Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry

Abstract

ABSTRACT Bacterial abundance is a fundamental metric for understanding the population dynamics of soil bacteria and their role in biogeochemical cycles. Despite its importance, methodological constraints hamper our ability to assess bacterial abundance in terrestrial environments. Here, we aimed to optimize the use of flow cytometry (FCM) to assay bacterial abundances in soil while providing a rigorous quantification of its limitations. Soil samples were spiked with Escherichia coli to evaluate the levels of recovery efficiency among three extraction approaches. The optimized method added a surfactant (a tetrasodium pyrophosphate [TSP] buffer) to 0.1 g of soil, applied an intermediate degree of agitation through shaking, and used a Nycodenz density gradient to separate the cells from background debris. This procedure resulted in a high (average, 89%) level of cell recovery. Recovery efficiencies did not differ significantly among sites across an elevation gradient but were positively correlated with percent carbon in the soil samples. Estimated abundances were also highly repeatable between technical replicates. The method was applied to samples from two field studies and, in both cases, was sensitive enough to detect treatment and site differences in bacterial abundances. We conclude that FCM offers a fast and sensitive method to assay soil bacterial abundance from relatively small amounts of soil. Further work is needed to assay differential biases of the method across a wider range of soil types. IMPORTANCE The ability to quantify bacterial abundance is important for understanding the contributions of microbial communities in soils, but such assays remain difficult and time-consuming. Flow cytometry offers a fast and direct way to count bacterial cells, but several concerns remain in applying the technique to soils. This study aimed to improve the efficiency of the method for soil while quantifying its limitations. We demonstrated that an optimized procedure was sensitive enough to capture differences in bacterial abundances among treatments and ecosystems in two field studies.

Keywords

• bacterial cell count
• ecosystem types
• extraction procedure
• flow cytometry
• soil