102 MICEOSCOPIC METHODS 



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. 



1. Loffier's Methylene-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 Loffier's blue by five minutes' exposure or 

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

 tissue elements are not overstained. 



2. Kiihne's Methylene-blue. 



Methylene-blue ... 1*5 gnu. 



Absolute alcohol . . . 10 c.c. 



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



Stain and decolorise as with Lomer's blue, or decolorise with very weak 

 hydrochloric acid (a few drops in a bowl of water). 



3. Oarbol- Thionin-blue. — Make up a stock solution consisting of 1 

 gramme of thionin-blue dissolved in 100 c.c. carbolic acid solution (1-40). 

 For use, dilute one volume with three of water, and filter. Stain sections 

 for five minutes or upwards. Wash very thoroughly with water, other- 

 wise a deposit of crystals may occur in the subsequent stages. Decolorise 

 with very weak acetic acid. A few drops of the acid added to a bowl 

 of water are quite sufficient. Wash again thoroughly with water. 

 Dehydrate with absolute alcohol. Thionin-blue stains more deeply 

 than methylene-blue, and gives equally good differentiation. It is very 

 suitable for staining typhoid and glanders bacilli in sections. Cover- 

 glass preparations stained by this method do not usually require 

 decolorisation. As a contrast stain, 1 per cent, watery solution of eosin 

 may be used before staining with the thiunin. 



