Density gradient separation of active and non-active cells from natural environments.
We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.
Whiteley
A S
AS
Centre for Molecular Ecology, University of Newcastle upon Tyne, UK.
Barer
M R
MR
O'Donnell
A G
AG
D000818:Animals; D001419:Bacteria / Q000166:cytology*; D002475:Cellobiose; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004275:DNA, Ribosomal / Q000032:analysis; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D011205:Povidone; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012623:Seawater / Q000382:microbiology*; D012822:Silicon Dioxide; D013778:Tetrazolium Salts
D004275:DNA, Ribosomal; D007202:Indicators and Reagents; D012336:RNA, Ribosomal, 16S; D013778:Tetrazolium Salts; C013574:iodonitrotetrazolium; D002475:Cellobiose; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D005947:Glucose