328 DAN H. JONES 



and subcultures. But after a prolonged series of alternate 

 replatings and flask cultivations, these spore-forming rods ap- 

 peared to be eliminated. It was a comparatively simple matter 

 to isolate the spore-forming rods from the azotobacter, but very 

 difficult to isolate the azotobacter from the spore-forming rods. 

 The spore-forming rods on subsequent cultivation proved to be 

 other species than azotobacter. 



As stated in the article "Further Studies of Some Azotobacter" 

 previously mentioned, the writer tested the thermal death point 

 of four varieties of this organism from a series of cultures ranging 

 in age from sixteen days to two years and two months, and in 

 every case the cultures heated to G5°C. or over, failed to give 

 any subsequent growth. 



In July, 1919, the stock cultures of azotobacter on Ashby's 

 agar that had remained untouched since 1914, or earlier, were 

 again tested for thermal death point in the recognized manner, 

 except that 10 cc. of Ashby's solution was used instead of water 

 or bouillon in the test tubes that were heated. In every case 

 a generous loopful of the culture was transferred to the test 

 tubes, which, on being stirred in, was not thoroughly broken up, 

 as a considerable number of macroscopic particles of culture 

 remained unbroken, thus favoring resistance to heat on the part 

 of the organisms constituting these particles. Again all cultures 

 heated to 65°C. and over failed to give any subsequent azoto- 

 bacter growth, while all controls gave good azotobacter develop- 

 ment. Thus it was concluded that these four varieties of azoto- 

 bacter even in cultures that had been kept for four years at 

 room temperature, did not produce heat-resistant endospores. 

 However, on plating out on beef peptone agar from these stock 

 cultures, some of them produced colonies of encapsulated spore- 

 forming rods both large and small. These on isolation and sub- 

 culture did not develop azotobacter characteristics. On Ashby's 

 agar or in Ashby's solution some would not grow at all and 

 others only to a very limited extent, while on beef media they 

 made good development. It was therefore concluded that the 

 resistant spore-formers, which were found present only in very 

 limited numbers, and not in all the cultures tested, were con- 

 taminations. 



