ENZYME PRODUCTION 277 



and keep careful records of these points, and also of the 

 age of the cultivation used in the final examination. 

 Examine the cultivations for the various products of 

 bacterial metabolism after forty-eight hours' growth, 

 and never omit to examine "control" (uninoculated) 

 tube or flask of medium from the same batch, kept for 

 a similar period under identical conditions. 



' If the results are negative, test further cultivations 

 at three days, five days, and ten days. 



1. Enzyme Production. 



(A) Proteolytic Enzymes. (Convert proteins into 

 proteose, peptone and further products of hydrolysis; 

 e. g., B. pyocyaneus.) 



Media Required: 



Blood-serum and milk-serum which have been carefully filtered 

 through a porcelain candle. 

 Reagents Required: 



Ammonium sulphate. 



Thirty per cent, caustic soda solution. 



Copper sulphate, 0.5 per cent, aqueous solution. 



One per cent, acetic acid solution. 



Millon's reagent. 



Glyoxylic acid solution. 



Concentrated sulphuric acid. 



METHOD. 



1. Prepare cultivations in bulk (50 c.c.) in a flask 

 and incubate. 



2 . Make the liquid faintly acid with acetic acid, then 

 boil. (This precipitates the unaltered proteins.) 



3. Filter. 



4. Take 10 c.c. of the filtrate in a test-tube and add 

 i c.c. of the caustic soda, then add the copper sulphate 

 drop by drop. 



Pink colour which becomes violet with more 

 copper sulphate = proteose and peptone. 



5. Saturate the rest of the filtrate with ammonium 

 sulphate. 



Precipitate = proteose. 



