THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 137 



mash in a manner similar to the method given for corn-Hver medium with 

 the exception that the Uver powder is omitted. Brain medium may be 

 suitable also (see also Chap. III). 



In any medium, after all gassing has subsided and spores have been 

 demonstrated microscopically, the tube should be sealed in the flame or 

 the stopper covered to protect the medium from evaporation and the 

 tube placed in a cool room or refrigerator. Viable subcultures may be 

 obtained from such tubes for months or even years in some instances. 

 Another method which has been used with success is worthy of mention. 

 This involves the storage of cultures on sterile soil : Dry fresh garden soil 

 and sift through a fine-mesh screen; add 5 per cent of CaCOs to neutralize 

 any acidity of the culture. Place soil in tubes in 2-in. columns, and auto- 

 clave overnight. Test each tube for sterility, using both aerobic and 

 anaerobic media. If sterile, add 2 or 3 ml of a well-sporulated culture 

 with a sterile pipet and dry the tube (preferably in a vacuum desiccator) . 

 To obtain an active culture from this stock (which may be stored at room 

 temperature) transfer a small amount of the soil to an enrichment medium 

 and heat-shock. By the soil stock method a relatively permanent source 

 is available from which cultures may be revived as needed without 

 destroying the stock culture. 



Serological Reactions 



The serological relationships of the sporeforming anaerobes have been 

 reviewed (McCoy and McClung, 1938; Smith, 1955). The toxin-anti- 

 toxin reaction is of value as a taxonomic aid with certain species. In 

 such an instance one takes advantage of the fact that relationships may 

 be established by the success or failure of the reaction of antitoxin, pre- 

 pared against the toxin of a kno\vn organism, with the toxin from the 

 unidentified strain. In some instances the anaerobic species are mono- 

 typic with respect to toxin formation. In other species this is not true, 

 and subgroups have been established within these species or species 

 groups on the basis of non-cross-neutralization tests. 



The problem of the complex relationships of the toxins produced by the 

 Clostridia is beyond the scope of this chapter, but those interested in the 

 details beyond those presented by Smith (1955) should consult Oakley 

 (1954) and Van Heyningen (1950, 1955). 



REFERENCES 



Barker, H, A. 1936. Studies upon the methane-producing bacteria. Arch. Mikrob., 

 7, 420-438. 



Brewer, J. H. 1940. Clear liquid mediums for the "aerobic" cultivation of anae- 

 robes. /. Am. Med. Assoc, 115, 598-600. 



. 1942. A new petri dish cover and technique for use in the cultiva- 



tipn of anaerobes and microaerophiles. Science, 95, 587. 



