PREPARATION OF MEDIA ii,,-21 



and peptone). Tube in deep columns and autoclave for 20 minutes at 

 15 pounds. The final medium should be an opaque whitish liquid. 

 Proteolysis is indicated by progressive clearing of the medium. 



Brain Medium. Secure fresh sheep (or calf) brains which are as 

 free as possible from injury. Using forceps clean blood and mem- 

 branous material from brain tissue. Add distilled water, in the 

 ratio of 100 ml. of water to 100 g. of brain, and boil slowly for one 

 half hour. Put brains through potato ricer. Add 1.0% peptone and 

 0.1% glucose to the resulting mixture and heat slightly to put peptone 

 in solution. Tube in deep columns while the mixture is stirred in 

 order to effect an even distribution of the brain tissue. Reduced 

 iron or a strip of black stove-pipe iron or iron wire may be added to 

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

 30 minutes at 15 pounds and check sterility by incubation at 37° C. 

 for a minimum of 24 hours. The finished medium has approximately 

 an equal amount of liquid broth above the brain particles. Proteoly- 

 sis is indicated by putrefactive odors, a disintegration of the particles 

 and a blackening reaction. 



Use: The blackening reaction of this medium has some diagnostic 

 significance (Hall and Peterson, 1924). This medium is also valu- 

 able for many species for the production of spores and hence as a 

 stock culture medium. 



Milk Agar for Testing Proteolytic Action. Reed and Orr (1941) suggest the follow- 

 ing medium: Mix equal parts of skim milk (reconstituted from powder) and a plating 

 agar (see their media in section on plating media for purification). Autoclave the 

 two media separately and mix just before pouring. Proteolysis is indicated by a wide 

 clear zone surrounding the growth. 



Medium for Production of H2S. Probably most, if not all, species 

 of anaerobes produce H2S, at least in trace amounts. From the dis- 

 cussion of McCoy, et al. (1926), Spray (1936), Pacheco e Costa (1940) 

 and Reed and Orr (1941), we conclude that there is, as yet, no stand- 

 ard medium for this reaction. The media listed below were found to 

 be satisfactory by Reed and Orr (1941); and it is recommended that 

 the exact method of preparation be listed in published reports for 

 any additional medium which may be devised. 



Medium 1 



Proteose peptone 20 g. Glucose 1 g. 



Na.HP04 2 g. Agar 2 g. 



Water 1000 ml. 



Dissolve ingredients, adjust to pH 7.6, and add 10 ml. of 2% lead 

 acetate. This results in a cloudy precipitate which, however, re- 

 mains after autoclaving in a reasonably stable suspension. 



Medium 2 



Proteose peptone 20 g. Glucose 1 g. 



NajHP04 2 g. Water 1000 ml. 



Dissolve ingredients, adjust to pH 7.6, and add 10 ml. of a 1.5% bis- 

 muth and ammonium citrate solution. This ordinarily produces a 

 solution which remains clear after autoclaving. 



Medium for the Formation of Indole and Skatole. The foUov/ing 

 medium will usually be found satisfactory: 



