VERMONT AGRICULTURAL REPORT. 135 



METHOD OP PREPARING TUBERCULIN. 



In the preparation of large quantities of tuberculin in the 

 biochemic laboratory of the Bureau of Animal Industry during 

 past years, the following method of procedure has been found 

 to give the most satisfactory results : The fluid upon which the 

 tubercle bacilli are allowed to grow is an extract made with dis- 

 tilled water from perfectly fresh meat which has been finely 

 chopped. One pound of meat is used to a liter of water, to 

 which is added i per cent, of peptone, one-fourth of i per cent, 

 of salt, and 7 per cent glycerin. The solutionis heated to boil- 

 ing, filtered, and placed in perfectly sterilized flasks. The 

 medium is then sterilized for three successive days in a steam 

 bath. After the cotton plugs of the flasks have become dry, 

 they are removed, dipped in paraffin, and replaced in the flasks 

 so as to make tight stoppers. When the culture medium so 

 prepared is found to be thoroughly sterile, it is inoculated. 

 The thorough sterilization of the media can be proved by allow- 

 ing the flask to stand for some days in a warm place, during 

 which time they should remain perfectly clear if there is no 

 contamination with ordinary bacteria from the air. 



INOCULATING THE MEDIA. 



The inoculation of the media in the flasks is accomplished 

 by taking up on the end of a platinum wire a small mass of 

 tubercle bacilli obtained originally from an animal that has died 

 of tuberculosis. The first cultures are made from dead ani- 

 mals, by transferring to a jelly made of glycerin and agar, or 

 blood serum, or potato, pieces of the diseased organ, lung or 

 spleen, of a guinea pig that has been infected by inoculation 

 with tuberculosis. The germs, after four to six weeks, are 

 found to have developed very readily, and to form a thick, 

 spongy layer on the surface of the jelly or potato. It is a very 

 easy matter to detach a small bit of this spongy growth from 

 the surface of the jelly and transfer it to the flask containing 

 the liquid media prepared in the manner already indicated. 



In order that the tubercle bacilli may grow readily, it is 

 necessary that they shall have a free supply of oxygen ; hence, 

 the mass of bacteria that are transferred to the flask should be 

 caused to float on the surface of the liquid. This can be readi- 

 ly accomplished by detaching a piece of the culture mass from 

 the platinum needle and floating it upon the surface of the 

 liquid without immersing it. If the inoculating piece is allowed 

 to become wet with the culture media, it will sink to the bot- 

 tom of the flask and the bacteria will not develop. The inocu- 

 lated flasks are then placed in the incubator, which is a double- 

 walled copper box. The space between the copper walls of the 



