Microbiology and flow cytometry

This material was originally published in the Purdue Cytometry CD-ROM Series.

Flow Cytometry and Microbiology

Aquatic Bacteria

Betsy R. Robertson and Don K. Button
Flow Cytometry Facility
Insitute of Marine Science
School of Fisheries and Ocean Sciences
University of Alaska Fairbanks
Fairbanks, Alaska 99775-1080, USA

email: brrob@ims.alaska.edu
email: dkbutton@ims.alaska.edu

Flow cytometry offers multiparameter analysis of bacteria on a cell-by-cell basis to characterize populations. It is particularly well-suited to natural populations of aquatic bacteria which are difficult to analyze by more traditional methods because of their small size (about 0.1µ m³) and resistance to cultivation. The primary focus of the laboratory has been on the characteristics and function of these organisms (1-4,8) which are a major component of the biomass in the ocean, and flow cytometry is improving our understanding of them. Versatility has allowed the analysis of bacterial populations from a range of diverse sources including various types of freshwater and marine systems, from surface to depth, in addition to hydrothermal vents, the Arctic ice edge, water wells, sewage, and sediments. Resulting data, used in conjunction with other techniques, have improved understanding of these organisms and their function. For example, strains believed to be typical have been isolated by extinction culture (5), cultivated, characterized, and used as standards for the evaluation of the size and activity of total populations present.

Bacterial biomass can be obtained from cell volume according to forward scatter intensity as specified by Rayleigh-Gans theory (7), cell density, and population. Values are central to formulations describing the nutrient processing ability of bacteria as well as for deriving appropriate theory on which the descriptions may be based.

Profiles of DAPI-DNA fluorescence intensity, along with population data, are used for cell cycle analysis. In combination with biomass data, information is obtained regarding cell mass accumulation during the growth cycle.

Other applications of flow cytometry in our laboratory include the identification of Hematodissidium spores (parasites) in tanner crab blood, detemination of the sex of kelp spores (6), confirmation of intact salmon brain nuclei in physiological studies of the smolting process, evaluation of the relative chlorophyll content of winter wheat chloroplasts, comparison of pigment fluorescence profiles of various tree pollens, evaluation of triploidy in salmon impinged by the Exxon Valdez oil spill, and evaluation of the effectiveness of filters used for water injection in oil wells on the North Slope of Alaska.

Data Examples

Fig. 1. Bacteria in Resurrection Bay, Alaska, stained with DAPI. Population: 6.1 x 10⁵/ml; mean cell volume, from Rayleigh-Gans theory: 0.107 µm³; apparent DNA content: 2.3 fg/cell.

Fig. 2. Distributions of marine isolate Cycloclasticus oligotrophus grown at two different rates (µ) under acetate-limiting conditions for cell cycle analysis.

Fig. 3. Mean cell volume, computed from forward scatter intensity according to Rayleigh-Gans theory, with corrections for relative refractive index of Cycloclasticus oligotrophus RB1, Marinobacter sp. strain T2, and E. coli. Profiles for bacteria in Resurrection Bay, Alaska, and in Lake Zurich, Switzerland, are based on data for marine isolate RB1. The volume indicated for the narrow profile of standard 0.6-µm microspheres (s) is overestimated due to the high relative refractive index of polylatex.

References
1. Button, D. K., and B. R. Robertson. 1989. Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry. Cytometry 10:558-563.

2. Button, D. K., and B. R. Robertson. 1993. Use of high-resolution flow cytometry to determine the activity and distribution of aquatic bacteria, p.163-173. In P. F. Kemp, B. F. Sherr, E. B. Scherr, and J. J. Cole (eds.), Handbook of methods in aquatic microbial ecology. Lewis Publishers, Ann Arbor, Michigan.

3. Button, D. K., B. R. Robertson, and F. Juttner. 1996. Microflora of a subalpine lake: bacterial populations, size, and DNA distributions, and their dependence on phosphate. FEMS Microbiol. Ecol.

4. Button, D. K., B. R. Robertson, D. McIntosh, and F. Juttner. 1992. Interactions between marine bacteria and dissolved-phase and beached hydrocarbons after the Exxon Valdez oil spill. Appl. Environ. Microbiol. 58:243-251.

5. Button, D. K., F. Schut, P. Quang, R. Martin, and B. R. Robertson. 1993. Viability and isolation of marine bacteria by dilution culture: Theory, procedures and initial results. Appl. Environ. Microbiol. 59:881-891.

6. Druehl, L. D., B. R. Robertson, and D. K. Button. 1989. Characterizing and sexing laminarialean meiospores by flow cytometry. Mar. Biol. 101:451-456.

7. Koch, A. L., B. R. Robertson, and D. K. Button. 1996. Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry. J. Microb. Met. In press.

8. Robertson, B. R., and D. K. Button. 1989. Characterizing aquatic bacteria according to population, cell size and apparent DNA content by flow cytometry. Cytometry 10:70-76.

Flow Cytometry Facility at the University of Alaska Fairbanks
The Flow Cytometer Facility at the University of Alaska Fairbanks has been in operation since 1986. Equipment includes an Ortho Cytofluorograf IIS equipped with two 5W argon lasers with UV capability (Coherent), adjustable focusing lens, 3.5-decade log circuitry, compensation module, sorter, and temperature controller. Data acquisition, analysis and storage have been upgraded with a Cyclops/Cicero system (Cytomation, Inc.) and 90 Mb Bernoulli storage media. A Coulter Counter (ZBI), epifluorescence microscope (Leitz, Dialux EB), and scanning spectrofluorimeter are available for standardization and verification of analytical results.

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