SPORE STAINING 129 



causes the penetration of the stain, and then cautiously 

 decolorising, it is possible to remove the colour from 

 everything 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 on 

 a slide which 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 colourless 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 six, 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 Moeller should be 

 made use of, and rarely fails. 



(b) Moeller' $ method. Prepare the jslide specimen in the 

 ordinary way. Treat with absolute alcohol for two minutes, and 

 then with chloroform for two minutes. Wash in water and treat 

 with a 5 per cent, solution of chromic acid for two minutes, wash, 

 and then stain with warm carbol-fuchsin for ten minutes. Wash, 

 decolorise carefully in 1 per cent, sulphuric acid, again wash and 

 counter-stain with Loffier's methylene blue for one minute ; 

 wash, dry, and mount. Some organisms, such as the B. mesen- 

 tericus, stain better if treated with the chromic acid for five to ten 

 minutes. 



M.B. 9 



