724 Syphilis 



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

 of paraffin 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 

 examined with a dark field illuminator to make sure that the organ- 

 isms 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 37C. 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 opales- 

 cence of the agar-agar caused by the extension into it of the grow- 

 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 

 cautiously broken at some appropriate part and the transplanta- 

 tion made from the opalescent part of the medium to fresh appro- 

 priate culture-media. By these means, after a few trials, pure 

 cultures of treponema were secured. 



The colonies were said never to be sharp, but always faintly 

 visible. There is no color and no odor. 



I By inoculating the organisms recently secured from human 

 lesions (by the method given) into monkeys (Macacus rhesus and 

 Cereopithicus callitrichus) Noguchi was able to produce typical 

 syphilis of the monkey, thus showing that the virulence of the 

 organisms was not lost in the cultivation. 



Zinsser, Hopkins and Gilbert* found it possible to grow Tre- 

 ponema pallidum in massive cultures in fluid media. They em- 

 ployed a flask with a long slender neck like a " specific gravity flask." 

 The flask was filled with slightly acid (0.2 to 0.8 per cent, acidity) 

 broth containing sheep-serum, ascitic fluid, horse-serum or rabbit- 

 serum, with an addition of autoclaved and hence thoroughly sterilized 

 tissue (kidney, liver, brain, lung or heart muscle) and covered with 

 sterile neutral paraffine oil. The culture contains the greatest 

 number of organisms after three weeks. To collect them for mak- 

 ing luetin, etc., the fluid in the flasks was poured into tubes and 

 '"Journal of Experimental Medicine," 1915, XXT, No. 3, p. 213. 



