112 MICROSCOPIC METHODS 



1. PitfieWs 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 ,, 



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



Carbol-fuchsin (vide p. 109) . . . . . 5 ,, 



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 ,, 



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. Zettnow's Method. 



An emulsion of a young agar culture is made in water ; a small amount 

 of this emulsion is brought into a large drop of water to which 1-2 loop- 

 fuls of 2 per cent, osmic acid solution have previously been added. From 

 this mixture cover-glass films are made, allowed to dry, and then fixed 

 by passing through a flame. 



Mordant. 

 Solution A 



Tannin .10 grms. 



Water 200 c.c. 



Solution B 



Tartar emetic ....... 2 grms. 



Water . . . . . . . 40 c.c. 



Warm solution A to 50-60 C. and then add 36-37 c.c. of solution B, 

 and heat till the precipitate which forms at first has dissolved. A small 

 portion of the mixture when cooled in a test-tube should be opalescent ; if 

 it is too opaque, add more of the tannin solution ; if it is clear, add 1 c.c. 

 more of solution B. The mordant when heated should contain no pre- 

 cipitate ; any sediment which forms should be removed by filtering the 

 hot solution through filter-paper. Place the cover-glass with film in a 

 watch-glass and cover with excess of mordant ; then heat on a hot plate 

 for 5-7 minutes at 100 C., allow to cool, and when the solution begins to 

 become turbid, remove the film and rinse it thoroughly in water. 



