MICROSCOPIC STUDY AND STAINING 101 



tian-violet made slightly alkaline by the addition of one-tenth per cent 

 sodium hydrate. 



The stain should be filtered directly upon the cover-slip. Warm 

 gently and leave on for one to two minutes. Wash in water. Mount in 

 balsam. 



VAN ERMENGEM'S METHOD. 1 This method requires the preparation 

 of three solutions. 



(1) Twenty per cent tannic acid solution 60 c.c. 



Two per cent osmic acid solution 30 c.c. 



Glacial acetic acid 4-5 drops. 



The cover-slip carrying the fixed preparation is placed in this solu- 

 tion for one hour at room temperature, or for five minutes at 100 C. 

 (boiling) . 



Wash in water. 



Wash in absolute alcohol. 



Immerse the cover-slip for one to three seconds in 



(2) Silver nitrate, 0.25-0.5 per cent solution. 

 Without washing, transfer to 



(3) Gallic acid 5 gm. 



Tannic acid 3 " 



Fused potassium acetate 10 " 



Distilled water 350 c.c. 



Immerse in this for a few minutes, moving the cover-slip about. 



Return to the silver nitrate solution until the preparation turns 

 black. 



Wash thoroughly in water. 



Blot and mount. 



SMITH'S MODIFICATION OF PITFIELD'S METHOD. 2 A saturated solu- 

 tion of bichloride of mercury is boiled and is poured while still hot into a 

 bottle in which crystals of ammonia alum have been placed in quantity 

 more than sufficient to saturate the fluid. The bottle is then shaken and 

 allowed to cool. Ten c.c. of this solution are added to 10 c.c. of freshly 

 prepared ten per cent tannic acid solution. To this add 5 c.c. carbol- 

 fuchsin solution. Mix and filter. 



To stain, filter the above mordant directly upon the fixed cover-slip 



1 Van Ermengem, Cent. f. Bakt., I, xv, 1894. 



2 Smith, Brit. Med. Jour., I, 1901, p. 205. 



