Differentiating between living and non-living prokaryotic cells: Development, evaluation, and application of a modified vital stain and probe method (mVSP).

摘要

The focus of this project was the development, testing, and application of a novel combined vital stain and molecular probe (VSP) approach that allows for the in situ assessment of bacterial cells. The modified VSP method evaluates cells based on (a) membrane integrity by employing the vital stain propidium iodide (PI), (b) rRNA content via eubacterial-specific oligonucleotide probes, and (c) DNA content using the general stain DAPI. Together they comprise a tool, which can simultaneously interrogate individual cells with all three components. Validation studies included starvation and nutrient addition as well as assessing the response of the staining properties of the mVSP in cells subjected to viral infection. These studies found that the mVSP could accurately determine changes in the physiological status of cells under controlled laboratory conditions and resulted in the first in situ investigation of the physiological status of cells in response to environmental conditions. Extensive field evaluations found that natural populations were composed of bacterioplankton cells expressing a range of physiological states that varied with space and time. Side-by-side comparisons with other activity measurements (CTC, leucine) supported these findings. The combination of field and lab studies resulted in the development of an accurate and reliable method for determining the physiological status of individual bacterial cells.