Direct estimation of biofilm density on different pipe material coupons using a specific DNA-probe

    Authors

    Y. C. Chang; M. Le Puil; J. Biggerstaff; A. A. Randall; A. Schulte;J. S. Taylor

    Comments

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    Abbreviated Journal Title

    Mol. Cell. Probes

    Keywords

    BO-PRO (TM)-3; drinking water; confocal laser scanning microscope; epifluorescent microscopy; fluorescence spectroscopy; HYDRODYNAMIC BOUNDARY-LAYERS; DRINKING-WATER; SURFACE MICROENVIRONMENTS; DISTRIBUTION-SYSTEM; RESPIRING BACTERIA; IMAGE-ANALYSIS; DISINFECTION; MICROSCOPY; BIOMASS; SUBSTRATUM; Biochemical Research Methods; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology; Cell Biology

    Abstract

    A variety of approaches to quantify biomass in biofilms without disruption due to detachment have been developed over the years. One basic approach is the combination of advanced microscopy with molecular staining. However, many stains (e.g. 4',6-diamino-2-phenylindole, acridine orange or live-dead stains) can be non-specific when corrosion products, precipitates, and pipe material are present. In addition, some pipe materials cause high background when using epifluorescent microscopy. The new refinement discussed in this presentation used fluorescence spectroscopy to obtain the spectra from four common distribution system pipe materials: PVC, 'concrete' lined cast iron, cast iron, and galvanized steel. The emission maximum for all four materials was between 500 and 550 nm, but emissions radically decreased around 575-600 nm. A molecular probe, BO-PRO(TM)-3 (Molecular Probes, Inc., Eugene, OR, USA) was identified which has an emission intensity maximum at 599 nm (red), with emission intensity 200 times greater when it is bound to DNA. The BO-PRO(TM)-3 has greatly reduced non-specific staining and background problems. In the preliminary experiment, using diluted waste water, a significant exponential relationship was found between stained surface area/total area ratio and fixed biofilm inventory measurements from scraping heterotrophic plate counts (SHPC) on R2A medium. In addition, the biofilm inventory on different pipe material coupons from pilot distribution systems was also correlated to the stained surface area fraction and SHPC. (C) 2003 Elsevier Ltd. All rights reserved.

    Journal Title

    Molecular and Cellular Probes

    Volume

    16

    Issue/Number

    5

    Publication Date

    1-1-2003

    Document Type

    Article

    Language

    English

    First Page

    237

    Last Page

    243

    WOS Identifier

    WOS:000180407000011

    ISSN

    0890-8508

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