Flow cytometry has recently established itself as a tool to track short-term dynamics in microbial community assembly and link those dynamics with ecological parameters. However, instrumental configurations of commercial cytometers and variability introduced through differential handling of the cells and instruments frequently cause data set variability at the single-cell level. This is especially pronounced with microorganisms, which are in the lower range of optical resolution. Although alignment beads are valuable to generally minimize instrumental noise and align overall machine settings, an artificial microbial cytometric mock community (mCMC) is mandatory for validating lab workflows and enabling comparison of data between experiments, thus representing a necessary reference standard for the reproducible cytometric characterization of microbial communities, especially in long-term studies. In this study, the mock community consisted of two Gram-positive and two Gram-negative bacterial strains, which can be assembled with respective subsets of cells, including spores, in any selected ratio or concentration. The preparation of the four strains takes a maximum of 5 d, and the stains are storable with either PFA/ethanol fixation at –20 °C or drying at 4 °C for at least 6 months. Starting from this stock, an mCMC can be assembled within 1 h. Fluorescence staining methods are presented and representatively applied with two high-resolution cell sorters and three benchtop flow cytometers. Benchmarked data sets allow the use of bioinformatic evaluation procedures to decode community behavior or convey qualified cell sorting decisions for subsequent high-resolution sequencing or proteomic routines.

流式细胞术最近已成为一种追踪微生物群落组装的短期动态并将这些动态与生态参数联系起来的工具。然而，商业细胞仪的仪器配置和通过对细胞和仪器的不同处理引入的可变性经常导致单细胞水平的数据集可变性。这对于处于较低光学分辨率范围内的微生物尤其明显。尽管比对珠通常对于最大限度地减少仪器噪音和调整整体机器设置很有价值，但人工微生物细胞计数模拟群落 (mCMC) 对于验证实验室工作流程和启用实验之间的数据比较是强制性的，因此代表了微生物群落可重复细胞计数表征的必要参考标准，特别是在长期研究中。在这项研究中，模拟群落由两种革兰氏阳性和两种革兰氏阴性细菌菌株组成，它们可以以任何选定的比例或浓度与相应的细胞亚群（包括孢子）组装在一起。四种菌株的制备最多需要 5 天，并且污渍可以在 –20 °C 下使用 PFA/乙醇固定或在 4 °C 下干燥至少 6 个月。从此库存开始，可以在 1 小时内组装 mCMC。介绍了荧光染色方法，并代表性地应用了两个高分辨率细胞分选仪和三个台式流式细胞仪。