4.1 Calibration and validation of the cell counting approach
The flow cytometry calibration plots for each microorganism are given in the supplementary material Figure S1. These results confirmed excellent linear relationship between biomass concentration and the cell counting results, with R2 ranging 0.979 – 0.983. Figure S1 also shows that cell counting with flow cytometry is more reliable/consistent when cell concentrations are low. Using the calibration relationship obtained from the single cultures, we validated the accuracy of the flow cytometry measurements using static coculture samples with known individual biomass concentrations. For each coculture pair, six samples with different compositions were tested.
The individual biomass concentrations for each microorganism in the cocultures measured using flow cytometry are plotted against the known concentrations in Figure 2 (a) ~ (d), with the detailed results provided in Supplementary Material Table S1. As shown in these figures, the measured individual biomass concentrations (converted from cell counting based on the calibration curves in Figure S1) show good agreement with the known concentrations. However, there are relatively large variations among the triplicates for each sample, especially for higher concentrations, which is consistent with the similar trend observed in the calibration curves in Figure S1. In addition, when the same sample was measured multiple times, the measurements showed same level of variations, suggesting the source of the variation was cell counting. One possible reason for such large variation is the non-uniform distribution of the cells in the liquid sample, and the small sample volumes (25 µL) for cell counting makes such variation more pronounced for higher concentrations, as observed in both calibration and validation experiments. Another possible reason, which we believe is more important for mixed culture samples, is the effect of sample fixation process on cell counting.
In this work, we had to optimize the sample fixation protocols multiple times in order to obtain the acceptable validation results. Figure 2 (e) and (f) compare the cell counting results for a same static coculture sample. Figure 2 (e) was obtained following the cell fixation protocol initially developed for the salt water pair, while Figure 2 (f) following the protocol optimized for the fresh water pair. The known and measured individual biomass concentration are provided in Table 2. The large measurement errors shown in Figure 2 (e) (-88.3% for the methanotroph and 18.0% for the microalgae) indicate that some methanotroph cells stuck to the microalgae cells and the flow cytometer could not separate them properly. With the optimized protocol, methanotroph cells were much better separated from microalgae cells, which resulted in significantly reduced measurement error (-7.9% for the methanotroph and -6.5% for the microalgae). Currently the flow cytometry has been commonly used to characterize the composition of synthetic microbiome. This example highlights the importance of performing validation experiments to confirm the appropriateness of the experimental protocol and the accuracy of the cell counting to avoid misleading results.