cess once more, and from the growth in this third. tube plant a streak on wort gelatine, and incubate at 20° C. 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 40° C., 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, 4° C.—e. g., Bacillus pyocyaneus, thermal death-point 55° C., and Bacillus mesentericus vulgatus, thermal death-point 60° C.—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 is found to be of even greater practical value when applied to 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 only 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. 113). It consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the space between the walls. This space is nearly filled with benzole (boilingpoint 80° C.), and to the tubulure is fitted a long glass tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube. 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. Fig. 113.-Benzole bath. 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 80° C. 4. When the thermometer again records 80° C., note the time, and fifteen minutes later remove the tube containing the mixture from the bath. 5. Make cultures upon suitable media; incubate. 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 37° C., 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 Bullock'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 tempera ture. 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. XIV. METHODS OF IDENTIFICATION. In order to identify an organism after nutrient media have been inoculated, and tube, plate, and other cultivations prepared, these are incubated under suitable conditions as to temperature and environment, are examined from time to time (a) macroscopi= cally, (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 identity 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 systematical, 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 .in 18..., from 1. Cultural Characters.—(Vide Macroscopical Exam ination of Cultivation, page 207.) |