FORMULA OF STAINS. 109 



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

 hour to several hours. They do not readily overstain. The tissue con- 

 taining the bacteria is then decolorised if necessary with ^-i 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 I 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 decolorisation, as 

 the tissue elements are not overstained. 



2. Kiihne's Methylene-blue. 



Methylene-blue . . 1.5 g r - 



Absolute alcohol . . . 10 c.c. 



Carbolic acid solution (I -20) . . 100 c.c. 



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 

 i gramme of thionin-blue dissolved in 100 c.c. carbolic acid solution 

 (1-40). For use, dilute I 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 is quite sufficient. Wash again 

 thoroughly with water. Dehydrate with absolute alcohol or aniline oil. 

 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. 



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 to prevent access of light. (6) Make a saturated solution of 

 gentian-violet in alcohol. When the stain is to be used, I part of (6) is 

 added to 10 parts of (a), and the mixture filtered. The mixture should 

 be made not more than twenty-four hours before use. Stain sections for 

 a few minutes ; then decolorise with methylated spirit. Sometimes it 

 is advantageous to add to the methylated spirit a little hydrochloric 

 acid (2-3 minims to 100 c.c.). This staining solution is not so much 

 used by itself, as in Gram's method, which is presently to be described. 

 Instead of aniline oil water, carbolic acid solution (1-20) may be used 

 in the same way. 



