Cultivation 



723 



placing a fragment of human tissue, containing it, deep down into 

 gelatinized horse-serum. The treponema grew together with the 

 contaminating organism and no pure culture was secured. Muhlens* 

 and Hoffmann,! using the same method, succeeded in securing pure 

 cultures of the treponema, but found them avirulent. 



Noguchi,!: taking advantage of the observations of Bruckner 

 and Galasesco§ and Sowade,|| that an enormous multiplication 

 of treponema occurred when material containing it was inoculated 

 into the rabbit's testis, performed a lengthy series of cultivation 

 experiments with the enriched material thus obtained. The 

 culture-medium used in these experiments was a "serum water," 

 composed of i part of the serum of the sheep, horse, or rabbit 

 and 3 parts of distilled water; 16 cc. of this mixture was placed 

 in test-tubes 20 cm. long and 1.5 cm. in diameter and sterilized for 

 fifteen minutes at ioo°C. each day for three days. 



To each of a series of such tubes a carefully removed fragment of sterile rabbit's 

 testis was added, after which the tubes were incubated at 37°C. for two days to 

 determine their sterility. To each tube the material from the inoculated rabbit's 

 testis, rich in the treponema, is added, after which the surface of the medium in 

 each receives a thick layer of sterile paraffin oil. As the most strict anaerobiosis 

 is necessary, the tubes are placed in a Novy jar, the bottom of which contains 

 pyrogallic acid. Noguchi first passes H gas through the jar, permitting it to 

 bubble through the pyrogallic acid solution for ten minutes. He then uses a 

 vacuum pump to exhaust the atmosphere in the jar, and lastly permits the alka- 

 hne solution (KOH) to flow down one of the tubes and mix with the pyrogallic 

 acid. 



In these cultures the pallidum grows together with such bacteria 

 as may have been simultaneously introduced. To secure the 

 cultures free from these bacteria Noguchi permitted the treponema 

 to grow through a Berkefeld filter, which for a long time held back 

 the other organisms. Later it was found that both bacteria and 

 treponema grow side by side in a deep stab in a serum-agar- tissue 

 medium, but that the bacteria grow only in the stab or puncture, 

 whereas the treponemata grow out into the medium as a hazy 

 cloud. By cautiously breaking the tube and securing material for 

 transplantation from the scarcely visible cloud, the organisms may 

 be transplanted to new media and pure cultures obtained. 



In a later paper, Noguchi** details the cultivation of the tre- 

 ponema from fragments of human chancres, mucous patches, and 

 other cutaneous lesions. The medium employed is a mixture of 

 2 per cent, slightly alkaline agar and i part of ascitic or hydrocele 

 fluid, at the bottom of which a fragment of rabbit kidney or testis 

 is placed. The medium is prepared in the tubes, after the addi- 

 tion of the tissue, by mixing 2 parts of the melted agar at so°C. with 



* Ibid., 1909, XXXV, 1261. 



t "Zeitschrift fiir Hygiene und Infektionsk.," 1911, Lxviii, 27. 

 J "Journal of Experimental Medicine," 1911, xiv, 99. 

 § " Compt.-rendu de la Soc. de Biol, de Paris," 1910, lxviii, 648. 

 II "Deutsche med. Wochenschrift," 1911, xxxvii, 682. 

 ** "Journal of Experimental Medicine," 1912, xv, i, p. go. 



