Toward absolute viability measurements for bacteria

Area of Science:

• Microbiology
• Biophysics
• Analytical Chemistry

Background:

• Assessing bacterial viability is crucial in various fields, including medicine and environmental science.
• Current methods for distinguishing between quiescent and dead bacterial cells can be limited in accuracy and comparability.
• Quantitative, time-based measurements offer a promising avenue for improved viability assessment.

Purpose of the Study:

• To develop a quantitative method for distinguishing quiescent from dead bacterial cells.
• To establish fluorescence lifetime as a referenceable and comparable metric for bacterial viability.
• To validate this method for Streptococcus mutans.

Main Methods:

• Utilized fluorescence lifetime imaging with an anionic, fluorescent membrane voltage probe.
• Employed a random forest machine-learning model for cell classification.
• Compared classification accuracy using lifetime variables, phasor variables, and a combination of five variables.

Main Results:

• Fluorescence lifetime imaging successfully distinguished between quiescent and dead Streptococcus mutans.
• Machine learning models, particularly those using all five variables, achieved high classification accuracy.
• The method demonstrated the potential for time-based, quantitative viability assessment.

Conclusions:

• Fluorescence lifetime of a membrane voltage probe is a viable marker for assessing quiescent bacteria.
• This quantitative approach offers a more precise and comparable method for bacterial viability.
• Further research on diverse bacterial species and fluorophores will enhance this technique.