We sampled biofilm (assemblage of living and dead autotrophs, heterotrophic microbes, and detritus on rocks) monthly from December 2010 to March 2011 at 36 study locations (18 thalweg and 18 edge) in the mainstem of Big Creek. Study locations were evenly distributed along the 3 km study segment. Three rocks were chosen haphazardly at each location, placed into plastics bags, and transported to the research station for processing. Rocks were scrubbed with a wire brush to remove all attached biofilm. The composite slurry from the combined 3 rocks was sub-sampled (20 - 50 mL), filtered onto pre-combusted (400 °C for 30 min) and pre-weighed glass-fiber filters (0.7 mm), and frozen immediately. Samples were then transported back to Idaho State University for further analysis. The planar area of sampled rocks was traced onto paper. Rock tracings were cut, weighed, and converted to planar area with linear regression. This resulted in c. 36 composite samples (18 thalweg and 18 edge) for each of the 4 sampling periods.

Based on standard methods (APHA, 1998, Davis et al. 2013), filters from each location were analyzed for chlorophyll-a (chl-a, measure of algal pigments and index of living primary producers) and ash-free dry mass (AFDM, measure of living and senesced primary producers). Filters were extracted in methanol for c. 12 h, analyzed for chl-a with a spectrophotometer and corrected for phaeophytin. After extraction, the filters and methanol extract containing the chl-a component were recombined, dried at 60 °C for at least 48 h, weighed and ashed at 550 °C for c. 4 h. Samples were then reweighed to calculate AFDM. Additionally, the chl-a values were divided by the corresponding AFDM values to generate the ratio of chl-a to AFDM, an indicator of biofilm quality by contrasting the portion of living autotrophs of biofilm (chl-a) to the portion of living and senesced autotrophs of biofilm (AFDM).