Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria
Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria
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DOI：
10.1128/aem.71.2.826-834.2005
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发表时间：
2005-02-01
影响因子：
4.4
通讯作者：
Janssen, PH
中科院分区：
文献类型：
--
作者：
Davis, KER;Joseph, SJ;Janssen, PH
土壤中有许多来自系统发育组的细菌，这些细菌的研究很少，因为在培养研究中很少分离出代表性的细菌。未能培养这些细菌的部分原因是，当接种到标准微生物培养基上时，土壤中的细菌细胞形成可见菌落的频率较低，导致活菌计数较低。我们研究了三个因素对活菌计数的影响，评估为固体培养基上的CFU数，以及分离的菌落形成细菌所属的系统发育组。这些因素是接种量、生长培养基和孵育时间。减少接种量导致活菌计数显着增加，但似乎并不影响很少孤立的群体成员的菌落形成。传统上用于土壤微生物研究的一些培养基返回的活菌计数较低，并且不会导致很少分离的群体的成员分离。新开发的媒体，相反，导致高活菌计数和隔离的许多成员很少孤立的群体，无论接种量。增加孵育时间长达3个月，允许结合使用适当的培养基，形成罕见分离群体成员的可见菌落。一旦被隔离，很少隔离的群体成员的纯培养物比通常隔离的群体成员需要更长的时间才能形成可见的菌落。使用这些新的培养基和延长的孵育时间，我们能够分离出许多酸杆菌门（亚门1、2、3和4）、芽单胞菌门（Gemmatimonadetes）、绿弯菌门（Chloroflexi）和浮游菌门（包括以前未培养的WPS-1谱系的代表）的成员，以及放线菌门（Actinobacteria）的红杆菌科（Rubrobacteridae）和酸性微杆菌科（Acidimicrobidae）的成员。
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.