The sealed chambers were placed on the surface of the sediment collected from the tidal flat and kept in a marine aquarium (Fig. 1B). A thin layer of air was left between the agar and the top membrane. In the aquarium, this space was filled with seawater. This design allowed us to observe the undisturbed agar surface after peeling off the top membrane.
A large number of colonies of varying morphologies were observed after 1 week of incubation in the chambers (Fig. 2A). Most of these (.99%) were microcolonies invisible to the naked eye. Addition of 0.01% casein increased the number of colonies in the chamber, and this supplement appeared supe- rior to starch or marine broth tested at a variety of concentrations (20).
In a series of microbial recovery experi- ments (20), we determined the fraction of cells that formed colonies inside the cham- bers compared with the standard petri dish method (Fig. 2B). The greatest microbial col- ony recovery in the chambers represented 40 6 13% of the cells inoculated and came from a sample obtained in June 2001. The number of microcolonies obtained in differ- ent months ranged from 2 to 40% of the cells inoculated, with an overall average of 22 6 13%. This is likely an underestimate, because the total direct microbial count included dead
cells, our colony-counting technique pro- duced conservative estimates (20), and the fairly dormant March sample skewed the re- covery results. Representative microorgan- isms from the chambers were successfully isolated in pure culture by passage to new chambers. Of the 33 colonies passaged, 23 produced sustainable growth in the chambers at the first attempt.
Unexpectedly, a significant number of mi- crocolonies appeared on the petri dishes (6 6 4% of the number of cells inoculated). We investigated their ability to produce sustainable growth in three independent trials. Each time, 27 to 30 microcolonies were passaged to a new petri dish. Most of the transfers (86 6 7%) did not result in microbial growth. It seems that the majority of microorganisms from the sediment could only undergo a limited number of divi- sions on a petri dish. The microcolonies that did grow after passage to petri dishes (14%) ap- peared to represent mixed cultures, and only those that produced rapidly growing macro- colonies, visible to an unaided eye, seemed capable of sustained growth on petri dishes. Counting visible colonies is the conventional method of performing microbial plate counts (24). Such petri-dish macrocolonies made up 0.054 6 0.051% of the inoculum, consistent with previous reports (15–17). Finally, ;300- fold as many microorganisms produced sustain- able growth in the growth chambers as in stan- dard petri dishes.
We attempted to isolate into pure culture some of the microorganisms grown in the diffusion chambers (20). The isolates were considered pure if no contaminants could be detected microscopically or by polymerase chain reaction amplification of 16S ribosomal RNA (rRNA) gene (20). Several passages were required to achieve purity. Passages typically produced hundreds of microcolo- nies per chamber, which was more than suf- ficient for the purposes of the present study.