M MANUAL OF MICROBIOLOGICAL METHODS 



use of infusions is diminishing owing to their replacement by media pre- 

 pared from peptones or other materials of more uniform composition, 

 better growth-promoting properties, and less complicated preparation 

 details. 



Beef -liver infusion, used principally for anaerobic types, is prepared as 

 follows : Remove fat from 500 g of fresh beef liver, grind, and heat, with 

 occasional stirring, in 1,000 ml of tap water for 1 hr in flowing steam. 

 Cool, and strain through cheesecloth. Restore filtrate to original 

 volume, and add 1 per cent of peptone and 0.1 per cent of K2HPO4. Dry 

 the tissue (at 55° C if possible) rapidly. Tube broth over several chunks 

 of tissue. Use the broth (before addition of peptone and phosphate) in 

 the original strength or diluted five times. Sterilize 20 min at 121°C. 

 Avoid longer heating of medium, for this diminishes its value with respect 

 to initiation of growth from small inocula. 



Brain medium is used to stimulate spore production by many Clostridia 

 and hence is a valuable stock culture medium. The blackening reaction 

 produced by certain species has some diagnostic value (Hall and Peterson, 

 1924). The medium is prepared as follows: Secure fresh sheep (or calf) 

 brains which are as free as possible from injury. Using forceps, remove 

 blood and membranous material from brain tissue. Add distilled water, 

 in the ratio of 100 ml of water to 100 g of brain, and boil slowly for }4 hr. 

 Put brains through potato ricer. Add 1.0 per cent of peptone and 0.1 

 per cent of glucose to the resulting mixture, and heat slightly to dissolve 

 peptone. Tube in deep columns while the mixture is stirred in order to 

 effect an even distribution of the brain tissue. It is sometimes recom- 

 mended that reduced iron (''iron reduced by hydrogen," Merck & Co., 

 Rah way. New Jersey), a thin strip of iron, or iron wire be added to the 

 tube before tubing the liquid mixture. Sterilize in autoclave for 30 min 

 at 121°C and check sterility by incubation at 37°C for a minimum of 24 hr. 

 The finished medium should have approximately an equal amount of 

 liquid broth above the brain particles. Proteolysis is indicated by 

 putrefactive odors, a disintegration of the particles, and a blackening 

 reaction. 



Yeast infusion may be prepared by several procedures. A satisfactory 

 one follows: Obtain fresh yeast, preferably starch-free, and add 10 per 

 cent by weight to several liters of tap water. Autoclave for 3 hr or more. 

 Allow cells to settle by standing for several days at room temperature. 

 Remove liquid infusion by siphon or with the Sharpies centrifuge. Steril- 

 ize the liquid, after removal from the cells, in screw-capped bottle, and 

 store indefinitely. If fresh yeast is not available, or if a simpler procedure 

 is preferred, a similar medium may be prepared by adding 0.5 per cent of 

 dehydrated yeast extract to distilled water. Care should be observed in 

 selection of the yeast extract, since not all are equal in growth-promoting 



