192 Methods of Observing Micro-organisms 



directly upon the spread (the liquid must always be filtered at the 

 time of use) and heated gently for three minutes, but not permitted 

 to boil. Wash with water and then stain in the following: 



Saturated alcoholic solution of gentian violet i c.c. 



Saturated solution of ammonium alum 10 



Filter the stain also directly upon the slide at the time of 

 using, and heat it for three to four minutes. Wash thoroughly 

 in water, dry, and mount in balsam. 



Van Ermengem's Method. Van Ermengem * has devised 

 a somewhat complicated method of staining flagella, which 

 has given great satisfaction. Three solutions, which he 

 describes as the bain fixateur, bain sensibilisateur, and bain 

 reducteur et reinforcateur, are to be used as follows: 



1. Bain fixateur: 



2 per cent, solution of osmic acid 1 part 



10-25 per cent, solution of tannin 2 parts 



The cover-glasses, which are very thinly spread, dried, 

 and fixed, are placed in this bath for one hour at the room 

 temperature, warmed until steam arises, and then kept hot 

 for five minutes. They are next washed with distilled 

 water, then with absolute alcohol, then again with distilled 

 water. All three washings must be very thorough. 



2. Rain sensibilisateur: 



5 per cent, solution of nitrate of silver in distilled water. 



The films are allowed to remain in this for a few seconds, 

 and are then immediately transferred to the third bath. 



3. Bain reducteur et reinforcateur: 



Gallic acid 5 grams 



Tannin 3 " 



Fused potassium acetate 10 " 



Distilled water 350 c.c. 



The preparations are kept in this solution for a few 

 seconds, then returned to the nitrate of silver solution until 

 they begin to turn black. They are then washed, dried, 

 and mounted. 



Mervyn Gorden modifies the method by allowing the 

 preparations to remain in the second bath for two minutes, 

 transferring to the third bath for one and a half to two 



* "Travaux du Lab. d'hygiene et des bact. de Gand.," t. I, p. 3. 

 Abstracted in the " Central bl. f. Bakt. u. Parasitenk.," 1894, Bd. xv, 

 p. 969. 



