100 MICROSCOPIC METHODS. 



{b) By the addition of alkalies, such as caustic potash or 

 ammonium carbonate, in weak solution. 



(c) By the employment of heat. 



{d) By long duration of the staining process. 



As decolorising agents we use chiefly mineral acids (hydro- 

 chloric, nitric, sulphuric), vegetable acids (especially acetic acid), 

 alcohol (either methylated spirit or absolute alcohol), or a com- 

 bination of spirit and acid, e.g. methylated spirit with a drop or 

 two of hydrochloric acid added, also various oils, e.g. aniline, 

 clove, etc. In most cases about thirty drops of acetic acid in a 

 bowl of water will be sufficient to remove the excess of stain from 

 overstained films and sections. More of the acid may, of course, 

 be added if necessary. 



Hot water also decolorises to a certain extent ; overstained 

 films can be readily decolorised by placing a drop of water on 

 the film and heating gently over a flame. 



When preparations have been sufficiently decolorised by an 

 acid, they should be well washed in tap water, or in distilled 

 water with a little lithia carbonate added. 



The methods embracing the use of a stain with a mordant, 

 and a decoloriser, are very numerous, and we can only enumerate 

 the best of them. 



We sometimes have to deal with bacteria which show a 

 special tendency to be decolorised. This tendency can be 

 obviated by adding a little of the stain to the alcohol, or aniline 

 oil, employed in dehydration. In the latter case a little of the 

 stain is rubbed down in the oil. The mixture is allowed to 

 stand. After a little time a clear layer forms on the top with 

 stain in solution, and this can be drawn off with a pipette. 



When methylene-blue, methyl-violet, or gentian-violet is used 

 the stain can, after the proper degree of decolorisation has 

 been reached, be fixed in the tissues by treating for a minute 

 with ammonium molybdate (2^ per cent in water). 



The Formulae of some of the more commonly used Stain Combinations. 



I . Ldffler's Methylene-blue. 



Saturated solution of methylene-blue in alcohol .... 30 c.c. 



Solution of potassium hydrate in distilled water (1-10,000) . . 100 c.c. 



(This dilute solution may be conveniently made by adding i c.c. of a 

 I per cent solution to 99 c.c. of water.) 



