The Elements of Bacteriological Technique

Appendix 492

INDEX 505

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 × 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 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.

~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.

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.

~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.

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