Tài liệu THE ELEMENTS OF BACTERIOLOGICAL TECHNIQUE A LABORATORY GUIDE FOR MEDICAL, DENTAL, AND TECHNICAL STUDENTS pptx

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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.

[Pg x]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
Hæmolytic Serum, 327—The Titration of
Hæmolytic Serum, 328—Storage of
Hæmolysin, 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



[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.
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.[Pg 2]
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.

[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
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[Pg 4] 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 × 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.
Fig. 1.—Bohemian flask.
Fig. 2.—Pear-shaped flask.
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.