Isolation and Cultivation 433 



nary pus cocci, taking the Gram's stain, appear blue-black; 

 the gonococci are dark brown. 



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 C. or, preferably, 40 C. In twenty-four 

 hours the colonies could be observed. Those upon the sur- 

 face showed a dark center, surrounded by a delicate granu- 

 lar zone. 



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

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

 tained 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. Col- 

 lecting the fluid in .this way we have very rarely had it con- 

 taminated, 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 removed 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, 



* "Archiv. fur Gynakologie," 1892. 



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

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



28 



