PROTECTIVE INOCULATIONS. 363 
From this crude tuberculin Koch has obtained a white precipitate 
with sixty-per-cent alcohol which has the active properties of the 
crude tuberculin as originally prepared. This is fatal to tuberculous 
guinea-pigs in doses of two to ten milligrammes. It is soluble in 
water and in glycerin, and has the chemical reactions of an albuminous 
body. In preparing it one volume and a half of absolute alcohol is 
added to one volume of the crude tuberculin, and, after stirring it to 
secure uniform admixture, this is put aside for twenty-four hours. 
At the end of this time a flocculent deposit will be seen at the bot- 
tom of the vessel. The fluid above this is carefully poured off; and 
an equal quantity of sixty-per-cent alcohol is poured into the vessel 
for the purpose of washing the precipitate. This is again allowed to 
settle, and the procedure is repeated three or four times, after which 
the precipitate is washed with absolute alcohol. It is then placed 
upon a filter and dried in a vacuum exsiccator. 
The “tuberculocidin ” of Klebs is a purified tuberculin obtained by 
precipitation with alcohol. The precipitate is washed in chloroform 
and then dissolved in a mixture of carbolic acid and glycerin. 
Bujwid (1894) prepares tuberculin as follows: He uses cultures on 
glycerin agar or in glycerin bouillon which have been kept at a suit- 
able temperature for five to eight weeks. The glycerin-agar cultures 
are treated with distilled water by which the tuberculin is extracted. 
After adding the water the test tubes are kept in a cool place for 
twenty-four hours, and this is repeated two or three times. The ex- 
tract from the agar cultures or the bouillon cultures is then sterilized 
by exposure for from five to ten minutes to a temperature of 100° C.; 
then filtered through a Chamberland filter; then evaporated at a low 
temperature to a syrup-like consistence. When this crude tuberculin 
is dropped into ten times its volume of strong alcohol a brown pre- 
cipitate is thrown down which contains the active principle. From 
the tubercle bacilli obtained by filtering his cultures Bujwid also 
obtained an active substance which in doses of two milligrammes 
caused an elevation of 2° C. in the temperature of an infected guinea- 
pig. This substance was obtained by digesting the bacilli for two 
months in glycerin and water (three per cent of glycerin), filtering 
and evaporating the extract, and precipitation in six volumes of 
ninety-five-per-cent alcohol. The precipitate when dried was in the 
form of a white powder. 
Helman (1894) obtains tuberculin from potato cultures. The sec- 
tions of potato are neutralized by leaving them for half an hour ina 
solution of one-half to one per cent of bicarbonate of soda, after which 
they are sterilized for twenty minutes in the autoclave at 120° C. The 
