King's College London Dental Institute, Infection Research Group, Guy's Campus, London SE1 9RT, UK.
Environ Microbiol. 2010 Apr;12(4):916-28. doi: 10.1111/j.1462-2920.2009.02135.x. Epub 2010 Jan 13.
Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.
从患有牙周炎的人类受试者中获得的龈下菌斑样本，其中包括以前无法培养的协同菌门成员，用于接种熟肉培养基（CMM）。通过荧光原位杂交（FISH）和定量 PCR（Q-PCR）监测 A 簇（无法培养）协同菌的存在。发现 A 簇协同菌可以与其他菌斑细菌一起在 CMM 中生长，并且添加粘蛋白和血清可以刺激生长。菌斑样本也用于接种血琼脂（BA）平板，通过与特定探针的菌落杂交，在厌氧孵育后揭示 A 簇协同菌的生长。通过菌落杂交鉴定的平板表面生长区域被收获并用于接种新鲜平板，从而富集 A 簇协同菌。其他菌斑细菌的交叉划线也用于刺激协同菌的生长。在早期传代中，没有看到离散的协同菌菌落，但经过 8 次传代和使用富集群落中存在的其他细菌的交叉划线后，出现了菌落，这些菌落仅由 A 簇协同菌细胞组成，这是通过 16S rRNA 基因 PCR 和克隆确定的。这是首次成功培养该门不可培养分支成员的报告。
