Flow-cytometric quanti fi cation of microbial cells on sand from water bio fi lters

Rapid quanti fi cation of absolute microbial cell abundances is important for a comprehensive interpre- tation of microbiome surveys and crucial to support theoretical modelling and the design of engineered systems. In this paper, we propose a protocol speci fi cally optimised for the quanti fi cation of microbial abundances in water bio fi lters using fl ow cytometry (FCM). We optimised cell detachment from sand bio fi lter particles for FCM quanti fi cation through the evaluation of fi ve chemical dispersants (NaCl, Triton-X100, CaCl 2 , sodium pyrophosphate (PP), Tween 80 combined with PP), different mechanical pre- treatments (low and high energy sonication and shaking) and two fi xation methods (glutaraldehyde and ethanol). The developed protocol was cross-compared using other established and commonly employed methods for biomass quanti fi cation in water fi lter samples (adenosine triphosphate (ATP) quanti fi cation, real-time quantitative PCR (qPCR) and volatile solids (VS)). The highest microbial count was obtained by detaching the bio fi lm from bio fi lter grains and dispersing clusters into singles cells using Tween 80 and sodium pyrophosphate combined with four steps of high energy sonication (27W, for 80 s each step); glutaraldehyde was shown to be the best fi xative solution. The developed protocol was reliable and highly reproducible and produced results that are comparable to data from alternative quanti fi cation methods. Indeed, high correlations were found with trends obtained through ATP and qPCR ( r ¼ 0.98 and r ¼ 0.91) measurements. The VS content was con fi rmed as an inaccurate method to express biomass in sand samples since it correlated poorly with all the other three methods ( r ¼ 0.005 with FCM, 0.002 with ATP and 0.177 with qPCR). FCM and ATP showed the strongest agreement between absolute counts with a slope of the correlation equal to 0.7, while qPCR seemed to overestimate cell counts by a factor of ten. The rapidity and reproducibility of the method developed make its application ideal for routine quan- ti fi cation of microbial cell abundances on sand from water bio fi lters and thus useful in revealing the ecological patterns and quantifying the metabolic kinetics involved in such systems. (http://creativecommons.org/licenses/by/4.0/).

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