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Confocal Microscopy Proposal
I propose to use the confocal microscopy technique to estimate the amount of living biomass available in the biofilm on stream substrates, such as rocks, over time. Benthic macroinvertebrates in the scraper functional feeding group (such as some mayflies and caddisflies) get most of their nutrition from scraping and eating this biofilm containing diatoms, blue-green algae, fungi, fine particulate organic matter, and dissolved organic matter. I hypothesize that the living microorganisms in this biofilm provide the best nutrition to these kinds of benthic macroinvertebrates.
Clean glass microscope slides would be placed in the study stream. The slides would be surrounded by a fine mesh cage that exludes benthic macroinvertebrates but allows water to freely flow over the slides. The slides would be incubated in the stream for varying lengths of time. At proscribed intervals a slide would be removed from the stream, but kept in ambient water and brought back the lab. The slide would be stained with a type of fluorescent dye which is only taken up by living microbial cells (or a suite of dyes specific to different types of microorganisms).
Estimates of microbial cell abundance and density would be made by counting a representative area of the slide using standard light microscopy. The slide would then be exposed to the proper laser and fluorescing microbial cells would be counted within the representative area. These methods would yield an estimate of the ratio of living biofilm organisms to total available biofilm organisms. Such a ratio could provide an indication of the quality of the biofilm for benthic macroinvertebrate scrapers.
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