microorganisms from environmental samples in diffusion chambers

Isolating “Uncultivable” Microorganisms in Pure Culture

in a Simulated Natural Environment

T. Kaeberlein, K. Lewis,* S. S. Epstein*†

The majority (.99%) of microorganisms from the environment resist culti- vation in the laboratory. Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives. We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a sim- ulated natural environment. Colonies of representative marine organisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. This observation may help explain the nature of microbial uncultivability.

The number of existing microbial species is estimated at 105 to 106 (1, 2), but only several thousand have been isolated in pure culture (3), because few microorganisms from envi- ronmental samples grow on nutrient media in petri dishes (4–16). Attempts to improve the recovery of microorganisms from environ- mental samples by manipulating growth me- dia have met with limited success (6, 15, 17–19), and the problem of uncultivability remains a major challenge (4).

We reasoned that uncultivable microor- ganisms might grow in pure culture if pro- vided with the chemical components of their natural environment. To allow access to these components, we placed microorganisms in diffusion chambers and incubated the cham- bers in an aquarium that simulated these or- ganisms’ natural setting.

Intertidal marine sediment was used as a source of microorganisms (20). The upper layer of the sandy sediment harbors a rich community of microorganisms, primarily aerobic organoheterotrophs, which reach densities of .109 cells/g (21) and are most- ly uncultivated (22, 23). These microorgan- isms were separated from sediment parti- cles, serially diluted, mixed with warm agar made with seawater, and placed in the dif- fusion chamber (20) (Fig. 1). The mem- branes allow exchange of chemicals be- tween the chamber and the environment but restrict movement of cells. After the first membrane was affixed to the base of the chamber, the agar with microorganisms was poured in, and the top was sealed with

another membrane (Fig. 1A). 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.