FORMULA OF % 



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 |-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 1 exposure or 

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

 elements are not overstained. 



2. Kuhne^s Methylene-blue. 



Methylene-blue . . . . i-5 gr. 



Absolute alcohol . . . 10 c.c. 



Carbolic acid solution (1-20) . . 100 



Stain and decolorise as with LofHer'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. Thionin- 

 blue stains more deeply than methylene-blue, and gives equally good differ- 

 entiation. 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, i 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 exactly 2 c.c. aniline oil to 98 c.c. 

 distilled water in a flask, and shake violently till as much as possible of the 

 oil has dissolved. Filter twice through the same paper 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, 25 parts of (6) is added to 

 75 parts of (#), and the mixture filtered. This mixture will not readily de- 

 compose and may be used for several months if kept from the prolonged action 

 of light, although it may require occasional filtering to remove accumulated 

 crystalline deposit. 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 for tissues, as in Gram's method, which 

 is presently to be described, but makes an excellent stain for most bacterial 

 films. 



5. Carbol-gentian-violet. i part saturated alcoholic solution of gentian- 



