80 llllill TEMPERATURE ORGANISM OF PERMEXTIXG TAX-BARK, 



dextrose there was a jjroduetion of acid and of gas in 5 days at 60° . * The 

 colonies that developed on the agar slopes contained the rod-shaped bacteria 

 already noted, and as these did not produce gas, either in agar or in broth, it is 

 evident that the gas-producer is difficult to obtain. Some seventy colonies had 

 been picked out and none of them were gas producers. In films prepared from 

 the condensed water, one could see the stout bacteria accompanied by long, thin, 

 faintly staining rods. In old nutrient agar cultures, and especially in Hansen's 

 fluid, thin rods with central granules or with terminal gi'anules were noted and 

 it therefore seems unlikely that the faintly staining rods are the gas-producing 

 bacteria. By the negative method of staining one could see here and there 

 structuifs which might be either spirochaetes or flagella but they were neither 

 sufficiently numerous nor sufficiently decided to be the active agent. Although 

 the large rod-shaped l)acteria were non-motile yet when appropriately stained 

 they were seen to have many peritrichous flagella and it is possible that a few 

 were shown by the negative stain . 



In dextrose brotli containing various nitrogenous nutrients such as meat- 

 extract, asparagin, urea or ammonium phosphate, acid and bleaching were pro- 

 duced in the first two and no gas was formed in any. The infecting material 

 was the condensed water of a gassy culture, so that it is probable that the active 

 organism does not grow freely in fluids. 



A repetition of the experiment with the same kind of infecting material 

 gave in the case of asparagin a production of gas by the 5th day. It was noted 

 that by this time the growth was very con.siderable, the broth being quite opa(|ue. 

 It was also noted that on the day following, the gas-bubbles were absent and the 

 volume of gas in the inner tube was less. The evolution of gas had apparently 

 stopped on the fifth day and whether this was due to a lack of nutrition or to 

 the sudden cooling of the culture on the fifth day during examination, further ex- 

 periment must decide. The active liquid was, on the fifth day, seeded into new 

 nsparagin broth and one day later this was bleached, showing a turbidity at the 

 surface. No gas developed even' after twenty days. 



It must not be considered that treatment of the bark is necessary to obtain a 

 growth of thermophilic organisms. These can be obtained directly from the 

 bark by rubbing fragments over an agar slope. These however do not produce 

 gas in the medium. 



Having apparently failed to obtain a pure culture of the active organism, it 

 was decided to try the effect of an infusion of tan-bark. Accordingly a quantity 

 of bark was mixed with twice its weight of water and heated at 60° for half an 

 hour, strained and filtered. The infusi(m was i]uite gummy and had an acidity 

 to phcnolphthalein of -)- 0.8.5°. One c.c. of the infusion was added to saccharose- 

 nutrient agar together with sodium carbonate to make the alkalinity = — 24°. 

 Upon the slopes colonies appeared and, at the same time, the medium was pierced 

 with gas bubbles. Small colonies showed up on the second day and these were 

 fished out and transferred to fresh slopes. The growths were all similar and 

 apparently the same as had been obtained previously. No gas was formed on 

 saeeharose nutrient agar, but gas was produced in the same agar with the ad- 



* An exceptional case. No gas was obtained in these media with the pure and 

 necessarily older races. But it was the occurrence of this gas-production in the sup- 

 posedly impure culture that prolonged the research imtil it became apparent that 

 no other kind of organism could be obtained. 



