Live-Cell Time-Lapse Imaging of Bdellovibrio bacteriovorus Predation on E. coli
0 조회수 • 3:21 분 • February 26th, 2026
Begin with a Petri dish containing a co-culture of Bdellovibrio bacteriovorus, a predatory bacterium, and E. coli, its prey, beneath an agarose pad.
The agarose pad keeps E. coli immobilized, while B. bacteriovorus remains motile and actively invades prey cells.
The predator expresses a red cytoplasmic marker and a green fluorescent tag fused to a DNA polymerase subunit for visualization.
Secure the dish in a holder and apply immersion oil to the base of the dish and the microscope objective for optimal resolution.
Mount the holder on the microscope and configure it for dual-channel fluorescence detection.
Use brightfield optics to locate the focal plane and select multiple non-overlapping imaging fields.
Set time-lapse parameters to capture both brightfield and fluorescence images sequentially at defined intervals.
This method enables observation of predation beginning with prey attachment and invasion, followed by prey cell transformation, filamentous growth, septation, progeny formation, and ending with prey cell lysis.
For Time-Lapse Florescence microscopy of the co-culture, place the dish in a Petri dish holder such that the dish will not move over the course of the experiment and place a one drop of immersion oil onto the inverted microscope objective and one drop on the bottom of the dish. Mount the holder onto the microscope stage within the inverted microscope chamber and in the microscope software set the magnification to 100X and the polychromic lens to GFP and mCherry. Using an eye piece in Brightfield, locate the focal plane and open the pointlist manager.
Move the stage and use the Mark Points button to store the coordinates of at least 10 positions of interest. Switch the camera valve from the eye piece to the monitor. Set the focal plane and click the Replace Point button in the pointlist manager to calibrate each point.
Open the experiment designer and unmark disease stacking box. Select the appropriate fluorescent channels and the optimal illumination settings and set the intervals between the image acquisition and the total time of the experiment. Enable focus maintenance for the chosen positions and select the data folder in which the image files should be automatically saved.
Recheck all of the settings in the microscope software control and start the time-lapse experiment. After the first hour of the experiment, check that all of the stage positions are still in focus. When the time-lapse experiment is finished, remove the Petri dish and discard the 35 millimeter dish with an agarose pad according to the appropriate bio-safety protocols.
Overview
This article details a method for observing the predatory behavior of Bdellovibrio bacteriovorus on E. coli using time-lapse fluorescence microscopy. The setup allows for the visualization of various stages of predation, including attachment, invasion, and lysis of the prey cells.
Key Study Components
Area of Science
- Microbiology
- Cell Biology
- Fluorescence Microscopy
Background
- Bdellovibrio bacteriovorus is a predatory bacterium known for its ability to invade and consume other bacteria.
- E. coli serves as a model prey organism in this study.
- Time-lapse fluorescence microscopy is a powerful technique for observing dynamic biological processes.
- The use of fluorescent markers allows for the visualization of specific cellular events during predation.
Purpose of Study
- To observe the predation process of B. bacteriovorus on E. coli in real-time.
- To document the stages of prey cell transformation and lysis.
- To refine microscopy techniques for better visualization of microbial interactions.
Methods Used
- Co-culture of B. bacteriovorus and E. coli in a Petri dish.
- Application of immersion oil for enhanced microscopy resolution.
- Configuration of the microscope for dual-channel fluorescence detection.
- Time-lapse imaging to capture sequential events during predation.
Main Results
- Successful visualization of the predation process from attachment to lysis.
- Documentation of the transformation of E. coli cells during predation.
- Identification of key stages in the life cycle of B. bacteriovorus.
- Establishment of a reliable method for future studies on microbial predation.
Conclusions
- The study provides insights into the dynamics of bacterial predation.
- Time-lapse fluorescence microscopy is effective for observing microbial interactions.
- Findings contribute to the understanding of predator-prey relationships in microbial ecosystems.
Frequently Asked Questions
Executive Industry Relevance
Real-time visualization of bacterial predation dynamics supports mechanistic de-risking in antimicrobial target validation by enabling direct observation of host-pathogen interactions. This approach provides quantitative, time-resolved data on bacterial lysis and progeny release, informing predictive confidence in antimicrobial screening assays. The method bridges discovery biology and translational microbiology by establishing a disease-relevant system for evaluating novel antibacterial strategies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by visualizing bacterial invasion and intracellular replication in real time.
- Operational Value: Supports functional target validation through direct observation of prey cell transformation and lysis kinetics.
- Predictive Value: Facilitates mechanistic de-risking of antimicrobial candidates by correlating molecular interventions with predation outcomes.
Screening & Assay Development
- Assay Readiness: Generates standardized, quantitative fluorescence readouts for high-content screening of antibacterial compounds.
- Reproducibility: Immobilized prey configuration ensures consistent field selection and focus maintenance across time-lapse experiments.
- Scalability: Multi-position pointlist manager enables parallel data collection from defined imaging fields for comparative analysis.
Translational & Preclinical Research
- Disease Relevance: Establishes a controlled host-pathogen model to study microbial predation dynamics relevant to infection mechanisms.
- Translational Continuity: Connects discovery-stage observations to preclinical evaluation of antibacterial efficacy and resistance development.
- Risk-Adjusted Decisions: Provides visual and quantitative endpoints for go/no-go decisions in early antimicrobial development.
Pipeline & Workflow Integration
The method integrates into antimicrobial discovery workflows by enabling real-time monitoring of bacterial viability and structural changes during compound exposure, supporting lead identification and optimization.
- Discovery Biology: Direct visualization of predation stages supports hypothesis testing and pathway clarification in host-microbe interactions.
- Screening: Dual-channel fluorescence detection allows simultaneous tracking of predator localization and prey DNA replication for multiplexed assay readouts.
- Analytics: Time-lapse capture of attachment, invasion, filamentous growth, septation, and lysis enables quantitative kinetic analysis of antibacterial effects.
- Translational Research: Real-time observation of prey cell lysis supports biomarker alignment with antimicrobial mechanism of action.
- Enterprise Reuse: Standardized microscopy setup and pointlist calibration allow reuse across multiple predation models and compound screens.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in antimicrobial action by providing direct visualization of bacterial predation dynamics.
- Operational Value: Immobilized prey configuration and focus maintenance enhance reproducibility and reduce experimental variability.
- Strategic Value: Enables data-driven go/no-go decisions by linking compound treatment to observable predation inhibition or enhancement.
- Portfolio Impact: Supports risk-adjusted prioritization of antimicrobial candidates based on real-time efficacy and resistance profiles.
Implementation Considerations
- Requires expertise in fluorescence microscopy, time-lapse imaging, and bacterial co-culture techniques.
- Dependent on inverted microscope with dual-channel fluorescence, immersion oil compatibility, and stage stabilization hardware.
- Necessitates standardization of agarose pad thickness and prey immobilization protocols across users and labs.
- Adaptation considerations include fluorescent marker compatibility with different bacterial strains and predator-prey systems.
- Practical limitations include phototoxicity risks from prolonged illumination and agarose diffusion affecting long-term immobilization.
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마지막 업데이트: 29 8월 2026