride solution, which must be changed, as it becomes coloured. If

desired, treat subsequently with weak sodium sulphide solution to

darken the stain and make it sharper. After cutting sections from

material thus prepared they must be well washed with water.

GRAM’S STAIN FOR BACTERIA.--This is prepared by shaking 15 drops of

aniline oil with 15 Gm. of water, filtering the solution and adding to

the filtrate 4 to 5 drops of saturated alcoholic solution of gentian

violet. Or shake 3·3 C.c. of aniline with 100 C.c. of distilled water

and, after filtering, add 11 C.c. of concentrated alcoholic solution of

gentian violet and 10 C.c. of absolute alcohol. After preparations have

been stained for 1 to 3 minutes in one of the above they are quickly

rinsed in absolute alcohol and then placed in Gram’s solution of iodine

in potassium iodine (iodine, 1 Gm.; potassium iodine, 2 Gm.; water, 300

C.c.), until they have acquired a brown colour. This takes about 1 to

3 minutes, and they are next washed in 90 per cent. alcohol until they

become pale yellow, then dehydrated, cleared, and mounted in balsam.

Counterstain with eosine or vesuvine if desired.

GRAM’S SOLUTION.--Iodine, 1 Gm.; potassium iodine, 2 Gm.; distilled

water, 300 Gm.

GRENACHER’S ALUM CARMINE.--Dissolve 5 Gm. of ammonium alum in 100 C.c.

of distilled water, add 1 Gm. of carmine, and boil for 20 minutes,

filter when cool, and add distilled water to make up to 100 C.c.

GRENACHER’S ALCOHOLIC BORAX CARMINE.--Dissolve 4 Gm. of borax in 100

C.c. of distilled water, then add 3 Gm. of carmine, and heat gently.

Finally, add 100 C.c. of 70 per cent. alcohol, filter the solution, if

necessary, before use. Pieces of tissues are stained in this for 1 to 3

days, and then transferred to 70 per cent. alcohol, containing 0·5 to 1

per cent. of hydrochloric acid.

HEIDENHAIN’S HÆMATOXYLIN METHOD.--Dissolve (_a_) hæmatoxylin, 1 Gm.,

in distilled water, 300 C.c.; (_b_) potassium chromate, 1 Gm., in

distilled water, 200 C.c. Small pieces of tissue hardened in alcohol

or picric acid are placed in (_a_) for 12 to 24 hours, and then

transferred for a similar length of time to (_b_). Wash thoroughly in

water, dehydrate in alcohol, and imbed in paraffin.

HENLE’S STAIN (_for nervous tissue_).--Sections are left in palladium

chloride solution (1:300 to 1:600) till they are of a straw colour,

then rinsed in water and stained with strong ammonia carmine.

HENNEGUY’S ALUM CARMINE.--Excess of carmine is boiled in saturated

solution of potash alum, and 10 per cent. of glacial acetic acid added

on cooling. Allow to settle for some days, and then filter.

HENNEGUY’S PERMANGANATE METHOD.--Treat sections for 5 minutes with 1

per cent. potassium permanganate solution, then wash in water and stain

with safranine, rubin, gentian violet, vesuvine, preference being given

to a safranine solution prepared with aniline water.

HERMANN’S PLATINO-ACETO-OSMIC MIXTURE.--Mix 15 parts of 1 per cent.

platinic chloride solution, 1 part of glacial acetic acid, and 2 or 4

parts of 2 per cent. osmic acid.

HERTWIG’S MACERATING FLUID.--Mix equal parts of 0·05 per cent. osmic

acid, and 0·2 per cent. acetic acid. Medusæ are treated with this

mixture for 2 or 3 minutes, then washed in 0·1 per cent. acetic acid

until free from osmic acid. Leave them for 24 hours in the dilute

acetic acid, then wash in water, stain with Beale’s carmine, and mount

in glycerine. For Actiniæ use 0·04 per cent. osmic acid and make both

solutions with sea water. Wash out with 0·2 per cent. acetic acid, and

stain with picro-carmine.

HESSERT’S METHOD FOR STAINING FLAGELLA.--Fix the film by treating

cover-glass preparations with a saturated alcoholic solution of

mercuric chloride, wash, and stain for 30 or 40 minutes in a hot 10 per

cent. aqueous solution of saturated alcoholic solution of fuchsine.

HOFFMANN’S BLUE STAIN.--Dissolve 1 Gm. of Hoffmann’s blue in 20 C.c.

of rectified spirit and 80 C.c. of distilled water, then add 0·5

C.c. of glacial acetic acid. As a nuclear stain immerse sections for

10 minutes or more, rinse in water, wash in 90 per cent. alcohol,

dehydrate, clear, and mount in balsam. To stain sieve areas, less time

is required, 5 to 10 minutes, rinse in distilled water, and mount in

glycerine; or dehydrate, clear, and mount in balsam.

HOYER’S SHELLAC INJECTION MASS.--Dissolve shellac in 80 per cent.

alcohol to the consistency of a thin syrup, and strain through muslin

of medium thickness. Colour with aniline colours in alcoholic solution,

or by means of vermilion or other pigment suspended in alcohol.

HOYER’S SILVER NITRATE GELATINE MASS.--Mix a concentrated solution of

gelatine with an equal volume of a 4 per cent. silver nitrate solution

and warm, then add a very small quantity of aqueous pyrogallic acid

solution to reduce the silver salt, and add chloral and glycerine as in

the carmine gelatine mass.

HOYER’S SILVER STAIN.--Add ammonia to a solution of silver nitrate of

known strength, until the precipitate formed just re-dissolves, then

dilute the solution until it contains 0·75 to 0·50 per cent. of the

salt.

KAISER’S BISMARCK BROWN STAIN. Sections are stained for 48 hours, at a

temperature of 60 C., in a saturated solution of Bismarck brown in 60

per cent. alcohol, and washed out in 60 per cent. alcohol containing 2

per cent. of H.C.L., or 3 per cent. of acetic acid.

KAISER’S NERVE STAIN.--This is a modification of Weigert’s process.

The material is hardened in Müller’s solution for 2 or 3 days, then

cut into slices 2 to 4 Mm. thick, and treated with the solution for 5

or 6 days more. Subsequently immerse in Marchi’s solution for 8 days,

then wash, pass through alcohol, and imbed in celloidin. Sections are

mordanted for 5 minutes in the following mixture:--Solutions of ferric

chloride, 1 part; distilled water, 1 part; rectified spirit, 8 parts.

Next wash in Weigert’s hæmatoxylin, and warm in a fresh quantity of

the same for a few minutes, wash with water, differentiate in Pal’s

solution, and neutralise the oxalic acid by washing in water containing

a little ammonia.

KAISER’S STAIN FOR THE SPINAL CORD.--Sections are stained for a few

hours in solution of náphthylamine brown, 1 part, in water, 200 parts,

and alcohol, 100 parts. Afterwards wash with alcohol and clear with

origanum oil.

KALLIN’S NEUROLOGICAL METHOD.--Dissolve hydroquinone, 5 Gm., sodium

sulphite, 40 Gm., and potassium carbonate, 75 Gm., in 25 Gm. of

distilled water. At the time of using, dilute this solution with

one-third to one-half its bulk of absolute alcohol; immerse sections

of silvered material for several minutes until reduction is complete.

Then place them in 70 per cent. alcohol for 10 to 15 minutes, and

subsequently leave in aqueous solution of sodium hyposulphite (1:5) for

24 hours or more. Finally dehydrate and mount. Carmine may be used as

an afterstain.

KLEINENBERG’S SOLUTION (_Improved Formula_).--Hæmatoxylin, 2-1/2 Gm.;

crystallised calcium chloride, 20 Gm. in 10 C.c. of distilled water;

alum, 3 Gm. in 16 C.c. of distilled water; rectified spirit, 240 C.c.

Dissolve the calcium chloride and alum in their respective quantities

of water by the aid of heat; mix the solutions and immediately dilute

with rectified spirit; after an hour filter and add the hæmatoxylin.

This makes a good working solution which keeps well. Of course it

contains the alumina in solution, not as alum but aluminium chloride.

If in special cases the colour is considered too strong, the dilution

(when staining in bulk) must be made with some of the solution to which

hæmatoxylin has not been added.

KOCH’S METHOD FOR STAINING FLAGELLA.--Immerse cover-glass preparations

in a 1 per cent. aqueous solution of hæmatoxylin, then transfer to a 5

per cent. solution of chromic acid or to Müller’s fluid; dry and mount

in balsam.

KOCH-EHRLICH, BACILLI.--Place sections, or cover-glass preparations,

for at least 12 hours in gentian violet, or fuchsine aniline water

(aniline water, 100 C.c.; concentrated alcoholic solution of gentian

violet, or fuchsine, 11 C.c.; absolute alcohol, 10 C.c.), then immerse

in a mixture of pure nitric acid (sp. gr. 1·42), 10 C.c., and distilled

water, 30 C.c., for some seconds. Rinse in 60 per cent. alcohol for a

few minutes, and then counterstain with vesuvine (vesuvine, 0·5 Gm.;

rectified spirit, 20 C.c.; distilled water, 80 C.c.) after gentian

violet; or methylene blue (methylene blue, 0·25 Gm.; rectified spirit,

20 C.c.; distilled water, 80 C.c.) after fuchsine. Finally rinse in

water, dehydrate, clear, and mount in balsam. According to Squire, who

points out that nitric acid is apt to injure delicate sections, Watson

Cheyne recommends that sections should be transferred from fuchsine

aniline water to distilled water, then rinsed in alcohol, and placed

in the following contrast stain for 1 or 2 hours:--Saturated alcoholic

solution of methylene blue, 20 C.c.; distilled water, 100 C.c.; formic

acid (sp. gr. 1·2), 1 C.c.

KÜHNE’S CARBOLIC METHYLENE BLUE.--Rub up 1·5 Gm. of methylene blue with

10 C.c. of absolute alcohol, and add 100 C.c. of a 5 per cent. aqueous

solution of carbolic acid.

KÜHNE’S METHYL VIOLET SOLUTION.--Dissolve 1 Gm. of methyl violet in 90

C.c. of distilled water and 100 C.c. of alcohol.

KÜHNE’S ANILINE OIL SOLUTIONS.--Rub up as much methylene blue, methyl

green, or safranine as will go upon the point of a knife, with 10 C.c.

of aniline, and allow to settle.

KÜHNE’S CARBOLIC FUCHSINE OR BLACK BROWN.--Dissolve 1 Gm. of fuchsine

or black brown in 10 C.c. of absolute alcohol, and add 100 C.c. of a 5

per cent. aqueous solution of carbolic acid.

KÜHNE’S MODIFICATION OF GRAM’S METHOD.--Stain nuclei with carmine,

then treat sections for 5 minutes in methyl violet solution, diluted

one-sixth with a 1 per cent. aqueous solution of ammonium carbonate,

or in a solution of Victoria blue, 0·25 Gm., in rectified spirit, 20

C.c., and distilled water, 80 C.c. Next rinse thoroughly in water and

transfer to Grain’s solution for 2 to 3 minutes; again rinse in water

and extract excess of stain with solution of yellow fluorescine, 1

Gm., in absolute alcohol, 50 C.c. Finally, pass through pure alcohol,

aniline, terebene, and xylol, and mount in balsam.

LÖFFLER’S SOLUTION.--Concentrated alcoholic solution of methylene blue,

30 C.c.; solution of (caustic potash) potassium hydrate (1:10,000), 100

C.c. Mix and filter shortly before use. Sections are stained for a few

minutes (tubercle sections for some hours), and excess of stain can be

removed by immersion for a few seconds in 0·5 per cent. acetic acid.

Dehydrate in absolute alcohol, clear in cedar oil, and mount in balsam.

Löffler found that most bacteria stained better in this solution than

in the weaker solutions used by Koch for turbercle bacillus.

LAVDOWSKY’S BILBERRY JUICE STAIN.--Well wash the fresh berries of

_Vaccinium myrtillus_, then express the juice and mix with twice its

bulk of distilled water, mixed with a little 90 per cent. alcohol. Heat

for a short time and filter whilst warm. Dilute the stain with 2 or 3

volumes of distilled water before use.

LEE’S FORMALDEHYDE SOLUTIONS.--(1) Mix 1 part of 40 per cent.

formaldehyde solution with two parts of 1 per cent. chromic acid

solution, and add 4 per cent. of acetic acid. (2) Mix 1 part of 40

per cent. formaldehyde solution with 4 parts of 1 per cent. platinic

chloride solution, and add 2 per cent. of acetic acid.

LEE’S OSMIC ACID AND PYROGALLOL STAIN.--Fix the tissues in Hermann’s

mixture or Flemming’s mixture for half an hour, then place in a weak

solution of pyrogallol, which may be prepared with alcohol in some

cases. Safranine may be used as a second stain.

MARTINOTTI’S PICRO-NIGROSINE STAIN.--Pathological objects are stained

for 2 or 3 hours or days, in a saturated solution of nigrosine in

saturated alcoholic picric acid solution. Then wash out in a mixture

of 1 part of formic acid with 2 parts of alcohol until the grey matter

appears clearly differentiated from the white to the naked eye.

MAYER’S ALUMINIUM CHLORIDE CARMINE.--Dissolve 1 Gm. of carminic acid

and 3 Gm. of aluminium chloride in 200 C.c. of water.

MAYER’S BERLIN BLUE INJECTION.--Add a solution of 10 C.c. of tincture

of ferric chloride in 500 C.c. of water, to a solution of 20 Gm. of

potassium ferrocyanide in 500 C.c. of water, allow to stand for 12

hours, decant, wash the deposit for 1 or 2 days with distilled water

until the washings come through dark blue, then dissolve the blue in

about a litre of water.

MAYER’S CARMALUM.--Dissolve 1 Gm. of carminic acid and 10 Gm. of alum

in 200 C.c. of distilled water; decant, or filter, and add a few

crystals of thymol, 0·1 per cent. of salicylic acid, or 0·5 per cent.

of sodium salicylate. A weaker solution contains 3 to 5 times as much

alum and 5 times as much water.

MERBEL’S CARMINE AND INDIGO FLUIDS (give a blue and red stain, and

are very selective).--To prepare the red fluid, take--Carmine, 2 dr.;

borax, 2 dr.; distilled water, 4 ozs. For the blue fluid, take--Indigo

carmine, 2 dr.; borax, 2 dr.; distilled water, 4 ozs. Mix each in a

mortar, and allow it to stand, then pour off the supernatant fluid.

If the sections have been hardened in chromic acid, picric acid, or a

bichromate, they must be washed in water till no tinge appears. Place

them in alcohol for fifteen or twenty minutes, then in the two fluids

mixed in equal proportions, after which wash them in a saturated

aqueous solution of oxalic acid, where they should remain a rather

shorter time than in the staining fluids. When sufficiently bleached,

wash them in water, to get rid of the acid, then pass them through

spirit and oil of cloves, and mount in balsam or dammar.

MITROPHANOW’S GOLD PROCESS FOR PRICKLE-CELLS AND INTERCELLULAR

CANALS.--Wash the tail of an axolotl larva with distilled water, place

for an hour in a watch-glassful of 0·25 per cent. solution of gold

chloride, containing 1 drop of hydrochloric acid; wash, and reduce in a

mixture of 1 part of formic acid with 6 parts of water.

MITROPHANOW’S MACERATION METHOD FOR EPITHELIUM.--Fix the embryo for 15

minutes in 3 per cent. nitric acid; then place for an hour in a mixture

of alcohol, 1 volume, and water 2 volumes, and finally treat with

stronger alcohol for 24 hours to separate the epidermis.

MÜLLER’S BERLIN BLUE FOR INJECTIONS.--Precipitate a concentrated

solution of Berlin blue by means of 90 per cent. alcohol. The

precipitate is very finely divided, whilst the fluid is perfectly

neutral and much easier to prepare than that of Beale.

NEILSEN’S SOLUTION OF METHYL VIOLET.--Dissolve fuchsine, 1 part, in

alcohol, 10 parts, and add a 5 per cent. watery solution of carbolic

acid, 100 parts.

NEISSER’S DOUBLE-STAINING FOR SPORE-BEARING BACILLI.--Cover-glass

preparations are immersed for 20 minutes in fuchsine aniline water

(concentrated alcoholic solution of fuchsine, 11 C.c.; absolute

alcohol, 10 C.c.; aniline water, 100 C.c.; then heat to 80° or 90° C.;

next rinse in water, alcohol, or weak acid, according to the nature

of the bacilli, counterstain with aqueous solution of methylene blue,

rinse in water, dry and mount in balsam). The spores are stained red

and the rest of the bacilli blue.

NISSL’S FUCHSINE STAIN FOR NERVE CELLS.--(1) Fresh material in pieces

measuring 1 C.c. are hardened in a “chromic solution in 70 per cent.

alcohol” for 2 days, then transferred to absolute alcohol for 5 days,

and afterwards cut. Stain the sections singly in a saturated solution

of fuchsine, warming in a deep watch-glass until vapours begin to be

given off. Next plunge the section into absolute alcohol for 1 or 2

minutes, then place it on a slide, flood with clove oil, and when no

more colour is given off, drain and mount in balsam.

OHLMACHER’S FORMALDEHYDE STAINING.--Formalin in a 2 to 4 per cent.

solution is used as a mordant for tar colours. The tissues may be

mordanted separately by treatment for 1 minute or longer, or the

formalin may be added to the stain. Dissolve 1 Gm. of fuchsine in 10

C.c. of absolute alcohol, and add to 100 C.c. of 4 per cent. formalin

solution. Or, add saturated alcoholic solution of gentian violet or

methyl violet 5 B. to the formalin solution, in the proportion of 1:10.

In the case of methylene blue, dissolve 1 G.m. in 100 C.c. of the

formalin solution. Sections stain in half a minute, and are said to

resist alcohol much more than if formalin were not used.

OPPITZ’S SILVER STAINING.--Reduction is very rapidly effected by

placing the preparations for 2 or 3 minutes in a 0·25 to 0·5 per cent.

solution of chloride of tin.

PAL’S HÆMATOXYLIN STAIN.--Dissolve 0·75 Gm. of hæmatoxylin in 90 C.c.

of distilled water and 10 C.c. of absolute alcohol. Just before use add

saturated solution of lithium carbonate in the proportion of 3 drops to

each 10 C.c. of hæmatoxylin solution. (See Weigert.)

PAL’S HÆMATOXYLIN METHOD.--Proceed at first as in Weigert’s process for

nerve fibre, omitting the copper bath, and stain in Pal’s hæmatoxylin

solution (see above) for 5 or 6 hours. Then wash the sections in

distilled water (containing a trace of lithium carbonate if the

sections are not deep blue), next treat for 15 to 30 seconds with

a 0·25 per cent. potassium permanganate solution, rinse in water,

and decolourise in Pal’s bleaching solution. (If black spots appear

replace in the permanganate solution, again bleach, and wash for 15

minutes in water.) The grey substance of the sections is decolourised

in a few sections; the sections should then be well washed out, and

may be double-stained with picro-carmine or acetic acid carmine (see

Schneider), Magdala red, or eosine. The nuclei may be stained with alum

carmine. Finally dehydrate, clear, and mount.

PAL-EXNER’S OSMIC ACID METHOD.--Spinal cord or brain in 0·25 inch

cubes is immersed in 0·5 per cent. osmic acid solution for 2 days,

the solution being changed each day; then wash in water, transfer to

absolute alcohol, and imbed in celloidin or paraffin. Place sections as

cut in glycerine, then wash in water, treat with potassium permanganate

and Pal’s solution, as in Pal’s hæmatoxylin method, counter-stain with

carmine, dehydrate, clear, and mount in balsam.

PLANT’S METHOD OF STAINING ACTINOMYCOSIS.--Sections are placed for 10

minutes in Gibbes’ magenta solution or carbolic fuchsine, at 45° C.;

next they are rinsed in water and placed in saturated aqueous solution

of picric acid, mixed with an equal volume of absolute alcohol, for 5

or 10 minutes; they are then washed once more, passed through 50 per

cent. alcohol into absolute alcohol, cleared in cedar oil, and mounted

in balsam.

RANVIER’S LEMON JUICE METHOD.--Soak pieces of fresh tissue in fresh

lemon juice until transparent (5 to 10 minutes), then rapidly wash

in distilled water, treat for 10 to 60 minutes with 1 per cent.

gold chloride solution, again wash and expose to light in a bottle

containing 50 C.c. of distilled water and 2 drops of acetic acid.

Reduction is complete in 24 to 48 hours. If it is not desired to retain

the superficial epithelium, reduction may be more completely effected

in the dark, by treatment with formic acid (sp. gr. 1·2), diluted with

3 times its volume of water. The lemon juice in the above process may

be replaced by an aqueous solution of citric acid (40 grains in each

ounce).

RANVIER’S PICRO-CARMINE.--Carmine, 1 part; distilled water, 10 parts;

solution of ammonia, 3 parts; mix and add of a cold saturated solution

of picric acid 200 parts.

RENAUT’S HÆMATOXYLIC EOSINE.--Mix 30 C.c. of concentrated aqueous

solution of eosine, 40 C.c. of saturated alcoholic solution of

hæmatoxylin (which has been kept for some time and precipitated),

and 130 C.c. of saturated solution of potash alum in glycerine (sp.

gr. 1·26). Stand for 5 or 6 weeks in a partially covered vessel,

protected from dust, until the alcohol is evaporated, and then filter.

The filtrate can be diluted with glycerine if desired. Mount objects

in this fluid diluted with 1 or 2 volumes of glycerine, or, stain

separately for some days or weeks and mount in balsam, after washing in

alcohol charged with a sufficient quantity of eosine.

RANVIER AND VIGNAL’S OSMIUM MIXTURE.--Fix tissues in a freshly-prepared

mixture of equal volumes of 1 per cent. osmic acid and 90 per cent.

alcohol, then wash out in 80 per cent. alcohol, next with water, and

stain for 48 hours with picro-carmine or hæmatoxylin. This method has

been applied to the histology of insects.

RENAUT’S GLYCERINE HÆMATOXYLIN.--To a saturated solution of potash alum

in glycerine, add a saturated solution of homatoxylin in 90 per cent.

alcohol drop by drop, so as to form a deeply coloured solution. Expose

to daylight for a week, and then filter. This solution, like Renaut’s

hæmatoxylic cosine, may be used for mounting unstained sections, which

after some time absorb the colour from the liquid and become stained.

SAFRANINE.--Safranine, 0·5 Gm.; rectified spirit, 20 C.c.; distilled

water, 80 C.c.

SCHÄFER’S ACID LOGWOOD SOLUTION is especially useful for certain

structures, as tendon, cells, &c. It is thus prepared:--A 1 per cent.

solution of acetic acid is coloured by the addition of 1·3 of its

volume of logwood solution.

SCHÄFER’S ANILINE DYES, whether in aqueous or alcoholic solutions,

give good results, and are prepared as follows:--Roseanilin or magenta

(1 gr. to 1 oz. of alcohol), red; acetate of mauvein (4 gr., alcohol

1 oz., acid nitric 2 drops), blue; aniline black (2 gr., water 1

oz.), grey-black; Nicholson’s soluble blue (1-6 gr., alcohol 1 oz.,

and nitric 2 m.), blue. These stains should be used weak; and after

sections are stained they should be passed through alcohol and oil of

cloves as rapidly as possible; otherwise the colour will dissolve out

before they can be mounted in balsam.

SCHULTZE (_Staining Bacilli_).--Stain sections and cover-glass

preparations for some hours in aqueous methylene blue solution,

differentiate in 0·5 per cent. acetic acid, dehydrate in alcohol, clear

in cedar oil, and mount in balsam.

SCLAVO’S STAIN FOR FLAGELLA.--Leave the preparations for 1 minute in

a solution of 1 Gm. of tannin in 100 C.c. of 50 per cent. alcohol;

wash in distilled water; transfer for 1 minute to 50 per cent.

phospho-molybdic acid; again wash, and stain for 3 to 5 minutes in

a hot saturated solution of fuchsine in aniline water. Then wash in

water, dry on filter paper, and mount in balsam.

SQUIRE’S PICRO-CARMINE.--(1) Dissolve 1 Gm. of carmine with a gentle

heat in 3 C.c. of strong solution of ammonia, and 5 C.c. of distilled

water, then add 200 C.c. of saturated aqueous solution of picric

acid, heat to boiling, and filter. (2) Dissolve 10 Gm. of carmine in

a solution of 1 Gm. of caustic soda in 1000 C.c. of distilled water;

boil, filter and make up to 1000 C.c. with water. Mix the solution with

an equal quantity of water, and add 1 per cent. aqueous solution of

picric acid so long as the turbidity produced disappears on agitation.

SQUIRE’S BLUEING OF SECTIONS.--After staining with hæmatoxylin, treat

for a few seconds with a solution of sodium bicarbonate (1:1000) in

distilled water.

VALENTINE (_Fuchsine_).--Ether shaken with a solution containing

fuchsine is coloured violet after adding ferrous iodide, but not before.

VICTORIA BLUE.--Victoria blue, 0·25 Gm.; rectified spirit, 20 C.c.;

distilled water, 80 C.c.

WEDL’S ORSEILLE OR ORCHELLA STAIN.--Mix 5 C.c. of acetic acid, 20

C.c. of absolute alcohol, and 40 C.c. of distilled water; then add

sufficient archil, from which excess of ammonia has been driven off, to

form a dark reddish fluid.

WEIGERT’S HÆMATOXYLIN.--Dissolve 1 part of hæmatoxylin in 10 parts of

absolute alcohol; then add 90 parts of distilled water and 1 part of

aqueous solution (1:70) of lithium carbonate.

WEIGERT (_Gram’s Method_).--In this modification aniline is substituted

for alcohol, in order to avoid prolonged washing with the latter, and

the process is conducted on a slide. The section is placed on a slide,

stained with a few drops of gentian violet aniline water, prepared

as in Gram’s method, the excess of fluid removed, and a few drops of

Gram’s solution applied. Subsequently remove the liquid by gently

blotting it off, then wash the section by allowing aniline to flow’

backwards and forwards over it, and when colour ceases to come away,

repeat the operation with xylol for about 1 minute, then mount in

balsam.

WEIGERT (_Staining in Actinomycosis_).--Immerse sections for 1 hour in

Wedl’s Orseille stain, then quickly rinse with alcohol and counterstain

with gentian violet. If it be desired to stain the mycelium also,

afterwards submit the sections to Weigert’s modification of Gram’s

method. See page 335.

WEIGERT (_Staining Brain Tissue_).--Pieces of brain and spinal cord are

hardened in bichromate solution, followed by alcohol, then imbedded in

celloidin or gum. If imbedded in celloidin, the pieces are subsequently

taken from the spirit in which they are immersed, and placed for one

or two days in saturated aqueous solution of copper acetate, diluted

with an equal bulk of water, the mixture being kept at about 40° C.

Afterwards transfer the pieces to 80 per cent. alcohol until required

for cutting. Or, the sections can be cut first, and then treated with

copper acetate. To stain the sections, after being well washed in 90

per cent. alcohol, they are transferred to Weigert’s hæmatoxylin and

left from a few hours to two days, according to the differentiation

required. When opaque and of a deep blue-black colour, they should be

well washed for two or three days in distilled water. Next decolourise

for 0·5 to 2 hours in a solution of 2 Gm. of borax and 2·5 Gm. of

potassium ferrocyanide in 200 C.c. of water. As soon as the grey and

white substances are sharply defined, again wash the sections in water

for half an hour, then dehydrate, clear, and mount in balsam.

WOODHEAD’S METHOD OF STAINING TUBERCLE BACILLI.--Take a small quantity

of sputum rich in bacilli, and spread it out by pressure between

two cover-glasses, so that a fairly thin film remains on each. Then

carefully slip one over the other until they come apart. Thoroughly

dry the covers, and pass them rapidly three times through the flame

of a spirit lamp, care being taken not to scorch the film, then float

them face downwards on the staining solution, which has been previously

prepared and filtered into a watch-glass. The stain should consist

of saturated alcoholic solution of basic fuchsine, 1 part; absolute

alcohol or rectified spirit, 10 parts; carbolic acid solution (5 per

cent.), 10 parts. Leave the preparations in the watch-glass for 12

to 24 hours, unless time is an object. In the latter case heat the

fluid gently until vapour is given off, then drop the films on the

surface, and leave them for 3 to 5 minutes only. Next transfer the

covers to an aqueous solution of sulphuric acid (25 per cent.), and

when decolourisation is complete, as evidenced by the pink colouration

not returning when the specimens are plunged into a bowl of tap-water

containing a single drop of ammonia solution, thoroughly rinse in the

slightly alkaline water and counter-stain in an aqueous solution of

methylene blue. Finally, wash in water, carefully dry and mount in

Canada balsam. The bacilli should stand out as bright red rods on a

blue background of cells.

ZIEHL-NEELSEN (_Staining Bacilli_).--Sections are removed from weak

spirit into Neelsen’s carbolic fuchsine and left for 10 or 15 minutes;

next decolourise in sulphuric acid (sp. gr. 1·84) or nitric acid (sp.

gr. 1·42) diluted with 3 volumes of water, rinse in 60 per cent.

alcohol, and wash in a large volume of water to remove the acid.

Tubercle and leprosy bacilli are the only micro-organisms that can

retain the stain after treatment with acid. If the presence of traces

of nitrous acid in the nitric acid be suspected, Squire recommends

the use of saturated aqueous solution of sulphanilic acid mixed with

one-third its bulk of nitric acid. The sulphanilic acid destroys any

free nitrous acid, which would otherwise exercise a bleaching action on

the fuchsine-stained bacilli. The sections may be counterstained with

a solution of 0·5 Gm. of methyl green (or 0·25 Gm. of methylene blue)

in 20 C.c. of rectified spirit and 80 C.c. of distilled water. Finally

dehydrate in absolute alcohol, clear in cedar oil, and mount in balsam.

APPENDIX D.

THE METRIC SYSTEM OF WEIGHTS AND MEASURES.

The initial unit of the Metric System is the Metre or unit of length,

which represents one ten millionth part of the earth’s quadrant, or

one forty-millionth part of the circumference of the earth around the

poles. The multiples and sub-divisions of this and all the other units

are obtained by the use of decimals, and for this reason the system is

also known as the _decimal system_. The multiples are designated by the

Greek prefixes, _deca_ = 10; _hecto_ = 100; _kilo_ = 1000; _myria_ =

10,000. For the sub-divisions Latin prefixes are employed, as follows:

_deci_ = 1/10; _centi_ = 1/100; _milli_ = 1/1000. Thus for measures of

length we have the following expressions, showing the abbreviations

commonly employed, and the equivalents in the ordinary English

standards of measurement--

1 Myriametre, Mm. = 10,000.0 M. = 6.2137 miles.

1 Kilometre, Km. = 1,000.0 M. = 0.6213 mile.

1 Hectometre, Hm. = 100.0 M. = 109.362 yards.

1 Decametre, Dm. = 10.0 M. = 32.8086 feet.

1 Metre, M. = 1.0 M. = 39.3704 inches.

1 Decimetre, dm. = 0.1 M. = 3.9370 "

1 Centimetre, cm. = 0.01 M. = 0.3937 "

1 Millimetre, mm. = 0.001 M. = 0.0393 "

From the unit of linear measure of metre is derived the unit of the

measure of capacity or LITRE. This represents the cube of one-tenth

part of a metre, or a cubic decimetre, and its multiples and

sub-divisions with their corresponding equivalents in Imperial fluid

measure are as follows:--

1 Myrialitre, Ml. = 10,000.0 L. = 2200.9667 imperial gallons.[91]

1 Kilolitre, Kl. = 1,000.0 " = 220.0966 " "

1 Hectolitre Hl. = 100.0 " = 22.0096 " "

1 Decalitre, Dl. = 10.0 " = 2.2009 " "

1 Litre, L. = 1.0 " = 35.2154 fluid ounces imperial.

1 Decilitre, dl. = 0.1 " = 3.5215 " " "

1 Centilitre, cl. = 0.01 " = 0.3521 " " "

1 Millilitre, ml. = 0.001 " = 0.0352 " " "

or

1 Cubic Centimetre, ccm. = 0.001 L. = 0.0352 " " "

The unit of weight in the metric system is the GRAMME. This is also

derived from the metre, and represents the weight of one cubic

centimetre, of water, or the quantity of distilled water, at its

maximum density, 4° C. (39·2° F.), which would fill the cube of

one-hundredth part of a metre. The relative value of the gramme,

together with its multiples and sub-divisions, as compared with the

English standards of weight, may be seen from the following table:--

1 Myriagramme, Mg. = 10,000.0 Gm. = 22.0461 pounds.

1 Kilogramme, Kg. = 1,000.0 " = 2.2046 "

1 Hectogramme, Hg. = 100.0 " = 3.5273 ounces avoir.

1 Decagramme, Dg. = 10.0 " = 154.3235 grains.

1 Gramme, Gm. = 1.0 " = 15.4323 "

1 Decigramme, dg. = 0.1 " = 1.5432 "

1 Centigramme, cg. = 0.01 " = 0.1543 "

1 Milligramme, mg. = 0.001 " = 0.0154 "

The expression _micro-millimetre_ is used for microscopic measurements,

and denotes the thousandth part of a millimetre. Of the measures of

capacity, the terms most commonly employed are the litre and the cubic

centimetre. Thus a decalitre may also be expressed as 10 litres, a

centilitre as 10 cubic centimetres, etc. Of the metric weights the

gramme and its fractional parts, with their respective prefixes, are

much used in analytical work. The kilogramme is largely employed in

commercial transactions, and is commonly abbreviated _kilo_.

As a comparison of the values of some of the more frequently employed

expressions of the metric and English systems, the following may be

found convenient for reference:--

1 mm. (millimetre) = 1/25 of an inch.

1 cm. (centimetre) = 2/5 of an inch.

1 inch = 25 millimetres or 2-1/2 centimetres.

1 mg. (milligramme) = 0.01543 grain (or approx. 1/64 grain).

1 gm. (gramme) = 15.4324 grains.

1 Kg. (“Kilo” or kilogramme) = 2 lbs. 3-1/4 ozs. av.

1 pound avoir. = 453,592 grammes.

1 ounce avoir. = 28,350 grammes.

1 grain = 0.06479 gramme or 64.79 milligrammes.

1 cc. (cubic centimetre) = 16.9 minims Imperial measure.

1 L. (litre) = 35.21 fluid ounces Imperial measure, or 33.815 fluid

ounces Wine measure.

1 fluid ounce Imperial measure = 28.350 grammes.

1 pint Imperial measure = 567.0 grammes.

1 gallon Imperial measure = 4.536 litres, or 10 lbs. avoir. of pure water

at 62° F. and under an atmospheric pressure of 30 inches of mercury.

It may be well to bear in mind that on the Continent liquids are always

weighed, not measured.

APPENDIX E.

COMPARISON BETWEEN THE CENTIGRADE AND FAHRENHEIT THERMOMETERS.

F. C.

212 100

200 93.3

150 65.6

112 44.4

110 43.3

108 42.2

106 41.1

105 40.5

104 40

103 39.4

102 38.9

101 38.3

100 37.8

99 37.2

98 36.7

96 35.6

94 34.4

92 33.3

90 32.2

88 31.1

86 30

84 28.9

82 27.8

80 26.7

78 25.6

76 24.4

74 23.3

72 22.2

70 21.1

68 20

66 18.9

64 17.8

62 16.7

60 15.6

58 14.4

56 13.3

54 12.2

52 11.1

32 0

25 -3.9

[Illustration: Dr. Culpeper’s Microscope 1738.]

INDEX.

Abbé on microscopical vision, 37

Abbé’s apertometer, 59

---- condenser, 176

---- stereoscopic eye-pieces, 64

---- test-plate, 164

Aberration, chromatic, 25

---- of the eye, chromatic, 33

---- spherical, 23

Abraxas grossulariata, 598

Absolute alcohol as a hardening reagent, 287

Acaras domesticus, 625

Accessories of the microscope, 197

Achromatic condenser, Beck’s, 180

---- ---- Gillett’s, 173

---- ---- method of using, 190

---- ---- Powell’s, 178

---- ---- Ross’s, 176

---- ---- Smith & Beck’s, 173

---- ---- Watson’s, 177

Achromatic objective, the, 152

Acineta, 495

Actiniæ, 527

Actinophrys-sol, 489

Adams’s book on the microscope, 8

Adipose tissue, 644

Ædogoniaceæ, 409

Aerobic spores, 399

Agar-agar, to prepare nutrient, 330

Air bubbles, 348

Alcyonella, 534

Algæ, 399

---- media for preserving, 343

---- red, 413

Alvarez’s discovery of bacillus, 392

Amici prism, the, 190

Amœba, 480

Amphibian changes, 669

Amphistoma, 570

Amyot finder, the, 205

Anacharis alsinastrum, 419

Anemones, sea, 526

Angle of vision, 72

Anguillula, 567

Animal structures, staining, 292

Annulosa, 562

Antennæ of insects, 584

Antenna of silkworm moth, 605

Anthrax bacillus, 369

Anthrozoa, 523

Apertometer, Abbé’s, 59

Aperture, definition of, 45

---- measurement of, 57

---- numerical, 57

---- table, 58

Aphides, 587

Aphrophora bifasciata, 618

Apis mellifica, 598

Aplysiidæ, 549

---- dipilans, 549

Apparatus for mounting, 352

Appendices, 673

Arachnidæ, 618

Aragonite, 232

Arcella, 483

Arenicola, 577

Argyroneta aquatica, 621

Artemiæ, 581

Arteries, 622

Artery-needle, 303

Arthropoda, 583

Arthrospores, 366

Ascidian, 669

Astroides calyculcaris, 529

Babè’s method of staining bacteria, 334

Bacillus, anthrax, 369

---- of plague, 372

---- ---- in rat’s blood, 372

---- splenic fever, 369

---- typhoid, 370

Bacteria, 317

---- aerobic, 399

---- classification of, 373

---- Cohn on multiplication of, 367

---- cultivation of, 327

---- ---- in tubes, 331

---- ---- on plates, 331

---- faculties of, 373

---- in butter, 393

---- in cheese, 393

---- in milk, 393

---- in sections of tissue, 337

---- invasion of potato-tubers by, 398

---- microscopical examination of, 333

---- phosphorescent, 373

---- reproduction of, 365

---- size of, 365

---- staining, 334

---- Winogradsky’s investigations of, 398

Bacterial action in tanning skins, 393

---- fermentations, 391

Bacteriological investigations, apparatus for, 318

---- ---- mounting media, 320

---- ---- reagents used, 320

---- microscope, the, 135

Bacteriology of the dairy, 393

Baker’s advanced student’s microscope, 123

---- collecting stick, 350

---- histological microscope, 125

---- micro-photographic apparatus, 217

---- microscope lamp, 191

---- microscopes, 120

---- Nelson condenser, 184

---- ---- model microscope, 120

---- objectives, 168

---- student’s condenser, 184

Baird, Dr., on daphnia, 581

Barnacle, 539

Bartley’s warm-stage, 281

Batrachospermæ, 409

Beck’s binocular dissecting microscope, 101

---- ---- National microscope, 99

---- complete microscope lamp, 202

---- compressor, 275

---- disc-holder, 198

---- large Continental model microscope, 98

---- microscopes, 95

---- objectives, 167

---- pathological microscope, 95

---- Star microscope, 101

Beggiatoa, 400

Benjamin Martin’s microscope, 5

Beroidæ, 519

Biaxial crystals, 228

Bilharzia hæmatobra, 573

Binocular microscope, advantage of, 69

---- ---- Carpenter on, 69

---- ---- Nachet’s, 62

---- ---- Pillischer’s, 128

---- ---- Riddell’s, 62

---- ---- Stephenson’s erecting, 71

---- ---- Wenham’s, 65

---- vision, 60

Bismarck-brown for staining protoplasm, 306

Bivalves, 538

Bleaching process, 315

Blood as a test, 263

---- circulation of, in frog’s foot, 665

---- ---- ---- tadpole, 665

---- corpuscles, 638

---- ---- double staining, 295

---- ---- size of, 640

---- crystals, 641

---- spectrum, 252

Bombay plague, 371

Bone, 658

---- of fish, 661

---- of reptilia, 660

---- structure of, 659

Borax, 231

Boring sponges, 513

Botterill’s live-trough, 276

Brachiopoda, 538

Branchipodidæ, 580

Brewster’s microscope, 11

Brittleworts, 427

Browning-Huggins micro-spectroscope, 245

Browning’s pocket lens, 76

Bryophyta, 444

Bryozoa, 531

Buchner’s experiments on yeast, 389

Bull’s-eye condensing-lens, 199

Butter, bacteria in, 393

Butterfly’s tongue, 605

---- wings, 610

Calc-spar, 231

Cambridge rocking microtome, 290

Camera lucida, the, 207

---- ---- the Abbé, 208

---- ---- the Wollaston, 207

---- Swift’s horizontal, 213

Canada balsam, 293

Carbonate of lead, 232

Carmine as a nuclear stain, 312

Cartilage, 655

Catheart’s freezing microtome, 291

Cedar oil, use of, 171

Cell, definition of, 358

Cell-making turn-table, Walmsley’s 340

Cells, epithelial, 636

---- for living objects, 276

---- for mounting, 340

---- live, 277

Cellulose, 357

---- staining, 314

Cements, 347

---- list of, 676

Centipedes, 578

Cercariæ, 571

Cereal parasites, 381

Chætophoraceæ, 409

Chara, fructification of, 417

---- mounting, 347

---- vulgaris, 415

Characeæ, 415

Cheese, bacteria in, 393

---- mite, 625

Chilinidæ, 551

Chitonidæ, 545

Chloride of gold as stain, 297

---- of palladium as stain, 298

Chromatic aberration, 25

---- ---- of the eye, 33

Chromic acid as hardening reagent, 288

Ciliata, 498

Circulation of the blood, 665

Cistula catenata, 558

Cladocera, 580

Clavatella prolifera, 521

Clearing agents, list of, 676

Clepsinidæ, 576

Clionæ, 513

Closterium, 424

---- lunula, 425

Cnidaria, 519

Cockchafer’s eye, 590

Coddington lens, the, 76

Codosiga, 497

Cœlenterata, 515

Cohn on multiplication of bacteria, 367

Cole’s direction for section cutting, 285

---- section-cutting microtome, 289

Collecting stick, Baker’s, 350

Collection of objects, 349

Compound microscope, 78

Compressor, Beck’s, 275

Compressorium, 274

---- Ross’s, 275

---- Rousselet’s, 275

Concave lenses, 23

---- surfaces, 17

Condenser, Abbé’s, 176

---- Baker’s Nelson, 184

---- ---- student’s, 184

---- Beck’s achromatic, 180

---- Gillett’s achromatic, 173

---- method of using, 190

---- Powell’s achromatic, 178

---- Ross’s achromatic, 176

---- Smith & Beck’s achromatic, 173

---- ---- substage, 193

Condenser, Swift’s, 183

---- Watson’s achromatic, 177

---- ---- parachromatic, 182

---- Webster-Collins, 186

---- Wenham’s immersion, 189

---- ---- parabolic, 186

Confervaceæ, 408

Conjugate foci, 17

---- real and virtual, 21

Continental microscopes, 130

Contrast stains, 313

Convex lens, 18

Copepoda, 580

Corals, 515, 525

---- true, 528

---- typical forms of, 533

Correction collar, Lister’s, 155

Coryne stauridia, 534

Cotton fibres, 474

Cover glass gauge, Zeiss’s, 165

Crinoids, 542

Critical angle, 14

Crookshank’s incubator, 324

---- method of staining bacteria, 335

Crustaceæ, 578

Crystals, formation and polarisation of, 239

Ctenophora, 518

Cuckoo-spit, 618

Culex pipiens, 596

Cultivation of bacteria, 327

---- of micro-organisms, 327

Cutleria dichotoma, 413

Cutting sections of hard woods, 316

Cuttle-fish, 556

Cyclops, 580

Cyclosis, phenomenon of, 359

Cyclostomata, 537

Cyclotus translucidus, 558

Cydippidæ, 518

Cymba olla, 557

Cymothordæ, 580

Dairy, bacteriology of, 393

Daphnia, enemies of, 581

---- ephippial eggs of, 580

Daphnia pulex, 580

De Bary’s investigations in parasitism, 395

Decalcifying and bleaching agents, list of, 677

Decalcifying solution as hardening reagent, 288

Demodex folliculorum, 627

Dental structure, 652

Dermestes lardarius, 627

Dermis, the human, 647

Desmidiaceæ, 420

---- reproduction of, 423

Diamond microscope, Pritchard’s, 9

Diaphragm, the, 194

---- the iris, 176

Diatomaceæ, 420, 427

---- fossilised, 437

---- Max Schultze’s researches, 430

---- where found, 428

Diatoms, mounting medium, 343

---- movements of, 431

Didymoprium grevelli, 420

Difflugia, 482

Digestive system of insects, 587

Dipping-tubes, 279

Disc-holder, Beck’s, 198

Dissecting-knives, 284

Dog-tick, 624

Double convex lens, 19

Draparnaldia glomerata, 409

Draw-tube, Swift’s, 116

---- Watson’s, 104

Drone fly, 594

Dytiscus marginalis, 607

Echinococcus, 565

Echinodermata, 539

Eggs of insects, 612

Elementary optics, 12

Embedding fluids, list of, 678

---- in paraffin wax, 285

Entomological specimens, mounting, 341

Entozoa, 562

Eosin stain, 315

Eozoon, 492

Epeira diadema, 619

Epidermis of plants, 455

Epithelial cells, 636

Epithelium, mounting, 295

Equisetaceæ, 449

Ergot of rye, 382

Eristalis tenax, 594

Erysiphe Tuckeri, 380

Eudorina, 406

Euglypta, 482

Eurotium repens, 383

Exposure table for photo-micrography, 213

Eye, chromatic aberration of the, 33

---- of cockchafer, 590

---- of fly, 588

---- of whirligig beetle, 608

---- the human, 30

Eye-piece, Abbé’s stereoscopic, 64

---- compensating, 147

---- ---- Zeiss’s, 147

---- Huyghenian, 139

---- Jackson’s micrometer, 143

---- Ramsden, 142

---- ---- micrometer, 145

---- Ross’s, 68

---- Wenham’s double, 63

---- Zeiss’s, 147

Eye-pieces, 139

---- achromatic, 149

---- magnifying powers of, 169

---- projections, 150

---- to clean, 259

Eyes of insects, 584

Favellidium, 415

Feet of insects, observation of, 604

Felices, 446

Fermentation experiments, 361

Fermentations, bacterial, 391

Ferns, 446