Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC+ cell sorting.
Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green II fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction (CTC+ cells). Parallel microcosms, maintained at either field water capacity or subjected to drying, indicated that the total extractable cell count remained between 10(8) and 10(9) g(-1) (dry weight). In contrast, the CTC+ active count fell threefold in dried microcosms (6% of total cell count) when compared to wetted microcosms (18% of total cell count). Specifically, these data highlighted an overall deactivation of microbial biomass during water stress, with 16S rDNA analyses of flow-sorted CTC+ cells demonstrating shifts within the active diversity. Flow cytometry coupled with cell purification techniques represents a significant tool for operationally defining an active and redundant microbial component within soil communities and is demonstrated during water stress.
Whiteley
Andrew S
AS
Molecular Microbial Ecology Laboratory, CEH-Oxford, Mansfield Road, Oxford OX1 3SR, UK. aswhi@ceh.ac.uk
Griffiths
Robert I
RI
Bailey
Mark J
MJ
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D005434:Flow Cytometry; D009997:Osmotic Pressure; D012988:Soil Microbiology; D013778:Tetrazolium Salts / Q000032:analysis*; D017186:Titrimetry / Q000379:methods; D014871:Water Microbiology
D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium