iii„-U MANUAL OF METHODS FOR PURE CULTURE STUDY 



nants. The following test is suggested: For most cultures, streak 

 a glucose nutrient agar slope and incubate it at 37° C; but for 

 anaerobic species having a lower or higher optimum temperature, 

 incubate a second agar slope at the temperature which is optimum 

 for the anaerobe. If the culture appears free of aerobic types, in- 

 vestigate the purity with respect to anaerobic contaminants. Make 

 repeated platings and scrutinize intensely the colonies which develop. 



Preliminary Microscopic Examination 



If the sample is suitable, one should make preliminary examina- 

 tion using the Gram stain. The conventional method of staining 

 a smear, heat fixed on a glass slide, should be used, except that the 

 decolorizer should be either 95% ethyl alcohol {'preferred) or 25 parts 

 acetone and 75 parts ethyl alcohol. The use of greater amounts 

 of acetone must be avoided because of the ease with which anaerobes 

 are decolorized. The usefulness of the Gram method is limited in 

 smears prepared from blood, fibrin or albumin. In samples of patho- 

 logic material, large, Gram-positive rods are likely to prove to be 

 anaerobic bacilli, but a final diagnosis must not be based on micro- 

 scopic observations unsupported by cultural tests. Of the strictly 

 aerobic Gram-positive species, Bacillus anthracis Koch is the only 

 usual pathogen. The characteristic morphology of Clostridium 

 perfringens (syn. C. welchii) and the regularity of its appearance in 

 certain clinical conditions frequently combine to give presumptive 

 evidence of value; similarly, the typical microscopic picture presented 

 by a spore-bearing C. ietani culture should be remembered when such 

 forms are encountered in pathologic material. All anaerobic species 

 are non-acid fast; therefore, this stain has no diagnostic importance. 



Microscopic Examination of Pure Cultures 

 GRAM stain 



If the organism in question will grow within this period, apply 

 the Gram stain to a 16-18 hour culture and observe the same caution 

 with reference to the decolorizer as noted above. Ordinarily the 

 stain is satisfactory when prepared from any enrichment medium 

 in which the organism will grow. In recording the Gram reaction 

 of a new species, state the medium from which the smear was made 

 and the age of the culture. 



examination for motility 



The majority of the spore-forming anaerobic bacilli are motile; 

 the most important exception is C. perfringens (C. u-elchii). The 

 technic by which the motility examination is made is often of utmost 

 importance in securing the correct results. Unless the culture is 

 known to he nonpathogenic, discard all cover slips and slides into a 

 disinfectant solution or sterilize by steam before washing. Use young 

 cultures (12-18 hours) except as noted. Accept the results of hang- 

 ing drop or wet-mount preparations under coverslips only if observa- 



