APPENDIX A.

THE PRINCIPLES OF BACTERIOLOGICAL TECHNIQUE.1

In order to obtain a satisfactory knowledge of the biological characters of bacteria, it is necessary to obtain them in pure cultures by artificial cultivation, and next to subject them to a microscopical examination. For the purpose of testing their pathogenic power a further procedure is requisite, i.e., experimental inoculation in animals.

I. CULTIVATION OF BACTERIA.

1. Sterilisation of Apparatus.-All glass vessels, cotton wool, and metal instruments should be rendered germ-free by dry heat (150° C. for one hour). This is best done by means of special apparatus, but an efficient steriliser may be improvised out of an ordinary biscuit-box, and heat obtained from a convenient source. Another useful method is to wrap the apparatus in cotton wool and to apply heat until the wool is singed.

2. Nutrient Media.-Two of the most widely employed media are gelatine and agar-agar. Both are transparent and can be rendered solid or liquid at will. But while gelatine liquefies at a comparatively low temperature (25° C.), and so cannot be used in the tropics, agar-agar remains solid at blood-heat-a temperature which corresponds to

1 For further details the reader is referred to Curtis's Essentials of Practical Bacteriology, and other works on the subject.

the optimum of most pathogenic bacteria. Many bacteria liquefy gelatine, but no organism has been known to liquefy agar-agar. For special purposes various substances are added to these media, e.g., glycerine, glucose, lactose.

Other nutrient media commonly employed are blood serum, bouillon, milk, and potato.

A culture medium said to be very convenient in the tropical countries is the fluid contained in the interior of unripe cocoanuts. It contains glucose, vegetable albumen, and salts, and has the additional advantage of being contained in a germ-free receptacle.

After being prepared, all media are placed in flasks or test tubes, plugged with cotton wool, and sterilised by steam for three successive days (see p. 29).

3. Inoculation of Media. This is conveniently done by means of a platinum needle, either straight or with a looped end and fastened to the extremity of a glass rod. The needle must be heated red-hot both before and after it is used. Similarly, the plug of the culture tube must be singed both before and after inoculation. Fluid culture media are inoculated by transferring a loopful of the material containing bacteria to the medium. When solid media are used a single "stab" is made with a straight needle, or, if the medium is solidified in a slanting position, the needle is simply "stroked" over the surface. Potato cultures are also inoculated by the latter method. After inoculation gelatine tubes are kept at 20° C. to 22° C. but all others at 37° C. In order to maintain these constant temperatures "incubators" are frequently employed; but in the tropics a place in the verandah could easily be found where the temperature corresponded to 37° C. It is essential that the cultures which are being incubated should be kept in the dark, as light is inimical to the development of all microbes.

To isolate bacteria in pure cultures gelatine or agar plates are made. The latter medium is melted and next carefully cooled to a temperature not destructive to bacteria. The liquid medium is now inoculated, and rapidly poured into sterilised shallow glass dishes ("Petri dishes"), and allowed to solidify. These dishes, or, as they are now generally called, plates, are then incubated for two or three days, when colonies result from the growth of each bacterium. Finally, inoculations are made from one such colony to a tube of culture medium, and pure cultures of organisms obtained.

When numerous bacteria are present in the given material the necessary dilution may be made by inoculating a second tube from the first, and a third from the second. Plates are then made with the third tube, and the resulting colonies, being few in number, can be easily isolated.

II. MICROSCOPIC EXAMINATION OF BACTERIA.1

In preparing specimens for the microscope it is essential that slides and cover glasses should be absolutely clean and free from the slightest trace of grease. They are best cleaned by soaking them in boiling nitric acid for ten minutes and then washing in water. They are subsequently kept in small closed jars containing alcohol.

1. Hanging Drop Preparation.-When it is desired to study the motility of an organism the method of cultivation in a drop of culture fluid attached to the under surface of a cover glass, and suspended over a hollow ground out of a glass slide, is very useful. A drop of culture fluid is transferred to the centre of a clean cover glass, which is then rapidly inverted over the hollow of the slide and fixed in

1 An extremely useful bacteriological test-case, containing all the necessary stains and instruments required for this purpose, may be had from Mr. Martindale, New Cavendish Street, W.

position by a ring of vaseline.

The organisms are most abundant at the edge of the hanging drop, where they must first be looked for.

2. Cover Slip Preparation.-In this method the material containing bacteria is spread out over a thin cover glass, dried, and stained for microscopical examination.

(a) Preparing the Film.—With a platinum loop heated to

redness in the flame, a small drop of water is placed in the centre of the cleaned cover glass, and then, with the same instrument, a little material from the surface of a solid culture is taken with due precautions and mixed with a drop of water. In the case of liquid cultures the addition of water is evidently unnecessary.

The emulsion is next evenly spread over the cover slip and allowed to dry in the air. After the film is dry the cover glass is rapidly passed three times over the flame, so as to coagulate the albumen and fix the film to the cover slip.

(b) Staining. This is effected by covering the film side

of the cover glass with a stain, which is poured off

after a few minutes, and the excess washed in

water.

The penetrating power of stains is increased by heating or by adding to the stain carbolic acid, aniline oil shaken up with water, and other "mordants".

The dyes most commonly used are carbol-fuchsin, methylene blue, and aniline gentian violet. They are all kept in saturated alcoholic solutions in order to prevent decomposition. It is absolutely necessary to filter the stains before use. Formula of Stains:-

(i.) Carbol-fuchsin is made by adding a saturated alcoholic solution of fuchsin to carbolic lotion (1 in 20) until the solution has lost its transparency.