136 THE VENOMS OF CERTAIN THANATOPHIDE^B. 



hour. The bulbs were 'then hermetically sealed by melted glass. The bouillon in 

 these tubes (with sterilized venom) remained perfectly clear and free from bacteria. 

 Microscopical examination was made at various periods, the last time after eighteen 

 months when it was still perfectly clear and showed no signs of putrefaction. 



A similar result was obtained in an experiment with another set of six glass 

 bulbs tilled with bouillon, and to which some Moccasin peptone, previously sterilized, 

 was added. These bulbs looked somewhat cloudy, but on examination of the con- 

 tents eighteen months later no bacteria, and no putrefactive changes were noted. 



As a control experiment six bulbs filled with pure sterilized bouillon were kept 

 for a similarly long period, and they all remained clear and free from change; while 

 a few bulbs filled with unsterilized bouillon showed great cloudiness, bacteria, and 

 putrefactive change. 



4th. Culture Experiments. The study of the morphology of the bacteria inhabit- 

 ing the venom was next undertaken. To this end numerous culture experiments 

 to isolate the bacteria from the venpm were made. As stated before, the perfectly 

 fresh venom contained only one form of these vegetable organisms, the micrococci, 

 and only to these latter attention was paid ; the rod bacteria and bacilli not 

 appearing except in venom which had began to putrefy. 



The micrococci contained in the venom showed the following behavior in pure 

 cultures : Of culture soils, the peptonized gelatine prepared after the formula of 

 Koch proved to be quite suitable. The isolation of the micrococci was made after 

 the methods of Sternberg and of Koch, as adopted in the pathological laboratory of 

 the University of Pennsylvania. For gelatine culture a minute quantity of venom 

 was smeared on the surface of the solidified jelly contained in a sterilized, small, 

 flat, well covered glass vessel. The micrococci liquefied the jelly, an effect not 

 peculiar to all bacteria. After twenty-four hours all over the inoculated surface 

 of the jelly were seen small turbid drops which contained the micrococci. With a 

 sterilized platinum wire the micrococci from one of the liquefying specks upon the 

 first culture were transplanted to the jelly in a second culture vessel. From this 

 second generation a minute quantity was transplanted to a third and fourth culture 

 vessel. The fourth and all the later generations yielded usually a pure crop of 

 micrococci. 



In impure cultures dumb-bell-shaped bacteria and sometimes large bacilli were met 

 with. These, however, could not be said to be peculiar to venom, as they are never 

 found in fresh venom. It may, therefore, be concluded that these cultures represent 

 the micrococci peculiar to, or at least those constantly inhabiting venom. More- 

 over, the micrococci in these cultures whenever they were successful, were the 

 only bacterium seen and were fully identical as to shape, measurement, and be- 

 havior to aniline dyes with those found in the fresh venom. 



Much better crops of the venom-micrococci were obtained in bouillon cultures in 

 Sternberg's glass-bulbs. The micrococci grow more rapidly and better in these 

 bulbs, because the bouillon can be heated up to the more suitable temperature of 

 40 C. ; while the jelly cultures could not be warmed to such a degree without melt- 

 ing solid gelatine. A variety of other culture soils and methods of isolation were 



