Howard-Jones, M. H. Skidaway Institute of Oceanography / Georgia Tech, email@example.com
Frischer, M. H. Skidaway Institute of Oceanography, firstname.lastname@example.org
Williams, S. C. Skidaway Institute of Oceanography, email@example.com
Verity, P. G. Skidaway Institute of Oceanography, firstname.lastname@example.org
DISTINGUISHING BETWEEN LIVING AND NON-LIVING CELLS : DEVELOPMENT OF A MODIFIED VITAL STAIN AND PROBE METHOD
Recently we developed a method combining DAPI, a vital stain propidium iodide (PI), and eubacterial specific 16S rRNA targeted oligonucleotide probes to identify cells with compromised membranes and those containing sufficient rRNA to be considered metabolically active. The VSP (vital stain and probe) allows for the differentiation between cells that are (1) alive, (2) alive and inactive, (3) dead, and (4) dead but recently active. However the current VSP technique requires separate treatment of PI (dead) and probed (active) cells. Systematic studies were performed with the goal of modifying the VSP so a single cell could be simultaneously interrogated with all VSP components. Results indicate that the modified method is most reliable when staining and probing are performed sequentially. Staining with PI prior to permeabilizing with Ethanol : Formaldehyde (1.9%) provides optimal probe hybridization while maintaining the staining properties of PI. Results also indicate that MeOH storage provides consistent PI staining and probe hybridization. Evaluations of the modified VSP were performed in reference to the established VSP and laboratory studies include E.coli, B.subtilis, Vibrio S14, and a Pseudomonas sp. The modified VSP was used to investigate proportions of living, dead, and moribund cells in samples from the South Atlantic Bight.
Day: Tuesday, Feb. 2
Location: Sweeney Center