The protocol simultaneously tracks bacterial swarming responses to different inducer concentrations on a semisolid agar. This is achieved in a cost-effective manner. This high throughput screening technique negates the need of agar with various inducer concentrations to observe the microchemotactic responses.
The technique is designed for drug screening and also to observe the micropathogen SD responses. Also, it can be used to study microresponses to various biochemical cues in the human host. This method can provide insight into bacterial chemotaxis.
Bacteria will respond to various concentrations of chemical attractants known as positive chemotaxis or chemical repellents known as negative chemotaxis. A carefully prepared set up is essential to avoid a variations between the gel angle of the lower layer to prevent the reproducability issue. To begin, label 13 by 13 centimeter square Petri dishes with stains and inducer names.
Then prop the dishes up over the edge of the lids. Add arabinose solution to warm bottom layer medium. Add 40 milliliters of warm bottom layer medium.
Allow the bottom layer medium to cure uncovered for one hour inside a laminar flow hood undisturbed. Once the bottom layer is completely solidified, remove the lids and lay the Petri dishes inside a laminar flow hood. Add 40 milliliters of warm upper layer medium using a motorized pipette filter.
Cure the double layer plates covered and undisturbed for one hour, then store them at four degree Celsius for 24 hours. Place the marked A4 paper under a well solidified gradient plate. Push the broader side of a 100 microliter pipette tip into the semisolid medium surface at the marked position and press the pipette tip until it reaches the bottom of the upper layer medium.
When the tip touches the bottom, stop applying any vertical force to the tip and gently rotate the tip to isolate the content of the cylindrical well. Horizontally move the pipette tip along a small distance to allow airflow into the narrow place set aside. Press the tip with the index finger to block the gas flow inside the tip.
Pull the tip out vertically, keeping the well content in the tip while pulling it out. Add 80 microliters of the overnight growth culture into every well. Take the swarm plates out of the incubator one at a time every 12 hours and place them in the gel imaging system.
Select gel imaging mode, expose the swarm plate to white light, and adjust the focal length to obtain a clear view of swarms. Enhance the brightness of the swarms for precise observation by adjusting the exposure time to 300 milliseconds. Adjust the threshold to minimize interference from the background light.
Save the image file for further analysis. Record the imaging time, inducer type, gradient orientation, and strains in a txt file. Click Process, then Shadows to enhance the sharpness of the image.
Click on Process followed by Batch to process the image. Then process an image as a reference by clicking on Process, Shadows, and South. Click Process, Batch, Macro to open the Batch Process window.
Look for the commands displayed in the window. Type the folder address of the original images in the output file address by clicking Process in the Batch Process window. Use the wand tracing tool to select swarms individually and double click on the wand tracing tool to adjust the tolerance until the generated line fits the swarm boundary correctly.
Click on Analyze, then Measure to export the area value. E.coli K12 YdeH was tested on arabinose gradient plates used to demonstrate the surface swarming process with overlap occurring between adjacent wells. A plate with three test wells enabled the formation of non-overlapping boundaries.
P.aeruginosa PAO1 YdeH swarming motility was promoted with increased arabinose concentration from the lowest concentration, but gradually restricted with higher concentrations arabinose concentrations. E.coli K12 wild type strains tested on resveratrol gradient plates within the zero to 400 microliter concentration range showed a modest restriction of the swarming motility with increasing resveratrol concentration. Swarm areas were quantified by ImageJ software.
The swarming curve displayed the multiple concentration responses. The most crucial part of this procedure is manufacturing a double layer swarm plate with a specific concentration range of inducers. This method enables researchers to investigate the induced surface swarming within a specific concentration range and to quantify the effects of other inducers and components on swarming.
