104 MICROSCOPIC METHODS 



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. Tan Ermengenis Method for Staining Flagella. 



The films are prepared as above described. Three solutions are here 

 necessary : 



Solution A. (Bain fixateur] 



Osmic acid, 2 per cent solution . . . 1 part 



Tannin, 10-25 per cent solution .... 2 parts 



Place the films in this for one hour at room temperature, or heat over 

 a flame till steam rises and keep in the hot stain for five minutes. 

 Wash with distilled water, then with absolute alcohol for three to four 

 minutes, and again in distilled water, and treat with 



Solution B. (Bain sensibilisateur] 



*5 per cent solution of nitrate of silver in distilled water. Allow 

 films to be in this a few seconds. Then without washing transfer to 



Solution C. (Bain reducteur et reinfor$ateur] 



Gallic acid . . . . . . . w> 5 grm. 



Tannin . . . . . . . . . 3 ,, 



Fused potassium acetate . . . . . . 10 ,, 



Distilled water . . . . . . 350 c.c. 



Keep in this for a few seconds. Then treat again with Solution B till 

 the preparation begins to turn black. Wash, dry, and mount. 



It is better, as Mervyn Gordon recommends, to leave the specimen in 

 B for two minutes and then to transfer to C for one and a half to two 

 minutes, and not to transfer again to B. It will also be found an 

 advantage to use a fresh supply of C for each preparation, a small 

 quantity being sufficient. The beginner will find the typhoid bacillus 

 or the bacillus coli communis very suitable organisms to stain by this 

 method. 



Although the results obtained by this method are sometimes excellent, 

 they vary considerably. Frequently both the organisms and flagella 

 appear of abnormal thickness. This is due to the fact that the process 

 on which the method depends is a precipitation rather than a true 

 staining. The pictures on the whole are less faithful than in the first 

 method. 



Staining of Spirochsete in Sections. The following im- 

 pregnation method, which is practically that of Ramon-y-Cajal 



