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.
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.
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