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Microscopy-based protocol for the quantification of cells viability for temperature-sensitive S. pombe.

Kim Kiat Lim1, Ee Sin Chen2

  • 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

STAR Protocols
|May 20, 2024
PubMed
Summary

This study introduces a microscopy method to accurately quantify microbial cell viability, overcoming limitations of traditional assays. The new protocol enhances precision for temperature-sensitive yeast viability studies.

Keywords:
GeneticsMicrobiologyMicroscopyModel OrganismsMolecular Biology

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Area of Science:

  • Microbiology
  • Cell Biology
  • Microscopy

Background:

  • Traditional colony-forming unit assays for microbial viability are labor-intensive and prone to significant experimental variability.
  • This variability hinders accurate quantification of microbial cell viability, particularly for sensitive strains.
  • Existing methods lack the precision required for detailed viability studies.

Purpose of the Study:

  • To present a novel microscopy-based protocol for precise quantification of microbial cell viability.
  • To establish a reliable method for assessing the viability of temperature-sensitive Saccharomyces cerevisiae (S. pombe).
  • To provide a detailed protocol adaptable for various microbial viability assessments.

Main Methods:

  • Development of a microscopy protocol using Phloxine B staining for individual yeast cell viability assessment.
  • Detailed steps for growing and treating temperature-sensitive S. pombe.
  • Utilizing Nikon NIS Elements Advanced Research (AR) software for image processing and data analysis.

Main Results:

  • The microscopy-based protocol offers improved accuracy and reduced variability compared to conventional methods.
  • Successful visualization and quantification of individual S. pombe cell viability.
  • Demonstrated efficacy of Phloxine B staining in differentiating viable and non-viable cells.

Conclusions:

  • The presented microscopy protocol provides a robust and accurate alternative for microbial cell viability quantification.
  • This method is particularly advantageous for studying temperature-sensitive organisms like S. pombe.
  • The protocol facilitates more precise research in microbiology and cell biology by improving viability assessment.