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    Direct Epifluorescence Enumeration of Native Aquatic Bacteria: Uses, Limitations, and Comparative Accuracy

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    The suitability and comparative accuracy of the acridine-orange epifluorescence direct-count (AODC) technique for determining the numbers, biomass, and metabolic state of aquatic bacteria are reviewed, The AODA method has been indirectly verified as the best available procedure for estimating the total numbers of native aquatic bacteria. It is quantitative, precise, and suitable for use with both pelagic and epibenthic bacteria in all freshwater and marine environments. The development of a standard method should be possible by making only a few technical improvements. However, AODC numbers cannot be converted directly to total carbon biomass because of natural variations in bacterial cell sizes. It is possible that better biomass estimates can be obtained by using combined AODC measurements of cell numbers and cell volumes, but the accuracy of this approach is still to be determined. The alternative, indirect estimation of lipopolysaccharide (LPS), appears, from limited tests, to be accurate for planktonic marine bacteria; however, interference from blue-green algal LPS may restrict its use in fresh waters and sediments.

    The distribution of dead, moribund, viable, and growing bacteria within the total population visualized by epifluorescence is unknown. However, recent indirect evidence suggests that most, if not all, native bacteria are viable, but that many are metabolically dormant. Neither the AODC technique nor any of the “living” biomass techniques (plate, membrane, or liquid growth techniques, ATP assays, autoradiography) now used can distinguish the functional states of native bacteria. Autoradiography combined with epifluorescence has the best potential for such measurement, but further assessment is necessary.

    The usefulness of the AODC method for descriptive surveys, correlative studies, impact assessment activities, and experimental studies of bacterial dynamics is summarized. Epifluorescence enumeration, in conjunction with activity measurements and filter fractionation procedures, holds considerable promise for examining the productivity and nutrient uptake dynamics of native aquatic bacteria.


    aquatic bacteria, epiflourescence enumeration, acridine orange, AODC technique, bacterial biomass, microbial dynamics

    Author Information:

    Daley, RJ
    Chief, National Water Research Institute, Environment Canada, West Vancouver, British Columbia

    Committee/Subcommittee: D19.24

    DOI: 10.1520/STP36001S