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Related Concept Videos

Microbial Growth Measurement: Direct Methods01:23

Microbial Growth Measurement: Direct Methods

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Direct methods for measuring microbial populations in a culture are essential tools in microbiology, providing quantitative data for various applications. Among these, microscopic counts, plate counts, and serial dilution are widely used techniques, each with unique principles and applications.Microscopic CountsMicroscopic counting involves the use of a Petroff-Hausser chamber, a specialized microscope slide with a grid and defined depth. By observing a liquid culture under a microscope,...
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Microbial Growth Measurement: Indirect Methods01:27

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Estimating microbial growth is essential for understanding population dynamics and environmental adaptations. Indirect methods provide valuable insights by measuring parameters such as turbidity, metabolic activity, and biomass, enabling efficient and reproducible assessments.During exponential growth, microbial cells scatter light proportionally to their biomass, a principle used in turbidity measurements. About one million cells per milliliter produce detectable scattering, which a...
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Related Experiment Video

Updated: Oct 20, 2025

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells
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Toward absolute viability measurements for bacteria.

Joy P Dunkers1, Hariharan Iyer2, Brynna Jones3,4

  • 1Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.

Journal of Biophotonics
|September 12, 2021
PubMed
Summary

This study introduces a new method using fluorescence lifetime imaging to differentiate between live, quiescent, and dead bacteria. This quantitative approach offers a reliable way to assess bacterial viability.

Keywords:
FLIMbacteriafluorescence lifetime microscopymachine learningmembrane potentialmicrobequiescenceviability

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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.