SPORE STAINING 113 



twenty-four hours. Rinse well in 1 per cent, acetic acid, pass 

 through alcohol and xylol, and mount in balsam. 



Spore Staining 



When spore- bearing bacteria are stained by the ordinary 

 methods the spores are just tinted, or remain uncoloured 

 with the outlines more or less stained. This seems to be 

 due to the fact that the spores are surrounded with a 

 slightly permeable membrane which inhibits the entrance 

 of the staining agent. By staining by some method which 

 causes the penetration of the stain, and then cautiously 

 decolorising, it is possible to remove the colour from every- 

 thing except the spores, the impermeable membrane of 

 which in the same way prevents the full action of the 

 decolorising agent. 



(a) Simple method. A film is prepared in the ordinary way. If 

 a cover-glass, it is floated on a watch-glass, or, if a slide, it is flooded 

 with carbol-fuchsin, and the stain is warmed for twenty minutes. 

 After being washed in water the preparation is rinsed for a second 

 or two in 1 per cent, sulphuric acid and again washed at once in 

 water. If there is still a good deal of the red colour remaining, 

 the film may be once more rinsed in the acid, but if nearly colour- 

 less it should be mounted in water and examined with the -in. 

 objective. If the spores alone are well stained the preparation 

 may be counter-stained with Loffler's methylene blue for two to 

 five minutes, washed, dried, and mounted. If, however, the 

 bacilli as well as the spores retain the red colour, the preparation 

 must be further decolorised in the acid, while if everything has 

 been decolorised, it may be re-stained with warm carbol-fuchsin. 



The spores sometimes stain better if the preparation be fixed 

 by passing through the flame twelve times instead of three, as is 

 usual. To obtain good preparations and ones showing the spores 

 in situ, the specimens should be made as soon as spores have 

 definitely developed in the cultures. 



Spore staining often requires a good deal of patience, and in 

 many instances it is difficult to obtain a satisfactory preparation 

 by this simple method, in which case that of MoeJler should be 

 made use of, and rarely fails. 



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