TUBERCULOSIS 167 
that from 6 to 29% of the animals that failed to show physical symp- 
toms were eliminating tubercle bacteria. 
Tuberculin. The tuberculin test is the best, and, ina large majority 
of tuberculous cases among animals and in man, the only means of 
positively detecting the disease in the living individual. Tuberculin* 
is the concentrated liquid, usually glycerinated bouillon, on which 
tubercle bacteria have grown until the products resulting from their 
multiplication including the disintegrated bodies of dead tubercle 
bacteria have become imparted to the medium in sufficient quantity 
to inhibit their further development. 
Tuberculin in the dose necessary to bring out its diagnostic effect 
is harmless for healthy animals. In the tuberculous animal it pro- 
duces a rise of temperature which, within certain limits, follows a 
definite course usually terminating in from 18 to 24 hours after the 
injection. Occasionally the temperature remains above the normal 
for a longer time. The temperature usually begins to rise in from six 
*Tuberculin is prepared as follows: 
“The preparation of the culture medium (glycerinated bouillon), distributing it in 
suitable flasks (500 cc. Erlenmeyer putting but 100 ce. of liquid in each) and inoculating 
it with the growth from a pure culture of tubercle bacteria that will produce a good 
tuberculin. Both the human and bovine varieties of the organisms are used for this 
ose. 
“The flasks are placed in an incubator at a temperature of 37° C. where they remain 
until the growth ceases. The length of time necessary to accomplish this depends upon 
the age and condition of the culture from which the inoculations were made. From 
six to ten weeks are required. 
“After the maximum growth is attained, the cultures are sterilized by heat, either 
by boiling in a closed water bath or heating to a higher temperature in an autoclav. 
“After sterilization, the cultures are filtered to remove the bacteria, and the filtrate 
is evaporated, over a water bath, to the desired degree of concentration. 
“The concentrated liquid is passed through a Pasteur or Berkefeld filter, standard- 
ized, bottled and labeled for distribution. It should be perfectly clear although its 
color may vary. If it is cloudy it should be rejected.” 
It will be seen from the method of preparation that tuberculin cannot possibly 
contain living tubercle bacteria. It is heated on two occasions to a temperature and for 
a length of time far in excess of that required to destroy them, hesides being passed 
through a filter capable of removing all bacteria. 
The original tuberculin or lymph of Koch was concentrated to one-tenth of the 
volume of the saturated culture. This gave a thick, syrupy liquid owing to the presence 
of the glycerin. The diagnostic dose which came to be recommended for cattle of 
medium weight was 0.25 cc. On account of its consistency as well as the minuteness 
of the dose, it was found to be practicable to dilute this quantity with seven parts of a 
diluent. A weak solution of carbolic acid was ordinarily used. The difficulties and 
the danger of contamination involved in making the dilutions in the field led to the 
method of diluting the tuberculin in the laboratory before sending it out. This has 
been the practice of the Bureau of Animal Industry for a number of years. Equally as 
good results are obtained by concentrating the saturated culture to the point where 
2 cc. contains an equivalent of the 0.25 cc. of the highly concentrated lymph. This 
process avoids the necessity of dilutions and, with the addition of a few drops of car- 
bolic acid, the weaker solution keeps perfectly. 
