MICROSCOPIC STUDY AND STAINING 117 



are made by smearing the organisms in a drop of animal serum, preferably 

 beef-blood serum. 



Dry in air and fix by heat. 



Stain for a few seconds with 



Saturated alcoholic solution of fuchsin or gentian-violet 5 c.c., in distilled 

 water 95 c.c. 



The cover-slip is flooded with the dye and the preparation held for a second 

 over a free flame until it steams. 



Wash off dye with twenty per cent aqueous copper sulphate solution. 



Blot (do not wash). 



Dry and mount. 



By this method permanent preparations are obtained, the capsule appear- 

 ing as a faint blue halo around a dark purple cell body. 



HUNTOON'S CAPSULE STAIN (applicable only to cultures, not to animal 

 exudates). This depends upon the precipitating action of lactic acid on 

 nutrose. Requires two solutions. 



1. Diluent. 3 per cent solution of nutrose in distilled water; place in Arnold 

 one hour, add a small amount of carbolic as preservative, and allow to settle. 



2. Stain and fixative. 2 per cent carbolic, 100 c.c.; concentrated lactic acid, 

 0.5 x;.c. ; 1 per cent acetic acid, 1 c.c. ; saturated alcoholic solution basic fuchsin, 

 1 c.c.; carbol fuchsin, 1 c.c. 



As to the dye employed, most anything but methylene blue or Bismarck brown 

 may be used. Methyl violet gives the most beautiful results but is not permanent 

 and will not photograph. I have found the above mixture the best for classroom 

 work. 



Make a thin film, employing solution 1 as diluent. Dry in air. Do not 

 fix. Cover with stain 30 seconds. Wash in water, dry, and examine. 



BUERGER'S METHOD. Cover-slip preparations are made by smearing in 

 serum as in Hiss' method. 



As the edges of the smear begin to dry, pour over it Zenker's fluid (with- 

 out acetic acid) and warm in flame for three seconds. 



(Zenkers fluid is composed of potassium bichromate 2.5 gm., sodium 

 sulphate 1 gm., water 100 c.c., saturated with bichloride of mercury.) 



Wash in water. 



Flush with ninety-five per cent alcohol. 



Cover with tincture of iodin, U. S. P., one to three minutes. 



Wash with ninety-five per cent alcohol. 



Dry in the air. 



Stain with anilin water gentian-violet two to five seconds. 



Wash with two per cent salt solution. 



Mount and examine in salt solution. 



Buerger, Med. News, Doc., 1904. 



