DIFFERENTIAL STAINING OF FILMS 207 



without passing it through the flame cover it with a 5 per cent, solution of 

 acetic acid, and leave for 30 seconds. 



2. Expose to the vapour of ammonia for a few seconds. 



3. Wash in water. 



4. Stain, wash, dry and mount. 



(/?) Vincent's method. 1. Dry the film by gently heating it, and, without 

 fixing, flood the film with the following solution : 



5 per cent, aqueous solution of carbolic acid, 6 c.c. 



Saturated aqueous solution of common salt. 30 ,, 



Glycerin (pure), - 30 



and allow it to act for 1-2 minutes. 



2. Wash in water, stain, etc. 



(7) Direct staining of blood-films. Lastly, simple staining with Losffler's 

 blue gives very good results with blood-films ; the red cells are sharply 

 differentiated from the micro-organisms, the former being stained pale 

 green and the latter deep blue. Carbol-thionin is also useful in that it 

 stains the nuclei of the leucocytes and the organisms but leaves the red cells 

 practically unstained. 1 



(b) Differential staining. 



In dealing with micro-organisms which retain the stain by Gram's method 

 it is easy to get a double-stained preparation. But when the organism 

 under investigation does not stain by this method more delicate processes 

 which often give less satisfactory results have to be employed. Finally, in 

 the search for and in the study of certain organisms, such for example as the 

 tubercle and leprosy bacilli, special methods, of which Ehrlich's is a type, 

 have to be adopted. They will be described in the chapter devoted to the 

 tubercle bacillus. 



A. Gram's method and its modifications. 



The procedure originally described by Gram has undergone various modifi- 

 cations : reference will be made to the more important of these. Meanwhile 

 the beginner must be warned against the danger of practising a large number 

 of methods. The secret of success lies in the thorough understanding of one 

 reliable procedure ; if this advice be neglected the result may be error and 

 failure and consequent discouragement. The method described under (/3) is 

 the one recommended. 



(a) Gram's method. 1. Flood the slide or cover-glass with aniline- 

 gentian-violet (p. 139). Let the stain act for 2-4 minutes. 



2. Pour off the stain and, without washing, flood the film with Gram's 

 iodine solution. Let it act for about a minute until the preparation assumes 

 a blackish tint. 



3. Wash in distilled water. 



4. Decolourize with absolute alcohol (p. 143) until the film assumes a pale 

 grey tint. 



5. Wash in distilled water. 



6. Flood the film with a solution of eosin : 



Water-soluble eosin, - 1 gram. 



Distilled water, - - 200 c.c. 



Allow the eosin to remain on for 1-2 minutes. 



7. Wash in distilled water and dry. 



8. If the preparation has been made on a slide, a drop of cedar-wood oil 



1 In the case of birds' blood, the nuclei of the red cells are deeply stained by these 

 dyes. 



