506 IMMUNOLOGY 



is not affected when the proteose is destroyed by trypsin or erepsin. 

 This indicates that the active substance is a protein. 



5. The active substance, in the presence of N/10 hydrochloric 

 acid, is not destroyed when heated under fourteen pounds pres- 

 sure at a temperature of 120° C. for four hours. It is hydro- 

 lyzed by acids having a strength of N/6 or above. 



6. Seibert attempted crystallization of the protein and suc- 

 ceeded in inducing a portion of the active substance to crystallize 

 in needle form. This active crystalline substance possesses the 

 property of an albumin. 



7. They observed that a greater yield of active substance is ob- 

 tained in filtrates of the old synthetic broth culture than from 

 extraction of tubercle bacilli. This probably explains why tuber- 

 culin OT is generally preferred to BE or TR. 



8. More recently Seibert and Munday have combined ultra- 

 filtration with trichloracetic acid precipitation in the preparation 

 of a purified active protein fraction. 



9. Masucci and McAlpine (1930) made use of the suggestions 

 of Seibert in preparing a tuberculin which Funk and Huntoon 

 (1930) have designated as MA-100 in accordance with the John- 

 son chart for the chemical analysis of the tubercle bacillus. 



TPT. OF Seibert and Munday. — The new tuberculin protein pre- 

 pared by Seibert and Munday (1932) is designated by them as 

 TPT. These letters indicate that the product is tuberculin pro- 

 tein precipitated by trichloracetic acid. In their opinion it is 

 superior in some respects to TPA which they prepared in 1931 

 by precipitating old tuberculin with ammonium sulphate. Seibert 

 and Munday describe their method of preparing TPT as follows: 

 ''Virulent tubercle bacilli (Saranac Lake Strain H37 has been 

 used in our work) are grown upon Long's synthetic medium for 

 eight weeks at 37.5° C. (more protein is obtained by allowing the 

 culture to grow for eight to twelve weeks instead of the usual four 

 to six weeks). The bacilli are removed by filtration through China 

 silk or a Buchner funnel and then through a Mandler filter. The 

 clear yellow tuberculin, preserved with 0.5 per cent phenol, 

 is then concentrated by ultrafiltration on a 12.5 per cent gun- 

 cotton-glacial-acetic-acid membrane, and washed with 0.5 per cent 

 phenol solution by continued ultrafiltration until the filtrate is 

 chloride- and iron-free, and then filtered. (It has not yet been 



