98 MICROSCOPIC METHODS 



of the stain to the alcohol, or aniline oil, employed in dehydra- 

 tion. 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. Ldfflers 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 Lbffler'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 gr. 

 Absolute alcohol . . . . 10 c.c. 

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



Stain and decolorise as with Loffler's blue, or decolorise with very 

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



3. Carbol-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 1 volume with 3 of water and filter. Stain sections for 

 five minutes or upwards. Wash very thoroughly with water, otherwise 

 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 thionin. 



4. Gentian-violet in Aniline Oil Water. Two solutions have here to 

 be made up. (a) Aniline oil water. Add about 5 c.c. aniline oil to 

 100 c.c. distilled water in a flask, and shake violently till as much as 

 possible of the oil has dissolved. Filter and keep in a covered bottle 



