4.2.3. Direct cell counts
Another approach towards absolute abundance data from soil communities are direct cell counts obtained through fluorescence microscopy \citep{Bloem1995} or fluorescence-activated cell counting \cite{Khalili_2019,Frossard_2016} of cells liberated from soil particle surfaces \cite{Riis_1998,Lentendu_2013}. Total counts help to assess the absolute abundance of microbial cells that fall within a certain range of parameters such as cell size and morphology. Cell counting approaches remain more straightforward for single-cell archaea and bacteria than for filamentous bacteria, fungi or other soil eukaryotes. The success of cell counting can be negatively affected by soil autofluorescence (low signal-to-noise ratio), partial separation of microbial cells from soil particles, or masking the detection of cells by overlaying soil particles. Nevertheless, assessing the number of cells in samples also subjected to sequencing may help to estimate changes in absolute abundance and to better interpret sequencing data (Fig. 3).
In addition, the observation and enumeration of target species of interest through marker-based approaches (e.g., FISH: fluorescence in situ hybridization) enables the quantification of absolute abundances of those species identified through sequencing. This practice not only allows soil ecologists to verify if the change observed in relative abundance indeed translates to shifts in the community by counting taxa of interest on filters \citep{Piwosz2020}, but also expands the interpretation of sequencing data to localize and visualize species of interest in situ (e.g., on roots \citep{Martin_2020}) and to elucidate ecological implications behind changing abundances of target species in soil samples. Applications of FISH in conjunction with amplicon sequencing to soil samples are surprisingly rare albeit such targeted localization and enumeration is a powerful tool to understand the dynamics of certain phylogenetic groups in soil on a quantitative basis.
4.2.4. Combining classical soil biogeochemical methods with amplicon sequencing
Traditional soil biogeochemical approaches enable the quantification of total microbial biomass in soil, including methods such as chloroform fumigation extraction (CFE)\cite{Brookes_1985}, phospholipid fatty acid (PLFA) profiling \cite{Frosteg_rd_1991,Frosteg_rd_2011,Buyer_2012} and ergosterol measurements \cite{JOERGENSEN_2008,Montgomery_2000}. In contrast to PCR-based methods, they assess the concentration of chemical microbial biomarkers in soil directly, thereby avoiding biases introduced by amplification of the target molecules. For example, such quantitative information regarding an increase or decrease in total microbial biomass between treatments would complement corresponding shifts in relative abundance data as observed via amplicon sequencing (Fig. 3). In addition to assessing total microbial biomass, PLFA measurements can also generate abundance information for microorganisms at a coarse phylogenetic resolution. The ability to obtain abundance profiles for bacteria, fungi, as well as distinguishing between gram-positive, gram-negative, and Actinobacteria, could be used as a “benchmark” for interpreting relative abundance data for more specific subsets of an amplicon dataset (i.e., \citealt{Drigo_2010}). A combined interpretation of datasets from biochemical and molecular methods with fundamentally different measurement principles, however, may not always be as straightforward as the combination of amplicon sequencing data with quantitative PCR (see section 4.2.1).