If the burner lias a pilot light this must be turned off or the flame 

 will be smoky. A smoky or yellow flame ruins the slide. On pro- 

 longed storage the shdes may become greasy and must be re- 

 cleaned. In the atmosphere of a large industrial city this may 

 occur in a few weeks. 



Sulfuric acid cleaning solution, if hot, gives off appreciable 

 amounts of sulfur oxides which may ruin the flagella stains. Re- 

 move all hot cleaning solution from the vicinity of the staining 

 place, preferably to another room. 



Draw a hne with a wax pencil transversely across the middle of 

 the slide. Be sure the pencil line reaches both edges. A heaping, 

 medium size loopful of the prepared suspension is placed on the 

 distal end of the cool or shghtly warm slide; the slide is tilted to 

 cause the liquid to run down to the wax line. If the liquid does 

 not run down readily the slide is not clean and results may not be 

 good. Two smears, side by side, are readily made on each slide. 

 The smear is allowed to dry in air and not fixed in any manner. 

 It is now ready to be stained. 



Preparation of Flagella Stain 



The stain formula given below has proven satisfactory for the 

 visualization of the flagella of all bacteria with a few exceptions 

 such as the polar flagella of some strains of Chromobacterium. 

 The latter may be visualized by doubling the concentration of 

 tannic acid in the stain, i.e., instead of using a stock solution of 

 3% tannic acid use a stock solution of 6% tannic acid. With the 

 higher concentration of tannic acid the staining time is longer, 

 close to double. The normal formula of each stock solution is as 

 follows: 



Basic fuchsin in 95% ethyl alcohol, 1.27o; tannic acid in distilled 

 water, 3.0%; sodium chloride in distilled water, 1.5%. 



The basic fuchsin may be purchased certified for flagella stain- 

 ing. It should either be pure pararosaniline acetate or a mixture 

 of pararosaniline hydrochloride and pararosaniline acetate, but not 

 over % parts of the hydrochloride. Basic fuchsin must have an 

 odor of acetic acid to be satisfactory. Allow about 1 day to insure 

 complete solution of the fuchsin. 



The tannic acid should preferably have a light yellow color. 

 To prevent molds from growing in the tannic acid solution addi- 

 tion of phenol to a concentration of about ^^ooo is effective. The 



