tannic acid and the sodium chloride solutions may be mixed with 

 equal parts of each or prepared as one solution with 1.5% tannic 

 acid and 0.757c sodium chloride. The stock solutions should be 

 kept in the refrigerator. 



To prepare the stain, mix together equal parts of the three stock 

 solutions, or add 2 parts of the tannic acid-salt solution to 1 part 

 of the dye solution. Keep the stain bottle tightly stoppered. The 

 stain is ready for use immediately. A precipitate develops in the 

 bottle on storage which should not be disturbed when the stain 

 is used. The stain solution undergoes a gradual change during 

 storage, requiring a longer staining time; the change is faster at 

 higher temperatures. At room temperature the stain solution is 

 satisfactory for about 1 week, in the refrigerator for 1-2 months, 

 and in the deep freeze indefinitely. If the stain is frozen, care 

 must be taken to mix thoroughly after thawing, since the alcohol 

 has separated from the water. The author keeps his stain solution 

 in the refrigerator and discards it when the staining time exceeds 

 that of the freshly prepared stain by more than about 5 minutes. 



Application of the Stain 



For application of the stain solution the slides are most con- 

 veniently placed on a board or rack. The author uses a board, 

 painted or stained black, about 3 inches wide and 20 inches long 

 with very short legs slightly higher in front than in back. This 

 gives a slight tilt to the board and the slides, and the staining solu- 

 tion is slightly deeper at the distal end of the slide than in the mid- 

 dle. With a Pasteur pipette, fitted with a rubber bulb and marked 

 at the 1-ml. level, 1 ml. of the stain is taken from the top of the 

 solution and is quickly applied to part of the slide holding the 

 smear. The stain solution must not spread beyond the wax line 

 or run off the slide. 



Staining Time 



The time required for staining the flagella varies normally be- 

 tween 5 and 15 minutes. A short staining time is required with 

 freshly prepared stain, warm stain, high room temperature, strong 

 air currents, thin stain layer, and pure pararosaniline acetate dye. 

 A longer staining time is required with old stain solutions, cold 

 stain, cold room, little air circulation, deep stain layer, and high 



