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Applied and Environmental Microbiology, October 1998, p. 3900-3909, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

B. R. Robertson,1,* D. K. Button,1,2 and A. L. Koch3

Institute of Marine Science1 and Department of Chemistry and Biochemistry,2 University of Alaska Fairbanks, Fairbanks, Alaska 99775, and Biology Department, Indiana University, Bloomington, Indiana 474053

Received 27 February 1998/Accepted 23 July 1998

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4',6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 µg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 103 cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

* Corresponding author. Mailing address: Institute of Marine Science, University of Alaska Fairbanks, Fairbanks, AK 99775-1080. Phone: (907) 474-7709. Fax: (907) 474-7204. E-mail: brrob{at}ims.uaf.edu.

Applied and Environmental Microbiology, October 1998, p. 3900-3909, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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