Visualizing Bacterial Motility Based on a Color Reaction

Detection of bacterial motility is important because motility is crucial for pathogenicity. The protocol described here is a reliable method for the detection of bacterial motility, because it is based on the color reaction of TTC with intracellular dehydrogenases of living bacteria. The TTC semi-solid agar method for detecting bacterial motility is simple, easy to operate and does not involve large and expensive instruments.

The method can be used by clinical microbiologists or researchers to investigate pathogenic bacteria. Demonstrating the procedure will be Xiyi Zhuang, a senior experimentalist from my laboratory. To begin, prepare the traditional semi-solid medium by adding 10 grams of Tryptose, 15 grams of sodium chloride and four grams of agar in an adequate volume of distilled water.

Adjust the pH to 7.2 and make up the final volume to 1, 000 milliliters. Autoclave the medium at 121 degrees Celsius for 20 minutes and dispense the medium into 10 milliliter test tubes to a height of three centimeters. To prepare the TTC medium, cool the autoclaved traditional medium to 50 degrees Celsius then add five milliliters of sterile 1%TTC solution to 100 milliliters of medium.

Mix and dispense the medium into 10 milliliter test tubes to a height of three centimeters. Pick single colonies of bacteria from agar plates and inoculate them into both the semi-solid media by puncture using inoculating needles. Remember to include non-modal strains as negative controls and modal strains as positive controls.

To observe the effect of agar concentration on bacterial motility, prepare semi-solid media containing different agar concentrations and inoculate them by puncture. Incubate the tubes at 37 degrees Celsius and observe the results after 24 to 48 hours of incubation. If only the puncture line is red, characterize the bacteria as non-modal.

If the red color spreads outward along the puncture line, characterize the bacteria as modal. Bacterial motility was compared for various strains on traditional and TTC semi-solid media and summarized in this table. After culturing for 24 hours on TTC semi-solid medium, Staphylococcus aureus showed growth along the inoculated line.

Whereas, Escherichia coli showed growth in all directions around the inoculated line. Highest motility for Escherichia coli was observed in TTC semi-solid medium prepared with 0.3%agar in which the medium turned almost entirely red. In contrast, the area of red diffusion decreased in 0.5%agar and the diffusion was prolonged with increasing agar concentration to 0.8%It is important to adjust the pH to 7.2 and cool the autoclaved medium to 50 degrees Celsius before inoculation using the needles.

Bacteria should be inoculated by puncture into the semi-solid medium containing TTC. It is a direct method for detecting bacterial motility. It is also possible to detect motility indirectly by electronic microscope and