856 PATHOGENIC MICROORGANISMS 



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 two to three 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- 

 ehaetes appear as black, untransparent bodies lying chiefly extra- 

 cellularly. 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 Spirochaeta pallida were at first unsuc- 

 cessful. In 1909 Schereschewsky 25 reported that he had suc- 

 ceeded in obtaining multiplication of the organisms on artificial 

 media as follows : Sterile horse serum in centrifuge tubes was coagu- 

 lated at 60 C. until it assumed a jelly-like consistency. It was then 

 placed in the incubator at 37.5 C. for three days before being used. 

 The cultures were planted by snipping off a small piece of tissue 

 from a syphilitic lesion, dropping it into such a tube, and causing 

 it to sink to the bottom by means of centrifugalization. The tube 

 was then tightly stoppered with a cork. In such anaerobic serum 

 cultures Schereschewsky claims to have grown the organisms for 

 several generations, though not in pure culture. 



Miihlens also obtained growth of Spirochaeta pallida in horse 

 serum agar by a method which is very similar to that of Schereschew- 

 sky. The most extensive and convincing work on treponema palli- 

 dum has been more recently by Noguchi. Noguchi 26 began his work 

 in 1910 and 1911. His first successful cultivations were made from 

 the syphilis-infected testicles of rabbits, and after many unsuccessful 

 attempts, with slightly varying media and technique, he finally suc- 

 ceeded in the following way: He prepared tubes (20 cm. high and 

 1.5 cm. wide), containing 10 c.c. of a serum-water made of distilled 

 water, three parts; and horse, sheep, or rabbit serum, one part. 

 These were sterilized by the fractional method in the usual way 

 (15 minutes each day). Into them was then placed a small piece 

 of sterile rabbit kidney or testicle and a bit of the testicle of a 

 syphilitic rabbit, in which many spirochaetes were present. The 

 fluid was then covered with sterile paraffin oil and placed in an 



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

 28 Noguchi Jour. Exp. Med., xiv, 1911; xvii, 1913. 



