5 2 BACTERIOLOGY 



(b) Use the following stain, heated till it steams : 



Saturated alcoholic solution of gentian violet or fuchsin 5 c.c. 



Distilled water 95 c.c. 



(c) Wash in 20 per cent solution of cupric sulphate crystals. 



(d) Dry and mount in Canada balsam. 



The methods of Hiss are recommended to be used for bac- 

 teria that have been cultivated on serum-agar with i per cent of 

 dextrose. They have shown that many streptococci have cap- 

 sules. The writer has had good success from the latter method, 

 with preparations of the pneumococcus from animal tissues. 



Staining of Flagella. Flagella are among the most difficult 

 of all objects to stain. The best-known method is that of 

 Loffler. It is important to use young cultures (4 to 10 hours 

 old), preferably on agar. 



(a) A small amount of the growth is gently mixed with a 

 large drop of distilled water on a clean slide, so that the water is 

 made very faintly cloudy. From the top of this drop one or 

 two transfers are made to a second drop with a small platinum 

 loop. From this second drop a loopful is transferred to a per- 

 fectly clean (flamed) cover-glass, spread with minimum manipu- 

 lation and dried quickly, high over the flame. 



(b) After drying, fixation is effected by passing through the 

 flame three times, holding the cover-slip between the thumb and 

 fore finger to avoid overheating. 



(c) The essential point in this method is the use of a mordant 

 as follows : 



Tannic acid, 10 per cent solution 20 c.c. 



Saturated solution of ferrous sulphate 4 c.c. 



Saturated alcoholic solution of fuchsin i c.c. 



This solution should be freshly prepared from pure substances, 

 and should be filtered at once after mixing. It may deteriorate 

 in a few hours but sometimes keeps for a few days or weeks. 

 A few drops are placed on the cover-glass, or the cover-glass is 



