On the Determination of the Size of Microbial Cells Using Flow Cytometry

Abstract

In most flow cytometers the determination of cell size is based on the measurement of light scattered by cells as they pass through an illumination zone. In conventional instruments the source of this illumination is a laser (Shapiro 1988). Given that both the cell volume and the DNA content of bacteria is some 1000-fold less than that of higher eukaryotic cells, however, laser-based flow cytometers have generally proved unsuitable for the study of microorganisms (Steen et al. 1990). In the Skatron Argus 100 flow cytometer, a high-pressure mercury arc lamp is used as the excitation source, and the machine makes use of an open flow chamber in which a jet impinges at an angle onto the surface of a microscope cover slip (Fig. 4.1). The result is a flat, laminar flow of water across the glas & surface. This flow has only two interfaces — the glass/water interface and the water/air interface — and of these only the former can collect particles that may cause background scattering of light (Steen et al. 1989). Furthermore the orientation of these surfaces perpendicular to the optical axis means that the surfaces themselves scatter only the minimum of light. Thus the system has a high signal-to-noise ratio and is therefore ideal for detecting light scattered by microorganisms (Boye et al. 1983).