DISEASES CAUSED BY SPIROCH^TES 855 



smeared with the edge of another slide as in making blood smears. 

 When the smear dries, which takes about a minute, it may be 

 immediately examined with an oil-immersion lens. The organisms 

 are seen unstained on a black background. 



DEMONSTRATION OF SPIROCH^TES IN TISSUES. Ordinary histo- 

 logical staining methods do not reveal the spirochaetes in tissue 

 sections. It is customary, therefore, to employ some modification of 

 Cajal's silver impregnation. The technique most commonly em- 

 ployed is that known as Levaditi's method, 2 * which is carried out as 

 follows : 



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

 2 to 4 millimeters. 



Fix in 10% formalin (4% formaldehyde) for twenty- four hours. Wash 

 in water. 



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.' 2 * The directions given by these 

 authors are as follows : 



Fix in formalin as in previous method. 



Dehydrate in 96% alcohol twelve to twenty-four 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 two to three hours at room temperature and 

 from four to six hours at 50 C. approximately. 



Wash rapidly in 10% pyridin. 



23 Levaditi, Comptes rend, de la soc. de biol., 59, 1905. 



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



