32 STAINING METHODS. 



solution of methylene blue ; and if the operation has been successfully 

 carried out the spores will be stained red and the protoplasm of the 

 bacilli in which they are present will be blue. 



Moller has recently (1891) published the following new method 

 of staining spores : 



The cover-glass preparation, dried in the air, is passed three times 

 through a flame or placed for two minutes in absolute alcohol ; it is 

 then placed in chloroform for two minutes and washed in water ; it 

 is now immersed in a five-per-cent solution of chromic acid for from 

 half a minute to two minutes and again thoroughly washed in 

 water ; next a solution of carbol-fuchsin is poured upon it and it 

 is heated over a flame until it commences to boil, for sixty seconds ; 

 the carbol-fuchsin solution is then poured off and the cover glass is 

 immersed in a five-per-cent solution of sulphuric acid until the 

 film is decolorized, after which it is again thoroughly washed in 

 water. It is then placed for thirty seconds in an aqueous solution of 

 methylene blue or of malachite green, and again washed in water, 

 after which the preparation should be dried and mounted in balsam. 

 As a result of this procedure the spores are stained dark red and the 

 protoplasm of the bacilli blue or green. 



METHODS OF STAINING FLAGELLA. Koch first succeeded in de- 

 monstrating the flagella of certain bacilli and spirilla by staining them 

 with an aqueous solution of hsematoxylon, and dilute chromic acid 

 as a mordant. Recently Loffler (1889) has succeeded in demonstrat- 

 ing, by an improved staining method, the presence of flagella in a con- 

 siderable number of species in which they had not previously been seen, 

 although generally suspected to be present. His method is as follows : 



Loffler 's Method. The following solution is used as a mordant : 



No. 1. 



Solution of tannin of twenty per cent, . . . 10 cc. 



Saturated (cold) solution of ferrous sulphate, . . 5 cc. 



Aqueous or alcoholic solution of fuchsin, . . 1 cc. 



(Or one cubic centimetre alcoholic solution of methyl violet.) 



No. 2. 



A one per-eent solution of caustic soda. 



No. 3. 



A solution of sulphuric acid of such strength that one cubic centi- 

 metre is exactly neutralized by one cubic centimetre of the soda 

 solution. 



According to Loffler, solution No. 1 is just right for staining the 

 flagellum of Spirillum concentricum, but for certain other bacteria it 

 is necessary to add to this some of No. 2 or of No. 3. Thus, for the 

 cholera spirillum from half a drop to a drop of the acid solution is 



