GRAM'S METHOD OF STAINING 25 



stain. Some bacteria retain the stain, whilst others do not, and 

 this fact is of great value in diagnosis. The diphtheria bacillus, 

 for instance, stains when treated in the way we shall describe, 

 and if an organism which presents the character of that bacillus 

 does not stain by the process it must be of some other species. 

 We shall append a table of the most important pathogenic 

 bacteria which stain and which do not stain in this way. 



Requisites. i. Aniline gentian violet, or carbol gentian 

 violet (see p. 30). 



2. Gram's iodine solution (see p. 31). 



3. Absolute alcohol or methylated spirit. 



Process. i. Spread, dry, and fix the film in the way de- 

 scribed above. Stain for two or three minutes in the aniline 

 gentian violet or carbol gentian violet. The process may be 

 hastened by gentle heat. 



2. Rinse in water to remove excess of stain and flood with 

 the iodine solution, and allow the latter to act for a minute. 

 It is an advantage to replace it by a second lot, but this is not 

 absolutely necessary. 



3. Wash off the iodine solution with alcohol, and continue 

 the application of the latter until no more colour comes away. 

 It is best to pour a little alcohol on the slide and rock the latter 

 from side to side for a minute or so, then to pour off the 

 alcohol and add a fresh supply, and continue this until the 

 alcohol comes off colourless.* 



The Gram staining proper is then finished, but if the organ- 

 isms present do not stain by the method the result will be that 

 everything is decolorized, and microscopic examination will 

 reveal nothing. This, of course, might mean that the organ- 

 ism did not stain by Gram, but it might also be due to some 

 error in technique, by which no organisms were put on the 

 slide to begin with or got washed off at a subsequent stage. 

 To exclude this possibility proceed to counterstain with some 

 stain very different in colour from the gentian violet. Prob- 

 ably the most convenient counterstain for Gram is J per cent, 

 solution of neutral red in water; stain for half a minute. 

 Dilute carbol fuchsin has the advantage of being always at 



* Some books contain the absurd statement that the decolorization is to be 

 carried out until the film is a pale blue or violet colour. No such rule can 

 be given. If there is nothing that retains Gram the film will be colourless 

 when finished ; if it is a thick smear of an organism retaining the stain it may 

 be a very deep violet. 



