214 MEDICAL BACTERIOLOGY 



ment of a particular case is determined by the signs, symptoms, localization, 

 extent and progress of the disease and by the fancies of the person using the 

 tuberculin. 



Tuberculin is of greatest value as a diagnostic agent. 



Koch's Old Tuberculin. (Tuberculinum original alt) "T. O. A." or "O. 

 T." is made by planting the tubercle bacillus in a flask of glycerin broth, 

 incubating at 37C. for 8 weeks. It is then placed in steam sterilizer for i 

 hour or autoclave at 1 5 pounds pressure for % hour and evaporated on a water 

 bath at 8oC. to 3/L its original volume. The bacteria are removed by nitra- 

 tion, first through paper and then through a Berkefeld filter, the filtrate, which 

 is the tuberculin, is put in containers and sterilized in steam sterilizer, i hour 

 each day for 3 successive days, or in the autoclave at 15 pounds pressure for 

 20 minutes. 



Deny's Bouillon Filtrate. (Bouillon Filtrate) "B. F." is prepared the same 

 as O. T. except that it is not heated at any time during its preparation. It is 

 evaporated to one-tenth its original volume at room temperature in vacua over 

 sulphuric acid. 



Koch's Bacillen Emulsion. "B. E." cultures are made as for the prepara- 

 tion of O. T., the pellicle of bacteria is caught on cheesecloth as the culture is 

 poured on it, they are freed of broth by pouring over them first salt solution, 

 then water. The bacteria are placed in dishes and desiccated in vacua over 

 sulphuric acid and weighed, then ground in a porcelain ball mill for about 6 

 weeks; 100 cc. of salt solution for each gram of bacteria is then put in the mill 

 and grinding continued for 2 days. The emulsion is allowed to stand at rest 

 for several days so that coarser particles precipitate, the supernatant fluid is 

 then collected, mixed with 50 per cent, of glycerin and standardized to contain 

 5 mg. of solids in each cubic centimeter. 



Tuberculin Residue "T. R." ("New tuberculin")- Bacilli are grown and 

 recovered from the culture, dried, weighed and powdered, the same as when 

 proceeding to make B. E. When sufficiently comminuted, 100 cc. of sterile 

 distilled water is added to each gram of dried bacteria, and the mass again 

 ground for 24 hours. The emulsion is then centrifugalized until the solids 

 precipitate. The supernatant fluid is siphoned off and discarded. The solid 

 residue is collected, desiccated in vacua over sulphuric acid, mixed with water 

 and after 24 hours centrifugalized as before. The supernatant fluid, second 

 watery extract, is collected and set aside; the residue again desiccated in vacua, 

 after which it is extracted with water for the third time, the same as before and 

 the watery extract set aside. 



By repeating in the same way several times more this extraction, and retain- 

 ing the extracts, eventually there is no residue; the entire bacillary substance 

 having been put in solution. 



These several water extracts (solutions) are then mixed. 



During the process, the amount of water added each time to residue must be 

 regulated so that, when combined, there will be a total volume 100 cc. of solu- 

 tion for each gram of bacteria with which the production was started. 



