1V46-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 



4. Wash off with 20% aqueous CuS04-5H20. 



5. Blot dry, and examine. 



Results: capsules, faint blue; cells, dark purple. 



Stains for Spirochaetes — Recommended Procedure 



rONTANA STAIN 



Preparation of ammoniacal silver nitrate: 



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

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

 centrated ammonia solution until the sepia precipitate which forms 

 redissolves. Then add drop by drop enough more of the silver 

 nitrate solution to produce 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% tannic acid in 1% 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 seconds) followed by Lugol's 

 iodine (see p. IV46-8) for the same period in staining bacterial smears. With a slight 

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



Carbol crystal violet (1 vol. 10% ale. crystal violet to 10 vol. 1% aq. phenol) 

 30 seconds; wash with water; the Lugol-Gram iodine solution (see p. IV46-8) 30 

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



Stain for Rickettsiae 

 macchiavello's method 



Staining solution: 0.25 g. basic fuchsin (90% dye content) dis- 

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

 proper phosphate buffer mixture. 



Staining schedule: 



1. Smear a bit of tissue on a slide. 



2. Dry in the air and fix with gentle heat. 



3. Pour the above staining fluid onto the slide through a coarse 



filter paper. Allow to stand 4 min. 



4. Rinse very rapidly with 0.5% aqueous citric acid. 



5. Wash quickly and thoroughly with tap water. 



6. Counterstain about 10 sec. with 1% aqueous methylene blue. 



7. Rinse in tap water. 



8. Dry and examine. 



Results: Rickettsiae, red; cell nuclei, deep blue; cytoplasm, light blue. 



