26 CLINICAL BACTERIOLOGY AND HEMATOLOGY 



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. 32). 



3. Absolute alcohol or methylated spirit. 



Process. — i. Spread, dry, and fix the film in the way described 

 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 colotir 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 organisms 

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

 thing is decolorized, and microscopic examination will reveal 

 nothing. This, of course, might mean that the organism 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 : dilute carbol fuchsin is best. Stain in 

 this for a quarter of a minute — not more, for the stain is a very 

 powerful one, and if allowed to act too long may displace the 

 stain from a Gram-staining organism : wash for a minute or so 



* 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. 



