Estimation of microbial viability using flow cytometry.

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold-standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so-called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

Characterization of an autostimulatory substance produced by Escherichia coli.

The recovery of dilute populations of stationary phase cells of Escherichia coli was studied using an automatic growth analyser. The addition of 30% supernatant from 2-d-old stationary phase cells of the organism reproducibly shortened the apparent lag times by 22-57.5%, depending on the age of the inoculum. True lag times, as determined by colony counts, of stationary phase cells were reduced by supernatant addition by 41-62%. The growth-stimulating substance was characterized and partly purified from supernatants: the active material was shown to be dialysable, heat-stable, acid- and alkali-stable and protease-resistant. Extraction with ethyl acetate or ion-exchange resins was not successful, but the active material could be quantitatively extracted with ethanol after saturation with salt. It is concluded that the active substance is a small, non-proteinaceous, non-ionic organic molecule. Separation of extracts by HPLC indicated that the stimulatory substance is weakly hydrophobic and has retention times similar to those of uracil. So far, however, the exact chemical identity of the active substance has not been elucidated.