c. If some varieties are missing prepare a second stock dish from other colonies on plate 3, and repeat the process until each morphological form or tinctorial variety has been secured in subculture.

_d._ Place the stock dishes in the ice chest to await the results of incubation. (If any of the subcultures fail, further material can be obtained from the corresponding emulsion; or if it has dried, by moistening it with a further drop of sterile distilled water.)

_e._ Incubate all the subcultures and identify the organisms picked out.

4. Differential Media.--

(a) _Selective._--Some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties; e. g., wort is the most suitable medium-base for the growth of torulae and yeasts and should be employed when pouring plates for the isolation of these organisms. To obtain a pure cultivation of yeast from a mixture containing bacteria as well, it is often sufficient to inoculate wort from the mixture and incubate at 37 C. for twenty-four hours. Plant a fresh tube of wort from the resulting growth and incubate. Repeat the process once more, and from the growth in this third tube plant a streak on wort gelatine, and incubate at 20C. The resulting growth will almost certainly be a pure culture of the yeast.

(b) _Deterrent._--The converse of the above also obtains. Certain media possess the power of inhibiting the growth of a greater or less number of species. For instance, media containing carbolic acid to the amount of 1 per cent. will inhibit the growth of practically everything but the Bacillus coli communis.

~5. Differential Incubation.~--

In isolating certain bacteria, advantage is taken of the fact that different species vary in their optimum temperature. A mixture containing the Bacillus typhosus and the Bacillus aquatilis sulcatus, for example, may be planted on two slanted agar tubes, the one incubated at 40C., and the other at 12 C. After twenty-four hours' incubation the first will show a pure cultivation of the Bacillus typhosus, whilst the second will be an almost pure culture of the Bacillus aquatilis.

6. Differential Sterilisation.--

(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the varying thermal death-points of bacteria. From a mixture of two organisms whose thermal death-points differ by, say, 4C.--e. g., Bacillus pyocyaneus, thermal death-point 55C., and Bacillus mesentericus vulgatus, thermal death-point 60C.--a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58 C. and keeping it at that point for ten minutes. The mixture is then planted on to fresh media and incubated, when the resulting growth will be found to consist entirely of the B. mesentericus.

(b) _Sporing Bacteria._--This method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores. In this case the mixture is heated in a water-bath at 80 C. for fifteen to twenty minutes. At the end of this time the non-sporing bacteria are dead, and cultivations made from the mixture will yield a growth resulting from the germination of the spores only.

Differential sterilisation at 80 C. is most conveniently carried out in a water-bath of special construction, designed by Balfour Stewart (Fig.

140). It consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the s.p.a.ce between the walls.

This s.p.a.ce is nearly filled with benzole (boiling-point 80C.; pure benzole, free from thiophene must be employed for the purpose, otherwise the boiling-point gradually and perceptibly rises in the course of time), and to the tubulure is fitted a long gla.s.s tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube (a tube half this length if provided with a condensing bulb at the centre will be equally efficient). The interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer. This latter dips into the water and records its temperature. A very small Bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of 80 C. The bath is thus always ready for use.

METHOD.--To use the apparatus.

1. Place some of the mixture itself, if fluid, containing the spores, or an emulsion of the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained in the benzole bath, taking care that the upper level of the liquid in the tube is at least 2 cm. beneath the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a few degrees after opening the bath and introducing a tube of colder liquid, but after a few minutes the temperature will have again reached 80C.

4. When the thermometer again records 80C., note the time, and fifteen minutes later remove the tube containing the mixture from the bath.

5. Make cultures upon suitable media; incubate.

[Ill.u.s.tration: FIG. 140.--Benzole bath.]

7. Differential Atmosphere Cultivation.--

(a) By adapting the atmospheric conditions to the particular organism it is desired to isolate, it is comparatively easy to separate a strict aerobe from a strict anaerobe, and _vice versa_. In the first case, however, it is important that the cultivations should be made upon solid media, for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic, and under these conditions the growth of the anaerobes will continue unchecked.

(b) When it is desired to separate a facultative anaerobe from a strict anaerobe, it is generally sufficient to plant the mixture upon the sloped surface agar, incubate aerobically at 37C., and examine carefully at frequent intervals. At the first sign of growth, subcultivations must be prepared and treated in a similar manner. As a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation.

(c) If, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media.

Incubate under suitable conditions as to temperature and atmosphere.

~8. Animal Inoculation.~--

Finally, when dealing with pathogenic organisms, it is often advisable to inoculate some of the impure culture (or even some of the original _materies morbi_) into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate. Indeed, with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result.

XVI. METHODS OF IDENTIFICATION AND STUDY.

In order to identify an organism after isolation, tube, plate, and other cultivations must be prepared, incubated under suitable conditions as to temperature and environment, and examined from time to time (a) ~macroscopically~, (b) by ~microscopical methods~, (c) by ~chemical methods~, (d) by ~physical methods~, (e) by ~inoculation methods~, and the results of these examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by any _one_ character or property, whether microscopical, biological or chemical, but that on the contrary its entire life history must be carefully studied and then its ident.i.ty established from a consideration of the sum total of these observations.

In order to give to the recorded results their maximum value it is essential that they should be exact and systematic, therefore some such scheme as the following should be adhered to; and especially is this necessary in describing an organism not previously isolated and studied.

SCHEME OF STUDY.

Designation:

Originally isolated by (_observer's name_) in (_date_), from (_source of organism_).

~1. Cultural Characters.~--(_Vide_ Macroscopical Examination of Cultivation, page 261.)

Gelatine plates, } Gelatine streak, } at 20C.

Gelatine stab, } Gelatine shake, }

Agar plates, } Agar streak or smear, } Agar stab, } Insp.i.s.sated blood-serum, } at 20 C. and 37C.

Bouillon, } Litmus milk, } Potato, }

Special media for the purpose of demonstrating characteristic appearances.

~2. Morphology~.--(_Vide_ Microscopical Examination of Cultivations, page 272.)

Vegetative forms: Shape.

Size.

Motility.

Flagella (if present).

Capsule (if present).

Involution forms.

Pleomorphism (if observed).

Sporing forms (if observed). Of which cla.s.s?

Staining reactions.