28 STAINING METHODS. 



can be carried through the most complicated staining methods without 

 the necessity of scratching the cover-glass, etc., to see which is the film 

 side. In grasping a cover-glass with a Cornet or Stewart forceps, be 

 sure that the tips are well by the margins of the glass, otherwise the 

 stain will drain off. In staining with slides, the grease pencil and the 

 glass tubing, as recommended under Blood Smears, will be found 

 useful. The dilute carbol fuchsin and Loffler's methylene blue are 

 probably the best routine stains. 



Loffler's Alkaline Methylene Blue. Saturated alcoholic solution 

 of methylene blue, 30 c.c.; one to ten thousand caustic potash solution, 

 100 c.c. (Two drops of a 10% solution KOH in 100 c.c. of water 

 makes a i : 10,000 solution.) 



Carbol Fuchsin (Ziehl-Neelsen). Saturated alcoholic solution 

 basic fuchsin, 10 c.c.; 5% aqueous solution carbolic acid, 100 c.c. 



Gram's Method. The most important staining method in bac- 

 teriological techni: and the one so rarely giving satisfactory results to 

 the inexperienced is Gram's stain. In using this method, the lollowing 

 points must be kept in mind: 



1. Laboratory cultures (subcultures) which have been carried 

 over for years frequently lose their Gram characteristics. 



2. Cultures which are several days old or dead or degenerated do 

 not stain characteristically. 



3. The aniline gentian violet deteriorates when exposed to light 

 in two or three days it should be kept in the dark. It should have a 

 rich, creamy, violet appearance. 



4. The iodine solution deteriorates and becomes light in color. 

 It should be of a rich port-wine color. 



5. The decolorizing with 95% alcohol -should stop as soon as no 

 more violet stain streams out. This is best observed over a white 

 background, washing at intervals. Do not confuse stain on forceps 

 for that on preparation. 



6. The preparation should be thin and evenly spread. Some 

 prefer carbol gentian violet to aniline gentian violet. (Saturated 

 alcoholic solution of gentian violet, one part; 5% aqueous solution of 

 carbolic acid, ten parts.) This tends to overstain. The following 

 stock solutions of Weigert are recommended: 



