GRAM'S STAINING METHOD 39 



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. As 

 a rule better preparations are obtained with dilute stains than with more concen- 

 trated ones. 



Loffler's Alkaline Methylene Blue. Saturated alcoholic solution 

 of methylene blue, 30 c.c.; i to 10,000 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 bacteri- 

 ological technic and the one so rarely giving satisfactory results to the 

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

 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 char- 

 acteristically. 



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 appear- 

 ance. 



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, i part; 5% aqueous solution of carbolic acid, 10 parts.) This tends to over- 

 stain. 



The formula for aniline gentian violet is i part of saturated alcoholic solution 

 gentian violet and 3 parts of aniline oil water (made by adding 2 c.c. aniline oil to 

 100 c.c. distilled water, shaking violently for three to five minutes and then filtering 

 several times to get rid of the objectionable oil droplets which, in a Gram-stained 

 preparation, show as confusing black dots). 



The following stock solutions of Weigert are recommended: 



No. i No. 2. 



Gentian violet. ...... 2 grams. Gentian violet. ... 2 grams. 



Aniline oil 9 c.c. Distilled water 100 c.c. 



Alcohol (95%) 33 c.c. 



These stock solutions keep indefinitely. Mix i c.c. of No. i with 9 c.c. of No. 2. 

 Filter. This keeps about two weeks and is the solution to pour on the preparation. 

 It may be kept on from two to five minutes. Some hasten the staining by steaming 



