A modified incubation method reduces analytical variation of soil hydrolase assays

•We modified the microplate soil enzyme assay by incubating soils out-of-plate.•Analytical precision increased by an average of 10% with the modified method.•Out-of-plate incubation can decrease analytical variation when it is high.

Given that analytical precision affects the number of biological replicates required to achieve adequate statistical power, and analytical variation is often high for soil assays, we investigated ways to reduce variation of soil hydrolase assays. For two mineral soils (sandy and loamy) and one organic soil (peat), we compared variation of fluorescence for acid phosphatase, β-glucosidase, and N-acetyl-glucosaminidase using incubation methods that differed in assay volume and the ability of reagents to mix. For fluorescence of β-glucosidase and N-acetyl-glucosaminidase from the mineral soils, which had coefficients of variation that exceeded 10% on average, well-mixed, larger volumes significantly reduced variation. This reduction was not observed for fluorescence of acid phosphatase from peat, which had consistently low variation. For all soils tested, thorough mixing of NaOH also reduced variation. Because the goal of enzyme assays is to estimate the total enzyme pool in a sample of soil, modifications to reduce analytical variation, such as those proposed here, should be considered. These modifications can potentially increase our ability to detect treatment differences within the heterogeneous soil matrix.