The overall goal of this procedure is to visualize and quantify bacterial surface motility called swarming in a standard and reproducible manner. This is accomplished by first preparing and curing the surface motility plates. The second step is to inoculate the surface motility assay plates with bacteria and incubate these plates in a controlled environment.
Next, the patterns of bacterial growth on the plate assays are imaged in real time. The final step of the procedure is to process the images for quantification. Ultimately, this procedure provides a standardized protocol for surface motility plate assay preparation to generate quantitative dynamic information such as swarm expansion rate or bio product density distribution for surface modal bacteria.
Many groups perform surface motility assays. The main advantage of this technique is we provide a systematic protocol that minimizes the multiple variables that can lead to inconsistency. This method can help answer key questions in the field of bacterial surface motility, such as how bacteria colonize different types of surfaces.
Though this method can provide insight into pseudomonas swarming motility with some modifications, it can also be applied to study other surface modal bacteria. Generally, individuals new to this method will struggle because small changes in protocol or laboratory environment can greatly influence swarm assay results. Demonstrating the procedure will be Morgan, a graduate student from my laboratory.
To begin the protocol, prepare an agri mix by combining 200 milliliters of FAB minus ammonium sulfate swarm medium, 0.9 grams of noble agar, and 0.2 grams of caino acids into a 500 milliliter media bottle. Use a stir plate to mix the media thoroughly. Next, sterilize the media in an autoclave set at 121.1 degrees Celsius for 22 minutes with a fast vent option.
Immediately after sterilization, tighten the media bottle cap to prevent water evaporation. Place the media onto a magnetic stir plate and cool the AED to 50 degrees Celsius in an ambient temperature environment with active stirring. If the agar is to be used at a later experimental time point, it can be kept warm in a 60 degree Celsius water bath or an incubator for 15 hours without active stirring.
When the media reaches approximately 50 degrees Celsius at two milliliters of filter sterilized glucose, using standard sterile techniques, mix thoroughly with the magnetic stir bar to prevent bubble formation in the media. In a hood, use a sterile serological pipette to aliquot 7.5 milliliters of the edia into individual 60 millimeter Petri dishes. If a larger swarming surface area is desired, aliquot 25 liters of edia into 100 millimeter Petri dishes.
Leave all dishes unstacked and check the hood with a bullseye level to ensure an even horizontal surface for the agar to solidify on. For 60 millimeter plates, set aside the dish lids and cure the uncovered agar in the hood for 30 minutes. Larger 100 millimeter dishes will require a longer curing time.
The humidity, airflow, and temperature of a given laboratory may necessitate testing the curing time for optimal swarming of your bacterium. After drying, the APLs should be immediately inoculated with cells that have been pre grown separately to the desired growth phase. To begin, pick an isolated bacteria colony from a fresh LB plate and inoculate into six milliliters of culture.Media.
Incubate the culture overnight at 37 degrees Celsius with horizontal shaking. On the next day, inoculate one to five microliters of the overnight culture over the dried swarm agar plates by poking the agar surface with a sterile toothpick or wire inoculation needle, depending on the bacteria strain, transfer the swarm assay plates into an incubator with a temperature set to either 30 degrees Celsius, 37 degrees Celsius, or 42 degrees Celsius. Invert the plates such that excess moisture condenses on the plate lid and not on the agar, equilibrate the bacteria at strain specific temperatures for either two or four hours just prior to time-lapse imaging.
Next, transfer the plates into the in vivo imaging unit such that the bacteria on the agri surface are facing down towards the inverted camera of the unit. Fill all Petri dish lids with a small amount of water. Then place each lid with waterside facing up on top of the inverted AER plates.
Inside the imaging unit, seal the imaging enclosure to maintain a constant humidity level, adjust the imaging settings of the imaging unit and commence time-lapse imaging of swarming activity. After real-time imaging, the swarming dynamics of the bacteria can be analyzed using standard image analysis such as Image J in a surface motility experiment. Assaying for the agar moisture content just prior to inoculation is crucial in achieving successful swarming results.
Optimally AER plates will facilitate a tight inoculation spot and enhance bacterial swarming activity, whereas over dried plates will suppress surface motility in addition to moisture content, the presence or absence of media additives can also affect the swarming activity of bacteria as a function of incubation temperature by utilizing bacteria strains, expressing luminescence or fluorescent proteins. The swarming competition dynamics between two different bacteria species can be captured in a time-lapse video. Furthermore, changes in local cell density and the biosynthesis rate of mobility promoting lipids within the bacteria swarm can also be ascertained and quantitated with time-lapsed fluorescent microscopy.
Following this procedure. Other methods such as confocal microscopy can be employ in order to answer questions about individual cell behavior to generate a detailed, microscopic and microscopic analysis of bacterial surface motility. After watching this video, you should have a good understanding of how to perform a standard and reproducible swarm assay and obtain a comprehensive analysis of bacterial surface motility by preparing, curing and inoculating surface motility assays and processing and analyzing the result in patterns of bacterial growth.
