634 APPLICATION OF METHODS OF BACTERIOLOGY 



Levaditis Method for Tissues. Fix bits of suspected tissue, 

 not over 2 m.m. thick, in 10 per cent, formalin for one or 

 two days. Wash out in 95 per cent, alcohol for eighteen to 

 twenty hours. Wash thoroughly in distilled water until the 

 tissues no longer float. 



Impregnate in: 



Nitrate of silver 1 gram 



Pyridin 10 grams 



Distilled water 100 c.c. 



Made up fresh. 



Allow to stand at room temperature for two or three 

 hours, followed by 50 C. for from four to six hours. Wash 

 rapidly in 10 per cent, pyridin, after which reduce the silver 

 ,by immersing the tissue for several hours in: 



Pyrogallic acid ...;.".,.... 4 grams 



Acetone 10 " 



Pyridin 15 " 



Distilled water 100 c.c. 



Made up fresh. 



Harden in alcohol. Embed in paraffin and cut sections 

 about 5 /*. thick; mount in balsam. The spirochetes appear 

 in tissues so treated as intensely black objects. 



Cultivation. 1 Certain of the spirochsetacese lend them- 

 selves to artificial cultivation; others have eluded all such 

 efforts. There are not as yet any standard methods of 

 artificial cultivation such as are used in the routine study 

 of "bacteria. Special methods have been devised by a 

 number of investigators and all have met with more or less 

 success. Practically all such methods depend for success 

 upon certain fundamental requirements: 



The culture fluids must consist essentially of sterile animal 

 juices or exudates; must contain bits of fresh animal tissue; 



1 See Noguchi, Jour. Exper. Med., 1918, vol. xxvii, p. 593. 



