STAINING OF SPIROCHETES IN SECTIONS 109 



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- 

 Ms 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. 



Stain. 



A saturated solution of silver sulphate is prepared by shaking 2-3 

 grms. of silver sulphate with 200 c.e. water. Kqnal volumes of the 

 saturated solution and of water are mixed in a test-tube, and commercial 

 ethylamine (33 per cent.) solutinn is ad.led drop by drop till the precipi- 

 tate whicli forms at first is again just dissolved. 



Cover the mordanted film with the stain and warm till steam rises freely ; 

 when the margins of the tilui turn black, cease heating and rinse immedi- 

 ately in water. Dry and mount in Canada balsam. 



Staining of Spirochetes in Sections. — The following im- 

 pregnation methods have been applied for this purpose by 

 ■Levaditi, and give excellent results : — 



(a) Levaditi' 's Original Method. 



(•1) The tissues, which ought to be in thin slices, about 1 mm. in thick- 

 ness, are best fixed in 10 per cent, formalin solution for twenty-four hours. 



