Our protocol provides a simple yet robust approach to identify and characterize competitive mechanisms between different strains of Vibrio fischeri bacteria. This method uses stable plasmids that encode different antibiotic resistance genes and fluorescent proteins that allow for differential selection and visual discrimination of each bacterial strain in a mixed culture. To begin, use a sterile toothpick to scrape the cells from the agar plate in the size of a grain of rice.
Resuspend the cells in a 1.5 milliliter microcentrifuge tube containing one milliliter of LBS broth. To break up the cell clumps, press them into the side of the tube and then pipette up and down vigorously. Close the tube and vortex for one to two seconds and repeat if the cell clumps are still visible until the sample is visually uniform.
Repeat these preparation steps from cell scraping to vortexing for all samples. Measure and record the optical density at 600 nanometers for all samples. To obtain an accurate OD measurement, dilute the samples up to 10-fold using LBS broth.
Then normalize each sample to the desired OD 600. To mix the reference strain and competitor strain in a one to one ratio, add 100 microliters of each strain normalized to OD 1.0 to a labeled 1.5 milliliter centrifuge tube and vortex for one to two seconds. As a control, mix the reference strain with a differentially tagged version of itself and vortex as well.
It is critical to achieve the correct starting ratio as it can considerably change the outcome of the experiment. To ensure success, this step may require optimization for a given bacterial species. After repeating this step for each biological replicate, there should be four biological replicates with differentially tagged reference strains as controls and four biological replicates with differentially tagged reference and competitor strains.
To make culture spots that will be used for fluorescence microscopy after the incubation, add 10 microliters of each control and experimental mixture onto a Petri plate containing LBS agar. After the spots have completely dried on the bench, incubate the plates at 24 degrees Celsius for 24 hours. To make culture spots that will be used to quantify the colony forming units for each strain at the end of the experiment, add 10 microliters of each control and experimental mixtures into 24 well plates containing one milliliter of LBS agar per well.
After the spots have completely dried, incubate the plates at 24 degrees Celsius for five hours. To perform a serial dilution of starting coincubation mixtures, rotate a 96 well plate 90 degrees so there are eight columns and 12 rows. Label each row with the strain ID, the replicate number, and the time starting with t zero.
Label the LBS agar plates supplemented with the appropriate antibiotic on which to spot the dilution series while making sure to identify which strain each antibiotic plate is selecting for. With a multi-channel pipette, add 180 microliters of LBS broth to each well leaving the first column empty for the undiluted coincubation mixture. Using a 200 microliter single channel pipette, transfer 100 microliters of the coincubation mixture from the tube to the first well of the first column, then discard the tip and repeat for all coincubation mixtures.
With a multi-channel pipette, transfer 20 microliters from column one to column two and pipette up and down to mix. Discard the tips and repeat for each column while being consistent with the number of pipetting up and down for each dilution. By the end, each row will contain a 10-fold serial dilution of the initial mixture.
Use a multi-channel pipette fitted with eight tips to aspirate five microliters from each well in a dilution series and spot onto the Kanamycin supplemented LBS agar plate that selects for the reference strain. Repeat this step to select for the competitor strain spotting onto the Chloramphenicol supplemented plate. After the spots have dried completely, place the plates in the incubator at 24 degrees Celsius.
After the coincubation spots and the 24 well plates have been incubated for five hours at 24 degrees Celsius, add one milliliter of LBS broth to each well. Resuspend by pipetting up and down until all cells have been resuspended being consistent with the vigor in which the cells are resuspended across all replicates. Once each coincubation spot is resuspended, prepare a 96 well plate for a serial dilution and LBS agar plates with the appropriate antibiotics on which to spot the dilution in the same way as previously done.
To complete the serial dilution for t final measurements in the 24 well plates, perform steps used for the serial dilution of the starting inoculum and spot each dilution on LBS agar plates as done previously. Proceed with imaging the coincubation spots on the Petri plates and data analysis as described in the manuscript. In the experimental treatment, the reference strain and competitor strain one are present at a proportion of 0.5 at the beginning and end of the experiment consistent with the control treatment.
Log RCI values were not statistically different from zero for the control treatment or when the reference strain was incubated with competitor strain one. These data suggest that there was no competition between the reference strain and competitor strain one. By contrast, when the reference strain was incubated with competitor strain two, the reference strain decreased from a proportion of 0.5 at the beginning to a proportion less than 0.01 at the end of the experiment.
Furthermore, log RCI values were significantly greater than zero when the reference strain was incubated with competitor strain two. Together, these data indicate that the proportion of the reference strain decreased in the presence of competitor strain two suggesting competition between the strains. When the reference strain was incubated with competitor strain one, CFUs of both strains increased over the course of five hours and were not significantly different suggesting that no competition occurred.
Also, approximately 3200%recovery was observed which was not statistically different from the control. However, when the reference strain was incubated with competitor strain two, reference strain CFUs were significantly lower than strain two CFUs in the control at five hours. Also, approximately 4%recovery was observed significantly lower than the control.
Altogether, these data indicate that strain two out competed the reference strain by killing its cells. The most important step in this procedure is achieving the correct starting ratio as it can significantly impact results.
