THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 131 



further observations on younger (4-6 hr) cultures. For these, examine 

 the third or fourth tube of a serial passage series, using the medium which 

 appears to give the best growth of the culture. Semisolid agar (0.5 per 

 cent) may be inoculated by stab and observed for clouding due to motility. 

 In some cases this procedure is complicated by breaking up of the agar as 

 a result of gas production. Because of the relatively small number of 

 species which are nonmotile, considerable caution should be exercised in 

 reporting cultures which appear to be nonmotile. Naturally occurring 

 nonmotile variants of motile species, however, have been encountered. 



Flagella stain. For material for preparation of flagella stains use young 

 cultures growing in the medium which is most favorable to the organism 

 being studied. A temperature lower than that generally considered 

 optimum is recommended (Leifson, 1951). If difficulty is encountered 

 in securing positive slides from cultures known or thought to be motile, 

 use the technic of Leifson, and consult the directions given by O'Toole 

 (1942) for suggestions in technic which refer particularly to anaerobic 

 bacteria. 



Capsule stain. For the capsule stain one may use any of the conven- 

 tional methods. The most important capsulated species is Clostridium 

 perfringens (C welchii). Material taken from artificially infected labora- 

 tory animals generally serves as the origin of smear preparations. If 

 stains from in vitro cultures are desired, the medium of Svec and McCoy 

 (Chap. Ill) is useful if other media prove unsuccessful. 



Demonstration of spores. Cultures surviving 20 min heating at 80°C 

 may be presumed to be sporeformers. It is, however, useful to demon- 

 strate the spores microscopically. The exact method of making the 

 spore stain is of little importance in comparison with other factors, as each 

 of the common methods (Dorner, Moeller, and malachite green) appears 

 satisfactory. One must, however, pay some attention to the medium in 

 which one expects to induce sporulation. Media containing fermentable 

 carbohydrates are not satisfactory, in general, for the pathogenic group. 

 The media naturally containing carbohydrate (e.g., corn mash or potato 

 infusion), on the other hand, appear ideal for most of the butyric-butyl 

 group. For the pathogens one should use the deep brain, beef heart, or 

 alkaline egg medium. In some instances spores may be demonstrated 

 within 24-28 hr after inoculation, but if the culture is negative at this 

 time, older cultures should be examined. Protection from evaporation 

 must be given cultures which are to be incubated longer than one week. 

 Clostridium perfringens (C. welchii) appears to be one of the most difficult 

 species in which to demonstrate spores microscopically with regularity. 

 If success is not attained using the above-mentioned media in cultures 

 having the characteristics of this organism, one may use the medium 

 recommended by Ellner (1956). 



