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

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-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-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.