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

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


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.

[Pg x]XI. ORDINARY OR STOCK CULTURE MEDIA 163



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
[Pg 1]
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.

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.

[Pg 3]
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 × 1.5 cm., to contain media for ordinary tube cultivations.
2. 18 × 1.3 cm., to contain media used for pouring plate cultivations, and also for
holding sterile "swabs."
3. 18 × 2 cm., to contain wedges of potato, beetroot, or other vegetable media.
4. 13 × 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

known as Roux's bottles (Fig. 5) are to be preferred.
Fig. 4.—Kolle's culture flask.
Fig. 5.—Roux's culture bottle.
Fig. 6.—Guy's culture bottle.
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.[Pg 6]
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,

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[Pg 9] 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.
Fig. 12.—Glass
blower's table with Enfer's foot bellows.
6. Sedimentation tubes 5×0.5 cm., for sedimentation reactions, etc., and for
containing small quantities of fluid to be centrifugalised in the hæmatocrit. 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[Pg 10] 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.

"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[Pg 12] 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.
Fig. 15.—Blood pipettes and hair-lip pin in a test-tube.
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[Pg 13] 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.
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.