Use of image-based flow cytometry in bacterial viability analysis using fluorescent probes

Area of Science:

• Microbiology
• Cell Biology
• Analytical Chemistry

Background:

• Accurate enumeration and viability assessment of bacterial populations are crucial in various scientific fields.
• Traditional methods for bacterial viability analysis can be time-consuming and may not provide morphological insights.
• Imaging flow cytometry (IFCM) offers a powerful platform for high-throughput single-cell analysis.

Purpose of the Study:

• To develop and validate a protocol for differentiating and quantifying live and dead bacterial cells in a mixed population.
• To analyze morphological features of bacterial cells alongside their viability status.
• To establish the reliability and accuracy of IFCM for bacterial cell concentration determination.

Main Methods:

• Utilized imaging flow cytometry (IFCM) combined with two fluorescent dyes: 5(6)-carboxyfluorescein diacetate (CFDA) for live cells and DRAQ7 for dead cells.
• CFDA stains viable cells green via esterase activity, while DRAQ7 stains membrane-compromised dead cells red.
• IFCM system captured images for morphological analysis and enumerated distinct live (green) and dead (red) cell populations.

Main Results:

• CFDA and DRAQ7 effectively differentiated live and dead bacterial cells based on distinct green and red fluorescence.
• The IFCM system accurately and precisely determined bacterial cell concentrations down to 1 × 10(3) cells/ml.
• Morphological features of both live and dead cells could be analyzed from the captured images.

Conclusions:

• The developed IFCM protocol provides a reliable, accurate, and precise method for bacterial viability analysis.
• This technique allows for simultaneous enumeration, viability assessment, and morphological characterization of bacterial cells.
• The combination of CFDA and DRAQ7 is effective for distinguishing live and dead bacteria in mixed samples.