Use of a microscope photometer to analyze in vivo fluorescence intensity of epilithic microalgae grown on artificial substrata

Abstract

An epifluorescence microscope photometer was used to develop a new, in vivo fluorimetric method for analyzing fluorescence intensities of epilithic microalgae grown on clay tiles in the field. This enabled a nondestructive, direct quantification of algal biomass on the substratum surface. Measurements of a chlorophyll a standard in ethanol (90%) with our fluorimetric method (exitation at 546 nm; emission, >590 nm) correlated well with those from conventional spectrofluorimetric and spectrophotometric methods. Biofilms were analyzed with the microscope photometer by measuring the in vivo fluorescence intensity of 70 spots distributed randomly over the tile surface. They were then analyzed by the two in vitro methods after photopigment extraction. Chlorophyll a content and in vivo fluorescence intensity correlated well. The regression curves were linear up to 6 (mu)g cm(sup-2) but were quadratic or hyperbolic at higher concentrations of up to 28 (mu)g cm(sup-2). The degree of scatter among individual measurements was higher in biofilms than chlorophyll a standards. This in vivo analysis is well suited to ecological experiments and has the advantage of measuring on an extremely small scale, which enables direct analysis of the microdistribution of epilithic microalgae in live biofilms. We demonstrated this by comparing fluorescence intensities of the grazing tracks of the snail Ancylus fluviatilis with those of ungrazed areas. Our in vivo analysis is also unique in enabling biofilms on artificial substrata to be removed, analyzed, and then returned intact in field or laboratory experiments.