730 THE SPIROCILETE OF SYPHILIS 



1. Pour into a small capsule of about 5 cm. diameter : 



1 per cent, aqueous solution of osmic acid, - 5 c.c. 



Crystallized acetic acid, ... * - 10 drops. 



Place the capsule inside a glass vessel closed with a ground-glass cover. 



2. Lay a well-cleaned slide on the capsule for a couple of minutes. 



3. Spread the material to be examined on this slide with a platinum spatula. 



4. Then, without drying the film, replace the slide on the capsule and leave it 

 exposed to the vapours for about 2 minutes. 



5. Dry the film quickly by gently heating it. 



6. Flood the preparation for a minute with a weak (pale red) solution of potassium 

 permanganate. 



7. Wash in distilled water. Blot with filter paper. 



8. Stain with Giemsa's solution as described above. 



9. Wash. Dry. Examine. 



The treponemata stain bluish-red even in the thick parts of the film. The flagella 

 are also rendered distinctly visible. 



(P) Flagellum staining. 



The flagella of the Treponema pallidum may be stained by Loeffler's original 

 method (p. 149) or modifications of it. 



Borrel and Burnett's method (p. 728) gives very good results as does also 

 Hoffmann and Halle's method. 



Schaudinn employed Loeffler's method, thus : 



1. Fix in absolute alcohol for 10 minutes. 



2. Mordant with fuchsin ink (p. 150) heating the solution gently for 30-60 

 seconds. 



3. Wash in distilled water, then in 75 per cent, alcohol. 



4. Stain with gentle heat for about 1 minute in a large drop of the following 

 solution : 



Aniline water, ------ - 100 c.c. 



Basic fuchsin, - - - Q.S. to saturate. 



1 per cent, soda solution, - 10 to 15 drops. 



5. Wash in distilled water. Dry. 



(7) Methods of staining sections. 



Bertarelli, Volpino, and Bovero's method. THese observers were the first 

 to successfully stain the Treponema pallidum in sections of syphilitic tissues. 

 The method, a modification of van Ermengem's method for staining flagella, 

 is based upon the reduction of silver nitrate in the tissues of the treponema. 

 The results obtained were, at the time, criticized by German writers, but 

 there can now be no doubt as to the true nature of the " Spirochsetes of silver " 

 as the treponemata were described. 



1. Fix the tissues in absolute alcohol (p. 188), embed in paraffin and cut 

 into very thin (ca 5/u) sections. 



2. Immerse the sections in a solution of silver nitrate for 2448 hours. 



Crystals of silver nitrate, - - - - - - 0*5 gram. 



Distilled water, - - 100 c.c. 



3. Wash in distilled water. 



4. Transfer the sections to van Ermengem's tanno-gallic solution (p. 149) 

 for a quarter-of-an-hour. 



5. Wash in distilled water. 



6. Return the sections to the silver solution until they assume a brownish- 

 yellow colour. 



7. Wash. Dehydrate in absolute alcohol. Wash in xylol and mount in 

 balsam. 



