6-2 Investigating bacterial motility by flagella

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Period 1

Materials

Young broth cultures (12-15 hours) of Enterobacter aerogenes (or other motile organism) and Staphylococcus epidermidis. These will serve as positive and negative controls (respectively) for the unknown.

Young broth culture of an unknown organism

3 tubes of Motility Medium

Demonstration (phase microscope) of Rhodospirillum, a motile, spiral-shaped bacterium.

  1. Note the demonstration of Rhodospirillum set up under the phase-contrast microscope (Figure 5-2).
  2. For each broth culture, prepare a wet mount as previously done. It is best to trap some air bubbles under the cover slip, as respiring organisms (such as the facultative anaerobes we are using) generally show increased motility where oxygen is available.
  3. If you use the regular light microscope, focus initially with the 10X objective, switching to the 40X objective and then (but only if needed) the 100X, oil-immersion objective. Adjust the light with the iris diaphragm; optimum results are achieved with a relatively low light intensity. (Alternately, excellent results can be had by the use the phase microscope with the 40X (middle) objective lens in place. Do not use oil with this lens!)
  4. Look for true motility which should be evident for the E. aerogenes culture. Do not be misled by Brownian motion or currents (see introduction). Tabulate results below.
  5. Discard the wet mount (without disassembling it!) into the disinfectant.
  6. Inoculate each broth culture into a separate tube of the semisolid Motility Medium. Use the needle and carefully stab-inoculate the medium about half-way down through the center. Incubate at 30 °C.

Period 2

  1. Observe the tubes of Motility Medium for growth away from the line of inoculation and the subsequent cloudiness throughout the medium as discussed in the introduction. In a well-lit room, hold all of the tubes together against a darker part of the ceiling (such as the space between the fluorescent light units) so that degrees of growth can be discerned easily. Ignore all surface growth and any growth that might be creeping down from the surface along the inner wall of the tube. Tabulate your results

Figure 6-1 Reactions in Motility Medium

The appearance of motile (A: Bacillus cereus) and non-motile (B: Klebsiella planticola) microbes in motility medium.

reaction of motile and non-motile microbes in motility medium

Figure 6-2 is a video of motile and non-motile bacteria in the microscope.

Figure 6-3 video of gliding bacteria in the microscope.

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