STAINING THE FLAGELLA; 53 



flagella staining (4). Make about ip cover-glass prepara- 

 tions on these from the agar culture and stain for flfigella. Use 

 Loeffler's method, but if it does not succeed, the second process 

 may be tried. 



81. Making Cover-glass Preparations for Flagella Stain. — 

 Place 2 loopfuls of sterilized, distilled water or normal salt solu- 

 tion on the centre of the cover-glass. Gently touch the sur- 

 face growth on the agar culture with the end of the platinum 

 needle and immerse it in the water on the cover-glass without 

 spreading the drop. The impregnated needle should carry 

 bacteria enough for 3 or 4 preparations. Then place the tray 

 of cover-glasses in the incubator to dry. The bacteria become 

 disseminated throughout the water by means of their power 

 of locomotion. When dry, they are ready for the staining 

 treatment. 



82. Staining the Flagella by Loeffler's Method. — The bac- 

 teria are fixed to the cover-glass by holding them, film upward, 

 between the thumb and forefinger, over a gas flame for about a 

 minute. They are then treated with the following mordant : — 



-J ■ TaBniG 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 motdant 

 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 mordant was 

 heated and allow the stain to act for from 5 to ro minutes. 

 Remove the cover-glass as before and thoroughly rinse in 

 water. If the stain is too deep, decolorize by rinsing the prepa- 

 ration for a few seconds in alcohol and again in water. It is 



