Live-Cell Bdellovibrio bacteriovorus imaging has been challenging due to the complex life cycle of these predatory bacteria. Our protocol allows monitoring of the stages of complete life cycle in real time. Although our protocol is based on a specific equalize strains, it may be easily adapted to a variety of bacterial strains including pathogenic strains.
To set up a B.bacteriovorus culture, combine one milliliter of fresh 24-hour cultured bacteria with three milliliters of overnight prey culture in a 250 milliliter flask containing 50 milliliters of calcium hippies buffer supplemented with antibiotics as needed. After a 24 hour incubation at 30 degrees Celsius and 200 revolutions per minute, check that the prey cells have been fully lysed by liquid mounted face contrast microscopy. Numerous multi-attack phased B.bacteriovorus cells should be present and no E.coli cell or bdelloplast should be visible.
Strain the B.bacteriovorus culture through a 0.45 micrometer pore size filter into a 50 milliliter conical tube and suspend down the filtrate in a centrifuge. Then re-suspend the B.bacteriovorus pellet in three milliliters of calcium hippies buffer to a final optical density at 600 nanometers of approximately 0.2 and incubate the bacteria at 30 degrees celsius and 200 revolutions per minute for 30 minutes. To prepare the host cells, select a single colony from the host strain of interest and to nodulate the bacteria in 10 milliliters of YT medium supplemented with antibiotics as necessary for an overnight incubation at 37 degrees Celsius and 180 revolutions per minute.
The next morning transfer two milliliters of the overnight culture to a two milliliter test tube and pellet the cells by centrifugation. Then re-suspend the pellet in 200 microliters of calcium hippies buffer. It is very important to fully re-suspend the whole cell pellet after centrifugation to make sure that the prey cell are separated at the single cell level.
To prepare the cells for time-lapse fluorescence microscopy, first, mix 200 milligrams of low fluorescent molecular grade agarose with 20 milliliters of calcium hippies buffer and dissolve the agarose in a microwave. Pour three milliliters of the melted agarose into a 35 millimeter glass bottom dish and allow the agarose to solidify. Using a laboratory micro spatula, carefully remove the agarose pad from the dish without disturbing the center pole of the pad and place the pad bottom side up on a Petri dish cover.
Place five microliters of the concentrated host cell suspension onto the flipped pad and use an inoculation loop to spread cells in the center pole. Next add five microliters of the concentrated B.bacteriovorus suspension onto the host cell coated surface without spreading and quickly return the gel to the 35 milliliter glass bottom dish top side down. Then cover the dish with a lid.
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 polychrome lens to GFP and Mcherry. Using an iPS in Brightfield, locate the focal plane and open the pointless manager.
Move the stage and use the mark points button to store the coordinates of at least 10 positions of interest. Switch the camera val from the iPS to the monitor. Set the focal plane and click the replace point button in the pointless to manager to calibrate each point.
Open the experiment designer and unmark disease stacking box. Select the appropriate fluorescent channels and the optimal elimination 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.
For processing of the time-lapse image data, transfer the experimental data to a computer loaded with the Fiji software and open an image in Fiji. In bio-formats import options, select view stack with hyperstack. In composite color mode, select auto scale.
By scrolling through the image stacks assess whether the cells and bdelloplasts remained focus over the course of the experiment. Whether the green fluorescent spots from the DNA and mNeon green are present within the B.bacteriovorus cells inside the bdelloplasts throughout the growth phase and whether all of the stages of the predatory lifecycle can be visualized. To analyze a particular B.bacteriovorus cell bdelloplast, use a selection tool to mark the cell of interest.
Then duplicate the chosen region. In the duplicated image, click process and smooth and for each fluorescent channel, click image, color, channels tools, and select the channel of interest. Then click image, adjust, and brightness contrast to adjust the brightness and contrast of the image in each channel.
To add a scale bar, select analyze, tools, and scale bar. To add a timestamp to the images, click images, stacks, and time stamper. To save the modified images, select TIF.
To save the data as an image sequence, AVI to produce a movie, or PNG to save a single image of the current frame. This Time-Lapse Fluorescence microscopy based system allows the real-time tracking of individual B.bacteriovorus cells and provides valuable information about each stage of the complex predatory lifecycle. The pillsy mCherry fusion enables the entire predatory cell to be labeled in the attack phase as well as in the early stage of the growth phase.
The transition from the attack to the replicative phase can be visualized not only by the host bdelloplast formation but also by the appearance of the replisome. The replisome is assembled at the invading cell pole and more than one replisome can be observed within a growing B.bacteriovorus filament as the reproduction phase progresses. After several copies of the chromosome are synthesized, the replication is terminated.
Finally, the multinucleated filament undergo septation and the progeny cells are released from the bdelloplast. The B.bacteriovorus cells should be material enough to actively search for their prey and the whole cell density should be high enough to allow predatory cell attachment. This platform may be useful in experiments involving multi-drug resistant pathogens and to facilitate improvements in the genetic engineering of the B.bacteriovorus as alive antibiotic in human and veterinary medicine.
