BACTERIOLOGICAL TECHNIC t ;y 



2. Carbolic Acid Solution 



Carbolic Acid, 20 cc. 



Distilled Water, I0 o cc. 



Filter. This mordant is rarely used by itself. 



C. Stains. We give here the more important stains, approximately in 

 the order of preferred use. 



i. Loeffler's Methylene Blue 



Stock Solution (saturated) Methylene Blue, 30 cc. 



1:10,000 Sol. KHO in Dist. Water, 100 cc. 



Mix, shake filter. This stain is much used as a general bacterial stain 

 and in the examination of blood, pus, etc. 



2. Aniline Gentian-Violet 



Aniline Water, 75 cc. 



Stock Solution Gentian-Violet, 25 cc. 



Mix, shake, filter. This is an excellent bacterial stain. 



3. Carbol-Fuchsin 



Stock Solution of Basic Fuchsin, 10 cc. 



5 per cent. Sol. Carbolic Acid, 100 cc. 



Mix, shake, filter. This is one of the most useful stains with the 

 so-called acid-proof microbes. It is also a spore stain, and is the most 

 commonly employed stain used in contrast or double staining. It is a 

 comparatively slow stain, but is quite permanent. 



4. Gram's Stain 



Gram's stain is used for diagnostic purposes, and is perhaps the best 

 known stain in the entire field of bacteriological technic. Its value de- 

 pends upon the fact that certain microbes, when stained and afterward 

 treated with a solution of iodine and washed in alcohol, give up the stain. 

 Such microbes are known as Gram-negative, whereas those which do not 

 give up the stain are said to be Gram-positive. 



The method of using this stain is somewhat complicated, requires care, 

 and, with a beginner, often yields disappointing results. Keeping in mind 

 the following will minimize the disappointments : 



a. Long-continued (one year or more) subcultures frequently lose the 

 Gram-stain behavior. 



b. Old cultures, that is, those which have been growing in the same 

 medium for several days or more, as a rule do not stain characteristically. 

 With such cultures the results are often neither negative nor positive, just 

 enough to be confusing and perplexing. 



c. The solutions used must be fresh. The gentian-aniline solution, as 

 well as the iodine solution, deteriorates quite rapidly. 



