CULTIVATION OF THE SPIROCILETE 737 



tissue before sowing they were readily found in it subsequently and had 

 evidently multiplied in the culture tubes. 



Levaditi and Macintosh have grown the treponema in collodion sacs. 

 The virus, obtained from monkeys, was sown in sacs filled with human blood, 

 and in these sacs, placed in the peritoneal cavity of a Macacus cynomolgus, 

 an impure culture of the treponema was obtained. Sub-cultures were 

 grown in series by placing the sacs in the peritoneal cavities of rabbits ; they 

 contained numerous anaerobic organisms were devoid of virulence and had 

 no immunizing properties. All attempts to isolate the treponema in pure 

 culture failed. 



[Noguchi 1 has devised a method by which he has been able to grow the 

 Treponema pallidum in pure culture outside the body. Moreover the inocula- 

 tion of these pure cultures has resulted in the development of typical syphilitic 

 lesions in the rabbit. 



[Medium. After trial of many culture media Noguchi finds that the most suitable 

 consists of a mixture of 1 part of sheep, horse or rabbit serum with 3 parts of distilled 

 water into which a piece of freshly excised normal rabbit tissue (kidney or testicle) 

 is placed. 



[The medium is tubed in quantities of 16 c.c. in each tube and sterilized for 15 

 minutes on each of 3 successive days at 100 C. A layer of sterile paraffin oil is 

 poured on the surface to render the medium anaerobic and to prevent evaporation. 

 The sterility of the contents of the tubes is determined by incubating the latter at 

 37 C. for 2 days. 



[Material. For sowing the medium Noguchi used portions of the testicles of 

 rabbits which had been infected with material from cases of syphilis in the human 

 subject. By using rabbit tissues the difficulty of contaminating organisms was 

 largely overcome. 



[Conditions of growth. For primary cultivations it is essential that the medium 

 shall be incubated under anaerobic conditions. The tubes may be placed in a 

 Bulloch's apparatus which is then exhausted over pyrogallol and the bell jar after- 

 wards filled with hydrogen. 



[The serum and tissue forming the culture medium must be slightly alkaline. 



[To remove contaminations Noguchi filters through Berkefeld filters as he finds 

 that the treponemata are filtrable after the fifth day. 



[Results. The rabbits providing the material for cultivation had been inoculated 

 with material from ten different sources. Cultivations were obtained from six of 

 these. Cultivation was difficult and many failures were experienced. Once only 

 did Noguchi obtain a pure culture at first trial. One strain had passed through 

 twenty-five sub-cultures. 



[The treponemata begin to multiply after about 48 hours' incubation and con- 

 tinue slowly to increase for 4 or 5 weeks. They attain their natural size in 10-12 

 days and later elongate and form tangled masses. 



[In appearance the cultivated treponemata are quite typical and quite indis- 

 tinguishable from treponemata obtained from human or experimental animal 

 sources. 



[With two of the cultivated strains Noguchi was able to produce lesions in the 

 testicles of rabbits which in every way resembled the lesions produced by the inocula- 

 tion of material from cases of human syphilis. ] 



SECTION V. SERUM DIAGNOSIS. 



Wassermann, Neisser and Bruck conceived the idea of applying to the 



diagnosis of syphilis the complement fixation method of Bordet and Gengou. 



As it was impossible to obtain cultures of the Treponema pallidum, Wassermann 



used as the antigen the liver of a congenitally syphilitic infant which, as is 



well known, contains very large numbers of the organism. He showed that 



an extract or fragment of such a liver will, in the presence of heated syphilitic 



f 1 Journal of Experimental Medicine, vol. xiv.] 



3A 



