126 MICROBIOLOGY 



for staining after the paraffin has been removed by benzine, 

 and the benzine removed by immersing in 95% alcohol. Bac- 

 teria are best shown, however, in tissues with fixation by ab- 

 solute alcohol or formalin. 



Staining with Loeffler's methylene blue. The hydrated 

 sections are placed in Loeffler 's methylene blue solution for 

 5 to 30 minutes, and then for a few seconds in a 1 per cent 

 solution of acetic acid.* They are then washed quickly in 

 water after which they are transferred to 95% alcohol and 

 then to absolute alcohol, cleared in xylol, and mounted in 

 Canada balsam. The number of seconds during which the 

 preparation remains in the acetic acid must be tested by trials; 

 The bacteria appear stained a dark blue, the nuclei of the 

 cells blue, and the cell bodies light blue. 



Thionin solution, carbol-fuchsin and gentian violet can 

 be used instead of Loeffler's methylene blue. Gram's method, 

 using a 3 per cent hydrochloric acid in alcohol as a tissue 

 decolorizer and allowing it to act for ten seconds, is valuable 

 in certain cases. 



Dyes used in staining bacteria. The cell body or proto- 

 plasm of bacteria reacts to stains very much as nuclear chro- 

 matin. There is, however, much variation in the intensity 

 with which they take the coloring matter. The best stains for 

 bacteria are the basic aniline dyes which are compounds de- 

 rived from the coal tar product aniline. The aniline dyes 

 which are used for staining purposes are divided into two 

 groups, according as the staining property depends upon the 

 basic or acid portion of the molecule. The former contain the 

 amido groups and are spoken of as nuclear stains. They color 

 the nuclear chromatin of both animal cells and bacteria. The 

 latter or acid portion contain the hydroxyl groups and do not 

 stain bacteria but are used chiefly for contrast coloring. They 

 are often mentioned in staining tissues as general stains. The 



* In place of acetic acid the following solution is thought by 

 some to be better. Distilled water 100 cc., cone, sulphuric acid 20 

 drops, oxalic acid 5% solution 10 drops. 



