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THE ELEMENTS OF BACTERIOLOGICAL TECHNIQUE
A Laboratory Guide for Medical, Dental, and Technical Students
by
J. W. H. EYRE, M.D., M.S., F.R.S. (EDIN.)
Director of the Bacteriological Department of Guy's Hospital, London,
and Lecturer on Bacteriology in the Medical and Dental Schools; formerly
Lecturer on Bacteriology at Charing Cross Hospital Medical School, and
Bacteriologist to Charing Cross Hospital; sometime Hunterian Professor,
Royal College of Surgeons, England
Second Edition Rewritten and Enlarged
Philadelphia and London
W. B. Saunders Company
1913
Copyright, 1902, by W. B. Saunders and Company Revised, entirely
reset, reprinted, and recopyrighted July, 1913
Copyright, 1913, by W. B. Saunders Company
Registered at Stationers' Hall, London, England
Printed in America
Press of
W. B. Saunders Company
Philadelphia
TO THE MEMORY OF
JOHN WICHENFORD WASHBOURN, C.M.G., M.D., F.R.C.P.
Physician to Guy's Hospital and Lecturer on Bacteriology in the
Medical School, and Physician to the London Fever Hospital
MY TEACHER, FRIEND, AND CO-WORKER
PREFACE TO THE SECOND EDITION
Bacteriology is essentially a practical study, and even the elements of
its technique can only be taught by personal instruction in the
laboratory. This is a self-evident proposition that needs no emphasis,
yet I venture to believe that the former collection of tried and proved
methods has already been of some utility, not only to the student in the
absence of his teacher, but also to isolated workers in laboratories far
removed from centres of instruction, reminding them of forgotten details
in methods already acquired. If this assumption is based on fact no
further apology is needed for the present revised edition in which the
changes are chiefly in the nature of additions--rendered necessary by
the introduction of new methods during recent years.
I take this opportunity of expressing my deep sense of obligation to my
confrere in the Physiological Department of our medical school--Mr. J.
H. Ryffel, B. C., B. Sc.--who has revised those pages dealing with the
analysis of the metabolic products of bacterial life; to successive
colleagues in the Bacteriological Department of Guy's Hospital, for
their ready co-operation in working out or in testing new methods; and
finally to my Chief Laboratory Assistant, Mr. J. C. Turner whose
assistance and experience have been of the utmost value to me in the
preparation of this volume. I have also to thank Mrs. Constant Ponder
for many of the new line drawings and for redrawing a number of the
original cuts.
JOHN W. H. EYRE.
GUY'S HOSPITAL, S. E.
_July, 1913._
PREFACE TO THE FIRST EDITION
In the following pages I have endeavoured to arrange briefly and
concisely the various methods at present in use for the study of
bacteria, and the elucidation of such points in their life-histories as
are debatable or still undetermined.
Of these methods, some are new, others are not; but all are reliable,
only such having been included as are capable of giving satisfactory
results even in the hands of beginners. In fact, the bulk of the matter
is simply an elaboration of the typewritten notes distributed to some of
my laboratory classes in practical and applied bacteriology;
consequently an attempt has been made to present the elements of
bacteriological technique in their logical sequence.
I make no apology for the space devoted to illustrations, nearly all of
which have been prepared especially for this volume; for a picture, if
good, possesses a higher educational value and conveys a more accurate
impression than a page of print; and even sketches of apparatus serve a
distinct purpose in suggesting to the student those alterations and
modifications which may be rendered necessary or advisable by the
character of his laboratory equipment.
The excellent and appropriate terminology introduced by Chester in his
recent work on "Determinative Bacteriology" I have adopted in its
entirety, for I consider it only needs to be used to convince one of its
extreme utility, whilst its inclusion in an elementary manual is
calculated to induce in the student habits of accurate observation and
concise description.
With the exception of Section XVII--"Outlines for the Study of
Pathogenic Bacteria"--introduced with the idea of completing the volume
from the point of view of the medical and dental student, the work has
been arranged to allow of its use as a laboratory guide by the technical
student generally, whether of brewing, dairying, or agriculture.
So alive am I to its many inperfections that it appears almost
superfluous to state that the book is in no sense intended as a rival to
the many and excellent manuals of bacteriology at present in use, but
aims only at supplementing the usually scanty details of technique, and
at instructing the student how to fit up and adapt apparatus for his
daily work, and how to carry out thoroughly and systematically the
various bacterioscopical analyses that are daily demanded of the
bacteriologist by the hygienist.
Finally, it is with much pleasure that I acknowledge the valuable
assistance received from my late assistant, Mr. J. B. Gall, A. I. C., in
the preparation of the section dealing with the chemical products of
bacterial life, and which has been based upon the work of Lehmann.
JOHN W. H. EYRE.
GUY'S HOSPITAL, S. E.
CONTENTS
PAGE
I. LABORATORY REGULATIONS 1
II. GLASS APPARATUS IN COMMON USE 3
The Selection, Preparation, and Care of
Glassware, 8--Cleaning of Glass
Apparatus, 18--Plugging Test-tubes and
Flasks, 24.
III. METHODS OF STERILISATION 26
Sterilising Agents, 26--Methods of
Application, 27--Electric Signal Timing
Clock, 38.
IV. THE MICROSCOPE 49
Essentials, 49--Accessories, 57--Methods
of Micrometry, 61.
V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER
MICRO-FUNGI 69
Apparatus and Reagents used in Ordinary
Microscopical Examination, 69--Methods of
Examination, 74.
VI. STAINING METHODS 90
Bacteria Stains, 90--Contrast Stains,
93--Tissue Stains, 95--Blood Stains,
97--Methods of Demonstrating Structure of
Bacteria, 99--Differential Methods of
Staining, 108.
VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES 114
Freezing Method, 115--Paraffin Method,
117--Special Staining Methods for
Sections, 121.
VIII. CLASSIFICATION OF FUNGI 126
Morphology of the Hyphomycetes,
126--Morphology of the Blastomycetes,
129.
IX. SCHIZOMYCETES 131
Anatomy, 134--Physiology,
136--Biochemistry, 144.
X. NUTRIENT MEDIA 146
Meat Extract, 148--Standardisation of
Media, 154--The Filtration of Media,
156--Storing Media in Bulk, 159--Tubing
Nutrient Media, 160.
XI. ORDINARY OR STOCK CULTURE MEDIA 163
XII. SPECIAL MEDIA 182
XIII. INCUBATORS 216
XIV. METHODS OF CULTIVATION 221
Aerobic, 222--Anaerobic, 236.
XV. METHODS OF ISOLATION 248
XVI. METHODS OF IDENTIFICATION AND STUDY 259
Scheme of Study, 259--Macroscopical
Examination of Cultivations,
261--Microscopical Methods,
272--Biochemical Methods, 276--Physical
Methods, 295--Inoculation Methods,
315--Immunisation, 321--Active
Immunisation, 322--The Preparation of
Haemolytic Serum, 327--The Titration of
Haemolytic Serum, 328--Storage of
Haemolysin, 331.
XVII. EXPERIMENTAL INOCULATION OF ANIMALS 332
Selection and Care of Animals,
335--Methods of Inoculation, 352.
XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE 370
General Observations, 371--Blood
Examinations, 373--Serological
Investigations, 378--Agglutinin,
381--Opsonin, 387--Immune Body, 393.
XIX. POST-MORTEM EXAMINATION OF EXPERIMENTAL ANIMALS 396
XX. THE STUDY OF THE PATHOGENIC BACTERIA 408
XXI. BACTERIOLOGICAL ANALYSES 415
Bacteriological Examination of Water,
416--Examination of Milk, 441--Ice Cream,
457--Examination of Cream and Butter,
457--Examination of Unsound Meats,
460--Examination of Oysters and Other
Shellfish, 463--Examination of Sewage and
Sewage Effluents, 466--Examination of
Air, 468--Examination of Soil,
470--Testing Filters, 478--Testing of
Disinfectants, 480.
APPENDIX 492
INDEX 505
[Illustration]
BACTERIOLOGICAL TECHNIQUE.
I. LABORATORY REGULATIONS.
The following regulations are laid down for observance in the
Bacteriological Laboratories under the direction of the author. Similar
regulations should be enforced in all laboratories where pathogenic
bacteria are studied.
_Guy's Hospital._
~BACTERIOLOGICAL DEPARTMENT.~
HANDLING OF INFECTIVE MATERIALS.
The following Regulations have been drawn up in the interest
of those working in the Laboratory as well as the public at
large, and will be strictly enforced.
Their object is to avoid the dangers of infection which may
arise from neglect of necessary precautions or from
carelessness.
Everyone must note that by neglecting the general rules laid
down he not only runs grave risk himself, but is a danger to
others.
REGULATIONS.
1. Each worker must wear a gown or overall, provided at his
own expense, which must be kept in the Laboratory.
2. The hands must be disinfected with lysol 2 per cent.
solution, carbolic acid 5 per cent. solution, or corrosive
sublimate 1 per mille solution, after dealing with
infectious material, and ~before using towels~.
3. On no account must Laboratory towels or dusters be used
for wiping up infectious material, and if such towels or
dusters do become soiled, they must be immediately
sterilised by boiling.
4. Special pails containing disinfectant are provided to
receive any waste material, and nothing must be thrown on
the floor.
5. All instruments must be flamed, boiled, or otherwise
disinfected immediately after use.
6. Labels must be moistened with water, and not by the
mouth.
7. All disused cover-glasses, slides, and pipettes after use
in handling infectious material, etc., must be placed in 2
per cent. lysol solution. A vessel is supplied on each bench
for this purpose.
8. All plate and tube cultures of pathogenic organisms when
done with, must be placed for immediate disinfection in the
boxes provided for the purpose.
9. No fluids are to be discharged into sinks or drains
unless previously disinfected.
10. Animals are to be dissected only after being nailed out
on the wooden boards, and their skin thoroughly washed with
disinfectant solution.
11. Immediately after the post-mortem examination is
completed each cadaver must be placed in the zinc
animal-box--_without removing the carcase from the
post-mortem board_--and the cover of the box replaced, ready
for carriage to the destructor.
12. Dead animals, when done with, are cremated in the
destructor, and the laboratory attendant must be notified
when the bodies are ready for cremation.
13. None of the workers in the laboratory are allowed to
enter the animal houses unless accompanied by the special
attendant in charge, who must scrupulously observe the same
directions regarding personal disinfection as the workers in
the laboratories.
14. No cultures are to be taken out of the laboratory
without the permission of the head of the Department.
15. All accidents, such as spilling infected material,
cutting or pricking the fingers, must be at once reported to
the bacteriologist in charge.
II. GLASS APPARATUS IN COMMON USE.
The equipment of the bacteriological laboratory, so far as the glass
apparatus is concerned, differs but little from that of a chemical
laboratory, and the cleanliness of the apparatus is equally important.
The glassware comprised in the following list, in addition to being
clean, must be stored in a sterile or germ-free condition.
~Test-tubes.~--It is convenient to keep several sizes of test-tubes in
stock, to meet special requirements, viz.:
1. ~18 x 1.5~ cm., to contain media for ordinary tube cultivations.
2. ~18 x 1.3~ cm., to contain media used for pouring plate cultivations,
and also for holding sterile "swabs."
3. ~18 x 2~ cm., to contain wedges of potato, beetroot, or other vegetable
media.
4. ~13 x 1.5~ cm., to contain inspissated blood-serum.
The tubes should be made from the best German potash glass,
"blue-lined," stout and heavy, with the edge of the mouth of the tube
_slightly_ turned over, but not to such an extent as to form a definite
rim. (Cost about $1.50, or 6 shillings per gross.) Such tubes are
expensive it is true, but they are sufficiently stout to resist rough
handling, do not usually break if accidentally allowed to drop (a point
of some moment when dealing with cultures of pathogenic bacteria), can
be cleaned, sterilised, and used over and over again, and by their
length of life fully justify their initial expense.
A point be noted is that the manufacturers rarely turn out such tubes as
these absolutely uniform in calibre, and a batch of 18 by 1.5 cm. tubes
usually contains such extreme sizes as 18 by 2 cm. and 18 by 1.3 cm.
Consequently, if a set of standard tubes is kept for comparison or
callipers are used each new supply of so-called 18 by 1.5 cm. tubes may
be easily sorted out into these three sizes, and so simplify ordering.
5. ~5 x 0.7~ cm., for use in the inverted position inside the tubes
containing carbohydrate media, as gas-collecting tubes.
These tubes, "unrimmed," may be of common thin glass as less than two
per cent. are fit for use a second time.
[Illustration: FIG. 1.--Bohemian flask.]
[Illustration: FIG. 2.--Pear-shaped flask.]
[Illustration: FIG. 3.--Erlenmeyer flask (narrow neck).]
~Bohemian Flasks~ (Fig. 1).--These are the ordinary flasks of the chemical
laboratory. A good variety, ranging in capacity from 250 to 3000 c.c.,
should be kept on hand. A modified form, known as the "pear-shaped"
(Fig. 2), is preferable for the smaller sizes--i. e., 250 and 500 c.c.
~Erlenmeyer's Flasks~ (Fig. 3).--Erlenmeyer's flasks of 75, 100, and 250
c.c. capacity are extremely useful. For use as culture flasks care
should be taken to select only such as have a narrow neck of about 2 cm.
in length.
~Kolle's Culture Flasks~ (Fig. 4).--These thin, flat flasks (to contain
agar or gelatine, which is allowed to solidify in a layer on one side)
are extremely useful on account of the large nutrient surface available
for growth. A surface cultivation in one of these will yield as much
growth as ten or twelve "oblique" tube cultures. The wide mouth,
however, is a disadvantage, and for many purposes thin, flat culture
bottles known as ~Roux's bottles~ (Fig. 5) are to be preferred.
[Illustration: FIG. 4.--Kolle's culture flask.]
[Illustration: FIG. 5.--Roux's culture bottle.]
[Illustration: FIG. 6.--Guy's culture bottle.]
[Illustration: FIG. 7.--Filter flask.]
An even more convenient pattern is that used in the author's laboratory
(Fig. 6), as owing to the greater depth of medium which it is possible
to obtain in these flasks an exceedingly luxuriant growth is possible;
the narrow neck reduces the chance of accidental contamination to a
minimum and the general shape permits the flasks to be stacked one upon
the other.
~Filter Flasks or Kitasato's Serum Flasks~ (Fig. 7).--Various sizes, from
250 to 2000 c.c. capacity. These must be of stout glass, to resist the
pressure to which they are subjected, but at the same time must be
thoroughly well annealed, in order to withstand the temperature
necessary for sterilisation.
All flasks should be either of Jena glass or the almost equally
well-known Resistance or R glass, the extra initial expense being
justified by the comparative immunity of the glass from breakage.
~Petri's Dishes or "Plates"~ (Fig. 8, a).--These have now completely
replaced the rectangular sheets of glass introduced by Koch for the
plate method of cultivation. Each "plate" consists of a pair of circular
discs of glass with sharply upturned edges, thus forming shallow dishes,
one of slightly greater diameter than the other, and so, when inverted,
forming a cover or cap for the smaller. Plates having an outside
diameter of 10 cm. and a height of 1.5 cm. are the most generally
useful. A batch of eighteen such plates is sterilised and stored in a
cylindrical copper box (30 cm. high by 12 cm. diameter) provided with a
"pull-off" lid. Inside each box is a copper stirrup with a circular
bottom, upon which the plates rest, and by means of which each can be
raised in turn to the mouth of the box (Fig. 9) for removal.
~Capsules~ (Fig. 8, b and c).--These are Petri's dishes of smaller
diameter but greater depth than those termed plates. Two sizes will be
found especially useful--viz., 4 cm. diameter by 2 cm. high, capacity
about 14 c.c.; and 5 cm. diameter by 2 cm. high, capacity about 25 c.c.
These are stored in copper cylinders of similar construction to those
used for plates, but measuring 20 by 6 cm. and 20 by 7 cm.,
respectively.
[Illustration: FIG. 8.--Petri dish (a), and capsules (b, c).]
[Illustration: FIG. 9.--Plate box with stirrup.]
~Graduated Pipettes.~--Several varieties of these are required, viz.:
1. Pipettes of 1 c.c. capacity graduated in 0.1 c.c.
2. Pipettes of 1 c.c. capacity graduated in 0.01 c.c. (Fig. 10, a).
3. Pipettes of 10 c.c. capacity graduated in 0.1 c.c. (Fig. 10, b).
These should be about 30 cm. in length (1 and 2 of fairly narrow bore),
graduated to the extreme point, and having at least a 10 cm. length of
clear space between the first graduation and the upper end; the open
mouth should be plugged with cotton-wool. Each variety should be
sterilised and stored in a separate cylindrical copper case some 36 by 6
cm., with "pull-off" lid, upon which is stamped, in plain figures, the
capacity of the contained pipettes.
[Illustration: FIG. 10.--Measuring pipettes, a and b.]
The laboratory should also be provided with a complete set of "Standard"
graduated pipettes, each pipette in the set being stamped and
authenticated by a certificate from one of the recognised Physical
Measurement Laboratories, such as Charlottenburg. These instruments are
expensive and should be reserved solely for standardising the pipettes
in ordinary use, and for calibrating small pipettes manufactured in the
laboratory. Such a set should comprise, at least, pipettes delivering 10
c.c., 5 c.c., 2.5 c.c., 2 c.c., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c.,
0.1 c.c., 0.05 c.c., and 0.01 c.c., respectively.
In the immediately following sections are described small pieces of
glass apparatus which should be prepared in the laboratory from glass
tubing of various sizes. In their preparation three articles are
essential; first a three-square hard-steel file or preferably a
glass-worker's knife of hard Thuringian steel for cutting glass tubes
etc.; next a blowpipe flame, for although much can be done with the
ordinary Bunsen burner, a blowpipe flame makes for rapid work; and
lastly a bat's-wing burner.
[Illustration: FIG. 11.--Glass-cutting knife. a. handle. b. double
edged blade. c. shaft. d. locking nut. e. spanner for nut.]
1. The glass-cutting knife. This article is sold in two forms, a bench
knife (Fig. 11) and a pocket knife. The former is provided with a blade
some 8 cm. in length and having two cutting edges. The cutting edge when
examined in a strong light is seen to be composed of small closely set
teeth, similar to those in a saw. The knife should be kept sharp by
frequent stroppings on a sandstone hone. The pocket form, about 6-cm.
long over all, consists of a small spring blade with one cutting edge
mounted in scales like an ordinary pocket knife.
2. For real convenience of work the blowpipe should be mounted on a
special table connected up with cylindrical bellows operated by a pedal.
That figured (Fig. 12) is made by mounting a teak top 60 cm. square upon
the uprights of an enclosed double-action concertina bellows (Enfer's)
and provided with a Fletcher's Universal gas blowpipe.
3. An ordinary bat's-wing gas-burner mounted at the far corner of the
table top is invaluable in the preparation of tubular apparatus with
sharp curves, and for coating newly-made glass apparatus with a layer of
soot to prevent too rapid cooling, and its usually associated
result--cracking.
[Illustration: FIG. 12.--Glass blower's table with Enfer's foot
bellows.]
6. ~Sedimentation tubes 5x0.5~ cm., for sedimentation reactions, etc., and
for containing small quantities of fluid to be centrifugalised in the
haematocrit. These are made by taking 14-cm. lengths of stout glass
tubing of the requisite diameter and heating the centre in the Bunsen or
blowpipe flame. When the central portion is quite soft draw the ends
quickly apart and then round off the pointed ends of the two test-tubes
thus formed. With the glass-cutting knife cut off whatever may be
necessary from the open ends to make the tubes the required length.
A rectangular block of "plasticine" (modelling clay) into which the
conical ends can be thrust makes a very convenient stand for these small
tubes.
~Capillary Pipettes or Pasteur's Pipettes~ (Fig. 13 a).--These little
instruments are invaluable, and a goodly supply should be kept on hand.
They are prepared from soft-glass tubing of various-sized calibre (the
most generally useful size being 8 mm. diameter) in the following
manner: Hold a 10 cm. length of glass tube by each end, and whilst
rotating it heat the central portion in the Bunsen flame or the blowpipe
blast-flame until the glass is red hot and soft. Now remove it from the
flame and steadily pull the ends apart, so drawing the heated portion
out into a roomy capillary tube; break the capillary portion at its
centre, seal the broken ends in the flame, and round off the edges of
the open end of each pipette. A loose plug of cotton-wool in the open
mouth completes the capillary pipette. After a number have been
prepared, they are sterilised and stored in batches, either in metal
cases similar to those used for the graduated pipettes or in large-sized
test-tubes--sealed ends downward and plugged ends toward the mouth of
the case.
[Illustration: FIG. 13.--Capillary pipettes. a, b, c.]
The filling and emptying of the capillary pipette is most satisfactorily
accomplished by slipping a small rubber teat (similar to that on a
baby's feeding bottle but _not perforated_) on the upper end, after
cutting or snapping off the sealed point of the capillary portion. If
pressure is now exerted upon the elastic bulb by a finger and thumb
whilst the capillary end is below the surface of the fluid to be taken
up, some of the contained air will be driven out, and subsequent
relaxation of that pressure (resulting in the formation of a partial
vacuum) will cause the fluid to ascend the capillary tube. Subsequent
compression of the bulb will naturally result in the complete expulsion
of the fluid from the pipette (Fig. 14).
[Illustration: FIG. 14.--Filling the capillary teat-pipette.]
A modification of this pipette, in which a constriction or short length
of capillary tube is introduced just below the plugged mouth (Fig. 13,
b), will also be found extremely useful in the collection and storage
of morbid exudations.
A third form, where the capillary portion is about 4 or 5 cm. long and
only forms a small fraction of the entire length of the pipette (Fig.
13, c), will also be found useful.
~"Blood" Pipettes~ (Fig 15).--Special pipettes for the collection of
fairly large quantities of blood (as suggested by Pakes) should also be
prepared. These are made from _soft_ glass tubing of 1 cm. bore, in a
similar manner to the Pasteur pipettes, except that the point of the
blowpipe flame must be used in order to obtain the sharp shoulder at
either end of the central bulb. The terminal tubes must retain a
diameter of at least 1 mm., in order to avoid capillary action during
the collection of the fluid.
[Illustration: FIG. 15.--Blood pipettes and hair-lip pin in a
test-tube.]
[Illustration: FIG. 16.--Blood-pipette in metal thermometer case.]
For sterilisation and storage each pipette is placed inside a test-tube,
resting on a wad of cotton-wool, and the tube plugged in the ordinary
manner. As these tubes are used almost exclusively for blood work, it is
usual to place a lance-headed hare-lip pin or a No. 9 flat Hagedorn
needle inside the tube so that the entire outfit may be sterilised at
one time.
For the collection of small quantities of blood for agglutination
reactions and the like, many prefer a short straight piece of narrow
glass tubing drawn out at either extremity to almost capillary
dimensions. Such pipettes, about 8 cm. in length over all, are most
conveniently sterilized in ordinary metal thermometer cases (Fig. 16).
~Graduated Capillary Pipettes~ (Fig. 17).--These should also be made in
the laboratory--from manometer tubing--of simple, convenient shape, and
graduated by the aid of "standard" pipettes (in hundredths) to contain
such quantities as 10, 50, and 90 c. mm., and carefully marked with a
writing diamond. These, previously sterilised in large test-tubes, will
be found extremely useful in preparing accurate percentage solutions,
when only minute quantities of fluid are available.
[Illustration: FIG. 17.--Capillary graduated pipettes.]
~Automatic ("Throttle") Pipettes.~--These ingenious pipettes, introduced
by Wright, can easily be calibrated in the laboratory and are
exceedingly useful for graduating small pipettes, for measuring small
quantities of fluids, in preparing dilutions of serum for agglutination
reactions, etc. They are usually made from the Capillary Pasteur
pipettes (Fig. 13, a). The following description of the manufacture of
a 5 c. mm. pipette will serve to show how the small automatic pipettes
are calibrated.
1. Select a pipette the capillary portion of which is fairly roomy in
bore and possesses regular even walls, and remove the cotton-wool plug
from the open end.
2. Heat the capillary portion near the free extremity in the by-pass
flame of the bunsen burner and draw it out into a very fine hair-like
tube and break this across. This hair-like extremity will permit the
passage of air but is too fine for metallic mercury to pass.
3. From a standard graduated pipette deliver 5 c. mm. clean mercury into
the upper wide portion of the pipette.
4. Adjust a rubber teat to the pipette and by pressure on the bulb
gradually drive the mercury in an unbroken column down the capillary
tube until it is stopped by the filiform extremity.
5. Cut off the capillary tube exactly at the upper level of the column
of mercury, invert it and allow the mercury to run out.
6. Snap off the remainder of the capillary tube from the broad upper
portion of the pipette which is now destined to form the covering tube
or air chamber, or what we may term the "barrel." This barrel now has
the lower end in the form of a truncated cone, the upper end being cut
square. Remove the teat.
7. Introduce the capillary tube into this barrel with the filiform
extremity uppermost, and the square cut end projecting about 0.5 cm.
beyond the tapering end of the barrel.
[Illustration: FIG. 18.--Throttle pipette--small capacity.]
8. Drop a small pellet of sealing wax into the barrel by the side of the
capillary tube and then warm the tube at the gas flame until the wax
becomes softened and makes an air-tight joint between the capillary tube
and the end of the barrel.
9. Fit a rubber teat to the open end of the barrel, and so complete a
pipette which can be depended upon to always aspirate and deliver
exactly 5 cm. of fluid.
Slight modification of this procedure is necessary in making tubes to
measure larger volumes than say 75 c. mm. Thus to make a throttle
pipette to measure 100 c. mm.:
1. Take a short length of quill tubing and draw out one end into a roomy
capillary stem, and again draw out the extremity into a fine hair point,
thus forming a small Pasteur pipette with a hair-like capillary
extremity.
2. With a standard pipette fill 100 c. mm. into the neck of this
pipette, and make a scratch with a writing diamond at the upper level
(a) of the mercury meniscus (Fig. 19, A).
[Illustration: FIG. 19.--Making throttle pipettes--large capacity]
Now force the mercury down into the capillary stem as far as it will go,
so as to leave the upper part of the tube in the region of the diamond
scratch empty (Fig. 19, B).
3. Heat the tube in the region of the diamond scratch in the blowpipe
flame, and removing the tube from the flame draw it out so that the
diamond scratch now occupies a position somewhere near the centre of
this new capillary portion (Fig. 19, C).
4. Heat the tube in this position in the peep flame of the Bunsen
burner, and draw it out into a hair-like extremity. Snap off the glass
tube, leaving about 5 mm. of hair-like extremity attached to the upper
capillary portion (Fig. 19, D). Allow the glass to cool.
5. Lift up the bulb by the long capillary stem and allow the mercury to
return to its original position--an operation which will be facilitated
by snapping off the hair-like extremity from the long piece of capillary
tubing.
6. Mark on the capillary stem with a grease pencil the position of the
end of the column of mercury (Fig. 19, E.)
7. Warm the capillary tubing at this spot in the peep flame of the
Bunsen burner, and draw it out very slightly so that when cut at this
position a pointed extremity will be obtained.
8. With a glass-cutting knife cut the capillary tube through at the
point "b," and allow the mercury to run out.
9. Now apply a thick layer of sealing wax to the neck of the bulb.
10. Take a piece of 5 mm. bore glass tubing and draw it out as if making
an ordinary Pasteur pipette.
11. Break the capillary portion off so as to leave a covering tube
similar to that already used for the smaller graduated pipettes. Into
this covering tube drop the graduated bulb and draw the capillary stem
down through the conical extremity until further progress is stopped by
the layer of sealing wax.
12. Warm the pipette in the gas flame so as to melt the sealing wax and
make an air-tight joint.
13. Fit an india-rubber teat over the open end of the covering tube, and
the automatic pipette is ready for use (Fig. 19, F).
~Sedimentation Pipettes~ (Fig. 20).--These are prepared from 10 cm.
lengths of narrow glass tubing by sealing one extremity, blowing a
small bulb at the centre, and plugging the open end with cotton-wool;
after sterilisation the open end is provided with a short piece of
rubber tubing and a glass mouthpiece. When it is necessary to observe
sedimentation reactions in very small quantities of fluid, these tubes
will be found much more convenient than the 5 by 0.5 cm. test-tubes
previously mentioned.
[Illustration: FIG. 20.--Sedimentation pipette.]
Pasteur pipettes fitted with india-rubber teats will also be found
useful for sedimentation tests when dealing with minute quantities of
serum, etc.
[Illustration: FIG. 21.--Fermentation tubes.]
~Fermentation Tubes~ (Fig. 21).--These are used for the collection and
analysis of the gases liberated from the media during the growth of some
varieties of bacteria and may be either plain (a) or graduated (b).
A simple form (Fig. 21, c) may be made from 14 cm. lengths of soft
glass tubing of 1.5 cm. diameter. The Bunsen flame is applied to a spot
some 5 cm. from one end of such a piece of tubing and the tube slightly
drawn out to form a constriction, the constricted part is bent in the
bat's-wing flame, to an acute angle, and the open extremity of the long
arm sealed off in the blowpipe flame. The open end of the short arm is
rounded off and then plugged with cotton-wool, and the tube is ready for
sterilisation.
CLEANING OF GLASS APPARATUS.
All glassware used in the bacteriological laboratory must be thoroughly
cleaned before use, and this rule applies as forcibly to new as to old
apparatus, although the methods employed may vary slightly.
~To Clean New Test-tubes.~--
1. Place the tubes in a bucket or other convenient receptacle, fill with
water and add a handful of "Sapon" or other soap powder. See that the
tubes are full and submerged.
2. Fix the bucket over a large Bunsen flame and boil for thirty
minutes--or boil in the autoclave for a similar period.
3. Cleanse the interior of the tubes with the aid of test-tube brushes,
and rinse thoroughly in cold water.
4. Invert the tubes and allow them to drain completely.
5. Dry the tubes and polish the glass inside and out with a soft cloth,
such as selvyt.
~New flasks, plates, and capsules~ must be cleaned in a similar manner.
~To Clean New Graduated Pipettes.~--
1. Place the pipettes in a convenient receptacle, filled with water to
which soap powder has been added.
2. Boil the water vigorously for twenty minutes over a Bunsen flame.
3. Rinse the pipettes in running water and drain.
4. Run distilled water through the pipettes and drain.
5. Run rectified spirits through the pipette and drain as completely as
possible.
6. Place the pipettes in the hot-air oven (_vide_ page 31), close the
door, open the ventilating slide, and run the temperature slowly up to
about 80 deg. C. Turn off the gas and allow the oven to cool.
Or 6a. Attach each pipette in turn to the rubber tube of the foot
bellows, or blowpipe air-blast, and blow air through the pipette until
the interior is dry.
Glassware that has already been used is regarded as _infected_, and is
treated in a slightly different manner.
~Infected Test-tubes.~--
1. Pack the tubes in the wire basket of the autoclave (having previously
removed the cotton-wool plugs, caps, etc.), in the vertical position,
and before replacing the basket see that there is a sufficiency of water
in the bottom of the boiler. Now attach a piece of rubber tubing to the
nearest water tap, and by means of this fill each tube with water.
2. Disinfect completely by exposing the tubes, etc., to a temperature of
120 deg. C. for twenty minutes (_vide_ page 37).
(If an autoclave is not available, the tubes must be placed in a
digester, or even a large pan or pail with a tightly fitting cover, and
boiled vigorously for some thirty to forty-five minutes to ensure
disinfection.)
3. Whilst still hot, empty each tube in turn and roughly clean its
interior with a stiff test-tube brush.
4. Place the tubes in a bucket or other convenient receptacle, fill with
water and add a handful of Sapon or other soap powder. See that the
tubes are full and submerged.
5. Fix the bucket over a large Bunsen flame and boil for thirty minutes.
6. Cleanse the interior of the tubes with the aid of test-tube brushes,
and rinse thoroughly in cold water.
7. Drain off the water and immerse tubes in a large jar containing water
acidulated with 2 to 5 per cent. hydrochloric acid. Allow them to remain
there for about fifteen minutes.
8. Remove from the acid jar, drain, rinse thoroughly in running water,
then with distilled water.
9. Invert the tubes and allow them to drain completely.
Dry the tubes and polish the glass inside and out with a soft cloth,
such as selvyt.
~Infected flasks, plates, and capsules~ must be treated in a similar
manner.
~Flasks~ which have been used only in the preparation of media must be
cleaned immediately they are finished with. Fill each flask with water
to which some soap powder and a few crystals of potassium permanganate
have been added, and let boil over the naked flame. The interior of the
flask can then usually be perfectly cleaned with the aid of a flask
brush, but in some cases water acidulated with 5 per cent. nitric acid,
or a large wad of wet cotton-wool previously rolled in silver sand, must
be shaken around the interior of the flask, after which rinse thoroughly
with clean water, dry, and polish.
~Infected Pipettes.~--
1. Plunge infected pipettes immediately after use into tall glass
cylinders containing a 2 per cent. solution of lysol, and allow them to
remain therein for some days.
2. Remove from the jar and drain. Boil in water to which a little soap
has been added, for thirty minutes.
3. Rinse thoroughly in cold water.
4. Immerse in 5 per cent. nitric acid for an hour or two.
5. Rinse again in running water to remove all traces of acid.
6. Complete the cleaning as described under "new pipettes."
When dealing with graduated capillary pipettes employed for blood or
serum work (whether new or infected), much time is consumed in the
various steps from 5 onward, and the cleansing process can be materially
hastened if the following device is adopted.
Fit up a large-sized Kitasato's filter flask to a Sprengel's suction
pump or a Geryk air pump (see page 43). To the side tubulure of the
filter flask attach a 20 cm. length of rubber pressure tubing having a
calibre sufficiently large to admit the ends of the pipettes.
Next fill a small beaker with distilled water. Attach the first pipette
to the free end of the rubber tubing, place the pipette point downward
in the beaker of water and start the pump (Fig. 22).
[Illustration: FIG. 22.--Cleaning blood pipettes.]
When all the water has been aspirated through the pipette into the
filter flask, fill the beaker with rectified spirit and when this is
exhausted refill with ether. Detach the pipette and dry in the hot-air
oven.
~Slides and cover-slips~ (Fig. 23), when first purchased, have "greasy"
surfaces, upon which water gathers in minute drops and effectually
prevents the spreading of thin, even films.
~Microscopical Slides.~--The slides in general use are those known as
"three by one" slips (measuring 3 inches by 1 inch, or 76 by 26 mm.),
and should be of good white crown glass, with ground edges.
~New slides~ should be allowed to remain in alcohol acidulated with 5 per
cent. hydrochloric acid for some hours, rinsed in running water, roughly
drained on a towel, dried, and finally polished with a selvyt cloth.
[Illustration: FIG. 23.--Slides and cover-slips, actual size.]
If only a few slides are required for immediate use a good plan is to
rub the surface with jeweler's emery paper (Hubert's 00). A piece of
hard wood 76x26x26 mm. with a piece of this emery paper gummed tightly
around it is an exceedingly useful article on the microscope bench.
~Cover-slips.~--The most useful sizes are the 19 mm. squares for ordinary
cover-glass film preparations, and 38 by 19 mm. rectangles for blood
films and serial sections; both varieties must be of "No. 1" thickness,
which varies between 0.15 and 0.22 mm., that they may be available for
use with the high-power immersion lenses.
Cover-slips should be cleaned in the following manner:
1. Drop the cover-slips one by one into an enamelled iron pot or tall
glass beaker, containing a 10 per cent. solution of chromic acid.
2. Heat over a Bunsen flame and allow the acid to boil gently for twenty
minutes.
NOTE.--A few pieces of pipe-clay or pumice may be placed in
the beaker to prevent the "spurting" of the chromic acid.
3. Turn the cover-slips out into a flat glass dish and wash in running
water under the tap until all trace of yellow colour has disappeared.
During the washing keep the cover-slips in motion by imparting a
rotatory movement to the dish.
4. Wash in distilled water in a similar manner.
5. Wash in rectified spirit.
6. Transfer the cover-slips, by means of a pair of clean forceps,
previously heated in the Bunsen flame to destroy any trace of grease, to
a small beaker of absolute alcohol.
Drain off the alcohol and transfer the cover-slips, by means of the
forceps, to a wide-mouthed glass pot, containing absolute alcohol, in
which they are to be stored, and stopper tightly.
NOTE.--After once being placed in the chromic acid, the
cover-slips must on no account be touched by the fingers.
~Used Slides and Cover-slips.~--Used slides with the mounted cover-slip
preparations, and cover-slips used for hanging-drop mounts, should, when
discarded, be thrown into a pot containing a 2 per cent. solution of
lysol.
After immersion therein for a week or so, even the cover-slips mounted
with Canada balsam can be readily detached from their slides.
_Slides._--
1. Wash the slides thoroughly in running water.
2. Boil the slides in water to which "sapon" has been added, for half an
hour.
3. Rinse thoroughly in cold water.
4. Dry and polish with a dry cloth.
_Cover-slips._--
1. Wash the cover-slips thoroughly in running water.
2. Boil the cover-slips in 10 per cent. solution of chromic acid, as for
new cover-slips.
3. Wash thoroughly in running water.
4. Pick out those cover-slips which show much adherent dirty matter, and
rub them between thumb and forefinger under the water tap. The dirt
usually rubs off easily, as it has become friable from contact with the
chromic acid.
5. Return all the cover-slips to the beaker, fill in _fresh_ chromic
acid solution, and treat as new cover-slips.
NOTE.--_Test-tubes, plates, capsules_, etc., which, from
long use, have become scratched and hazy, or which cannot be
cleaned in any other way, may be dealt with by immersing
them in an enamelled iron bath, containing water acidulated
to 1 per cent. with hydrofluoric acid, for ten minutes,
rinsing thoroughly in water, drying, and polishing.
PLUGGING TEST-TUBES AND FLASKS.
Before sterilisation all test-tubes and flasks must be carefully plugged
with cotton-wool, and for this purpose best absorbent cotton-wool
(preferably that put up in cylindrical one-pound packets and interleaved
with tissue paper--known as surgeons' wool) should be employed.
1. For a test-tube or a small flask, tear a strip of cotton-wool some 10
cm. long by 2 cm. wide from the roll.
2. Turn in the ends neatly and roll the strip of wool lightly between
the thumb and fingers of both hands to form a long cylinder.
3. Double this at the centre and introduce the now rounded end into the
open mouth of the tube or flask.
4. Now, whilst supporting the wool between the thumb and fingers of the
right hand, rotate the test-tube between those of the left, and
gradually screw the plug of wool into its mouth for a distance of about
2.5 cm., leaving about the same length of wool projecting.
[Illustration: FIG 24..--Plugging test-tubes: a, cylinder of wool
being rolled; b, cylinder of wool being doubled; c, cylinder of wool
being inserted in tube.]
The plug must be firm and fit the tube or flask fairly tightly,
sufficiently tightly in fact to bear the weight of the glass plus the
amount of medium the vessel is intended to contain, but not so tightly
as to prevent it from being easily removed by a screwing motion when
grasped between the fourth, or third and fourth, fingers, and the palm
of the hand.
For a large flask a similar but larger strip of wool must be taken; the
method of making and inserting the plug is identical.
III. METHODS OF STERILISATION.
STERILISING AGENTS.
Sterilisation--i. e., the removal or the destruction of germ life--may
be effected by the use of various agents. As applied to the practical
requirements of the bacteriological laboratory, many of these agents,
such as electricity, sunlight, etc., are of little value, others are
limited in their applications; others again are so well suited to
particular purposes that their use is almost entirely restricted to
such.
The sterilising agents in common use are:
~Chemical Reagents.~--_Disinfectants_ (for the disinfection of glass and
metal apparatus and of morbid tissues).
~Physical Agents.~ HEAT.--(a) _Dry Heat:_
1. Naked flame (for the sterilisation of platinum needles, etc.).
2. Muffle furnace (for the sterilisation of filter candles, and for the
destruction of morbid tissues).
3. Hot air (for the sterilisation of all glassware and of metal
apparatus).
(b) _Moist Heat:_
1. Water at 56 deg. C. (for the sterilisation of certain albuminous
fluids).
2. Water at 100 deg. C. (for the sterilisation of surgical instruments,
rubber tubing, and stoppers, etc.).
3. Streaming steam at 100 deg. C. (for the sterilisation of media).
4. Superheated steam at 115 deg. C. or 120 deg. C. (for the disinfection
of contaminated articles and the destruction of old cultivations of
bacteria).
FILTRATION.--
1. Cotton-wool filters (for the sterilisation of air and gases).
2. Porcelain filters (for the sterilisation of various liquids).
METHODS OF APPLICATION.
~Chemical Reagents~, such as belong to the class known as antiseptics (_i.
e._, substances which inhibit the growth of, but do not destroy,
bacterial life), are obviously useless. Disinfectants or germicides (_i.
e._, substances which destroy bacterial life), on the other hand, are of
value in the disinfection of morbid material, and also of various pieces
of apparatus, such as pipettes, pending their cleansing and complete
sterilisation by other processes. To this class (in order of general
utility) belong:
Lysol, 2 per cent. solution;
Perchloride of mercury, 0.1 per cent. solution;
Carbolic acid, 5 per cent. solution;
Absolute alcohol;
Ether;
Chloroform;
Camphor;
Thymol;
Toluol;
Volatile oils, such as oil of mustard, oil of garlic.
Formaldehyde is a powerful germicide, but its penetrating vapor
restricts its use. These disinfectants are but little used in the final
sterilisation of apparatus, chiefly on account of the difficulty of
effecting their complete removal, for the presence of even traces of
these chemicals is sufficient to so inhibit or alter the growth of
bacteria as to vitiate subsequent experiments conducted by the aid of
apparatus sterilised in this manner.
NOTE.--Tubes, flasks, filter flasks, pipettes, glass tubing,
etc., may be rapidly sterilised, in case of emergency, by
washing, in turn, with distilled water, perchloride of
mercury solution, alcohol, and ether, draining, and finally
gently heating over a gas flame to completely drive off the
ether vapor. Chloroform or other volatile disinfectants may
be added to various fluids in order to effect the
destruction of contained bacteria, and when this has been
done, may be completely driven off from the fluid by the
application of gentle heat.
~Dry Heat.~--The _naked flame_ of the Bunsen burner is invariably used for
sterilising the platinum needles (which are heated to redness) and may
be employed for sterilising the points of forceps, or other small
instruments, cover-glasses, pipettes, etc., a very short exposure to
this heat being sufficient.
_Ether Flame._--In an emergency small instruments, needles, etc., may be
sterilised by dipping them in ether and after removal lighting the
adherent fluid and allowing it to burn off the surface of the
instruments. Repeat the process twice. It may then be safely assumed
that the apparatus so treated is sterile.
[Illustration: FIG. 25.--Muffle furnace.]
_Muffle Furnace_ (Fig. 25).--Although this form of heat is chiefly used
for the destruction of the dead bodies of small infected animals, morbid
tissues, etc., it is also employed for the sterilisation of porcelain
filter candles (_vide_ p. 42).
Filter candles are disinfected immediately after use by boiling in a
beaker of water for some fifteen or twenty minutes. This treatment,
however, leaves the dead bodies of the bacteria upon the surface and
blocking the interstices of the filter.
To destroy the organic matter and prepare the filter candle for further
use proceed as follows: