82 HIGH TEMPERATCRE ORGANISM OP FERMENTING TAN-BARK, 



growth of the active gas jiroduccr. The familiar spore-bearing rods persisted 

 in coining up and no other orgjuiism could be isolated. 



A bottle of active stack-bark originally covered with N/5 soda had been set 

 aside in the incubator at 37° and had grown a good crop of Aspergillus. The 

 same mould aijpeared in the original sample of stack-bark and had apparently 

 survived the action of the soda and the lengthy exposure at (50° . With the idea 

 that possibly this might be the active agent, a tube of saccharose nutrient agar 

 was infected with the spores and incubated at 37° . In three days, gas bubbles 

 appeared in the medium under a dense mycelial growth. It was also sown in 

 combination with the thermophilic rod and incubated at 60" but there wai; no 

 growth of mould apparent and no gas foi-med. An attempt to acclimatise the 

 Aspergillus to 60° by gradually raising the temperature from 37° was a failure 

 and the conclusion was come to that the Aspergillus was not responsible for the 

 fermentation of tlic sa<'cliarose. 



It had been noted that the fluid taken' directly from an alkaline bark liciuor 

 and sown on saccharose nutrient agar, gave a good growth of bacteria and a 

 medium blown up with gas bubbles. The turbid condensed water at the base 

 of the slope, when transferred to a second agar-slope, sometimes gave rise to gas 

 production but generally did not. Colonies picked from the first tube never 

 prodiu-ed gas. Thus the gas production was fugitive. 



The failure to obtain a gas-forming organism in pure culture and indeed 

 to obtain any organism capable of growing at 60° other than the drumstick rod 

 led to the idea that probably an alkaline condition of the agar was necessary for 

 the evolution of gas, just as it was necessary to stimulate the growth of the cells 

 from the bark. Possibly enough alkali was contained in the large loop used for 

 seeding the tubes from the alkaline bark liquids, and as this disappeared on 

 subculture so did the gas t'orniation. 



The saccharose nutrient agar had an acidity to phenolphthalcin of -|- 'l.'l° 

 and tubes of this were treated with increasing quantities of normal sodium car- 

 bonate and seeded with the condensed water of an active culture. Growth and 

 gas were obtained on media having an alkalinity of — 14.4" and over, but not in 

 media ranging from -\- 2.2° to — 12°. Tiie limiting amount for growth ap- 

 peai'ed to be about — 24°, for witli this alkalinity and with — 26.3° the agar 

 had to be seeded several times. 



Some of the races which had been isolated at different times, and were pre- 

 sumably pure, were grown on agar brought to approximately — 13° with Na2C03. 

 Out of 16 races, two failed to gi'ow, three gave a growth of cells and no gas 

 while the remainder produced growth with gas. The inactive bacteria could not 

 be distinguished IVdUi the otlicrs. 



The gas seems to be produced from the sugar, for tubes of alkalised nutrient 

 agan without sugar gave luxuriant growths without gas when seeded with a cul- 

 ture which gave gas in the presence of sugar. 



Thus we arrive at the fact that the drum-stick/ rod is the gas producer, and 

 that the production of gas from saccharose by the pure races of the bacterium 

 depends upon an alkaline condition of the medium. 



An experiment was made with saccharose nutrient agar coloured with litmus 

 and treated with increasing quantities of sodium carbonate. The slopes were 

 stroked with an active race. 



