Rapid Assessment by Flow Cytometry

Judith A. Noble-Wang, Shangtong Zhang, Daniel Price, and Donald G. Ahearn

1. Introduction

Conventional methods for the evaluation of antimicrobials and disinfecting solutions with microorganisms involve culture-based techniques, which are time-consuming and underestimate the number of viable organisms. Rapid detection and viability measurements of microorganisms in homogenous and heterogenous microbial populations have been greatly enhanced by recent advances in the use of fluorescent stains in flow cytometry (FCM) (1-5). FCM has been applied to enumerate, differentiate, and identify microorganisms, determine protein and DNA content of cells, analyze the physiological state of individual cells, and analyze the interaction of drugs, antibiotics, and antimicrobials with microbial cells (1-14). Four physiological states of cells can be distinguished by FCM: (1) reproductively viable, (2) metabolically active, (3) intact, and (4) permeabilized (15).

FCM permits a rapid and quantitative measurement of the optical characteristics of cells as they pass through, in a single file, a focused beam of light (4). As cells are carried within a fast-flowing fluid stream and through the focus of exciting light, three parameters are measured: forward angle light scatter, side angle light scatter, and fluorescence emitted by dyes that have specific interaction with intracellular components of individual cells. FCM data that are presented in histogram and dot plots can be generated to give information on a variety of properties of interest among cells in the population as a whole.

FCM offers major advantages in multiparameter data acquisition and multivariate data analysis, high-speed analysis, and cell-sorting capabilities. Disadvantages may be associated with the cost, which is usually over $100,000 (US) for a typical laser-

based flow cytometer with just analyzing capabilities. Another disadvantage is that skilled personnel are usually required to operate these complex instruments so as to get optimum performance (3). A schematic overview of flow cytometry is presented in Fig. 1.

FCM analyses with viability dyes, such as propidium idodide and oxonols, can assess cell viability after treatment with antimicrobial and disinfecting solutions. A breakdown in membrane integrity and a loss of transmembrane potential are indicators of cell death. Propidium iodide (absorbance: 535 nm; emission: 617 nm) has been widely used to indicate membrane integrity (2,4,7,14). Propidium iodide stains nucleic acids but will not diffuse appreciably into intact cells; therefore, this dye preferentially stains dead cells that have permeable membranes (4). Changes in membrane potential can be detected with some oxonol dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol (Ox). Oxonols are lipophilic anionic dyes, and their accumulation within cells is favored by a reduction in the magnitude of membrane potential that enables dye molecules to concentrate within the cell by association with intracellular macromolecules (5).

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