No abstract

Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed: by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3-dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful.

The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.

No abstract

Flow cytometry was used to study the lag, exponential, stationary and death phases of non-fixed cultures of Escherichia coli. Fluctuations in the forward angle scatter signal (FALS) were compared with cell size as measured by scanning electron microscopy at low temperature and image analysis. A correlation between FALS and cell size was not observed, although a correlation (r = -0.8) was obtained between FALS and the age of the culture for the first eleven days of incubation. Marked increases in FALS were observed during the lag phase, which were attributed both to changes in size and changes in structure or chemical composition. The distribution of FALS for all culture phases was asymmetric, and was associated with the cell size distribution.