108 MICROSCOPIC METHODS. 



be cleaned in the mixture of sulphuric acid and potassium bi- 

 chromate described on page 89. 



/ 



i . Pitfield V Method as modified by Richard Muir. 



Prepare the following solutions : 



A. The Mordant. 



Tannic acid, 10 per cent watery solution, filtered . 10 c.c. 

 Corrosive sublimate, saturated watery solution . 5 c.c. 

 Alum, saturated watery solution . . . .5 c.c. 

 Carbol-fuchsin (vide p. 104) 5 c.c. 



Mix thoroughly. A precipitate forms, which must be allowed to deposit, 

 either by centrifugalising or simply by allowing to stand. Remove the clear 

 fluid with a pipette and transfer to a clean bottle. The mordant keeps well 

 for one or two weeks. 



B. The Stain. 



Alum, saturated watery solution . . . . .10 c.c. 

 Gentian-violet, saturated alcoholic solution . . . 2 c.c. 



The stain should not be more than two or three days old when used. It may 

 be substituted in the mordant in place of the carbol-fuchsin. 



The film having been prepared as above described, pour over it as much 

 of the mordant as the cover-glass will hold. Heat gently over a flame till 

 steam begins to rise, allow to steam for about a minute, and then wash well in 

 a stream of running water for about two minutes. Then dry carefully over the 

 flame, and when thoroughly dry pour on some of the stain. Heat as before, 

 allowing to steam for about a minute, wash well in water, dry, and mount in a 

 drop of xylol -balsam. 



This method has yielded the best results in our hands. 



2. Loffler^s Method. 

 Two solutions must be made up as follows : 



A. The Mordant* 



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

 Ferrous sulphate, cold saturated aqueous solution . 5 c.c. 

 Fuchsin, saturated alcoholic solution i c.c. 



Mix well, set aside for a few days and filter always before using. This mor- 

 dant improves with age. 



B. The Stain. Either carbol-fuchsin or aniline gentian-violet will be 

 found to be eminently satisfactory if filtered before using. 



Make a film as above described, and holding the cover-glass in a pair of 

 forceps, pour on as much of the mordant A as the cover-glass will hold. Heat 

 it carefully above a flame till steam begins to rise and then move the prepara- 

 tion gently in and out of the hot-air column over the flame for about 2 minutes. 

 Wash well in distilled water till every trace of mordant appears to be gone. 



