The reason for failure to obtain growth of an obligatory anaerobe (Actinomyces necrophorus) on plate cultures incubated in an anaerobic jar.

Abstract

In their work on Bacillus (Actinomyces) necrophorus, Mohler and Morse 1 reported difficulty in inducing the organism to grow on plate cultures incubated anaerobically although they were successful in cultivating it on the same media in other ways. They do not discuss the cause of the difficulty.

Recently, in attempting to plate out some cultures of this organism we have experienced the same difficulty. In “hormone” agar shake cultures, good, though not vigorous, growth was easily obtained, but when the same medium was inoculated and poured into Petri dishes and incubated in a Brown anaerobic jar, failure to obtain growth has nearly always resulted.

In preparing the plate cultures, the inoculated medium necessarily is exposed to the air from the time it is poured into the plates until anaerobic conditions become established in the jar. Since the ratio of exposed surface to the volume of the medium is large, there is opportunity for the dissolving of a considerable volume of atmospheric gases, and it was thought that in this fact the cause of the failures might lie.

To test this hypothesis, fluid cultures were exposed to the air in shallow layers in flasks, and subcultures into cooked meat medium were made at intervals. It was found that, although most of the subcultures grew, there always was a period of bacterial lag, the duration of which varied directly with the time of the exposure up to 3 or 4 hours. From this time up to 8 hours, which is the limit of our observations, very little difference was noted in the subcultures. Whereas one loopful of young, normal culture sufficed to give, regularly, a vigorous subculture within 18 hours, a similar amount of the same culture exposed for 30 minutes to 1 hour gave subcultures showing a bacterial lag of 24 to 36 hours or more, and some cultures exposed for 3 to 4 hours have shown lag periods as long as 96 hours.