PREPARATION OF MEDIA 43 



Thiogly collate broth, originally used for the growth of anaerobic bac- 

 teria, is now being used also for other types with regard to oxygen rela- 

 tionships. The liquid medium may be prepared as follows: Dissolve 

 15 g of peptone (preferably a pancreatic digest of casein), 5 g of glucose, 

 5 g of yeast extract, 0.75 g of L-cystine, 2.5 g of NaCl, 0.75 g of agar, and 

 0.1-0.5 g of sodium thioglycollate in 1 liter of water; adjust pH to 7.2. 

 Sterilize for 15 min at 121°C. The small amount of agar which is neces- 

 sary to maintain a low oxidation-reduction potential does not affect 

 appreciably the fluidity of the medium. This medium is unsatisfactory 

 for stock cultures unless CaCOs is added. If a dye to indicate the 0/R 

 potential is desired, add 0.002 g of methylene blue or 0.001 g of resazurin 

 per liter. If the medium to be used contains an indicator dye, for 

 obligate anaerobes, the dye should show the medium to be reduced 

 except in the upper layer. Therefore, unless the dye indicates that the 

 medium has been reoxidized to a considerable extent, it is unnecessary to 

 follow the usual practice of heating the medium for exhaustion of oxygen 

 immediately prior to inoculation. This medium should not be stored in 

 the refrigerator after preparation, as it absorbs more oxygen at lower 

 temperatures. For special purposes, such as the sterility testing of bio- 

 logical products in which inactivation of mercurials presents a problem, 

 dehydrated media are available which meet the specifications of the 

 National Institutes of Health and other agencies. 



Sodium caseinate agar. This medium is often used for the enumera- 

 tion of bacteria, including the Actinomycetes, in soil. It is prepared as 

 follows: sodium caseinate, 1.0 g; glucose, 1.0 g; MgS04, 0.2 g; K2HPO4, 

 0.2 g; FeS04, trace; distilled water, 1,000 ml; agar, 15 g. It is adjusted to 

 pH 7.0. Before pouring plates or dispensing in other containers, it is 

 shaken gently to disperse precipitate. 



Infusion broths may be prepared by extraction of either lean skeletal 

 muscle or heart muscle in the cold or by heat. In the former case the 

 following is typical : Add 1 ,000 ml of water to 400-600 g of lean veal or 

 beef tissue which has been finely ground after removing as much fat as 

 possible. Allow to infuse overnight at refrigerator temperature, remove 

 scum of fat, squeeze infusion through mushn cloth, and restore volume to 

 1,000 ml. Add 5 g of peptone, ^nd heat for 20 min at 100°C; filter 

 through paper; adjust pH to desired value. For infusions prepared by 

 heat, use the same proportion of meat to water and boil over free flame 

 for 15 min, with or without a previous overnight infusion period in a 

 refrigerator. Add peptone, and if desired as the basal medium for blood 

 agar, add 5 g NaCl to 1,000 ml, and continue as above. For aerobic 

 organisms adjust the pH to 7.0-7.2, but for anaerobic types adjust to 7.6 

 and tube the liquid deep over a 1- to 2-cm column of desiccated tissue 

 particles saved from the infusion. Autoclave for 20 min at 121°C. The 



