GRAM'S STAINING METHOD 33 



border of the cover-glass should be sealed with gold size or some other cement. 

 Some prefer to mount directly in water without preliminary drying. It is good 

 practice to make a rule to always keep the smeared side of the preparations up 

 never allowing it to be reversed. By this simple rule, preparations 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 margin of the glass, 

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

 glass tubing, as recommednded under Blood Smears, will be found useful. The dilute 

 carbol fuchsin and Lb'ffler'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. ; 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 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 

 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 gen- 

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

 overstain. 



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). 

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