Abstract
The accuracy of flow cytometric (FCM) quantifications of microbial populations in sediments varies with FCM settings, cell extraction and staining protocols, as well as sample types. In the present study, we improve the accuracy of FCM for enumerating microorganisms inhabiting diverse lake and marine sediment types based on extensive tests with FCM settings, extraction buffer chemical compositions, cell separation methods, and staining procedures. Tests on the FCM settings, (e.g., acquisition time, rates of events) and salinity of extraction solutions show minor impacts on FCM enumerations and yields of cell extraction, respectively. Existing methods involving hydrofluoric acid (HF) treatment to release sediment-attached cells into solution prove effective on both marine and freshwater samples. Yet, different staining techniques (direct staining of cell extracts, staining of membrane-filtered cell extracts) produce clear differences in cell number estimates. We demonstrate that, while labor-intensive membrane-staining generates high cell staining efficiency and accurate cell counts that are consistent across FCM and epifluorescence microscopy-based (EFM) quantification methods, accurate cell counts determined by more time- and labor-efficient direct staining require consideration of dye concentration, sample dilution, and lithology. Yet, good agreement between the two staining methods can be achieved through sample-specific adjustments of dye concentrations and sample dilutions during direct staining. We thus present a complete protocol for FCM-based cell quantification, that includes all steps from the initial sample fixation to the final enumeration, with recommendations for buffer compositions, direct and membrane-based staining procedures, and the final FCM assay. This protocol is versatile, accurate, and reliable, as is evident from good agreement with cell quantifications by EFM and quantitative polymerase chain reaction (qPCR) of 16S rRNA genes across a wide range of sedimentary sample types.
Highlights
Microorganisms are ubiquitous in marine and freshwater sediments and play important roles in global elemental cycles, including the carbon and nitrogen cycles
We demonstrate the efficacy of the optimized direct staining method based on diverse surface and subsurface sediment sample types from freshwater lakes and marine environments, where flow cytometric (FCM)-based cell counts with direct staining show good agreement with ones after membrane staining, but are reproducible by other cell quantification methods, including epifluorescence microscopy-based (EFM) and quantitative polymerase chain reaction (qPCR)
Since little is known about how hydrofluoric acid (HF) treatment might affect cell recovery and FCM counts on freshwater cell extracts, we evaluate the efficiency of HF treatment on lake sediments by (1) determining cell recovery rates based on sediment spikes with known numbers of E. coli cells, and (2) comparing the cell extraction efficiency of HF-based and density-gradient centrifugation-based assays (Histodenz) using natural samples
Summary
Microorganisms are ubiquitous in marine and freshwater sediments and play important roles in global elemental cycles, including the carbon and nitrogen cycles. Previous estimates of microbial abundance in subseafloor sediment based on different techniques vary from 2.9–50 × 1029 cells, of which the lower boundary is comparable to global cell numbers in seawater and soil, whereas the upper boundary approaches the total microbial abundance elsewhere on Earth (Whitman et al, 1998; Lipp et al, 2008; Kallmeyer et al, 2012; Parkes et al, 2014). A reliable and fast quantification method is critical for estimating microbial population size in both marine and freshwater sediment. The results derived from different techniques often show limited agreement, even when the same samples are studied (Lloyd et al, 2013; Buongiorno et al, 2017)
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