BACTERIOLOGICAL TECHNIC. 75 



Mix. and filter. Let stand for twenty-four hours before using. Renew 

 in three weeks. This stain is much used to distinguish between bacteria 

 and nuclear substances. Allow the stain to act for from two to ten minutes. 



7. Flagella Staining. 



Care is necessary in staining flagellae. Numerous methods have been 

 recommended, but Pitfield's method, as modified by Muir, is perhaps the 

 best and at the same time comparatively simple. The following solutions 

 are required: 



a. Mordant. 



Tannic Acid (10 per cent. Aq. Sol.), 10 c.c. 



Sat. Aq. Sol. Mercuric Chlor., 5 c.c. 



Sat. Aq. Sol. Alum, S c.c. 



Carbol-Fuchsin, S '^■c- 



Mix, shake, filter or centrifuge. This solution does not keep longer 

 than one week. 



b. Slain. 



Sat. Aq. Sol. Alum, 10 c.c. 



Stock Sol. Gentian-Violet, 2 c.c. 



Mix, filter. Carbol-fuchsin may be used instead of gentian-violet. This 

 stain will not keep longer than a few days. 

 The method is as follows: 



1. Drop on mordant. Leave for one minute, with gentie heat. 



2. Rinse in water for two minutes. 



3. Dry carefully at slight warmth. 



4. Stain for one minute with gentle heat. 



5. Wash, dry, and mount in Canada balsam. 



In making the cover-glass preparation, take a loopful from a young 

 aqueous subculture of some motile bacillus and touch it on the carefully 

 cleaned cover and allow the drop to spread by rotating and tilting the cover. 

 Do not use the loop more than is necessary. Flagellae are very delicate and 

 easily destroy* d. Dry very carefully, and do not pass through flame more 

 than three times. 



8. Spore Staining. 



As already stated, spores (endospores) of microbes stain with great diffi- 

 culty, for which reason a contrast is effected negatively; that is, the rest of the 

 cell is quickly stained, leaving the unstained, highly refractive spore to appear 

 like a bit of glass within the colored frame. This is in many ways the most 

 satisfactory way of demonstrating the presence of spores. The spores may. 



