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Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth
Published on: November 24, 2017
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Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth
Matthew Howell1, Jeremy J Daniel1, Pamela J B Brown2
1Division of Biological Sciences, University of Missouri.
Journal of Visualized Experiments : Jove
|December 30, 2017
Summary
Target-specific dyes and time-lapse microscopy visualize essential bacterial processes. This study provides protocols for labeling and imaging bacterial pathogens like Agrobacterium tumefaciens to understand cell growth and division.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Essential bacterial proteins govern core cellular processes like DNA replication, protein synthesis, and cell division.
- Target-specific dyes serve as vital probes to investigate these complex cellular mechanisms.
- Understanding bacterial protein function is crucial for developing effective antimicrobial strategies.
Purpose of the Study:
- To present protocols for cell labeling and time-lapse microscopy in the bacterial plant pathogen Agrobacterium tumefaciens.
- To demonstrate how target-specific dyes can reveal defects in membrane structure, cell wall biosynthesis, and chromosome segregation.
- To illustrate the utility of time-lapse microscopy in observing morphological changes linked to essential protein function.
Main Methods:
- Utilizing lipophilic dyes for membrane structure visualization.
- Employing fluorescent d-amino acids (FDAAs) to map peptidoglycan biosynthesis sites.
- Applying cyanine DNA stains for live-cell imaging of DNA replication and segregation during cell growth.
- Implementing time-lapse microscopy to monitor cellular morphology changes upon essential protein depletion.
Main Results:
- Lipophilic dyes effectively visualize membrane structures and identify blebs.
- FDAAs pinpoint sites of cell wall biosynthesis, revealing potential growth defects.
- Nucleic acid stains highlight issues in DNA replication or chromosome segregation.
- Time-lapse microscopy of protein depletion mutants showed characteristic morphological changes (e.g., filamentation, rounding).
Conclusions:
- Target-specific dyes combined with time-lapse microscopy offer a powerful approach for characterizing essential cellular processes in bacteria.
- The provided protocols are adaptable for studying diverse bacterial species, including other plant pathogens.
- This methodology aids in dissecting the roles of essential proteins in bacterial survival and pathogenesis.
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