Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry

Abstract

Forward scatter signals (0–20°) from a flow cytometer can be converted into cellular volumes or cellular wet or dry biomasses by the calculations presented here. The results are applicable to bacteria, and including very small bacteria (0.02–0.6 μm 3), rods as well as cocci. The physical limitations and the mathematical basis of the calculations are discussed to justify the application of the proposed formula for practical use in microbial ecology and physiology of extremely small aquatic bacteria as well as the more well-studied forms. Although the method was developed for the small bacteria present in natural populations that are difficult to size by alternative methods, it will also be useful for laboratory cultures of bacteria when present in very low concentrations as well as for the characterization of subpopulations and different growth phases. Bacteria, in general, are small enough so that inclusion of the phase shifts of the light passing through the bacteria (the Mie theory) is superfluous. That theory is, however, useful for instrumental calibration with latex microspheres and for some blue-green algae. Even the interference between rays scattered from different parts of the cell (the Rayleigh-Gans theory), although significant, is minor, particularly from elongated particles which are oriented by the fluidics. The computer programs, provided here, correct for intraparticle interference and generate cell volumes (or wet or dry masses) from the forward scatter intensity. The result of the calculations of the light scattered into the collection system of the flow cytometer is presented. From analysis of these values a rule is developed to relate the observed intensities, when scaled by an empirical constant and raised to a power between 0.5 and 0.75, to the cell biomass.