GRAM-STAINING METHOD 119 



less Gram-negative. Even strongly Gram-positive organ- 

 isms may be decolorised by prolonged treatment with 

 alcohol. Most of the moulds, filament forms, yeasts and 

 cocci, the acid-fast bacilli (tubercle, etc.) and sporing 

 bacilli are Gram -positive, while the vibrios, spiral forms, 

 protozoa and many non-sporing bacilli are Gram-negative 

 (see p. 121). 



The Gram -staining Method 



The original method was to stain in an anilin -gentian -violet 

 solution (alcoholic solution of gentian violet, 30 c.c. ; anilin 

 water, 100 c.c. Anilin water is prepared by vigorously shaking 

 2 c.c. of anilin with 100 c.c. of distilled water and filtering). Films 

 are stained for 3-5 minutes and sections for 10-15 minutes. 

 The stain is then drained off and the preparation rinsed with 

 Lugol's iodine solution (see below), and some of the iodine solu- 

 tion is allowed to remain in the preparation for 1-3 minutes, 

 after which it is treated with alcohol films for about half a 

 minute, sections until practically decolorised. The alcohol 

 (absolute or methylated spirit) may be renewed once or twice. 

 After decolorisation the preparation may be counter-stained with 

 weak fuchsin, neutral red, eosin, Bismarck brown, etc. Carbol- 

 gentian violet or carbol-thionine blue, crystal violet, etc., may 

 also be employed instead of anilin gentian violet. 



Owing to some degree of uncertainty in the results 

 obtained by the original method, the American Society of 

 Bacteriologists recommend one of the two following 

 methods for films of cultures : 



METHOD 1. 

 Gentian violet solution ..... 1 minute 



(This is prepared by grinding 5 grm. of 

 gentian violet with 10 c.c. of 95 per cent, 

 alcohol in a mortar. Add 2 c.c. of anilin oil 

 and 88 c.c. of water. Filter.) 

 Iodine solution (Lugol's). . . . . 1 minute 



(Iodine 1 grm., potassium iodide 2 grm., 

 water 300 c.c.) 



