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Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94 Page 13 of 19

different types of bacteria using flow cytometry, which was consistent with our current results .

Figure 6. Effect of spike-in on the α diversity and β-diversity analysis. (A) α diversity indicated by the Shannon index and Chao1 index;
(B) β-diversity Principal coordinates analyses (PCoA, Bray-Curtis’ dissimilarities) from relative abundance analysis; (C) β-diversity from
***
spike-in analysis. P < 0.001.

when using whole cells. For samples that have already been extracted and sequenced, the spike-in method
can be applied starting from library preparation instead of extraction from the beginning to investigate the
influence of microbial loads, especially when sample quantities are very low.

In this study, we observed significant differences in the estimation of microbial loads between flow
cytometry and the spike-in method, which may be attributed to the overestimation of cells in flow
cytometry and variations in DNA extraction efficiency.

When using the Bacterial Viability and Counting Kit for flow cytometry, sample preparation steps like
sonication and vortexing may cause foam formation and microbubbles, potentially leading to an
overestimation of cell counts [38-40] . Different levels of overestimation have been observed when measuring
[14]

Overall, plate count resulted in bacterial counts approximately 7-8 times less than those obtained through
flow cytometry, suggesting that the actual bacterial load likely falls within this range. While it is
acknowledged that not all bacteria grow on the YCFA medium, our prior experience indicates that the
majority of bacterial taxa can be successfully cultivated using this method .
[22]

Notably, DNA extraction variability is a major factor contributing to experimental variation, leading to a
reduced relative abundance of gram-positive bacteria in the samples. This issue has also been observed in
numerous previous studies [41-43] . We used bead-beating for DNA extraction to ensure effective cell
disruption, especially for hard-to-lyse microbes like gram-positive bacteria [43-45] .