DISEASES CAUSED BY SPIROCH^TES 599 



Dehydrate in 96% alcohol twenty-four hours. Wash in water. 

 Place in a 3% silver-nitrate solution at incubator temperature (37.5 C.) 

 and in the dark for 3 to 5 days. Wash in water for a short time. 

 Place in the following solution (freshly prepared) : 



Pyrogallic acid 2-4 grams. 



Formalin 5 c.c. 



Distilled water 100 " 



Leave in this for twenty-four to forty-eight hours at room temperature. 

 Wash in water. 

 Dehydrate in graded alcohols. 

 Embed in paraffin and cut thin sections. 



The sections may be examined without further staining, or, if desired, 

 may be weakly counterstained with Giemsa's solution or hematoxylin. 



A modification of this method which has been much recommended is 

 that of Levaditi and Manouelian. 1 The directions given by these authors 

 are as follows: 



Fix in formalin as in previous method. 



Dehydrate in 96% alcohol 12 to 24 hours. Wash in distilled water. 



Place in a 1% silver-nitrate solution to which 10% of pyridin has been added 

 just before use. 



Leave in this solution for 2 to 3 hours at room temperature and from four 

 to six hours at 50 C. approximately. 



Wash rapidly in 10% pyridin. 



Place in a solution containing 4% of pyrogallic acid to which 10% of C. P. 

 acetone, and 15% (per volume) of pyridin have been added just before use. 

 Leave in this solution 2 to 3 hours. 



Wash in water, dehydrate in graded alcohols, and embed in paraffin by the 

 usual technique. 



Examined after treatment by either of these methods, the spiro- 

 chaetes appear as black, untransparent bodies lying chiefly extracellu- 

 larly. They are characteristically massed about the blood-vessels of the 

 organs and only exceptionally seem to penetrate into the interior of 

 the parenchyma cells. 



Attempts at cultivating Spirochseta pallida were at first unsuccessful. 

 Recently Schereschewsky 2 has reported that he has succeeded in ob- 

 taining multiplication of the organisms on artificial media as follows: 

 Sterile horse serum in centrifuge tubes was coagulated at 60 C. until 

 it assumed a jelly-like consistency. It was then placed in the incubator 



1 Levaditi et Manouelian, Comptes rend, de la soc. de biol., 60, 1906. 



2 Schereschewsky, Deut. med. Woch., N. S., xix and xxix, 1909. 



