Isolation and Cultivation 313 



Isolation and Cultivation. The cultivation of the gono- 

 coccus is difficult and requires considerable bacteriologic 

 skill. 



The organism does not grow upon any of the ordinary 

 culture media, and grows very scantily upon any artificial 

 medium. Wertheim* succeeded in cultivating it by diluting 

 a drop of gonorrheal pus with human blood-serum, mixing 

 this with an equal part of melted 2 per cent, agar-agar at 

 40 C., and pouring the mixture into Petri dishes, which, 

 as soon as the medium became firm, were stood in the 

 incubator at 37 or, preferably, 40 C. In twenty-four hours 

 the colonies could be observed. Those upon the surface 

 showed a dark center, surrounded by a delicate granular 

 zone. 



Young f had excellent success with a hydrocele-agar pre- 

 pared as follows : "The fluid (hydrocele or ascitic) is obtained 

 sterile, the locality of the puncture being carefully sterilized 

 by modern surgical methods, the sterile trocar covered at 

 its external end with sterilized gauze so as not to be infected 

 by the operator's hand, and the fluid collected in sterile 

 flasks, the sterile stoppers being then replaced. Collecting 

 the fluid in this way we have very rarely had it contaminated, 

 often keeping it several months before using it. The fluid 

 is mixed with ordinary nutrient agar. A number of common 

 slants are put in the autoclave for five minutes. This 

 liquefies the agar and at the same time thoroughly sterilizes 

 the tubes and cotton stoppers. The slants are then put 

 in a water-bath at 55 C. so as not to coagulate the albumin 

 when mixed with the agar. The stopper having been re- 

 moved from a small flask of hydrocele fluid, the top of the 

 flask is flamed and the albuminous fluid is then poured into 

 an agar tube (the top of which has also been flamed) in 

 proportions a little more than one to two." The medium 

 can be allowed to solidify in tubes or can be poured into 

 Petri dishes. 



When one of the colonies was transferred to a tube of 

 human blood-serum, or of one of the above-described mix- 

 tures obliquely coagulated, isolated little gray colonies occur, 

 later becoming confluent and producing a delicate smeary 

 layer upon the medium. The main growth is surrounded by 



* " Archiv. fur Gynakologie," 1892. 



t "Contributions to the Science of Medicine by the Pupils of Wil- 

 liam M. Welch," Baltimore, 1900, p. 677. 



