A variety of approaches to quantify the biofilm 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 (4',6-diamino-2-phenylindole (DAPI), 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, cast iron, "concrete" lined cast iron, and galvanized iron. 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-PRO3 was identified which has an emission intensity maximum at 599nm (far red), with emission intensity 200 times greater when it is bound to DNA. The BO-PRO3 has greatly reduced non-specific staining and background problems. In the preliminary experiment, using diluted wastewater, a significant linear relationship was found between stained surface area/total area ratio and fixed biomass measurements from heterotrophic plate counts (HPC) on R2A medium and total direct count (TDC). In addition, the biomass on different pipe material coupons from pilot distribution systems was also correlated to the stained surface area fraction and HPC. Includes 10 references, tables, figures.
