130 MANUAL OF MICROBIOLOGICAL METHODS 



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

 either 95 per cent ethyl alcohol {'preferred) or 25 parts of acetone and 75 

 parts of ethyl alcohol. The use of greater amounts of acetone must be 

 avoided because of the ease with which anaerobes are decolorized. 

 Decolorization of young cultures of Clostridia is relatively common even 

 when alcohol is used with caution. The presence of numerous gram- 

 negative cells does not rule out Clostridia. The usefulness of the gram 

 method is limited in smears prepared from blood, fibrin, or albumin. In 

 samples of pathologic material, large, gram-positive rods are likely to 

 prove to be anaerobic bacilli, but a final diagnosis must not be based on 

 microscopic 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. tetani culture should be remembered when such forms are encountered 

 in pathologic material. All anaerobic species are non-acid-fast; there- 

 fore, 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- to 18-hr culture and observe the same cau- 

 tion 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 sporeforming anaerobic 

 bacilli are motile ; the most important exception is Clostridium perfringens 

 (C. welchii) . 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 cul- 

 tures (12-18 hr) except as noted. Accept the results of hanging drop or 

 wet-mount preparations under cover slips only if observation reveals posi- 

 tive motility. If motility is doubtful or appears to be negative, initiate 

 other procedures. For example, use a flattened 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 



