Storage treatment and diluent cause inconsistencies in commonly used fluorometric microplate enzyme assays conducted with saltmarsh soils

Abstract Fluorometric enzyme assays are a tool for determining potential enzyme activity in soils. A wide array of methodological approaches is adopted by different studies, making comparisons difficult. An important consideration is whether assay conditions replicate the environment of the samples or optimize potential enzyme activity. Related is whether buffer or distilled water is used as a diluent when creating the soil slurry used in the assay. Buffer is favored for maintaining a stable pH while using distilled water more closely replicates environmental conditions by allowing the sample to regulate its own pH. Furthermore, sample storage before and after slurry creation is a factor that varies widely. Using soil from two saltmarshes, highly dynamic ecosystems experiencing extreme conditions such as waterlogged soils and high salinity, we compared the impacts of creating slurries with phosphate buffer, distilled water, and saline saltmarsh creek water and of storing slurries at 4°C and −20°C before analysis. Assay outcomes were highly sensitive to these pre‐assay treatments and that sensitivity was large enough to affect relative comparisons among samples. There were notable differences between diluents that could drive these outcomes. First, pH fluctuations occurred when buffer was not the diluent. Second, the actual pH of the assays differed by as much as 1.04. Third, when pH‐dependent 4‐methylumbelliferone was the fluorophore, quench effects were greater in distilled water controls than in assays with soil particles present because of pH differences arising in an unbuffered system. In conclusion, the methodological differences tested here could compromise comparability among studies.