Acidobacteria, Rubrobacteridae and Chloroflexi are abundant among very slow-growing and mini-colony-forming soil bacteria.

Easily visible colonies of bacteria continued to form on plates inoculated with soil and incubated for 24 weeks. Using two different media, 13% and 29% of easily visible colonies appeared after more than 12 weeks. In addition, 10% and 18% of all colonies had diameters of 25-200 µm (mini-colonies), which could not be readily seen with the unaided eye. Members of soil bacterial groups that are only rarely cultured, such as members of the subclass Rubrobacteridae of the phylum Actinobacteria, members of subdivisions 1 and 2 of the phylum Acidobacteria and members of three subphyla of the phylum Chloroflexi, were more abundant among the easily visible colonies and mini-colonies that developed after > 12 weeks of incubation. Our results indicate that there is a hidden culturable diversity of soil bacteria that may require laboratory study at colony sizes and incubation periods outside those commonly anticipated by most microbiologists. Working at these scales increases the likelihood of obtaining cultures from groups of soil bacteria that have generally eluded laboratory study by cultivation methods.

Plasma 1,5 anhydroglucitol levels, a measure of short-term glycaemia: assay assessment and lower levels in diabetic vs. non-diabetic subjects.

An assay of plasma 1,5-anhydroglucitol was evaluated. Assay CVs, effects of four plasma freeze-thaw cycles, glucose up to 80 mmol/L and triglycerides up to 20 mmol/L were acceptable. 1,5-anhydroglucitol levels were significantly lower in diabetic vs. non-diabetic subjects and correlated inversely with renal function, but not with HbA1c.

Effect of pH on isolation and distribution of members of subdivision 1 of the phylum Acidobacteria occurring in soil.

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.

Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria.

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.

Detection and cultivation of soil verrucomicrobia.

Only one isolate each of the class "Spartobacteria" (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class "Spartobacteria" and were related to "Chthoniobacter flavus." Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class "Spartobacteria" were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class "Spartobacteria" were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class "Spartobacteria" was inhibited by other culturable bacteria.