396 QUEENSLAND AGRICULTURAL JOURNAL. [1 May, 1899. 
_ It will be noticed that there is no albumen material present, while the 
nitrogenous element is supplied in the asparagin. 
Having prepared a stock of the above material, it is transferred to a 
number of 50 cc. Erlenmyer’s conical shaped flasks which have been previously 
chemically cleaned, plugged with cotton wool and sterilised. 
Itis necessary that, in order to obtain as large a surface of the fluid 
exposed to the air, only 20 ce. of the nutrient media be placed in each flask. 
The flasks and their contents are sterilised on four successive days for 20 
minutes at each operation, in a steam steriliser at 212 degrees Fahr., or for 30 
minutes on two successive days in an autoclave or steam digestor, under a 
pressure, of 27 lb, on the square inch. On removal they are allowed to cool down, 
when each flask is ready to be inoculated with the pure culture of the tubercle 
bacillus, which to perform for the first time is an extremely difficult and tedious 
operation. Night after night, for many weeks at a time, I have worked away 
by myself in the laboratory in order to perfect this operation. 
If we take a trace of the growth from the surface of an agar-agar culture~ 
of tubercle bacilli, and place it into the flask, it gradually sinks to the bottom 
of the fluid; if this flask of inoculated media is placed in an incubator we have 
to wait many months before we see a perceptible increase in the growth of the 
colony of tubercle bacilli, the reason being that the organisms have not had 
free access to oxygen, which is absolutely essential for their rapid development. 
Although this specific bacillus is recognised as one of the slowest growing 
Organisms, we have succeeded in obtaining most luxuriant cultivations by 
growing it on the surface of this fluid media. In the first place this is only 
accomplished by inoculating from an old and very dry agar-agar culture, a very 
large number of flasks, special care being exercised to make the minute traces 
of growth of bacilli float. After the flasks have been in the incubator for 
about three weeks it is possible to find, out of LOO flasks, perhaps one flask 
with an extremely delicate film, almost imperceptible to the naked eye, floating 
over a small portion of the surface fluid. Should a microscopical examination 
of this film, atter staining with special aniline dyes, result in showing nothing but 
a pure culture of the tubercle bacillus, immediate steps are taken to transfer, by 
meaus of a flattened platinum needle, portions of this film to the surface of 
fluid media in each of the other flasks, which are afterwards returned to the 
incubator where they remain at 98'6 degrees Fahr. for another 10 weeks. On 
examination at intervals during this period, it will be noticed that the film, 
which has grown all over the surface of the fluid media, becomes crinkled, 
breaks up gradually ; then a new film forms, and so on, until the growing 
bacilli have exhaisted all the nutrient properties in the fluid media, at the same 
time generating or setting free their poisonous products, the tuberculin, The 
next part of the process is to carefully destroy the vitality of every bacillus in 
the culture and then separate them from the fluid by passing the contents of 
each flask through a Pasteur-Chamberland filter, undér a pressure of 500 lb. on 
the square inch. 
This porcelain filter, the manufacture of which is practically a French 
Government secret, is so uniform and fine in texture that the very smallest 
spores of bacilli or micrococci knowu to bacteriologists cannot be forced through 
even under this enormous pressure. 
This filtrate is now placed in a series of Florence flasks, fitted with 
perforated rubber corks, and connected one with another to a Sprengel’s 
exhaust pump by means of glass and thick-walled rubber tubing. The flasks 
thus connected are placed in a steam steriliser and kept heated up to a tempera- 
ture of 120 degrees Fahr., while the exhaust pump assists in evaporating the 
contents of the flasks zz vacuo until the fluid is so concentrated that the 
glycerine, originally in the proportion of 7 per cent., amounts to 50 per cent. 
The next part of the programme is the standardising, and requires the 
greatest care and attention, inasmuch as it can onl v be performed by a large 
series of physiological experiments on guinea-pigs. 
