THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii,,-l,5 



tioii reveals positive motility. If motility is doubtful or appears 

 to be negative, initiate other procedures. For example, use a flat- 

 tened capillary tube sealed at each end. Heat glass tubing, of 

 small diameter, and flatten a small area. Prepare a capillary 

 tube from the flattened section. Draw a small amount of culture 

 into this tube and seal the tube in the flame on both sides of the drop 

 of culture. Examine this preparation with the high power objective. 

 If the motility is still recorded as negative, make further observations 

 on younger (4-6 hour) cultures. For these, examine the 3rd or 

 4th tube of a serial passage series, using the medium which appears 

 to give the best growth of the culture. Because of the relatively 

 small number of species which are non-motile, considerable caution 

 should be exercised in reporting cultures which appear to be non- 

 motile. Naturally occurring non-motile 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. If difficulty is encountered in securing positive slides 

 from cultures known or thought to be motile, 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 conventional methods. 

 The most important capsulated species is Clostridium perfringens 

 (C loelchU). Material taken from artificially infected laboratory 

 animals generally serves as the origin of smear preparations. If 

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

 McCoy (See Leaflet II) is useful if other media prove unsuccessful. 



DEMONSTRATION OF SPORES 



Cultures surviving 20 minutes heating at 80° C. may be presumed 

 to be spore-formers. It is, however, useful to demonstrate 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 patho- 

 gens one should use the deep brain, or beef heart, or alkaline egg 

 medium. In some instances spores may be demonstrated within 

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

 time, older cultures should be examined. Protection from evapora- 

 tion must be given cultures which are to be incubated longer than 



