Question

How disease disease occurrence depends on inoculum size and incubation period?

Answer 1

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes(including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.

Soils contain phylogenetic groups of bacteria that are globally distributed and abundant in terms of the contributions of individuals of those groups to total soil bacterial communities (3, 10, 23). However, until recently, no representatives of many of these groups were available for detailed study due to their apparent inability to grow in or on laboratory media. Part of the reason for this is that only a few (often only about 1%) of the >109 bacterial cells in each gram of soil seem able to form colonies on laboratory media (5, 14, 28). This means that many groups of soil bacteria cannot be easily studied due to the inability of microbiologists to grow representatives in the laboratory. Some isolates of these groups have recently been cultured by the use of new culture media and extended incubation periods to increase the numbers of colonies formed and by the selection of isolates from plates receiving only small inocula and yielding only small numbers of colonies (12, 15, 24). These approaches were chosen empirically in previous studies. Among the isolates obtained were many members of the phylum Acidobacteria as well as some members of the phyla Verrucomicrobia and Gemmatimonadetes and of the subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria (12, 15, 24). These groups are very poorly studied due to the paucity of cultured representatives from soils.

The aim of the present study was to investigate the effects of growth medium, inoculum size, colony density, and incubation time on the appearance of colonies of members of these poorly studied groups of soil bacteria on plates of solid growth media inoculated with soil