600 PATHOGENIC MICROORGANISMS 



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 Schereschewsky. The most ex- 

 tensive and convincing work on trepon- 

 ema pallidurn has been done recently by 



Noguchi. Noguchi l began his work in 

 FIG. 130. SPIROCH^TA PAL- -mm j inn TT- c. e i i 



o , 1910 and 1911. His first successful cul- 



LIDA. Spleen, congenital syph- 

 ilis. (Levaditi method.) ' tivations were made from the syphilis- 

 infected testicles of rabbits, and after 



many unsuccessful attempts, with slightly varying media and 

 technique, he finally succeeded in the following way: He pre- 

 pared 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 spirochsetes were 

 present. The fluid was then covered with sterile paraffin oil and placed 

 in an anaerobic jar. After 10 days at 33.5 C. the spirochsetes had 

 multiplied considerably, in all but one case, together with bacteria. He 

 obtained pure cultures from these initial cultivations after much diffi- 

 culty, by a number of methods. At first he succeeded only by allowing 

 the spirochsetes to grow through Berkefeld filters, which they did on 

 the fifth day. A better method more recently adopted by him consists 

 in preparing high tubes of three parts of very slightly alkaline or neutral 

 agar to which a piece of sterile tissue has been added. These tubes are 

 then inoculated from the impure cultures with a long pipette. Close 

 to the tissue and along the stab the spirochsetes and bacteria will grow 

 and, after about ten days to two weeks, the spirochsetes will have wan- 

 dered away from the stab and will be visible as hazy colonies. They can 



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



