104 MICROSCOPIC METHODS 



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 over-stained films and sections. More of the acid may, of 

 course, be added if necessary. 



Hot water also decolorises to a certain extent ; over-stained 

 films can often 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 lithium carbonate added. 



Different organisms take up and retain the stains with various 

 degrees of intensity, and thus duration of staining and decoloris- 

 ing must be modified accordingly. 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 (2J per cent, in water). 



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



1. Lojfler's Methylene-blue. 



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

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



(This dilute solution maybe conveniently made by adding 1 c.c. of a 

 1 per cent, solution to 99 c.c. of water. ) 



Sections may be stained in this mixture for from a quarter of an hour 

 to several hours. They do not readily overstain. The tissue containing 

 the bacteria is then decolorised if necessary with ^-1 per cent, acetic acid, 

 till it is a pale blue-green. The section is washed in water, rapidly 

 dehydrated with alcohol or aniline oil, cleared in xylol, and mounted. 



The tissue may be contrast-stained with eosin. If this is desired, 

 after decolorisation wash with water, place for a few seconds in 1 per 

 cent, solution of eosin in absolute alcohol, rapidly complete dehydration 

 with pure absolute alcohol, and proceed as before. 



