HIDEYO NOGUCHI 471 



column is bent, it breaks across and exposes a surface upon which colonies of spirochetes, if 

 present, may be seen more clearly. A capillary pipette may now be introduced into the hazy 

 areas, and if darkfield examination proves that the organisms are present, a number of sub- 

 cultures are made. Repeated subculturing results finally in bacteria-free cultures. 



In the case of pure material, introduced with the tissue at the bottom of the tube, growth 

 begins about the tissue and gradually extends upward to the border of the aerobic zone, 

 which is about 3 cm. below the surface (cf. Fig. 77). The anaerobic jar is no longer neces- 

 sary, the requisite anaerobiosis being produced by the tissue, and the entrance of air being 

 excluded by the high column of solid agar. 



Animal serum, particularly that of the rabbit, may be used in place of the ascitic fluid. 

 The tissue, however, seems to be essential for growth, at least until the parasites have been 

 under cultivation for a very long time. 



Neither of the media described is ideal for the isolation of fresh strains of treponema, 

 and only a limited number of trials at cultivation is ever successful, hence much patience is 

 required. Once the organism is obtained in culture, however, it grows more readily, and 

 after a period of years on culture medium it becomes saprophytic and may grow without 

 the addition of the fresh tissue. The precautions at first required become less and less im- 

 portant until a great many cultural conditions may prove suitable. Zinsser, Hopkins, and 

 Gilbert' isolated a strain of T. pallidum by the methods outlined, and after the tenth genera- 

 tion were able to grow it on various media. Gates^ grew some of the author's twelve-year-old 

 strains under anaerobic conditions on the surface of a blood-agar plate. The virulence of 

 the organism is lost very early in the course of cultivation, and pathogenicity tests to deter- 

 mine whether a strain is actually T. pallidum must be made soon after isolation. 



Treponema pallidum and other anaerobic spirochetes which have been under cultiva- 

 tion for a long time can be grown in fluid medium under a vasehne seal, as first recommenderl 

 by Legros^ and recently advocated by Gates and Olitsky after a careful study of the condi- 

 tions producing anaerobiosis. A piece of kidney is placed at the bottom of the tube or flask, 

 and removal of the oxygen from the medium may be indicated in a control container to 

 which methylene blue is added. When the last trace of oxygen has been removed from the 

 control tube, the others are inoculated. 



The writer used for the isolation of the saprophytic treponemas {T. microdciilium, T. 

 ma-crodentium, T. refringens, T. mucosum, T. calligyrum, T. genitalis) the same technique 

 as for the pathogenic types, the initial inoculations being made into fluid medium and the 

 impure cultures purified by repeated subculture on soUd medium. An anaerobic jar is nec- 

 essary for the fluid cultures. The saprophytic types grow more rapidly than the pathogenic 

 forms, growth becoming visible within forty-eight to seventy-two hours in the case of T. 

 mucosum, and within four to five days in the case of T. refringens, T. macrodentium, and 

 T. calligyrum. In the case of the larger type of mouth treponema {T. macrodentium), it is 

 desirable to make several transplants of the impure culture on fluid medium before attempt- 

 ing purification. The purification is a tedious process because of the presence of gas-forming 

 and acid-producing bacteria which break up the medium and render it turbid. Smith,s in 

 using this medium for the cultivation of spirochetes in sputum, has found that the gas-form- 

 ing organisms can be eliminated by first inoculating the sputum into the groin of a guinea 



' Zinsser, H., Hopkins, J. G., and Gilbert, R.: /. Exper. Med., 21, 213. 1915. 



* Gates, F. L.: ibid., 37, 311. 1923. 



3 Legros and Besson, A.: Technique microbiologique el serother. (6th ed.), p. 97. 191 1 



'i Gates, F. L., and Olitsky, P. K.: /. Exper. Med., 33, 51. 1921. 



s Smith, D. T.: Am. Rev. Tuberc., 16, 584. 1927. 



