STAINING THE FLAGELLA 65 



Around the edge where the drop was applied to the cover 

 glass a heavy line of bacteria will be found, and if the right 

 amount of culture was added to the water, many will be found 

 scattered within the ring, some of them isolated so they can 

 be easily studied. A little practice with this will enable one 

 to make good preparations altogether free from background. 



96. Staining the flagella by Loeffler's method. Place 2 loop- 

 fuls of sterilized distilled water or normal salt solution on the 

 center of the cover glass. Gently touch the surface 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 dissemi- 

 nated throughout the water by means of their power of loco- 

 motion. When dry they are ready for the staining treatment. 



The bacteria 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. 



Tannic acid, 20 % solution 10 cc. 



Sulphate of iron, saturated solution 5 cc. 



Fuchsin, saturated alcoholic solution . . . . i cc. 



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 mordant 

 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 with 

 carbol fuchsin for staining. Heat this as the mordant was 

 heated and allow the stain 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 



