GRAM'S STAIN 143 



carbolic solution and treated afterwards with a special mordant containing 

 iodine, are not decolourized by absolute alcohol and similar decolourizing 

 agents. The anthrax bacillus is an example of this group. 



On the other hand, other bacteria when treated in the same way are readily 

 decolourized with absolute alcohol, e.g. the typhoid bacillus. 



Bacteria then are classified with reference to these reactions into two 

 groups, termed gram-positive (organisms which retain the stain) and gram- 

 negative (organisms which are decolourized). The anthrax bacillus is said 

 to be gram-positive, and the typhoid bacillus gram-negative. 



The mordant has the following composition : 



Gram's (or Lugol's) solution. 



Iodine, - 1 gram. 



Potassium iodide, - 2 grams. 



Distilled water, - 300 c.c. 



In the original method absolute alcohol was used as the decolourizing agent. 

 But pure aniline oil (Weigert) or acetone alcohol (Nicolle) are now sometimes 

 used in its place. 



Acetone alcohol. 



Absolute alcohol, - 5 parts. 



Acetone, - 1 part. 



According to Nicolle, a bromine- bromide, iodine- bromide, or bromine- iodide 

 solution may any of them be used in place of Gram's solution. They are all 

 prepared in the same proportions as Gram's iodine-iodide solution. 



Gram's stain has undergone many modifications, and is used as a double 

 stain for films, sections, etc. These modifications will be dealt with in a 

 special chapter and for the present the use of this classical method as a means 

 of diagnosis will alone be considered. 



Technique. 1. Prepare a film on a slide or cover-glass. 



2. Stain for 30 to 60 seconds with carbol-gentian-violet. 



3. Blot up the excess of stain (but do not wash), drop two or three large 

 drops of Gram's solution on the film and let it act for 20 to 30 seconds. The 

 preparation will have now assumed a brown tint. 



4. Wash in water and dry. 



5. Pour absolute alcohol over the film a drop at a time until no more 

 violet stain comes away usually 20 to 30 seconds (Notes (a) and (b) infra). 



6. Wash in water quickly. 



7. Examine the film in water. If the organisms are gram-positive they 

 are stained deep violet, but if gram-negative decolourized : sometimes some 

 of the organisms will be decolourized while others are still stained violet ; 

 in that case a further washing in alcohol will complete the reaction. 



[Many bacteriologists prefer to counterstain the film. For this purpose, 

 after washing in water (Stage 6) the film is flooded with some weak staining 

 solution the colour of which is in sharp contrast with violet. Dilute carbol- 

 fuchsin (1-5 or 1-10) or bismarck brown (p. 136) is convenient ; the former 

 is allowed to act for about J minute, while bismarck brown requires rather 

 longer (2 minutes). Wash in water, blot and dry. Gram-positive organisms 

 are as in the former case stained violet, while gram-negative organisms being 

 decolourized by the alcohol take the counterstain and appear pink or 

 brown as the case may be.] 



To keep the cover-glass preparation, dry and mount in balsam. If the 

 film was made on a slide it merely requires to be dried. 



To sum up : Prepare and fix a film, stain, treat with iodine solution, wash> 

 dry, treat with alcohol, wash [counterstain, wash,] dry and examine. 



