i62 Methods of Observing Micro-organisms 



The cover-glass must be cleaned most painstakingly; too much 

 heating in fixing must be avoided. After using and washing off 

 the mordant, the preparation should be dried before the applica- 

 tion of the anihn-water-fuchsin solution. 



Pitfield's Method. — Pitfield* has devised a single solution, at once 

 mordant and stain. It is made in two parts, which are filtered and 

 mixed : 



(A)- 



Saturated aqueous solution of alum lo cc. 



Saturated alcoholic solution of gentian violet . i " 



(B)- 



Tannic acid i gram 



Distilled water lo cc. 



The solution should be made with cold water, and immediately- 

 after mixing the stain is ready for use. The cover-slip is carefully 

 cleaned, the grease being burned off in a flame. After it has cooled, 

 the bacteria are spread upon it, well diluted with water. After 

 drying thoroughly in the air, the stain is gradually poured on and 

 by gentle heating brought almost to a boil; the slip covered with 

 the hot stain is laid aside for a minute, then washed in water and 

 mounted. 



Smith's Modification of Pitfield's Method.^ — A boiling saturated solution of 

 bichlorid of mercury is poured into a bottle in which crystals of alam have 

 been placed in quantity more than sufficient to saturate the fluid. The 

 bottle is shaken and allowed to cool; lo cc. of this solution are added to 

 the same volume of freshly prepared tannic acid solution and 5 cc. of car- 

 bol fuchsin added. ■ Mix and filter. The filtrate, which is the mordant, is 

 caught 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 cc. 



Saturated solution of ammonium alum 10 " 



Filter the stain 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 ErmengemJ has devised a some- 

 what complicated method of staining flagella, which has given great 

 satisfaction. Three solutions, which he describes as the hain fixa- 

 ieur, bain sensibilisateur, and bain reducteur et reinforgateur, are to 

 be used as follows: 



I . Bain fixateur: 



2 per cent, solution of osmic acid i part 



10-25 pw 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 



* "Medical News," Sept. 7, 1895. , 



t "Brit. Med. Jour.," 1901, i, p. 205. 



X "Travaux du Lab. d'hygiene et des bact. de Gand.," 1. 1, p. 3. Abstracted 

 in the "Centralbl. f. Bakt. u. Parasitenk.," 1894, Bd. xv, p. 969. 



