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Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid
Omar Zmerli1,2, Sara Bellali1, Gabriel Haddad1,2
1Institut Hospitalo-Universitaire Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille, France.
Computational and Structural Biotechnology Journal
|July 28, 2023
Summary
This study introduces a new scanning electron microscopy (SEM) assay using phosphotungstic acid (PTA) staining to rapidly distinguish between live and dead microbes. The SEM-PTA method shows promise for microbiome and antimicrobial testing.
Area of Science:
- Microbiology
- Microscopy
- Biotechnology
Background:
- Traditional electron microscopy stains enhance ultrastructure visualization but have limited use in microbiological viability.
- Phosphotungstic acid (PTA) is a common negative stain in scanning electron microscopy (SEM).
Purpose of the Study:
- To investigate the feasibility of a novel SEM-PTA assay for differentiating viable from dead microbes.
- To establish optimal sample preparation conditions for the SEM-PTA viability assay.
Main Methods:
- Bacteria were stained with varying PTA concentrations and incubation times to optimize the assay.
- The developed SEM-PTA protocol was applied to diverse samples, assessing bacterial viability under different conditions.
- Results were compared against traditional culture methods for validation.
Main Results:
- A 5-minute, 10% PTA staining effectively distinguished viable (hypo-dense) from dead (intensely stained) microorganisms.
- High Tungsten (W) peaks on EDX spectra confirmed the staining differences.
- SEM-PTA viability counts after stress (freezing, freeze-drying, oxygen exposure) correlated well with culture results.
Conclusions:
- This study presents the first report on PTA staining for differentiating live and dead bacteria using SEM.
- The SEM-PTA assay is a rapid, cost-effective, and efficient method for viability assessment.
- This technique offers an open-view of samples and has potential applications in microbiome studies and antimicrobial susceptibility testing.
