CHAPTER IX. 



METHODS OF ISOLATING THE BACTERIAL PEOTEIDS. 



Hankin employed the following process iu preparing 

 his anthrax proteid : 



" The cultures are made in 0.1 per cent. Liebig's extract 

 of meat solution, to which some fibrin is added. The Lie- 

 big's extract is very difficult to sterilize, and must be heated 

 for two or three hours in the steam sterilizer on two or 

 three successive days. The fibrin must be added only after 

 this has been done, and then the flaslc is re-sterilized by 

 repeated heating to boiling-point, for a short time only on 

 each occasion. If the fibrin were added at first it would 

 be decomposed by the prolonged boiling. By the above 

 method this only occurs to a slight degree, a mere trace of 

 peptone being present in the sterilized culture-fluid. After 

 sterilizing, this is inoculated with the blood of an animal 

 dead of anthrax, and kept at the ordinary temperature. 

 The anthrax forms a typical growth on the masses of fibrin, 

 and samples of the liquid removed on successive days show 

 a gradual increase in the strength of their biuret reaction. 

 After about a week the liquid is filtered and the albumose 

 extracted. The reason for not keeping the flask at a tem- 

 perature of 37° is that the albumose is gradually decom- 

 posed into peptone by the anthrax ferment present, and 

 this change takes place more rapidly at the higher tempera- 

 ture. For instance, I have found scarcely a trace of albu- 

 mose in a culture which had been kept at 37° for a week, 

 and which gave a strong biuret reaction. The albumose 

 is separated from the culture-liquid thus prepared by satu- 

 ration with ammonium sulphate. It is better to acidulate 

 it slightly by adding a little acetic acid. The bulky pre- 

 cipitate of albumose which then appears is filtered off, and 

 the salt separated from it by dialysis. An excess of thymol 



