SIMPLE STAINING 217 



3. Treat for a few seconds with dilute hydrochloric acid (1-1000). 



4. Transfer rapidly to lithia solution (p. 210). 



5. Wash again carefully in distilled water. Blot up the excess of water 

 and leave the section exposed to the air until it is nearly dry. 



6. Dehydrate as rapidly as possible in absolute alcohol. 



7. Clear in clove oil and xylol. 



8. Mount in Canada balsam. 



(5) Kuhne's method B. This method is not recommended. It is very 

 tedious and only stains a few species of micro-organisms. 



1. Stain for about 30 minutes in carbol-blue. 



2. Wash in distilled water. 



3. Treat with dilute hydrochloric acid (1-1000) until the tissue is pale blue. 



4. Wash in lithia solution (p. 210). 



5. Wash for several minutes in distilled water and blot up the excess. 



6. Dehydrate very rapidly in absolute alcohol lightly tinted with methylene blue. 



7. Pour off the alcohol and treat with aniline oil similarly tinted with blue for 

 about 2 minutes. 



8. Replace the tinted aniline oil with ordinary aniline oil for about 2 minutes. 



9. Clear with clove oil and then with two lots of xylol to ensure the removal of 

 all traces of aniline oil. 



10. Mount in Canada balsam. 



(e) Staining with thionin. Method recommended. 1. Stain with carbol- 



thionin (p. 138) for several minutes. 



2. Wash in distilled water and blot up the excess. 



3. Dehydrate very rapidly in absolute alcohol. 



4. Clear in clove oil and xylol. 



5. Mount in Canada balsam. 



(0 Gram's method for the typhoid bacillus. 1. Stain for a few hours in 

 aniline-gentian-violet (p. 139). 



2. Wash the section in distilled water. 



3. Transfer for 1 minute to a 1 per cent, solution of hydrochloric acid. 



4. Wash carefully in distilled water : blot up the excess. 



5. Dehydrate very rapidly in absolute alcohol. 



6. Clear in clove oil and xylol. 



7. Mount in Canada balsam. 



By this method the bacilli alone are stained. 



(rj) Nicolle's tannin method. Method recommended. 1. Stain the section 

 for 2 or 3 minutes in Lceffler's or Kuhne's blue. 



2. Wash in distilled water. 



3. Treat for a few seconds in a 10 per cent, aqueous solution of tannin. 



4. Wash in distilled water and blot up the excess. 



5. Dehydrate rapidly in absolute alcohol. 



6. Clear in clove oil and xylol. 



7. Mount in Canada balsam. 



B. Differential staining. 

 1. Methods applicable to gram-positive organisms. 



To demonstrate the presence of gram-positive organisms in a tissue in 

 which they are present the background (the animal tissue) is first stained with 

 an acid dye which has but little affinity for micro-organisms, then by Gram's 

 method. The bacteria being the only structures stained violet stand out 

 sharply from the other tissues. 



The background may be stained with one of several dyes. 



For double staining, eosin, fluorescein, carmine (Orth's), vesuvin, Boehmer's 

 hsematoxylin, aurantia, hsematein, etc., are used. 



