Other, potentially more time-consuming approaches for more direct quantification of microbial abundance include flow cytometric counting of cells in combination with sequencing. Previously conducted using direct plate or cell counts, flow cytometry is relatively higher throughput and more accurate. In this approach, cells are separated from large soil particles and incubated with nucleic acid stain prior to flow cytometric enumeration. Counting of cells using flow cytometry may circumvent overestimation of microbial diversity related to extracellular DNA by counting only intact cells (48, 49)⁠. Additionally, fluorescence microscopy using common nucleic acid stains (e.g. DAPI, Sybr-green) can be applied to achieve direct cell counts. However, this approach is relatively lower throughput and less accurate than flow cytometric enumeration (45)⁠.
An additional approach to improve the quantitative nature of amplicon sequencing is Catalyzed Reporter Deposition coupled to Fluorescence in situ hybridization (CARD-FISH). Recently, Piwosz and colleagues combined CARD-FISH with sequencing (50)⁠. Although the use of CARD-FISH is more labor-intensive than amplicon sequencing alone, it allows sequencing to become more quantitative through direct cell counts and phylogenetic staining of microorganisms of interest. This approach is restricted to the analysis of bacteria and archaea, as high-throughput sequencing data and CARD-FISH analysis of eukaryotic organisms correlate poorly with one another (50)⁠. The correlation between datasets was higher for bacteria and taxa that were highly underrepresented in the sequencing data could be captured using CARD-FISH. Some of these approaches remain limited at present due to the sheer biological diversity of soil ecosystems. However, with an expanding view of the diversity of microorganisms and growing number of published reference genomes, more accurate quantitative approaches are within reach. In combining sequencing with quantitative measurements, one can obtain absolute abundances of organisms in a given sample, making investigations of complex microbial communities more robust.