What is flow cytometry and how is it used in microbiology?

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I came across this explanation while trying to understand how labs study bacteria quickly. Turns out, flow cytometry is used to analyze individual microbes, check if they’re alive, and even sort them. It’s faster than traditional methods and super detailed. This answer gave me a solid understanding of how it really works in microbiology labs.

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Anttilampinen 2025-06-10T09:59:53+00:00 1 Answer 6 views
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  1. fleming
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    2025-06-10T10:03:01+00:00
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    What is flow cytometry and how is it used in microbiology?

    How it Works

    1. Sample Preparation: Cells (e.g., bacteria, yeast, algae, or host cells infected with microbes) are suspended in a fluid.
    2. Hydrodynamic Focusing: The cell suspension is injected into a faster-moving sheath fluid within the flow cytometer. This forces the cells to align in a single-file stream.
    3. Interrogation Point: The single-file stream passes through one or more focused laser beams.
    4. Light Scattering: As each cell passes through the laser, it scatters light:
      • Forward Scatter (FSC): Light scattered at a narrow angle to the laser beam. The intensity of FSC is roughly proportional to the cell’s size.
      • Side Scatter (SSC): Light scattered at approximately 90 degrees to the laser beam. The intensity of SSC is related to the cell’s internal complexity or granularity (e.g., presence of granules, shape of the nucleus).
    5. Fluorescence Detection: If the cells are stained with fluorescent dyes (fluorochromes) or express fluorescent proteins (like GFP), the laser excites these fluorochromes, causing them to emit light at specific wavelengths. Detectors (photomultiplier tubes – PMTs) capture the emitted fluorescence.
    6. Data Acquisition: Detectors convert the light signals (scatter and fluorescence) into electronic signals, which are processed and stored by a computer. Each cell passing through generates a multi-parameter dataset.
    7. Data Analysis: Software is used to visualize and analyze the data, often displayed as histograms (single parameter) or dot plots/scatter plots (two parameters), allowing identification and quantification of different cell populations based on their scatter and fluorescence properties.

    Applications in Microbiology

    Flow cytometry has numerous applications in microbiology due to its speed, sensitivity, and ability to analyze individual cells within heterogeneous populations:

    1. Microbial Identification and Enumeration:
      • Rapidly counting bacteria or yeast cells in samples (e.g., water quality testing, industrial fermentation monitoring).
      • Differentiating between different microbial species or groups based on size, shape, and staining properties (e.g., using fluorescent DNA stains or specific antibodies).
    2. Viability and Physiological State Assessment:
      • Distinguishing between live and dead cells using viability dyes (e.g., propidium iodide, SYTOX Green) that only enter cells with compromised membranes.
      • Assessing metabolic activity, membrane potential, or enzyme activity using specific fluorescent probes.
    3. Antimicrobial Susceptibility Testing (AST):
      • Rapidly determining the effect of antibiotics on bacterial growth or viability, potentially providing faster results than traditional culture-based methods.
    4. Detection of Specific Pathogens:
      • Using fluorescently labeled antibodies or nucleic acid probes (Fluorescence In Situ Hybridization – FISH) to detect specific pathogens in clinical samples (e.g., blood, urine) or environmental samples.
    5. Analysis of Microbial Communities (Microbiome Studies):
      • Characterizing the composition of complex microbial communities based on differences in cell size, granularity, and staining properties, often complementing sequencing-based methods.
    6. Phage-Bacteria Interactions: Studying viral infection dynamics by monitoring changes in bacterial populations or detecting phage components.
    7. Biofilm Analysis: Analyzing cells dispersed from biofilms to understand their heterogeneity and physiological states.
    8. Cell Sorting (Fluorescence-Activated Cell Sorting – FACS): More advanced flow cytometers can physically sort cells based on their measured properties. This allows researchers to isolate specific microbial subpopulations (e.g., cells expressing a certain gene, viable cells, specific species from a mix) for further study or cultivation.

    Advantages

    • Speed: Analyzes thousands of cells per second.
    • Quantitative: Provides precise measurements of cell properties.
    • Multi-parameter: Measures multiple characteristics simultaneously.
    • Single-cell resolution: Analyzes individual cells within a population, revealing heterogeneity.
    • Sensitivity: Can detect rare cell populations.

    Limitations

    • Requires cells to be in suspension.
    • Resolution may be limited for very small bacteria.
    • Initial equipment cost can be high.
    • Requires expertise for operation and data analysis.

    Overall, flow cytometry is a versatile and powerful tool that provides rapid, quantitative insights into the properties and functions of individual microbial cells, significantly advancing research and diagnostics in microbiology.

    Source: Shapiro, H.M. Practical Flow Cytometry; Journal articles in Applied and Environmental Microbiology, Cytometry Part A

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