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Flow cytometry is a sensitive method for the detection and quantification of physiological and structural conditions of bacterial cells, including probiotics and other bacteria of biotechnological importance to the food sector. Double staining with propidium iodide (PI) and/or carboxyfluorescein diacetate (cFDA) allows the differentiation of three subpopulations in the cytogram: dead cells (PI positive, cFDA negative = red cells), viable cells (cFDA positive, PI negative = green cells), and altered cells (PI positive, cFDA positive = green and red cells). To procedure with double staining, suspend the encapsulated cell with phosphate buffer saline (PBS), harvest by centrifugation, add 10 μL/mL of cFDA and 10 μL/mL of PI to the suspension, and incubate for 15 min at 37 °C (one by one). Afterward, centrifuge the sample, discard the supernatant, and resuspend with 1 mL of PBS. The flow cytometer collects the signs of cFDA in FL1 and PI in FL3 band-pass filters, acquiring a total of 10,000 events for each sample. Pass an unmarked sample before marked sampled to define the parameters of cytometer gates. Evaluate graphs generated with data acquired for each evaluated sample.

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Evaluation of the Physiological Status of Encapsulated Probiotic Bacterial Cells Using Flow Cytometry

  • 章节

  • 发表日期：

    2025年

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  • Thatyane Mariano Rodrigues de Albuquerque,
  • Karoliny Brito Sampaio,
  • Maiara da Costa Lima,
  • Evandro Leite de Souza

摘要

Flow cytometry is a sensitive method for the detection and quantification of physiological and structural conditions of bacterial cells, including probiotics and other bacteria of biotechnological importance to the food sector. Double staining with propidium iodide (PI) and/or carboxyfluorescein diacetate (cFDA) allows the differentiation of three subpopulations in the cytogram: dead cells (PI positive, cFDA negative = red cells), viable cells (cFDA positive, PI negative = green cells), and altered cells (PI positive, cFDA positive = green and red cells). To procedure with double staining, suspend the encapsulated cell with phosphate buffer saline (PBS), harvest by centrifugation, add 10 μL/mL of cFDA and 10 μL/mL of PI to the suspension, and incubate for 15 min at 37 °C (one by one). Afterward, centrifuge the sample, discard the supernatant, and resuspend with 1 mL of PBS. The flow cytometer collects the signs of cFDA in FL1 and PI in FL3 band-pass filters, acquiring a total of 10,000 events for each sample. Pass an unmarked sample before marked sampled to define the parameters of cytometer gates. Evaluate graphs generated with data acquired for each evaluated sample.