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Preparation protocol for epifluorescence microscopy when working close to the detection limit.
Rebecca McCerery1, Lewis Hall2, Jithin Omanakuttan2
1Department of Geography and Environmental Sciences, Faculty of Engineering and Environment, Northumbria University, Newcastle-upon-Tyne, NE1 8ST, United Kingdom.
Letters in Applied Microbiology
|February 22, 2025
Summary
Reducing laboratory contamination is crucial for accurate cell counting in low biomass samples. This study details methods to minimize background noise in epifluorescence microscopy, enhancing microbial detection reliability.
Area of Science:
- Microbiology
- Microscopy
- Environmental Science
Background:
- Accurate cell counting in low biomass samples is challenging due to background contamination.
- High background noise significantly impacts the reliability of microbiological assessments near detection limits.
Purpose of the Study:
- To identify and eliminate sources of laboratory contamination during cell mounting for epifluorescence microscopy.
- To improve the accuracy of cell enumeration in low biomass environmental samples.
Main Methods:
- Assessed microscope slides and coverslips for contamination before and after cleaning protocols (autoclaving, detergent, ethanol).
- Tested mounting solutions (DAPI, PBS, immersion oil) for contamination before and after autoclaving and filtration (0.2 µm).
- Implemented a combined cleaning and triple-filtration method to reduce background microbial load.
Main Results:
- Reduced background contamination by nearly two orders of magnitude (from ~1x10^4 to ~300 cells/filter paper).
- Successfully validated the method on low biomass glacial sediment samples, improving detection near the epifluorescence microscopy limit.
Conclusions:
- A rigorous cleaning and filtration protocol significantly minimizes background contamination in epifluorescence microscopy.
- This approach enhances the reliability of cell counting for low biomass samples, crucial for environmental microbiology studies.
