470 THE SPIROCHETES 



the bacteria. The piece of tissue acts as an oxygen absorber, as first pointed out by 

 Theobald Smith, and also furnishes nutriment. Other treponemas have been grown 

 by the writer by the same methods, including T. pertenue' of yaws, and several organ- 

 isms which are normal inhabitants in the human mouth^ and about the genital region. ^ 



Preparation of the fluid medium. — A rabbit kidney (testicle or heart muscle may also be 

 used, but liver is unsuitable because often contaminated with bacteria and containing car- 

 bohydrates which give rise to acid) , removed under aseptic conditions, is cut into about six- 

 teen pieces, and a piece of the tissue is put into each of several tall tubes (14-20X 200 mm.). 

 The material for inoculation, which must be relatively free from bacteria, as is the case with 

 testicular tissue of the rabbit after several rabbit passages of a strain of pathogenic trepo- 

 nema, is added at this point, and the tubes subsequently two-thirds filled (about 15 cc. 

 is required) with ascitic fluid or serum water (one part sheep, horse, or rabbit serum and three 

 parts distilled water). A layer of liquid paraffin is added to prevent evaporation of the me- 

 dium. The tubes are now placed in a sealed jar, in which the air is replaced by hydrogen,'' 

 and left undisturbed at a temperature of 37° C. for two to three weeks. Success is by no 

 means constant in the case of the pathogenic treponemas, and when growth is obtained it is 

 usually impure and must be purified either by the use of a filter {T. pallidum is not directly 

 filterable but will grow through a filter into a suitable culture medium before the bacteria 

 are able to pass through, i.e., in about five days), or by subculture on solid medium. 



Preparation of the solid medium. — This medium is suitable for impure material, either 

 direct from human lesions or from an impure fluid culture. It is also the medium which the 

 writer uses for the routine subculture of all pure treponema cultures. 



The tissue is placed at the bottom of the tube, as before, and to the tubes is added about 

 15 cc. of a mixture of 2 per cent nutrient agar (two parts), melted and cooled to about 50° 

 C, and ascitic fluid (one part), the mixture being kept warm during the course of the distri- 

 bution. If the material for inoculation is pure, it is best added before the mixture of agar 

 and ascitic fluid: if impure, it is inoculated after solidification of the medium. A piece of 

 tissue from a lesion may be forced to the bottom of the tube with a platinum loop. A fluid 

 inoculum is introduced with a capillary pipette. To furnish the necessary compression force, 

 a heavy metal syringe may be attached to the capillary with a piece of pressure tubing, but 

 care must be taken not to spHt the medium. A layer of liquid paraffin is always added to 

 prevent evaporation of the medium. The tubes are incubated at 37° C. for two to three 

 weeks. 



The bacteria grow along the stab canal and on the surface of the medium, but if the me- 

 dium is clear enough a faint haze is seen to radiate from the stab canal toward the sides of 

 the tube. Not infrequently the growth is invisible, however, owing to the quality of the me- 

 dium, and in this case darkfield examination must be relied on for detection of growth. 

 The surface of the medium is sterilized by sublimate alcohol, which is poured off. The tube 

 is cracked about the center (by making a small scratch and applying a red-hot glass rod), 

 and the upper half removed, exposing the agar column, which is again sterilized with sub- 

 limate alcohol, care being taken to absorb excess moisture with sterile gauze. When the agar 



" Noguchi, H.: Miinchen. mcd. Wchnschr., 58, 1550. 191 1. 



^ Noguchi, H.: /. Exper. Med., 15, 81. 191 2; 16, 194. 1912. 



3 Noguchi, H.: ibid., 15, 466. 191 2; 17, 89. 1913. 



'' I used a combination of hydrogen gas, vacuum, and pyrogallic acid to replace the air {ibid., 

 14, 102. 191 1), but other types of anaerobic apparatus have since been devised, e.g., those of Mcin- 

 tosh and Fildes (Lancet, 1, 768. 1916), Brown {J. E.xper. Med., 33, 677. 1921), and Boez (/. Boot., 

 13, 227. 1927). 



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