FIXING, IMBEDDING, AND STAINING 53 



the fungi. Carbol fuchsin is also largely employed in the staining 

 of bacteria. The alkaline stain most widely employed is perhaps 

 Loeffler's alkaline methylene blue. This solution consists of: 



Alcoholic solution methylene blue 30 cc. 



1 per cent solution potassium hydrate .... 1 cc. 

 Distilled water . 100 cc. 



Other excellent stains are Ehrlich's anilin-water fuchsin and gentian 

 violet. These are made by adding to 10 cc. of distilled water an 

 excess of anilin water ; this being shaken until no more will dis- 

 solve, and then filtered. To such solutions are then added 1 cc. 

 of the saturated solution of gentian violet or of fuchsin. 



In order to stain effectively the flagella of bacteria rather com- 

 plex methods are necessary. No method is satisfactory unless 

 every precaution is taken to have (1) the cover slips thoroughly 

 clean ; (2) the organism from a young (twelve to twenty hours), 

 vigorous culture, on a suitable medium ; and (3) the bacteria 

 evenly and thinly disposed upon the slip. Where experience has 

 not taught one to what extent to dilute a loop of bacteria for the 

 best staining effects, it is well to arrange the covers in series of 

 from four to five. Place a minute drop of water upon each slip, 

 then diffuse the bacteria from the culture in the first drop, and 

 with a loop from the first drop pass to the second, third, etc., in 

 turn, first diffusing the contents of the loop, then sweeping the 

 needle across the cover and passing to the next. These are dried 

 and fixed to the slip as previously indicated. 



There are several important methods of staining flagella. Loeffler's 

 method, or some modification of it, is frequently employed. This, 

 like most flagella methods, involves two chief operations, viz., mor- 

 danting and staining. The mordant consists of : 



20 per cent tannic acid solution . 1° cc. 



Saturated solution of ferrous sulfate 5 cc - 



Saturated solution of fuchsin, aq. or alcoholic . 1 cc. 



This mordant may be generally used as above, or it may be necessary 

 to add an acid (in the case of certain alkali-producing organisms) 

 or an alkali (certain acid-producing organisms), according to Loeffler, 

 this being done by adding to the mordant a fractional percentage 

 of weak stock solutions of a caustic alkali and an acid. 



