114 ROYAL SOCIETY OF CANADA 



inoculated and then some were closed with sealing-wax and others were 

 left unsealed; all were incubated at 20°. 



All showed growth in one day and abundant growth in 5 days, the 

 growth in the tubes not sealed being only a little more abundant than 

 in the sealed tubes. 



The tubes not sealed all became pigmented and those with lactose 

 1^, 5^ and 10;^ passed through the deep blue pigmentation and were 

 wine or rust color in 36 days. 



The sealed cultures developed no pigment at all as long as they 

 remained sealed. Cultures 5^ and 10/c were unsealed after 6 days and 

 soon developed a rich pigment. Cultures 1^/, and 10^ were unsealed 

 after 34 days and in 24 hours developed a strong blue pigment, but the 

 period of pigmentation was much shorter than in tubes not sealed. 

 When, after 34 days, 1^ and 10;^ were unsealed, cultures were made 

 and both found to be alive. 



Two tubes of agar with 1^ lactose, not sealed, were bright blue to 

 Prussian blue in 3 days. One of them was then sealed and in 3 days 

 more at 20° C. the agar was completely decolored and on softening the 

 wax in the flame, there was a strong inward pressure drawing the plug 

 into the tube. The other tube of agar with 1^ lactose was not sealed at 

 all and remained pigmented for 18 days or more. 



With a limited supply of oxygen, the organism grew well and 

 consumed oxygen in its growth but did not produce pigment. If a 

 pigmented culture of the organism was limited as to oxygen, it soon 

 consumed the pigment already formed and produced no more. Tliis 

 indicated that chemically the pigment body was rich in oxygen. 



Oxygen Requirements for Pigmentation. — If two tubes of liquid 

 media favorable for pigment formation be inoculated and put at a 

 favorable temperature and if one tube be inclined in an almost horizontal 

 position so as to give a large surface exposed to air, and if the other 

 be kept upright, then the inclined tube will develop a richer pigment 

 throughout and the upright tube less pigment, mainly limited to a sur- 

 face zone. 



There was no growth in the closed arm of the fermentation tubes, 

 see experiments under gas formation. 



Gai Production. — Beef peptone bouillon modified with b^ sucrose, 

 maltose, and lactose was sterilized in fermentation tubes; inoculated; 

 and incubated at 25° to 27° C. The three tubes of sucrose bouillon 

 became turbid in the open arm in 24 hours. This turbidity increased 

 and a white ropy sediment formed. The liquid in the connecting tube 

 became turbid but in the closed arm it remained clear. In the maltose 

 bouillon the growth was greater than in the sucrose bouillon and no gas 

 appeared in either in 20 days. 



