This method was designed to quantify contact-dependent competition via the type VI secretion system between bacterial strains at the single cell level. This technique uses total cell area rather than cell counts to quantify bacterial competitions, making it easy for researchers without proprietary image analysis software access. This method can be easily modified to quantify competition between diverse culturable microbes.
Additionally, the experimental setup can be optimized for use on either upright or inverted microscopes. To begin with, prepare the agarose pad solution by dissolving 2%low melt agarose into marine PBS. Heat the solution briefly for about 30 seconds in the microwave and vortex until the agarose is completely dissolved.
Keep this solution in a 55 degree Celsius water bath until ready to use. Next, wrap a piece of lab tape around a glass slide five times. Repeat wrapping of tape on the same slide, such that the distance between the two tape pieces is slightly smaller than the coverslip width.
To ensure a flat agarose pad surface, pipette warm agarose solution between the two pieces of tape and immediately put a coverslip on top such that the coverslip makes contact with the liquid and pushes out any bubbles in the agarose solution. Let the agarose pad solidify at room temperature for at least one hour. Then cut this agarose pad with a razor blade into four 5 square millimeter pads to be used for imaging.
To prepare the strains for co-incubation, streak out each strain from frozen stocks onto the LBS agar plates supplemented with the appropriate antibiotics and incubate overnight at 24 degrees Celsius. The next day, pick two colonies from each strain and resuspend the colonies in antibiotic supplemented LBS medium. Incubate resuspended colonies overnight at 24 degrees Celsius with shaking at 200 rotations per minute.
The next morning, subculture each biological replicate one into 1, 000 times fresh LBS medium without antibiotics and incubate until cells reach an optical density of about 1.5 at 600 nanometers. Measure and record the optical density at 600 nanometers for all samples. Normalize each sample to an optical density of one by diluting the culture with LBS medium.
Mix the two competing strains at an equal ratio by adding 30 microliters of each normalized strain to a labeled 1.5 milliliter tube. Vortex the mixed strain culture for one to two seconds. Mix the competing strains for each biological replicate and treatment to generate a total of four mixed strain tubes.
Concentrate each mixed culture threefold by centrifuging to ensure sufficiently dense competing cells for contact-dependent killing during co-incubation. Discard the supernatant, resuspend each pellet in 20 microliters of LBS medium and perform concentration procedures for each sample. If imaging on an inverted microscope, begin by spotting two microliters of a mixed culture onto the number 1.5 coverslip bottom of a 35 millimeter Petri dish and place the agarose pad over the co-incubation spot.
Place a 12 millimeter circular glass coverslip over the agarose pad. Repeat spotting and placing of coverslip for the remaining three mixed cultures, which will result in four dishes to be imaged. Before proceeding ahead, let the slides sit on the bench top for about five minutes to allow the cells to settle on the agar pad to avoid movement during the imaging process.
Begin by focusing on cells using DIC to minimize photobleaching effects. Based on the average size of a single bacterial cell, use a 100X oil objective. Adjust the exposure time and acquisition settings for each channel with minimal background detection.
Select at least five fields of view and acquire images in each appropriate channel. Open the image analysis software and import the image files to be analyzed. Convert the image to grayscale, separate the channels, and begin by thresholding and creating a binary mask of the pre-processed image.
Select Analyze, then from the dropdown menu, select Set Scale and enter the appropriate values for the microscopy setup. Next, go to set measurements and select Area. Under analyze tab, select analyze particles using the default settings.
If debris is present in the sample, the size or circularity could be adjusted to filter out non-cell particles. Select Show and then Outlines so that the output of filter out analysis will include a numbered outline of all particles analyzed. Export the measurements into spreadsheet software for further analysis and graphing.
The representative fluorescence images of each experimental treatment are shown. Concentrating mixed culture before spotting resulted in rounded or disappeared target cells over two hours, indicating successful inhibition. When the mixed culture is not concentrated, cells remain dispersed on the slide.
Without sufficient cell-to-cell contact, the target strain is not inhibited by the lethal strain. The target cells neither disappeared nor became rounded when co-incubated with a T6SS mutant in crowded or dispersed conditions. Thus, the target was uninhibited in either treatment.
The particle analysis results were graphed and analyzed for both target strain and inhibitor strain. Greater than 100%of the initial target area at the final time point represents a net increase in the target and lower than 100%of the initial target area indicated a net decrease in the target. The net growth of the inhibitor strain was observed across all treatments.
However, the percent of the initial area for the inhibitor strain was significantly higher when a wild-type inhibitor was co-incubated with the target in crowded conditions compared to all other treatments. To get clear images, the agarose pad needs to be as flat as possible. Cut tape pieces before wrapping them around the slide to ensure they are the same length.
