MICROSCOPIC STUDY AND STAINING 99 



substance is added in excess to distilled water and allowed to dissolve 

 to its full extent. The solution is then filtered and diluted to twice its 

 volume. 



Cover-glass preparations are made by spreading the bacteria on a 

 cover-slip in a drop of animal serum as in preceding method. They are 

 allowed to dry in the air and fixed by heat as usual. The dye is then 

 poured upon the preparation and allowed to remain for a few seconds. 

 It is then washed off with a twenty-five-hundredth per cent solution 

 of potassium carbonate in water, and studied in this solution. The 

 cover-shp inverted on a slide may be rimmed with vaseline to prevent 

 evaporation. 



Buerger's Method.' — 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. 



Wadsworth's Method.^ — 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, moimt in balsam. 5. Alcohol, 95 per cent, decolorize. 



6. Fuchsin, dilute aqueous solution. 



7. Wash water, two seconds. 



8. Dry, mount in balsam. 



1 Buerger, Med. News, Dec, 1904 ' Wadsworth, Jour. Inf. Dis., 1906. 



