Distinguishing between Living and Nonliving Bacteria in the Marine Environment: Evaluation of a Combined Molecular Probe and Vital Staining Procedure

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Bacteria are thought to be second only to phytoplankton in the role living organisms contribute to the transformation of organic carbon in aquatic ecosystems. In open ocean waters, the biomass of living bacteria has been reported to exceed that of all other living plankton, and even in continental shelf and coastal waters it is significant. However, these estimates are all derived from the assumption that all DAPI stained cells are metabolically active cells. This hypothesis no longer appears tenable: a large fraction of apparent bacteria are apparently inactive, moribund, or dead. Such a conclusion threatens to precipitate another paradigm shift in conceptual models of ecosystem function. At the very least, it would imply that a comparatively few bacteria in situ are growing (and respiring) much faster than previously thought, and that a large fraction of `apparent` bacteria is really a component of detritus. We have developed a novel combination of a fluorescent Vital Stain and molecular Probes (VSP) which quantitatively identifies those cells with compromised membranes, and those cells containing sufficient rRNA to be considered metabolically active. While neither approach alone might be considered adequate to specify active from inactive, moribund, or dead cells, together they potentially comprise a powerful tool to investigate the relative importance of these cell types in situ, and how they change in response to environmental pressures. We have already demonstrated the validity of the VSP procedure using E. coli, here we will examine its applicability to marine Gram positive, Gram negative, and oligophiles. We will then test the accuracy of the VSP to quantify living, non living, and starved cells in response to nutrients and viruses, and begin to document patterns of living and non living cells in natural communities. There is also promise of determining the numerical importance of inactive but living cells. The results will not only provide a stringent evaluation of the method for use in the marine environment, but the field data on active cells vs. those with compromised membranes will provide the first quantitative basis for a revised conceptual model of the role of bacteria in transformations between DOC, detritus, and bacteria. +n ±! ?n ¼f + _ Ûª? ÑOh ª' +'ª?0 P + Õ - Ã ? 0 T x À - Þ , R:\WWUSER\TEMPLATE\NORMAL.DOT n Bacteria are thought to be second only to ph S u m m a r y I n f o r m a t i o n ( ++++++++++++ 3 Ð ++++++++++++ ++++++++++++ ++++++++++++ ytoplankton in the role living organisms contribute to the transformation of organic carbon in aquatic ecosystems James William Ammerman Joanne MCcreary @ ?Nk+ ? @ -+Y+ª) @ ? -¹ ? L i @ dª Microsoft Word 6.0 7 Õªe =- e f f f f f f p f ? 1 z z z z z z z z Á t t t ? = ? = + ? T 3 Ó ? z z z z z ? z z z z z z z z z Á P X z Á z N z