452 STATE BOARD OP AGRICULTURE 



gas formation to a number of different tubes of media both solid and liquid, 

 and incubated under sterile paraffin at 55°C. A considerable number of 

 these media showed growth within 15 to 18 hours as evidenced by vigorous 

 gas production, but even after several da3^s no colonies could be observed 

 even in the clearest media in which gas was formed. The plain agar em- 

 ployed was exceptionally translucent j^et no visible growth was evident. 

 The fact remained, however, that groAvth was present or gas would not have 

 continued to form, consequently a very careful examination was made using 

 a hand lens and an artificial light, manipulating the tube this way and that 

 to get the effect of both transmitted and reflected fight. The result of this 

 effort was that quite large lenticular colonies could be seen only when the 

 tube was held a certain way between the observer and the light. The "low 

 visibility" of these colonies was due to the fact that they had about the 

 same refractivity as the agar in which they were growing. Having con- 

 vinced different members of the laboratory staff after much trouble that 

 colonies we^x actually present, these colonies were variously described as 

 appearing like "ghosts" of colonies, fike "the place where small gas bubbles 

 had been," or "like pieces of something in the agar." After the observer 

 knew what to look for these colonies were quite easily discernible to the 

 unaided eye. Barlow called the gas bubbles forming in his corn agar shakes 

 "gas colonies" and made his isolations from a well isolated gas bubble. It 

 has been found impossible to make a satisfactory photograph of these colonies, 

 but young cultures show gas formation. 



This organism seemed to grow best in a tube shake culture in certain media 

 which favored it when placed under anaerobic conditions. 



Visible colony formation has never been observed in anaerobic plate cul- 

 tures using paraffin, or pyrogallic acid and sodium hydroxide; in fact, anaerobic 

 tube and plate cultures have never shown growth using the latter method, 

 even when the most favorable media have been employed. It is quite evi- 

 dent that an anaerobic condition could never be obtained employing an ordi- 

 nary aerobe to partially or totally exhaust the oxygen, as with Giltner's 

 H-tube, especially if the culture were placed immediately at 55°. An 

 attempt was made to obtain surface growth by first exhausting the oxygen 

 by placing the H-tube containing the thermophile and B. subtilis at the 

 optimum temperature for the latter organism until a maximum growth had 

 been obtained, then placing the culture tube at 55°, but without success. 

 No surface growth, as was determined by direct microscopical examination 

 and by culturing, could be obtained by any of the ordinary anaerobic meth- 

 ods. The possibilities of obtaining surface growth, however, have not been 

 exhausted. In many media in which Str. BIG grew but had not been ob- 

 served to form gas, growth was not evident for several weeks. Perfectly 

 clear broth was inoculated and covered with sterile paraffin. No gas or 

 clouding was ever evident but after about a month at 55° a moderate amount 

 of whitish, flaky sediment was found which upon examination proved to con- 

 sist of bacteria and spores. The odor of the cultures in all media which 

 showed gas production, after about a month at 55° was from a rather pleasant 

 fruity odor to one that was more or less disagreeable according to the medium 

 and to the observer; in fact the peculiar odor was practically the only gross 

 test for growth in media in which the organism was multiplying but in which 

 its development was not evident to the eye. 



A list of the media follows in which this organism has been cultured. 

 The several media in which growth was evident by gas formation or by odor 

 only, is indicated by a + sign. 



