98 MICROSCOPIC METHODS. 



2. Kit fine's Methylene-blue. 



Methylene-blue, . . . 1.5 gr. 



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 gram of thionin-blue dissolved in loo 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, (b] Make a saturated solution of 

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

 added to 10 parts of (#), 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. 



5. Carbol-Fuchsin (see p. 102). This is a very powerful stain, and, 

 when used in the undiluted condition, ^-i minute's staining is usually 

 sufficient. It is better, however, to dilute with three or four times its 

 volume of water and stain for a few minutes. Methylated spirit with 

 or without a few drops of acetic acid is the most convenient decoloris- 

 ing agent. Then dehydrate thoroughly, clear, and mount. 



Various other staining combinations might be given, but 

 the above are the best and most widely used. If the 

 reader has thoroughly grasped the remarks made above 

 on the general principles which underlie the staining 

 of bacteria, he will be able to use any combination to 

 which his attention may be directed. We may only add 



