Determination of Bacterial Biomass from Flow Cytometric Measurements of Forward Light Scatter Intensity

B.R. Robertson1, D.K. Button1

1 University of Alaska Fairbanks, Fairbanks, Alaska
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.9
DOI:  10.1002/0471142956.cy1109s09
Online Posting Date:  May, 2001
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This unit presents methods for measuring biomass of bacteria by flow cytometry. Such measurements are an integral part of determining growth rates and kinetics. This unit discusses calibration and standardization methods for bacterial studies using flow cytometry and provides a detailed discussion of the components involved. The serious microbiologist will appreciate the advantages flow cytometry brings to many aspects of microbial study, and this unit is no exception.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Cell suspension at ≤107 cells/ml
  • Formaldehyde (formalin, supplied as 37% formaldehyde), filtered (0.2‐µm pore) just before use
  • DAPI/Triton X‐100 staining solution (see recipe)
  • 0.96‐µm‐diameter fluorescent microspheres (Polysciences)
  • Internal standard microsphere mixture: 0.6‐ and 0.9‐µm fluorescent microspheres (Fluoresbrite Plain Microspheres; Polysciences) in filtered water, with 0.9‐µm spheres at a known concentration of 108/ml as determined by Coulter counter ( appendix 3A)
  • 1/10 dilution of internal standard microsphere mixture (0.9‐µm spheres at 107/ml)
  • Basal medium (carbon‐source free) with osmolarity similar to that of the bacterial sample, filtered (0.2‐µm pore)
  • Sheath fluid (water or basal medium), filtered (>0.1‐µm pore; Millidisk cartridges, Millipore)
  • Radiolabeled cells for calibration standard (Robertson et al., )
  • BASIC software program (Table 11.9.1)
  • Curve‐fitting software (e.g., SigmaPlot; Jandel Scientific)
  • Syringe filters, 0.2‐ and 1.0‐µm pore
  • Flow cytometry tubes
  • 10°C incubator
  • Flow cytometer with:
  •  UV excitation
  •  Flat‐sided quartz flow cell
  •  Photomultiplier detector for forward light scatter (FS) with the beam blocker positioned to eliminate laser beam while maximizing FS collection at >20°, 3.5‐decade dynamic range provided by a calibrated logarithmic amplifier or by a linear circuit
  •  429‐nm long‐pass dichroic filter to separate UV light scatter from DAPI fluorescence
  •  310‐ to 370‐nm band‐pass filters to isolate UV light scatter in both forward and orthogonal directions
  •  450‐ to 490‐nm band‐pass filter for isolating DAPI fluorescence
  •  Data analysis software that gives values for FS intensity proportional to amplitude of the input signal
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Literature Cited

Literature Cited
   Button, D.K. and Robertson, B.R. 1993. Use of high‐resolution flow cytometry to determine the activity and distribution of aquatic bacteria. In Handbook of Methods in Aquatic Microbial Ecology (P.F. Kemp, B.F. Sherr, E.B. Scherr, and J.J. Cole, eds.) 163‐173. Lewis Publishers, Ann Arbor Mich.
   Button, D.K., Robertson, B.R., and Jüttner, F. 1996. Microflora of a subalpine lake: Bacterial populations, size, and DNA distributions, and their dependence on phosphate. FEMS Microbiol. Ecol. 21:87‐101.
   Button, D.K., Robertson, B.R., Lepp, P.W., and Schmidt, T.M. 1998. A small, dilute, high‐affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl. Environ. Microbiol 64:4467‐4476.
   Heller, W., Nakagaki, M., and Wallach, M.L. 1959. Theoretical investigations on the light scattering of colloidal spheres. V. Forward scattering. J. Chem. Phys. 30:444‐450.
   Koch, A.L. 1961. Some calculations on the turbidity of mitochondria and bacteria. Biochim. Biophys. Acta. 51:429‐441.
   Koch, A.L. and Ehrenfeld, E. 1968. The size and shape of bacteria by light scattering measurements. Biochim. Biophys. Acta. 165:262‐273.
   Koch, A.L., Robertson, B.R., and Button, D.K. 1996. Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry. J. Microbiol. Methods 27:49‐61.
   Robertson, B.R. and Button, D.K. 1989. Characterizing aquatic bacteria according to population, cell size and apparent DNA content by flow cytometry. Cytometry 10:70‐76.
   Robertson, B.R., Button, D.K., and Koch, A.L. 1998. Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl. Environ. Microbiol. 64:3900‐3909.
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