The protocols demonstrated in this video can be reproduced in any lab in the world. In combination, they represent a systematic and powerful approach to characterizing bacterial flagellum motility. Motility is a crucial aspect of bacterial dissemination and survival, and these three methods are useful and powerful tools for the analysis of both individual and collective motility.
The techniques described here can readily be applied to study an array of bacteria, including those classified as pathogenic in nature. When attempting this procedure for the first time, keep in mind that if the swarm plates are prepared incorrectly, inconsistent behavior or patterns that mimic but do not truly reflect collective motion may be observed. Grow overnight cultures of the desired motility deficient strain in five milliliters of Lennox broth at 30 degrees Celsius with horizontal shaking, then subculture in fresh LB under the same conditions to exponential phase.
Inoculate six microliters of the culture into the center of a soft agar plate, pushing the loaded pipette tip into the agar to gently expel the contents. Incubate the plate at 30 degrees Celsius until motility flares are evident, emanating from the inoculation point or the periphery of the motility rings. Use a sterile wire loop to lift the cells from the flare region and streak them onto an LB hard agar plate in order to purify single colonies.
To confirm that the isolated suppressor mutants have restored motility, culture them on soft agar plates for strains of interest. Include wild-type and the starting motility deficient strain for comparison. After incubating the culture plates at 30 degrees Celsius for eight to 10 hours, record the diameter of the outermost ring to establish which of the isolates have substantially restored motility.
Prepare an exponential phase culture of the strain of interest. Then pellet 10 milliliters of cells by centrifugation at 2000 times G for three minutes, and resuspend them in 10 milliliters of filter sterilized motility buffer, or MB.Wash the cells two more times in MB and resuspend them in one milliliter of MB after the final centrifugation. Transfer the cell suspension into a one milliliter syringe and attach a 23 gauge needle to the end.
Assemble an identical syringe and needle apparatus and attach the two together via six inches of polyethylene tubing, tightly sheathed over each needle tip. Shear the flagella by gently passing the cell suspension back and forth from one syringe to the other 50 times with one minute pauses between every 10 passes. Then centrifuge the sheared cells at 2000 times G for three minutes and resuspend them in 500 microliters of MB.To prepare a cell fixation chamber, stack an 18 by 18 millimeter cover slip over a glass microscope slide separated by double-sided tape.
Add 0.01%polylysine solution to the top of the chamber to flush it. Tilt the bottom edge onto a task wipe to draw the solution through the chamber, then leave it at room temperature for 10 minutes. Wash the chamber three times with 40 microliters of MB and add 40 microliters of sheared cell suspension to the top of the chamber.
Allow the cells to attach to the cover slip for 10 minutes, then gently flush the chamber with 40 microliters of MB to remove unattached cells. Transfer the slide to the microscope stage and use phase contrast microscopy and a 100X objective to scan the population for cells that are fixed in place and rotating on a single axis. Open the microscope software, make sure that the cells are in focus, and begin video acquisition to record the cell rotation for one minute.
From video playback, quantify the number of complete rotations per minute and the number of times the cell changes direction. Inoculate six microliters of a mid-exponential culture by spotting it on top of the agar. Leave the lid off for five to 10 minutes and replace it when the inoculum has dried into the agar surface.
Then incubate the plate at 30 degrees Celsius for eight hours. Pour approximately 30 milliliters of swarm agar into the right chamber of a dual compartment petri dish, ensuring that it is level with the plastic divider between chambers, but not overflowing into the left. After the agar is hardened, fill the left chamber with approximately 30 milliliters of swim or swarm agar.
Before it sets, use a sterile pipette tip to gently drive the agar over the border and connect the two sides with a one millimeter tall agar bridge that spans the entire length of the divide. Allow the plate to dry at room temperature. As you pour the agar, pay attention to its depth.
If it is too high, it will overflow. If it's too low, you will be unable to bridge the two chambers. This protocol was used to isolate pseudo revertant flares in a double mutant E.coli strain with impaired motility.
One of these revertants displayed motility close to wild-type. The double mutant parent and isogenic wild-type strain were used as controls. Video captures were analyzed to calculate rotations per minute, and the fraction of time that motors rotate in a clockwise direction, or tumble bias.
The yhjH mutant showed a few rotations per minute and a lower tumble bias compared to wild-type E.coli, as expected. Both the double mutant and its suppressor showed motor behavior similar to wild-type. The border crossing assay was used to compare the abilities of the wild-type and the suppressor isolate to swarm and then to move across the border and swarm on agar supplemented with kanamycin.
Both strains showed similar rates of swarming from an identical inoculation point, but crossover of the swarm to the antibiotic chamber was consistently greater for the wild-type than the suppressor. The difference between the two strains was more pronounced at higher kanamycin concentrations. The swarm assays can be tricky to set up.
You may need to spend some time optimizing the drawing conditions to suit your bacteria of interest, as well as your lab conditions during media preparation. The techniques in this protocol have been applied to an array of flagellated bacteria, and can likely be applied to a plethora of untested species.
