128 CULTURE MEDIA AND THEIR STERILIZATION 



the tubes so that they stand in an upright position, moisten the rubber 

 ring with formalin solution and then screw on the lid. Prepared in 

 this manner culture tubes may be kept for many months. Gelatin 

 tubes, however, if kept in this fashion for a long time may lose their 

 property of being liquefied by liquefying bacteria. 



FIG. 60 FIG. 61 



Erlenmeyer flask. Pasteur flask. 



Preparation of Nutrient Bouillon from Fresh Meat. The basis of 

 most solid artificial culture media in use is nutrient bouillon. This 

 in itself forms an excellent culture soil for the majority of patho- 

 genic bacteria. On account of the great advantages offered by 

 solid culture media in isolating bacteria in pure cultures, bouillon is 

 frequently combined with agar-agar or gelatin. Nutrient bouillon 

 may be prepared by either one of two methods. Take one pound 

 (500 grams) of finely chopped, lean, boneless meat (generally beef, 

 for special purposes veal, pork, horse or dog meat), add two quarts 

 (1000 c.c.) of water, and allow it to stand for twelve to eighteen 

 hours in the refrigerator or in winter in the cold. Filter through 

 muslin and thoroughly express the juice which remains in the 

 meat. Boil to precipitate the coagulable albumins, then filter and 

 make up to 1000 c.c. Add 10 grams of dried beef peptone and 5 

 grams of common salt (NaCl) to the filtrate. Boil again until it is 

 entirely dissolved. Test the reaction with litmus paper. It will be 

 found to be acid. Add enough of a solution of sodium hydrate to 

 make the bouillon very slightly alkaline to litmus. Boil again and 

 filter clear. Distribute into test-tubes, about 10 c.c. in each, or small 

 flasks, about 50 to 100 c.c. in each. Sterilize by continuous sterili- 

 zation in the ordinary steam sterilizer or autoclave. It is sometimes, 

 though not generally, necessary to procure culture media of a very 

 definite reaction. In this case the bouillon is titrated with a one- 

 twentieth normal solution of caustic soda or sodium hydrate (NaOH). 

 A small portion of the uncorrected bouillon is taken, diluted with 

 nine times the amount of distilled water, and titrated exactly with 

 the Y sodium hydrate solution until neutral or faintly acid to a 

 phenolphthalein reaction indicator. The amount of the caustic soda 



