University of California Berkeley, Department of Chemical Engineering, 201 Gilman Hall, Berkeley, California 94720, USA.
Appl Environ Microbiol. 2011 Mar;77(6):2130-40. doi: 10.1128/AEM.01203-10. Epub 2011 Feb 4.
Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.
三种方法被用于培养与海洋海绵 Haliclona (gellius) sp. 相关的细菌：琼脂平板培养、液体培养和漂浮滤器培养。使用了多种贫营养培养基，包括含有水相和有机海绵提取物、细菌信号分子和铁载体的培养基。分析了超过 3900 个分离株，并鉴定了 205 个操作分类单元 (OTU)。含有低浓度黏蛋白或蛋白胨和淀粉混合物的培养基最适合多样性的分离，而常用的海洋肉汤并没有导致分离株的多样性增加。抗生素的添加通常会导致平板上的多样性减少，但与其他培养基相比，会产生不同的细菌。此外，琼脂平板、液体培养和漂浮滤器分离株的多样性模式有显著差异。几乎 89%的所有分离株都是α变形菌；然而，也分离到了在培养研究中较少遇到的门的成员，如浮霉菌门、疣微菌门和δ变形菌门。海绵相关细菌被分为三个不同的组。第一组代表从先前分析的海绵组织的克隆文库中获得的 OTU(组 1)。此外，我们区分了从海绵组织中获得但未从海绵分离株中获得的 OTU(组 2)，还有一些 OTU 未从海绵组织中获得但从海绵分离株中获得的 OTU(组 3)。归入组 1 的 17 个 OTU 代表了先前从 Haliclona (gellius) sp. 海绵组织中生成的大型克隆文库中存在的所有细菌 OTU 的 10%至 14%，这高于以前报道的海绵相关细菌的可培养性分数。这 17 个 OTU 中有 6 个未从琼脂平板中获得，这强调了使用多种培养方法可以增加海绵可培养微生物的多样性。
