Determining Motility of Bacteria Using TTC Agar
In microbiology, it is important to be able to distinguish between different species of bacteria. One way to do this is to determine whether or not the bacteria are motile or not. Motility is the ability some microorganisms possess that allows for independent movement of the cell using metabolic energy. This is not the same thing as mobility, which describes the ability of an object to be moved. According to the lab manual, motility is movement with purpose and is genetically determined, requiring the use of at least one flagellum. The whip-like structures allow the cell to move toward desired areas or away from areas of danger. A semi-solid motility agar containing tetrazolium salt (TTC) was used to determine the motility of Pseudomonas aeruginosa (P.a.) and Staphylococcus aureus (S.a.). The oxidized TTC is colorless and soluble, so it mixes evenly throughout the media and acts as a metabolic indicator. When the bacteria come in contact with the TTC, it is reduced and changes color. The reduced TTC is insoluble and red in color. A negative result is indicated if the media turns red only along the stab line, while red spreading through the media will indicate a motile organism. It is hypothesized that P. aeruginosa is motile while S. aureus is not.
Materials/Methods
*3 TTC agar tubes *Inoculation needle
*Sharpie marker *Pure culture of S. aureus
*Pure culture of P. aeruginosa *Can for incubation
The three tubes of TTC agar were labeled with initials, the date of the experiment, the temperature, the exercise number, the lab class/time, and the organism being inoculated into the tube. One tube was labeled P.a., one tube S.a., and the final tube labeled for negative control. Aseptic technique was carefully followed throughout the experiment. The sterile needle was dipped into the pure culture broth then inserted straight into the media of the correctly labeled TTC tube. Special care was taken to be sure that the needle was inserted and retracted along the same path because too much wiggling of the needle would result in a false positive. These steps were followed with all three TTC tubes, with the negative control being inoculated last. All three TTC tubes were placed in a can and incubated for a week at 37˚C. The results were recorded during the following lab.
Results
In order to determine whether or not samples of bacteria are motile, motility agar containing TTC was utilized. The negative control showed no evidence of growth and no color change to the media. The TTC tube inoculated with P. aeruginosa had clear evidence of growth and the media had red color radiating away from the initial stab mark. The TTC tube inoculated with S. aureus had clear evidence of growth and the media had red coloring only along the stab line.
Conclusion/Discussion
Only certain species of bacteria are motile. This knowledge is helpful in attempting to identify bacteria. In order for bacteria to be motile, they must have the genes that cause them to have one or more flagella. The results of the negative growth control included in the test show that the aseptic technique was clean, and the other results can be trusted. The radiating of the red color away from the stab line in the P. aeruginosa tube suggests that the bacteria moved through the semisolid media. The red color appeared only where bacteria were. The only way the color could be away from the stab line is if the bacteria moved away from the initial stab. This supports the hypothesis that P. aeruginosa is motile. The results of the S. aureus tube show the red color remained only along the stab line. This suggests that the result is negative and supports the hypothesis that S. aureus are non-motile. There were no unexpected results with this experiment and all results support the hypotheses.
