STAINING AND EXAMINATION OF MICRO-ORGANISMS 53 



this purpose it is necessary to use a stronger dye and to 

 employ heat to assist its power of penetrating the spore. 

 Ehrlich's fuchsine solution (see p. 45) is best adapted 

 for this purpose, and after the cover-glass has been treated 

 in the usual way with the stain, it is held over a small 

 flame, care being taken to move it backwards and for- 

 wards the whole time. The cover-glass must be removed 

 directly it begins to steam, and on no account must the 

 liquid be allowed to boil, as this will spoil the prepara- 

 tion. The cover-glass is then washed in the ordinary 

 way and is ready for examination. The spores will be 

 found to have now assumed a strong red colour, and by 

 the greater intensity of their tint may be distinguished 

 from the bacilli. This, however, does not exhibit the 

 spores so perfectly as when the bacilli are stained a 

 different colour. For this purpose recourse is had to a 

 method of double-staining, as described below. 



Double-staining. The cover-glass stained as above 

 is treated with a decolorising agent, being washed with 

 a 5 per cent, solution of acetic acid until experience 

 indicates that the bacilli will have lost all their colour, 

 whilst the spores, which both take up and part with 

 the dye less readily, will remain tinted, although 

 less intensely than before. The preparation is then 

 thoroughly washed with water and treated with the 

 ordinary aqueous solution of methylene blue. The 

 spores, which are not affected by the latter aqueous 

 stain, will still remain red whilst the bacilli have 

 assumed the blue colour. 



The above process may be modified in detail as re- 

 commended by Gimther, thus : instead of heating the 

 rover-glass preparation in the flame, it is placed prepara- 

 tion-side do wn wards in a watchglass filled close to the 

 brim with the freshly prepared Ehrlich's solution. The 

 .solution with the cover-glass is now gently heated over 



