EFFECT OF NITROGEN, ETC. 37 



was removed bj' the aid of pyrogallic acid and sodium hydrate, thus 

 leavino- practically an atmosphere of nitroo;en. The jar was i)laced in 

 ditiused lioht at a temperature of from 18- to 25^ C. At the expira- 

 tion of thirty-tive days it was opened and the beef broth was as clear 

 as if it had not been inoculated, showing that no growth had taken 

 place in the absence of oxygen. Twenty-four hours after the jar was 

 opened the tubes were clouded as deeply as if the inoculation had been 

 made the day the jar was opened instead of thirty-tive days prior to 

 that time. Hence, while nitrogen will not enal)lc the organism to grow, 

 its life is not destroyed by the action of this gas, and when inocula- 

 tions were made from these cultures into callas the disease promptly 

 appeared, and in forty-eight hours the inoculated leaves and llower 

 stalks had rotted off. Agar-poured plates made from the clouded tubes 

 and from the diseased portion of the inoculated calla showed the same 

 characteristic pure cultures composed of radiating colonies. To deter- 

 mine how nuich longer the organism w^ould live in the absence of oxy- 

 gen, cotton -plugged tubes of beef broth, Uschinsky's solution, and a 

 mixture of Dunham's and Uschinsky's solutions (half and half) were 

 inoculated with the calla organism and were kept in an atmosphere of 

 nitrogen two hundred and seventy-live days, in the manner described 

 above. At the expiration of this time the tubes, all of which were 

 clear, were exposed to the air at room temperature, i. e., IS ' to 25° C, 

 the same temperature at whicli they had been kept in the atmosphere 

 free from oxygen. The atmosphere in the jar would not support com- 

 bustion at the moment it was opened, indicating that the ox3'gen had 

 not diffused into it. In twenty -four hours after exposing the tubes to 

 the air the Uschinsky solution and the mixture of the Uschinsky and 

 Dunham solutions w^ere all clouded, but the beef-broth solutions were 

 not clouded. The clouding increased for several days in those tubes 

 in which it had begun, ))ut no growth appeared in the beef broth even 

 after several weeks of exposure to the air. Poured plates and inocu- 

 lations into healthy callas from the clouded tubes showed that this was 

 the calla organism. 



EFFECT OF CARBON DIOXID. 



Freshly inoculated tubes of slant agar, Uschinsky's solution, nitrate 

 bouillon, and common bouillon were placed in an air-tight jar into 

 which carbon dioxid was passed. Before the gas entered the jar con- 

 • taining the tubes it was passed through solutions of potassium per- 

 manganate, sodium hydrate, and distilled water. After being filled 

 and exhausted six times, to insure an atmosphere of pure carbon 

 dioxid, the jar was tilled with the gas, sealed, and allowed to stand for 

 fourteen days. At the expiration of this time it was opened and the 

 tubes were examined. The slant agar showed a thin, pure white growth 

 the whole length of the streak and a small amount of whitish precipi- 

 tate in the fluid in the angle fon^ncd l)y the agar and the side of the 



