This protocol allows for visualization and tracking of interspecies bacterial interactions at the single cell level over time. This techniques main advantage is its applicability to studying bacterial interactions, including cell tracking, gene expression and viability staining. Additionally, the conditions can be modified to study different organisms.
To begin, prepare agarose pads by melting 2%low melt agarose in 10 milliliters of bacterial growth media in a clean 50 milliliter Erlenmeyer flask. Microwave the flask in short intervals until the agarose is in solution, then let it cool in a 50 degree Celsius water bath for at least 15 minutes. Align a silicon cut out with the opening in a 35 millimeter dish, then flip the dish and lightly tap the silicon cut out with a spatula to secure it to the dish and to remove any air bubbles.
Once the agarose has cooled, pipette 915 microliters of molten agarose into the mold and let the pad dry. Warm the microscope environmental chamber to the experimental temperature at least two hours prior to setting up the experiment. Prepare humidity wipes by tightly rolling up a lint-free wipe.
Place the rolled wipe inside a sterile Petri dish and add 500 microliters of sterile water directly to the wipes. Warm the wipes, pads and a sterile 35 millimeter dish to the experimental temperature in order to equilibrate all materials. Once the pads are dry, pipette one microliter of co-culture evenly across the bottom of a pre-warmed sterile 35 millimeter glass coverslip dish.
Remove the silicone cut out from the agarose mold using sterile tweezers, tilt it to the side and slip a slightly bent spatula under the edge of the agarose pad to drop it onto a sterile petriplate lid. Transfer the pad to the dish with the bacterial cells bottom side down by sliding a 90 degree angled spatula under the pad and placing it on top of the inoculated cover slip. Use the spatula to make the pad flush against the cover slip and gently press out any air bubbles.
Remove excess moisture from the moist wipe and place it around the edge of the dish, making sure it does not touch the pad. The sample is now ready for imaging. Prior to inoculated pads, set up the microscope by performing color illumination to align all image frames.
Once the inoculated dish is ready, view the dish with 100X objective and focus on the bacterial results. Click the phase option in the imaging software and adjust the percentage of DIA LED light emitted by selecting the light source and sliding the bar on the light percentage scale. Alternatively, manually enter an exposure time.
Save the settings for the phase channel by right clicking on the phase channel and selecting the Assign Current Microscope Setting option. If using fluorescence, select the fluorescence channel of interest and set the percentage of fluorescence light emitted, then adjust the exposure time as previously described. Alternatively, change the bid depth to adjust the dynamic range by selecting one of the other options in the drop down menu.
Save the settings for the fluorescence channel by right clicking on the fluorescence channel and selecting the Assign Current Microscope Setting option. In the XY tab, click the box in the point name column to save the selected XY position of interest. Then click the PFS box at the bottom of the tab to turn on the perfect focus system.
Focus the cells and click the arrow in the PFS column to save the focus of the XY position. Repeat this process for all other XY positions. Save the settings for the XY positions under the phase tab as previously demonstrated.
Click the time tab and check the box to select the acquisition interval, frequency and imaging duration. Then click the wave length tab and check the box to select channels to be used in imaging and channel interval frequency. When fished with the settings, click Run Now to initiate the time lapse imaging.
Compare to the P.aeruginosa cells in monoculture that remain grouped in rafts when in co-culture with S.aureus, P.aeruginosa has increased single cell motility towards S.aureus colonies. P.aeruginosa single cells will surround and eventually invade S.aureus colonies. Too much or too little humidity during the experiment and incorrectly dried pads are two common problems that lead to drift due to the pad shifting and dragging the cells out of the field of view.
Photo bleaching and photo toxicity can cause cells to first, loose their fluorescence then stop dividing and eventually die in subsequent frames. Cells that are initially too close in proximity may not have sufficient time to establish a gradient of secreted signals to which other species can respond. Bacterial cell viability can be visualized by adding propidium iodide to the agarose pads.
Green cells indicate live cells actively expressing GFP, while red cells indicate dead propidium iodide stained cells after being treated with P.aeruginosa cell free supernatant. The movement of individual P.aeruginosa cells are tracked from the frame in which a cell leaves the raft through the frame in which the cells reaches the S.aureus cluster. The distance between the P.aeruginosa raft and the S.aureus cluster, provide the Euclidean distance, while the total track lengths provide the accumulated distance.
Directedness of each cell is calculated as a ratio of the euclidean distance to the accumulated distance. Wild type P.aeruginosa moves toward wild type S.aureus with significantly higher directedness than toward S.Aureus lacking agr-regulated secreted factors which are necessary for directional motility. When attempting this protocol, it is important to keep in mind that each investigator will need to optimize conditions for drying agarose pads in live imaging based on geographical location, laboratory environment and their particular experiment.
This technique reveal interactions between Pseudomonas aeruginosa and Staphylococcus aureus, previously obscured during our investigations of their behavior in bulk culture, bringing us closer to understanding how these organisms interact during infections.
