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Author Spotlight: An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection
Published on: June 30, 2023
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An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection
Elijah I Christenson1, Qingyang Zhang2, Ruth Plymale3
1Department of Biology, Ouachita Baptist University.
Journal of Visualized Experiments : Jove
|July 17, 2023
Summary
This study presents a new microplate method to track bacterial growth and phage interactions over 96 hours. The developed protocol quantifies phage infection metrics for slow-growing bacteria like actinomycetes.
Area of Science:
- Microbiology
- Molecular Biology
- Bioinformatics
Background:
- Bacteriophages (phages) are crucial microbial predators influencing bacterial population dynamics in natural ecosystems.
- Understanding phage-bacteria interactions is vital, especially with slow-growing hosts like actinomycetes, but requires reliable long-term quantification methods.
- Existing spectrophotometric methods face challenges with small volumes and extended incubation periods.
Purpose of the Study:
- To develop and validate an easy, reliable method for quantifying long-term bacterial growth in the presence of phages.
- To adapt standard 96-well microplates for co-culturing phages and slow-growing bacteria over extended durations (96 hours).
- To establish a protocol for analyzing phage infection metrics using spectrophotometric data.
Main Methods:
- Adapted a 96-well microplate format for co-culturing bacteria and phages without sub-sampling for 96 hours.
- Utilized spectrophotometric absorbance measurements (optical density) recorded every 8 hours to monitor bacterial growth.
- Employed R software for analyzing optical density data to calculate infection metrics.
- Incorporated modifications to mitigate evaporation and lid condensation in microplate assays.
Main Results:
- Successfully quantified long-term bacterial growth and phage interactions in a 96-well microplate format.
- Generated infection metrics including percent growth inhibition, relative virulence, and the Stacy-Ceballos index.
- Demonstrated the effectiveness of the adapted protocol for extended-duration growth curve experiments.
Conclusions:
- The outlined procedure provides an effective and robust method for conducting and analyzing extended-duration microplate growth curve experiments involving phages and slow-growing bacteria.
- These protocols facilitate microplate-based assays crucial for studying phage-bacteria interactions in environmental and clinical microbiology.
- The method addresses technical challenges associated with long-term incubation of small volumes, enhancing high-throughput capabilities.
