GRAM'S METHOD AND ITS MODIFICATIONS 103 



4. Gentian-violet in Aniline Oil Water. — Two solutions have here to 

 be made up. (a) Aniline oil water. Add about 5 c.e. aniline oil to 

 100 c.c. distilled water in a flask, and shake violently till as much as 

 possible of the oil has dissolved. Filter and keep in a covered bottle 

 to prevent access of light, (b) Make a saturated solutiou of gentian- 

 violet in alcohol. When the stain is to be used, 1 part of (b) is added 

 to 10 parts of {a), and the mixture filtered. The mixture should be made 

 not more than twenty-four hours before use. Stain sections for a few 

 minutes ; then decolorise with methylated spirit. Sometimes it is 

 advantageous to add to the methylated spirit a little hydrochloric acid 

 (2-3 drops to 100 c.c). This staining solution is not so much used by 

 itself as in Gram's method, which is presently to be described. 



5. Carbol- Gentian-violet. — 1 part of saturated alcoholic solution of 

 gentian- violet, is mixed with 10 parts of 5 per cent, solution of carbolic 

 acid, the mixture being well shaken. It is used as No. 4. 



6. Carbol-Fuchsin (see p. 105). — This is a very powerful stain, and, 

 when used in the undiluted condition, £-1 minute's staining is usually 

 sufficient. It is better, however, to dilute with from five to ten times 

 its volume of water and stain for a few minutes. In this form it has a 

 very wide application. Methylated spirit with or without a few drops 

 of acetic acid is the most convenient decolorising agent. Then dehydrate 

 thoroughly, clear, and mount. 



Gram's Method and its Modifications. — In the methods 

 already described, the tissues, and more especially the nuclei, 

 retain some stain when decolorisation has reached the point to 

 which it can safely go without the bacteria themselves being 

 affected. In the method of Gram, now to be detailed, this does 

 not occur, for the stain can here be removed completely from 

 the ordinary tissues, and left only in the bacteria. All kinds 

 of bacteria, however, do not retain the stain in this method, 

 and therefore in the systematic description of any species it is 

 customary, to state whether it is, or is not, stained by Gram's 

 method — in other words, whether it is Gram-positive or Gram- 

 negative. It must also be mentioned that some tissue elements 

 may retain the stain as firmly as any bacteria, e.g., keratinised 

 epithelium, calcified particles, the granules of mast cells, and 

 sometimes altered red blood corpuscles, etc. 



In Gram's method the essential feature is the treating of the 

 tissue, after staining, with a solution of iodine. This solution 

 is spoken of as Gram's solution, and has the following com- 

 position : — 



Iodine ... .1 part. 



.Potassium iodide . 2 parts. 



Distilled water . . 300 „ 



The following is the method : — 



1. Stain in aniline oil gentian-violet or in carbol-gentian-violet [vide 

 supra) for about five minutes. 



