DIFFERENTIAL STAINING 219 



(i) Double staining. 



A. Method recommended. 1. Treat the section for about 30 seconds with 

 the dilute solution of eosin (p. 218) until it acquires a pink colour. 



2. Wash in distilled water. 



3. Stain the section on the slide for about 30 seconds with carbol-gentian- 

 violet or carbol-crystal- violet (p. 138). It now assumes a violet colour. 



4. Pour off the violet and treat the section with Gram's iodine for 30 

 seconds or so, renewing the solution two or three times until the section is 

 black. Wash in distilled water. 



5. Wash with absolute alcohol (or absolute alcohol and aniline oil) until 

 the pink colour of the ground-work reappears. 



6. Clear with clove oil and xylol. 



7. Mount in balsam. 



The background is stained pink and those organisms which retain the 

 stain by Gram's method are stained violet. 



B. Kiihne's method. 1. Stain the section for 5-15 minutes in Kiihne's 

 blue or ammoniacal blue. 



2. Wash in distilled water. 



3. Treat with Gram's solution for 2 or 3 minutes. 



4. Wash in distilled water. 



5. Decolourize in a saturated solution of fluorescein in absolute alcohol. 



6. Treat with pure absolute alcohol, clove oil and xylol. 



7. Mount in balsam. 



Bacteria are stained blue while the ground-work is faintly stained with 

 fluorescein. 



(ii) Triple staining. 



A. Method recommended. 1. Stain for about 5 minutes with Orth's 

 picrocarmine. 



2. Pour off the stain and fix in the fixing solution for about 30 seconds. 



3. Wash in distilled water. 



4. Stain with carbol-gentian-violet or carbol-crystal-violet for 30 seconds. 



5. Replace the stain with Gram's solution for 30 seconds. Wash in dis- 

 tilled water. 



6. Decolourize in absolute alcohol or absolute alcohol and aniline oil. 



7. Treat the section in turn with absolute alcohol slightly tinted with picric 

 acid, clove oil and xylol. 



8. Mount in balsam. 



B. Nicolle's method. This method is applicable to sections fixed on the 

 slide with Mayer's albumin. 



1. Stain with Orth's alcohol-carmine for 15 minutes. 



2. Wash in distilled water. 



3. Stain in carbol-gentian-violet (p. 138) for 6 seconds. 



4. Substitute Gram's strong solution (p. 209) for the gentian-violet and 

 treat for 4 or 6 seconds, renewing the solution twice during the process. 



5. Decolourize with alcohol-acetone (1 to 3). 



6. Transfer to picric acid in absolute alcohol for a second or two. 



7. Clear in clove oil and xylol. 



8. Mount in balsam. 



C. Claudius' method. 1. Fix the section on the slide with Mayer's albumin. 



2. Stain for 10-15 minutes in Orth's alcohol-carmine. 



3. Wash in distilled water. 



4. Stain for 2 minutes in a 1 per cent, aqueous solution of methyl violet 

 or in carbol-gentian-violet. 



