Bacterial mock communities as standards for reproducible cytometric microbiome analysis

Area of Science:

• Microbiology
• Cytometry
• Ecological Dynamics

Background:

• Flow cytometry is increasingly used for microbial community assembly studies.
• Instrumental variability and cell handling introduce significant data noise, especially for microorganisms.
• Existing alignment beads do not fully address single-cell data variability in microbial flow cytometry.

Purpose of the Study:

• To develop and validate a microbial cytometric mock community (mCMC) as a reference standard.
• To enable reproducible, comparable data across different flow cytometry experiments and instruments.
• To improve the accuracy of single-cell microbial characterization, particularly in long-term studies.

Main Methods:

• Assembled a mock community using two Gram-positive and two Gram-negative bacterial strains, including spores.
• Developed protocols for strain preparation, fixation (PFA/ethanol or drying), and long-term storage (≥6 months).
• Applied fluorescence staining methods and tested the mCMC on high-resolution cell sorters and benchtop flow cytometers.

Main Results:

• The mCMC can be prepared within 1 hour from stable stocks.
• Demonstrated successful application across diverse flow cytometry platforms, yielding benchmarked datasets.
• Validated the mCMC's utility for bioinformatic analysis of community behavior and cell sorting decisions.

Conclusions:

• The microbial cytometric mock community (mCMC) is an essential tool for standardizing microbial flow cytometry.
• mCMC facilitates reproducible data acquisition and inter-laboratory comparisons, crucial for microbial ecology.
• This standard supports downstream applications like high-resolution sequencing and proteomics by ensuring data quality.