MICROSCOPIC STUDY AND STAINING 99 



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 percent carbolic, 100 c.c.; concentrated lactic acid, 0.5 

 c.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 3p seconds. Wash in water, dry, and 

 examine. 



BUERGER'S METHOD. 1 Cover-slip preparations are made by smear- 

 ing in serum as in Hiss' method. 



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

 (without acetic acid) and warm in flame for three seconds. 



(Zenker's 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. 



WADS WORTH'S METHOD. 2 Wadsworth has devised a method of 

 staining capsules which depends upon the fixation of smears with forma- 

 lin. After such fixation capsules may be demonstrated both with simple 

 stains and by Gram's method. The technique is as follows: 



Smear preparations, made as usual, are treated as follows: 



1. Formalin, 40 per cent, two to five minutes. 



2. Wash in water, five seconds. 



SIMPLE STAIN. DIFFERENTIAL STAIN (Gram's Method). 



3. Ten per cent aqueous gentian-violet. 3. Anilin gentian-violet, two minutes. 



4. Wash water, five seconds. 4. Iodin solution, two minutes. 



5. Dry, mount in balsam. 5. Alcohol, 95 per cent, decolorize. 



6. Fuchsin, dilute aqueous solution. 



7. Wash water, two seconds. 



8. Dry, mount in balsam. 



i Buerger, Med. News, Dec., 1904 > Wadsworth, Jour. Inf. Dis., 1906. 



