Cultivation on Various Media. Koch inoculated solid blood serum with tubercular matter from an infected lymphatic gland of a guinea-pig, and noticed the first signs of growth in ten or twelve days in the form of whitish, scaly patches. These enlarged and coalesced with neighbouring patches, forming white, roughened, irregular masses. Nocard and Roux showed that by adding 5/8 per cent. of glycerine to the media commonly used in the laboratory, such as nutrient agar or broth, the best growth is obtained.
On glycerine broth or glycerine agar abundant growth appears at the end of seven or eight days. By continuous sub-culture on glycerine agar the virulence of the bacillus is diminished. But in fifteen days after inoculation of the medium the culture equals in extent a culture of several weeks' age on blood serum.
Sub-cultures from glycerined media will grow in ordinary broth without glycerine (Nocard, Roux, Crookshank).
In alkaline broth to which a piece of boiled white of egg was added Klein obtained copious growths, and found that continued sub-culturing upon this medium also lessens the virulence.
Description of Cultivations:—On glycerine agar minute white colonies appear in about six days, raised and isolated, and coalescing as time advances, forming a white lichenous growth, fully developed in about two months.
On glycerine broth a copious film appears on the surface of the liquid, which if disturbed falls to the bottom of the flask as a deposit.
Spore Formation. In very old cultivations spore-like bodies can be observed both in stained and unstained preparations, but neither the irregular granules within the capsule nor the unstained spaces between the granules are spores (Babes and Crookshank). That the bacilli possess spores is believed on account of the following facts:
1. That tubercular sputum, when thoroughly dried, maintains its virulent character (Koch, Schill, Fischer, etc.). No sporeless bacillus is known which can survive through drying.
2. That tubercular matter and cultures survive temperature up to 100° C. Non-spore-bearing bacilli and micrococci are killed by being exposed for five minutes to a temperature of 65–70° C., whereas spores of other bacilli withstand much higher temperatures.
3. Tubercular sputum distributed in salt solution does not lose its virulence by being kept at 100° C. for one or two minutes; sporeless bacilli certainly would (Klein).