624 APPENDIX: LABORATORY METHODS 



with aliquot parts of chipped ice and cold tap water. The mixture is 

 stored in earthenware jars in the cold room for 3 to 5 days and is 

 stirred at intervals. After most of the ice has melted, the mixture is 

 transferred to aluminum kettles, heated to boiling, strained through 

 cheesecloth, and the cake of coagulated meat pressed as nearly dry as 

 possible in a meat press. The strained liquid part of the infusion is col- 

 lected in 5-gallon Pyrex carboys and autoclaved at 120° for 45 minutes. 

 Air is then forced into the autoclave to keep the pressure at 15 to 20 

 pounds until the temperature falls to about 80° or 90°. The crude, ster- 

 ilized extract is stored in the cold room. 



b. Preparation of medium. The crude infusion is filtered in the cold 

 room through soft filter paper. It is siphoned from the carboy to avoid 

 disturbing the layer of partly solidified fat which collects on the sur- 

 face. Unless the infusion is to be used for producing Type III SSS, it 

 is best to remove the glycogen as follows : 



The infusion is evaporated to about one-tenth its original volume, and 

 the glycogen precipitated by the addition of two volumes of 95 per cent 

 alcohol to one of infusion. The glycogen is separated by centrifugation. 

 The glycogen-free infusion is then distilled under reduced pressure in a 

 water-bath to free it from alcohol. 



The glycogen-free, alcohol-free infusion is then made up to its origi- 

 nal volume with tap water, and to it is added peptone (Witte or Pfan- 

 stiehl) to a concentration of 1 per cent. The solution is filtered through 

 soft paper into a 5-gallon Pyrex carboy and autoclaved as described 

 above. 



The infusion-peptone solution may be diluted with an equal volume of 

 tap water before autoclaving and use. This step results in a higher yield 

 of polysaccharide per volume of infusion, but requires the handling of 

 larger amounts of fluid. Prior to inoculation, the medium is stored in the 

 incubator room for a sufficient time in order that it may reach the 

 proper temperature. 



A 30 to 40 per cent solution of glucose is added to the medium in 

 amounts to give a final concentration of 1 per cent. The glucose is ster- 

 ilized separately to prevent the formation of colored products which are 

 difficult to separate from the capsular polysaccharide. 



The alkali solution used in neutralizing the acid formed during 

 growth is sterilized in the Arnold sterilizer for 30 minutes in cali- 

 brated Pyrex flasks fitted with a stopper bearing an air filter and a 

 glass tube which reaches to the bottom of the flask. The other end of the 

 tube is fastened to a rubber tube of sufficient length to reach from the 



