Staining 



423 



Staining the organism in the tissues was found to be a more 

 difficult matter, for the Giemsa stain scarcely showed it at all. 

 Bertarelli and Volpino* endeavored to stain sections by a 

 modification of the van Ermengem method for flagella and 

 had some success, but the demonstration of the organisms 

 in tissue was not really successful until Levaditif devised 

 the method of silver impregnation. This consists in hard- 

 ening pieces of tissue about i mm. in thickness in 10 per 



sjF. -' 



Fig. 125. Treponema pallidum impregnated with silver. Film 

 prepared from the skin of a macerated, congenitally syphilitic fetus. 

 X 750 diameters (Flexner). The dense aggregation of organisms may 

 indicate agglutination. 



cent, formol for twenty-four hours, rinsing in water, and 

 immersing in 95 per cent, alcohol for twenty-four hours. 

 The block is then placed in diluted water until it sinks to 

 the bottom of the container, and then transferred to a 

 I -5~3 per cent, aqueous solution of nitrate of silver in a blue 

 or amber bottle and kept in a dark incubating oven at 37 C. 

 for from three to five days. Finally it is washed in water and 

 placed in a solution of pyrogallic acid 2-4 grams ; formol, 5 c.c. ; 



* "Centralbl. f. Bact. u. Parasitenk.," Orig., 1905, XL, p. 56. 

 t "Compt. rendu de la Soc. de Biol. de Paris," 1905, ux, p. 326. 



