DISEASES CAUSED BY SPIROCHETES 599 



The fresh tissue is cut into small pieces which should not be thicker 

 than 2 to 4 millimeters. 



Fix in ten-per-cent formalin (f our-per-cent formaldehyde) for twenty- 

 four hours. 



Wash in water. 



Dehydrate in 96-per-cent alcohol twenty-four hours. 



Wash in water. 



Place in a 3-per-cent silver-nitrate solution at incubator temperature 

 (37.5° C.) and in the dark for three to five days. 



Wash in water for a short time. 



Place in the following solution (freshly prepared) : 



PyrogalHc acid 2-4 grams. 



Formaliii 5 c.c. 



DistiUed water 100 " 



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

 perature. 



Wash in water. 



Dehydrate in graded alcohols. 



Embed in paraffin and cut thin sections. 



The sections may be examined without further staining, or, if de- 

 sired, may be weakly counterstained with Giemsa's solution or hema- 

 toxylin. 



A modification of this method which has been much recommended is 

 that of .Leviiditi and Manouelian} The directions given by these 

 authors are as follows: 



Fix in formalin as in previous method. 



Dehydrate in 96-per-cent alcohol twelve to twenty-fom- hours. 



Wash in distilled water. 



Place in a 1-per-cent silver-nitrate solution to which 10 per cent of 

 pyridin has been added just before use. 



Leave in this solution for two to three hoiu-s at room temperature 

 and from four to six hours at 50° C. approximately. 



Wash rapidly in lO-per-cent pyridin. 



Place in a solution containing 4 per cent of pyrogaUic acid to which 10 

 per cent of C. P. acetone, and 15 per cent (per volume) of pyridin 

 have been added just before use. Leave in this solution two to three 

 hours. 



^Levadili et Manoudian, Comptes rend, de la soc. de biol., 60, 1906. 



