4 8 



for about a minute. They are then treated with the follow- 

 ing mordant : 



Tannic acid, 20% solution 10 c.c. 



Sulphate of iron, saturated solution 5 c.c. 



Fuchsin, saturated alcoholic solution I c.c. 



This should be filtered before using. 



Place the fixed cover-glass preparation in a large test tube, 

 cover it with the mordant and carefully heat over a gas flame 

 or in a water bath until steam is given off. Allow the mor- 

 dant to act for from 3 to 5 minutes. Then remove the cover- 

 glass with a bent wire loop and fine forceps and thoroughly 

 rinse it in water. Then place it in a similar tube and cover 

 it with carbol-fuchsin for staining. Heat this as the mor- 

 dant, and allow it to act for from 5 to 10 minutes. Remove 

 the cover-glass as before and thoroughly rinse in water. If 

 the stain is too deep decolorize by rinsing the preparation for 

 a few seconds in alcohol and again in water. It is then 

 ready for the microscopic examination either in water or it 

 may be allowed to dry and be mounted in balsam. If the 

 first preparation fails add 2 drops of a 10% solution of sul- 

 phuric acid to the mordant. 



The flagella should appear as fine, hair-like appendages 

 radiating from the bacteria. 



73. Staining the flagella by Van Ermengem's 

 method. The films are prepared as described above. 

 Three solutions are necessary : 



Solution A. (Fixing bath). 



Osmic acid, 2% solution i part. 



Tannin 10-25% solution 2 parts. 



Place the films in this for one hour at room temperature, 

 or heat over a flame till steam rises, and keep in the hot 

 stain for five minutes. Wash with distilled water, then with 

 absolute alcohol for 3 to 4 minutes, and again in distilled 

 water, and treat with Solution B. 



