MORDANTS AND DECOLORISING AGENTS 105 



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 Formulas of some of the more commonly used Stain Combinations. 



1. Loffl.crs Mclhylcne-blue. 



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

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



(This dilute solution may be 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. 



Films may be stained with Loffler's blue by five minutes' exposure or 

 longer in the cold. They usually do not require decolorisatiou, as the 

 tissue elements are not overstained. 



2. Kuhne's Methylene-blue. 



Methylene-blue . . . . 1*5 grm. 

 Absolute alcohol . . . . 10 c.c. 

 Carbolic acid solution (1-20) . . 100 ,, 



