Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge

Abstract

Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz® solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high.

Flow-cytometric quantification of microbial cells on sand from water biofilters

“…This method constitutes a considerable improvement on previously developed protocols for biomass quantification on sand samples from water biofilters (MagicKnezev and van der Kooij, 2004). The protocol developed allows direct cell quantification free from potential biases introduced by assumptions such as equal average ATP content per cell (Frossard et al, 2016) and overcomes the limitations associated with qPCR quantification methods such as DNA extraction efficiency and yield (Bremen et al, 1999;Feinstein et al, 2009), and primer specificity. We optimised cell detachment for FCM quantification using a combination of chemical surfactants and ionic dispersants, together with low and high-energy sonication as a mechanical pretreatment, and different fixative methods.…”

“…A previous study, carried out on samples collected from several environments (sediments, soils, and sludge), directly compared cell abundances measured via the ATP assay and FCM on suspensions obtained after cell detachment via three cycles of sonication in a fixative solution (Frossard et al, 2016). The study showed that estimates based on the ATP assay yielded significantly higher average microbial abundances than the FCM method; the slope of the correlation between FCM and ATP was on average 0.36.…”

Quantitative PCR provides a simple and accessible method for quantitative microbiome profiling

“…We believe the practicability and accessibility of many previously proposed solutions represent obstacles for the wider adaptation of quantitative microbiome profiling in human microbiome research. In particular, the flow cytometry-based approach requires considerable expertise for reproducible results, considering flow cytometric enumeration of microbial cells was initially restricted to pure cultures (10) and still remains challenging when performed in complex matrices (11).…”

Effects of initial microbial biomass abundance on respiration during pine litter decomposition

“…Microbial biomass measurements are often influenced by microbial composition. Techniques used to measure microbial biomass abundance include phospholipid fatty acids (PFLAs) [60], substrate induced respiration (SIR) with selective inhibition of bacteria or fungi [61], DNAbased approaches such as qPCR [62,63], growth-based measurements [64], and particle counts by flow cytometry [65,66]. These techniques are affected by species' characteristics [24,67].…”