1144-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Use: Satisfactory for the determination of those characters usually 

 revealed by litmus milk. Of diagnostic aid in the search for C. 

 perfringens, due to the fact that this organism gives a stormy fer- 

 mentation. 



Note: This test is not strictly specific for C. perfringens as certain non-pathogenic 

 motile species of the butyric-butyl group also give this reaction. They may be separated 

 from C. perfringens by virtue of the non-motility of the latter. Robinson and Stovall 

 (1939) recommend the addition of 1.0 ml. of 20% Na^SOj solution and 0.1 ml. of 8% 

 FeClj solution to 10 ml. of milk as an additional aid in the diagnosis of C. perfringens. 

 This organism produces a blackening reaction. 



Medium for Liquefaction of Gelatin. For some species standard 

 nutrient gelatin plus 0.25% glucose may serve as a base medium for 

 testing for liquefaction of gelatin. If the organism in question will 

 grow in such a medium, it is recommended for use. For other species 

 choice may be made between the two formulae which follow: 



Gelatin Medium of Reed and Orr (19^1). Dissolve the following ingredients in 

 1,000 ml. of distilled water: 



Gelatin 50 g. Na2HP04 2 g. 



Peptone 10 g. Glucose 1 g. 



Sodium thioglycollate 1 g. 



Gelatin Medium of Spray (1936). Dissolve the following ingredients in 1,000 ml. of 

 distilled water: 



Difco Nutrient Gelatin 128 g. 



Glucose 1 g. 



Dissolve gelatin in water taking care not to scorch the gelatin. Include a strip of No. 

 26 gauge black stove-pipe iron in each tube. 



Use: Either of the above media may be used for the pathogenic group. The medium 

 of Spray has the additional advantage of being a presumptive medium for C. his- 

 tolyiicujti as this organism gives an orange to wine-red color within the first 48 hours of 

 incubation. 



Other Media for Testing Proteolytic Action. The action on gelatin 

 represents action on a simple and incomplete protein and positive 

 action is not necessarily an indication that the organism can hydrolyze 

 the complex proteins. The beef heart infusion represents one of the 

 media in which putrefactive action on complex proteins may be re- 

 corded. Coagulated serum slants, prepared in the usual manner, 

 inoculated and incubated in an anaerobic jar, represent another type 

 of protein to be tested. Evidence of proteolytic action in this 

 medium is shown by partial or complete liquefaction of the medium. 

 For action on coagulated egg albumin include a small cube of the 

 white of a hard boiled egg in a tube of 1% peptone and 0.2% glucose 

 broth or other liquid medium. Disintegration of this cube during the 

 incubation is evidence of proteolytic action. Peptonization of 

 litmus milk reveals caseinolytic ability. In addition to the above 

 three other media are recommended. It may not be necessary to 

 use all of these but more than one should be included in taxonomic 

 studies because of possible differential reactions. 



Alkaline Egg Medium. Mix the yolk of two and the whites of four 

 eggs (preferably in Waring Blendor). Add 1,000 ml. of distilled 

 water and 12 ml. of 1 A^ NaOH. Stir well or mix in Waring Blendor. 

 Add one part of the above to 5 parts of nutrient broth (beef extract 



