382 J. RUSSELL ESTY 



does not occur on inoculation. By using media containing 

 different percentages of oxygen, certain strains of Clostridium 

 Welchii develop under less oxygen tension than others, but 

 above a certain amount of free oxygen in the medium, growth 

 never occurs. Repeated artificial cultivation for several genera- 

 tions tends to increase the optimum free oxygen allowed, as 

 shown by the fact that the growth approaches nearer and 

 nearer the surface in agar stab cultures after each additional 

 transfer. 



Anaerobiosis is effected by simply sterilizing all media under 

 a thin film of oil (albolin) . If the medium has remained in the 

 laboratory more than one month after sterilization, it is boiled 

 for one half hour previous to inoculation or resterilized in the 

 auto-clave at ten pounds pressure for fifteen minutes. Anerobic 

 conditions are obtained with plate cultures by covering the 

 surface of the solidified medium with a thin layer of paraffin at 

 the lowest temperature at which this flows. Media which have 

 been sterilized without albolin permit the growth of Clostridium 

 Welchii if inoculated directly after sterilization since this organism 

 develops so rapidly at 37° that free oxygen cannot be absorbed 

 sufficiently in a short time to inhibit development. When 

 grown in this manner its life is extremely short as oxygen is 

 soon absorbed in sufficient amount to destroy it. Although a 

 film of oil renders media suitable for anaerobic growth for a 

 longer time than if sterilized without it, simply boiling the media, 

 or resterilization before using, is the only requisite for the growth 

 and isolation of Clostridium Welchii. 



2. Food requirements. Clostridium Welchii grows upon all of 

 the ordinary culture media, although 1 per cent glucose or 

 maltose stimulates its growth when added to plain meat-extract- 

 peptone agar or broth. To maintain the activity of vegetative 

 forms, transplantations are necessary at least every forty-eight 

 hours. In sugar-free broth, sporulation occurs within two to 

 four days. In broth containing a fermentable substance, all 

 vegetative forms of the organism die within three to five days 

 provided it is present in the vegetative form. 



