102 PHARMACEUTICAL BACTERIOLOGY 



b. Stain 



Sat. Aq. Sol. Alum, 10 cc. 



Stock Sol. Gentian- Violet, 2 cc. 



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 gentle 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 destroyed. 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 

 difficulty, 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, however, be stained by the usual acid-fast or 

 acid-proof methods, care being observed in decolorizing. Stain with 

 hot carbol-fuchsin for a few minutes, wash, and decolorize quickly with 

 3 per cent, hydrochloric acid in 95 per cent, alcohol, and then use a con- 

 trast stain, as gentian- violet or methylene blue. The red spores will then 

 appear in the violet or blue frame. 



9. Capsule Staining 



The gelatinous capsule of microbes is also stained with great difficulty, 

 and requires special methods and experience to yield anything like satis- 

 factory results. The methods of Welch and Hiss are quite satisfactory. 

 The capsule is, however, generally visible without any staining because 

 of the light contrast that naturally exists. Certain substances, as glacial 

 acetic acid (Welch method), cause the capsule to enlarge and take up the 

 stain more readily. Certain staining methods bring out the capsule of 

 certain microbes, as, for example, the Gram method as applied to pneu- 

 monia sputum. 



