METHODS OF OBSERVING BACTERIA. 101 



and boil it for at least fifteen minutes, after which it is 

 decolorized, either with 3 per cent, hydrochloric or 2-5 per 

 cent, acetic acid, washed in water, and counter-stained blue. 



Fiocca suggests the following rapid method: "About 

 20 c.cm. of a 10 per cent, solution of ammonium are 

 poured into a watch-glass, and 10-20 drops of a saturated 

 solution of gentian violet, fuchsin, methyl blue, or saf- 

 ranin added. The solution is warmed until vapor begins 

 to rise, then is ready for use. A very thinly-spread cover- 

 glass, carefully dried and fixed, is immersed for three to 

 five minutes (sometimes ten to twenty minutes), washed 

 in water, washed momentarily in a 20 per cent, solution 

 of nitric or sulphuric acid, washed again in water, then 

 counter-stained with a watery solution of vesuvin, chrys- 

 oidin, methyl blue, malachite green, or safranin, according 

 to the color of the preceding stain. This whole process 

 is said to take only from eight to ten minutes, and to give 

 remarkably clear and beautiful pictures." 



Method of Staining Flagella, This is much more 

 difficult than the staining of either the bacteria or their 

 spores, because each species seems to behave differently 

 in its relation to the stain, so that the chemistry of the 

 micro-organismal products must be taken into considera- 

 tion. 



The best method introduced is that of Loffler. In it 

 three solutions are used : 



A. A 20 per cent, solution of tannic acid, 10 ; 

 Cold saturated aqueous solution of ferrous sulphate, 5 ; 

 Alcoholic solution of fuchsin or methyl violet, i. 



B. A i per cent, solution of caustic soda. 



C. An aqueous solution of sulphuric acid of such strength 



that i c.cm. will exactly neutralize an equal quan- 

 tity of Solution B. 



Some of the bacteria to be stained are mixed upon a 

 cover-glass with a drop of distilled water. This is the 

 first dilution, but is too rich in bacteria to allow the 



