Many bacteria have flagella that make them capable of swimming through water-based environments. Motility can be determined by observing cells in a wet mount. However this determination can be difficult because bacteria are small enough that they are bounced around by water molecules. This random movement, called Brownian motion, can be confused with self-propelled motility.
Another way to determine motility is with a Motility Agar stab. Motility Agar is soft agar in a test tube (without a slanted surface). Cells are stab-inoculated into the agar (the top surface is not inoculated). Non-motile bacteria will only grow where they were inoculated. Motile bacteria will grow along the stab and will also swim out away from the stabbed area. Thus, a negative result is indicated by growth in a distinct zone directly along the stab. A positive result is indicated by diffuse (cloudy growth), especially at the top and bottom of the stab.
+ - Positive (cloudy, diffuse growth)
- = Negative (well-defined growth along the stab).
V = Variable
CAUTION: This test only works with organisms that can grow anaerobically (e.g., facultative anaerobes). Obligately aerobic organisms will grow on the top of the agar but will not grow in the stab.
1. Obtain a motility agar tube from the back shelf.
2. Use an inoculating pick. Straighten the pick as much as possible.
3. Make a stab inoculation (about 2/3 of the way into the agar) from your unknown stock culture. Try to make the stab (in and out) as straight as possible. Straightness is important because you will be evaluating the amount of growth away from the stab. A messy stab will be difficult to evaluate.
4. Incubate at appropriate temperature for 24 to 48 hours (up to 72 hours).
5. Observe your culture by holding it up to a light source. One good way to evaluate a motility agar is to have someone else stand at a distance (for instance, halfway across the room) and hold your Motility Agar to the light. If you can see the region of growth from halfway across the room, the growth must be in a well-defined zone. This would indicate a NEGATIVE result. If you have a hard time seeing the growth from halfway across the room, this might indicate a positive result (depending on your eyesight).