640 APPENDIX: LABORATORY METHODS 



removing the sodium sulfate by repeated precipitations of the SHI with 

 alcohol and acetic acid in the presence of sodium acetate the product 

 was found to contain 2.4 per cent of nitrogen. This was removed by 

 twice precipitating the SHI as the barium salt with barium chloride. 

 The barium was removed by repeated precipitations from 20 per cent 

 sodium acetate solution with acetic acid and alcohol. The SIII was iso- 

 lated as the neutral sodium salt. 



V. Preparation of Bacterial Enzymes Capable of Decomposing 

 Capsular Polysaccharides (Dubos, 336 and Dubos and Bauer 338 ) 



The bacteria are grown in a solution of 2 per cent casein hydrolysate 

 (pH 7.0) at 37° under conditions of strict aerobiosis ; the cells from the 

 16-hour-old culture, separated by centrifugation, are resuspended in 

 small amounts of distilled water. 



A medium is prepared consisting of 0.1 per cent capsular polysac- 

 charide and 0.1 per cent NaCl in distilled water. This medium is dis- 

 tributed in 25 cc. amounts in large Erlenmeyer flasks (1 liter capacity) 

 to provide for aerobic conditions, and each flask is inoculated with the 

 cells recovered from 500 cc. of the culture of the SIII bacillus in the 

 casein hydrolysate medium. The material is incubated for 12 to 18 

 hours at 37° and the cultures tested to ascertain the disappearance of 

 the specific polysaccharide and the absence of contaminants. The cul- 

 tures are now frozen and thawed repeatedly to secure the release of the 

 endocellular enzyme. 



The enzyme is ultimately separated from the cell debris by filtration. 

 However, since the cell suspension is very viscous, it is first subjected 

 to the following treatment. The cell suspension is made alkaline to pH 

 10.0 by the addition of sodium borate. Equimolecular concentrations of 

 dibasic sodium phosphate and calcium chloride are then added to bring 

 about a heavy precipitate of calcium phosphate which facilitates the 

 clarification of the material by centrifugation. The supernatant fluid 

 which contains all the enzyme in solution is now passed through a Seitz 

 filter, then through a Berkefeld (V) filter. The potency of this filtrate 

 is such that 0.002 to 0.004 cc. are required to decompose 0.01 mg. of 

 the capsular polysaccharide under the conditions of the test. 



For the purpose of further purification of the enzyme preparation, 

 the solution is passed through collodion membranes made and calibrated 

 according to the description given by Bauer and Hughes. 90 Membranes 



