Soil bacterial biodiversity characterization by flow cytometry: The bottleneck of cell extraction from soil

The importance of soil biodiversity is increasingly recognized in agriculture and natural resource research and development. Yet, traditional soil biodiversity assessments are costly and time-consuming, limiting the extent and frequency of sampling and analysis in space and time. Flow cytometry (FCM) is a powerful technique to characterize cell communities due to its high robustness and accuracy, requiring only a short time for the characterization. Therefore, FCM could expand soil research capabilities by allowing the characterization of different aspects of bacterial biodiversity. However, this implementation of FCM requires the previous dispersion, separation and purification of bacteria from complex soil matrices. Moreover, soil monitoring programs or evaluation of soil management practices require high-throughput analysis. In this context, soil processing protocols need to consider not only an adequate recovery of undamaged, representative and pure soil bacteria, but also short-time processing requirements. Although soil processing protocols have been reported over time, to our knowledge, there is no recommended soil extraction protocol for high-throughput analysis of bacterial biodiversity by FCM. We reviewed the state-of-art of the use of flow cytometry in scientific research and the protocols used for the extraction of bacteria from soil. We analysed the literature to take stock of the diversity of methodologies for soil processing and applications of flow cytometry in bacterial characterization considering abundance, diversity, community structure and functional properties. This review provides several lines of evidence of the use of flow cytometry for soil bacterial biodiversity (SBB) characterization, highlighting its potential for soil monitoring and studies on soil bacterial community dynamics. The review also highlights and discusses the most relevant constraints and research gaps that need to be considered for high-throughput analysis of SBB by FCM, such as evaluation of scale-down, new reagents for and methods of purification, threshold of bacterial recovery efficiency and selection of a standardized and validated protocol. We proposed a protocol for soil bacterial extraction for high-throughput analysis of SBB by FCM and we provided detailed databases of systematized information that would be useful to the scientific community.