MICROSCOPIC METHODS 111 



Spore Stain. — The spores of yeasts and of bacteria are 

 not readily penetrated by stains, but when they are stained 

 they are somewhat more resistent to deeolorization than the 

 vegetative cells ; this.makes possible a staining method which 

 will show spores of one color and vegetative cells of another. 

 The material to be examined is spread and fixed in the 

 manner already described. It is then flooded with some 

 powerful stain, usually carbol fuchsin. The mount is then 

 heated until the carbol fuchsin steams for a period of five 

 minutes. Care must be used to replace stain with fresh 

 as rapidly as it evaporates so that the mount does not be- 

 come dry over any part. The excess stain is washed off 

 with water, then with dilute (usually 5 per cent) acetic 

 acid, until the mount is light pink in color. Usually a few 

 seconds will be sufficient. It is then thoroughly washed in 

 water to remove all traces of acid. This procedure leaves 

 the spores red but the vegetative rods colorless. The latter 

 are stained by adding some LoefHer's methylene blue. 

 When this has acted for a few seconds the mount is 

 washed, dried, and examined. The spores will ordinarily 

 appear red and vegetative cells blue. With the yeasts the 

 spores will appear red and the asci blue. 



Study of Acid Fast Bacteria. — A few species of bacteria, 

 some of them of considerable economic importance, such 

 as the organism causing tuberculosis, are said to be acid fast 

 or acid proof. Like bacterial spores they resist the pene- 

 tration of stains, but when once the stain has penetrated 

 they are not easily decolorized, in fact, they will withstand 

 the decolorizing action of strong acids or alcohol for a 

 considerable length of time. The method of procedure is 

 essentially the same as in staining spores, except that as a 

 decolorizing agent a strong solution of acid, usually sul- 

 phuric acid, is used. Other bacteria or materials present 

 may be counterstained by methylene blue. The acid fast 

 bacteria appear as red rods in a blue field. 



