11,4-22 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Tryptone (Bacto) 20 g. Sodium thioglycollate (for 



NejHPO, 2 g. pathogenic group only) Ifg. 



Glucose 1 g. Agar 1 g. 



Water 1000 ml. 



Application of test (see Roessler and McClung, 1943): Place 2 drops offthe culture 

 (withdrawn by pipette) in a spot plate; add 2 drops of vanillin (5% in|95% ethyl 

 alcohol) and then 3 drops of concentrated HCl. The addition of one drop of 0.1% 

 NaN02 causes the violet-pink of skatole to become dark purple but the orange"color 

 characteristic of indole is not changed. 



Medium for Nitrate Reduction. (See Reed, 1942). As certain 

 species reduce nitrites as well as nitrates, there should be included a 

 test for the presence (or disappearance) of nitrates as well as the ap- 

 pearance of nitrites. A negative nitrite test is of no significance. 

 The medium of Reed and Orr (1941) is satisfactory: 



Tryptone (Bacto) 20 g. Agar 1 g. 



Na.HP04 2g. KNOj 1 g. 



Glucose 1 g. Water 1000 ml. 



Adjust pH to 7.6 before autoclaving. 



4. OTHER MEDIA OF VALUE 



Medium for demonstration of capsules and spores. It is sometimes 

 inconvenient to use animal autopsy material for demonstration of 

 capsules. Svec and McCoy (in press) recommend the following 

 medium for demonstration of capsules and spores of C. perfringens. 

 Presumably it will be suitable for other species. 



Casein hydrolysate (acid) 35 ml. K2HPO4 5 g. 



Ovalbumin hydrolysate (acid) . . 15 ml. Sodium thioglycollate 1 g. 



Yeast water (prepared by auto- (NH4)2S04 2 g. 



claving 20% wet weight of Tryptophane 12 mg. 



yeast in water) 100 ml. Glucose 2.5 g. 



Sodium lactate 5 ml. Distilled water to make 1000 ml. 



Adjust pH to 7.4 and sterilize 25 minutes at 15 pounds. 



To prepare acid hydrolysates : Autoclave 200 g. casein (or egg 

 albumin), 110 ml. concentrated HCl and 170 ml. distilled water for 

 45 minutes at 12 pounds. If desired, decolorize with norite. 



Medium for spore production by butyric-butyl group. If cultures of 

 this group do not sporulate readily on plain corn mash (prepared ac- 

 cording to directions for corn-liver medium except that the liver 

 powder is omitted), use potato infusion prepared as follows: 



Irish potatoes 200 g. (NH4)2S04 1 g. 



Glucose 5 g. CaCOj 3 g. 



Tap water to make 1000 ml. 



Peel potatoes and add water. Steam for one half hour or boil 

 slowly until soft and put through potato ricer. Add other ingredients 

 and bring up to original volume. Cool and tube, with stirring, so as 

 to obtain an even distribution of the potato particles. 



Medium for toxin production. In Leaflet III there is mention of 

 the fact that beef heart infusion or glucose meat infusion is satis- 

 factory for toxin production by most toxigenic species. Another 

 medium, proposed by Reed, Orr, and Baker (1939), may be recom- 

 mended for the gangrene group. This is prepared from commercially 

 available ingredients as follows: 



