Related Experiment Video
Updated: Jun 22, 2025
Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells
Published on: September 5, 2013
Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing
Shengbin He1, Yajing Chen1, Jingtong Wang1
1Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education, Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi 530021, P. R. China.
A new ratiometric fluorescent method using GelRed and SYTO 9 effectively distinguishes live from dead bacteria. This sensitive technique aids in food safety and drug development by accurately assessing bacterial viability.
Area of Science:
- Microbiology
- Biotechnology
- Analytical Chemistry
Background:
- Bacterial viability assessment is crucial for food safety and antimicrobial research.
- Traditional methods for viability testing can be limited in sensitivity and accuracy.
Purpose of the Study:
- To develop a novel, sensitive, and accurate ratiometric fluorescent method for bacterial viability assessment.
- To establish a method for distinguishing between live and dead bacteria using DNA-binding dyes.
Main Methods:
- Utilized GelRed as a DNA-binding stain, noting live bacteria exclude it while dead bacteria absorb it.
- Employed cations to minimize nonspecific adsorption of GelRed.
- Developed a double-staining technique with SYTO 9 (a membrane-permeable dye).
- Established a ratiometric fluorescent assay for quantifying dead bacteria.
Main Results:
- The GelRed/SYTO 9 method demonstrated high sensitivity, detecting dead bacteria at levels as low as 0.1% in Escherichia coli O157:H7.
- A strong linear correlation (R² = 0.97) was observed between ratiometric fluorescence and the theoretical dead bacterial ratio.
- The method proved effective in evaluating the germicidal efficacy of heat treatments, showing bacterial death at 50 °C.
Conclusions:
- The developed ratiometric fluorescent method offers superior sensitivity, accuracy, and safety compared to traditional viability assays.
- This technique has broad applications in microbial research, pathogen detection, and drug development.
- The method shows potential for adaptation to mammalian cell viability studies.
