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Motility in bacteria is achieved by any of several mechanisms. The most widespread mechanism is flagellar movement which allows travel in a liquid medium and is mediated by special threadlike organelles extending from the cell surface called flagella. Most rods and spirilla are motile by means of flagella; cocci are usually non-motile. A somewhat modified version of the bacterial flagellum is responsible for the movement of the bacteria known as spirochetes. These organisms possess an axial filament, consisting of two sets of flagella-like fibrils anchored at the two poles of the cell. Another type of movement observed for bacteria is known as gliding motility. It is the sole method of move-ment for certain of the cyanobacteria and myxobacteria. These organisms can move slowly over solid surfaces. Motility seen for any organism in this course will be due to flagella, the main focus of this experiment.

Virtually all bacteria which possess flagella are motile. Flagellation is a genetically-stable morphological trait of these cells. Certain environmental and nutritional conditions favor flagellar movement which can cease with increasing age (of the culture), temperature and concentration of waste products. Thus, optimal conditions for growth of the organism should be provided when one wishes to detect motility. The presence of flagella, as well as their number and distribution on the cell, are important characteristics for purposes of identification and classification of bacteria. When one or more flagella arise only from one or both ends of a rod or spiral-shaped cell, the arrangement is termed polar. When flagella arise randomly over the entire surface of the cell, the arrangement is termed peritrichous.

Motility is important in that an organism can swim toward optimal concentrations of nutrients and away from toxic substances. As an organism moves (runs), stops (twiddles), moves off in another direction, stops, etc. - in what superficially looks like a random fashion - one finds that the longest runs are in the direction toward the nutrient or away from the toxic substance. This type of purposeful movement is called chemotaxis. Other forms of tactic response include phototaxis (movement toward optimal light concentration or wavelength) and magnetotaxis (orientation and movement along lines of magnetic force). For more information of motility, see the chapter in the microtextbook on surface structures

Unlike flagella of eucaryotic cells (protozoa, algae, etc.), bacterial flagella are beyond the resolving capability of our lab microscopes. Flagella stains have been developed which coat the flagella to make them visible with the microscope and much practice is necessary to get consistently good results. Were we to have access to an electron microscope, we would be able to see the flagella (if present) and their arrangement for the organisms in this and other experiments in this course.

In our lab, determining the presence or absence of flagella is done by indirect methods, as we detect whether or not motility is evident under growth conditions made as favorable as possible for the organisms. With the wet mount, an actively-growing, young culture is required for observation. Motile organisms are usually easily seen as they move among each other in separate directions. One must, of course, discount Brownian motion, the movement due to bombardment of submicrosopic particles in the liquid, where the cells (alive or dead) appear to remain in one position but shake somewhat. One must also not confuse true motility with movement in a current of liquid where all cells (again, alive or dead) appear to be swept in one direction. We will also utilize tubes of Motility Medium wherein only motile bacteria can move away from the line of inoculation in the low (0.5% or less) concentration of agar; descendants of cells which have migrated throughout the medium show up as evidenced by turbidity (cloudiness) in the medium. One advantage of using Motility Medium is that a culture of any age can be used for inoculation - as long as it is pure and viable! In observing the tubes, one must always ignore growth at the surface of the medium and also between the medium and the wall of the tube. Such growth is not necessarily evidence of actual motility. Certain motile organisms, such as many which are strict aerobes, may grow with difficulty in the depths of the medium and ultimately give a false-negative reaction. Generally, one seeks to confirm microscopically any negative reaction seen in the tube.

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

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.

Above is a video of bacterial motility by flagella as view microscopically.

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