STAINING METHODS 31 



3. Remove, and wash three times in tap water. 



4. Stain 1-15 minutes, at 37°C, in the above diluted Giemsa stain. 



5. Mount in water for oil immersion examination. 



Note: If it is desired to mount in balsam, the staining time must be 

 increased to several hours. 



Results: The deeper colors (blue and violet) tend to be localized in the 

 chromatinic material comprising the nuclear structures. 



STAINS FOR SPIROCHAETES—RECOMMENDED PROCEDURE 



Fontana Stain 



Preparation of ammoniacal silver nitrate: 



Dissolve 5 g of AgNOs in 100 ml of distilled water. Remove a few 

 milliliters, and to the rest of the solution add drop by drop a concentrated 

 ammonia solution until the sepia precipitate which forms redissolves. 

 Then add drop by drop enough more of the silver nitrate solution to pro- 

 duce a slight cloud which persists after shaking. It should remain in 

 good condition for several months. 



Staining schedule: 



1. Prepare smear, and fix with heat. 



2. Pour on a solution of 5 per cent tannic acid in 1 per cent phenol, and 

 allow to steam 30 sec. 



3. Wash 30 sec in running water. 



4. Cover with a drop of the above ammoniacal silver nitrate, heat gently 

 over a flame, and allow it to stand 20-30 sec after steaming begins. 



5. Wash in tap water. 



6. Blot dry, and examine. 



Results: Spirochaetes, dark brown or black, in a dark maroon field. 



STAINS FOR SPIROCHAETES— ALTERNATE PROCEDURE 

 Tunnicliff's Stain 



Tunnicliff has employed carbol gentian violet (3 to 4 sec) followed by Lugol's iodine 

 (see page 16) for the same period in staining bacterial smears. With a slight modifi- 

 cation this proves a good spirochaete stain. The modification is: 



Carbol crystal violet (1 vol of 10 per cent ale crystal violet to 10 vol of 1 per cent 

 aq phenol) 30 sec; wash with water; the Lugol-Gram iodine solution 30 sec; wash 

 with water; safranin 30 sec; wash with water and dry. 



STAIN FOR RICKETTSIAE 



Macchiavello's Method 



Staining solution: 0.25 g of basic fuchsin (90 per cent dye content) dis- 

 solved in 100 ml of distilled water, buffered to pH 7.2-7.4 with the proper 

 phosphate buffer mixture. 



