Cultivation 767 



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

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

 acid. 



In these cultures the organism 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 seruni-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 trep- 

 onema from fragments of human chancres, mucous patches, and 

 other cutaneous lesions. The medium employed is a mixture of 

 2 per cent, shghtly 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 ? parts of the melted agar at so°C. with 

 I part of the ascitic or hydrocele fluid. After solidification a layer 

 of paraflSn oil 3 cm. deep is added. 



A considerable number of tubes should be prepared at the same 

 time and incubated for a few days prior to use to determine sterility. 

 The bits of human tissue are snipped up with sterile scissors in 

 salt solution containing i per cent, of sodium citrate and should 

 be kept immersed in this fluid from the time of securing to the 

 time of planting, so as not to become dried. A bit of the tissue 

 should be emulsified in a mortar with citrate solution and exam- 

 ined with a dark field illuminator to make sure that the organisms 

 to be cultivated are present. 



If they are found, and the material shown to be adapted to culti- 

 vation, each of the remaining bits of tissue is taken up by a thin 

 blunt glass rod and pushed to the bottom of a culture-tube and 

 into each tube several drops of the emulsion examined are intro- 

 duced by means of a capillary pipet, also inserted deeply into the 

 medium. The tubes are next incubated at 37°C. for two or three 

 weeks. In successful tubes, in which the medium has not been 

 broken up by gas-producing bacteria, there is a dense opaque 

 growth of bacteria along the line of puncture, and a diffuse opal- 

 escence of the agar-agar caused by the extension into it of the growr 

 ing treponemata. A capillary tube cautiously inserted into the opal- 

 escent medium withdraws a particle that can be examined with 

 the dark field illuminator. When such observation shows the cause 

 of the opalescence to be, in fact, the treponema, the tube can be 



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



