MICROSCOPIC EXAMINATION AND STAINING METHODS 105 



2. Wash in water. 



3. Stain with Gabbett's methylene-blue for one-half to one 

 minute. 



4. Wash and examine. 



The acid-fast organisms will be red in a blue field. 



Flagella Stain. The flagella of bacteria are not visible in 

 ordinary stained mounts, and can be demonstrated only by a 

 special technic. Young, twelve- to eighteen-hour cultures of 

 bacteria should be used for their demonstration. A tube con- 

 taining a few cubic centimeters (5) is inoculated with sufficient 

 quantity of the growth carefully removed from the agar surface 

 to produce a slight turbidity. Incubate for an hour in the ther- 

 mostat. Drop two or three drops without mixing or spreading 

 on a clean cover-slip. Dry and then fix in the flame. Many 

 methods of staining flagella have been suggested; the two follow- 

 ing are probably the best: 



Van Ermengem's Method. 1. Place the film for one hour in the 

 following solution: 



Osmic acid, 2 per cent 1 part 



Tannin, 10-25 per cent, solution 2 parts 



2. Wash in water, then absolute alcohol, then place in the 

 following solution for a few seconds only: 



Silver nitrate, 0.05 per cent, in distilled water. 



3. Wash in the following solution for a few seconds: 



Gallic acid 5 gm. 



Tannin 3 gm. 



Fused potassium acetate 10 gm. 



Distilled water 350 c.c. 



4. Wash in silver nitrate solution until film turns black. 



5. Wash in water and examine. 



Loffler's Method. 1. Prepare film, fix, and apply the following 

 mordant, heating for five minutes over a water-bath: 



Tannic acid (25 per cent, aqueous solution) 10 parts 



Saturated solution ferrous sulphate 5 parts 



Fuchsin (saturated alcoholic solution) 1 part 



