Techniques to examine Motility
Flagella are bacterial structures that allow directed movement, called motility. Motility enables bacteria to move towards favorable environments and away from unfavorable ones and is sometimes important in the characterization and identification of bacteria. Arrangement of flagella varies among species. A flagellum may occur singly at one end, or there may be more than one flagella at one or both ends (polar). Flagella may occur in tufts, or they can be arranged all around the cell (peritrichous). Not all motile bacteria have flagella and many bacteria are non-motile. There are different ways to examine motility or motility organelles. You should begin with the motility part of the SIMS test and if that is positive, do a wet mount to confirm motility and/or do a fagella stain to see if you can see flagella.
1. SIM medium is used to indicate motility. This special media relies on the ability of motile bacteria to move through a tube of semisolid medium. The growth of motile bacteria in such a tube will produce turbidity throughout the solid medium, whereas non-motile organisms will grow only along the line of inoculation. The media we will use is Sulfur Reduction/Indole Production/Motility media (SIM tubes) It is useful not only to determine motility but also tests for two metabolic pathways. Motility should be assessed first and if the SIM motility test appears positive do a confirmatory wet mount or hanging drop .
Inoculating the SIM tubes involves a technique you have not yet practiced. It is very similar to inoculations using the flame sterilized loop, but you will use an inoculating needle (the wire extending from the handle will not have a loop on the end). After picking up a visible amount of your isolate on the tip of a flame sterilized inoculating needle, you will stab it deeply into the center of the medium in the SIM tube, stopping just before or the bottom or stab it until the you are almost to the end of the needle.
Withdraw the needle through the same inoculation channel. If you don't have an inoculating needle, you may use a loop.
Inoculate a positive control organism using the same technique.
Incubate for 24-72 hours (or as appropriate)
Motile organisms will exhibit growth radiating from the stab inoculation line.
Non motile organisms will exhibit growth only along the stab inoculation line.
The interpretation of the metabolic pathways examined in this medium are described in Enzyme tests
2. Special Flagella Staining techniques can be used on non-living organisms following procedures similar to the Gram's stain. These techniques use special mordant stains to increase the width of the flagella allowing them to be seen with the light microscope.
To perform the flagella special stain, find the directions in: Special Stains: Endospore, Acid fast, Capsule, and Flagella. This stain is tricky to perform successfully so only use it when your other tests for motility are ambiguous or if you wish to determine arrangement of flagella in a clearly motile bacterial isolate.
3. Hanging Drop Materials NOT Available in S10
Motility in living cultures can be observed by the Hanging Drop Technique
. Use a loopful of an isolate either from a broth culture or from a tube of water into which a small amount of growth on solid medium has been transferred. The presence of directed movement (rather than Brownian motion) implies the presence of flagella. A hanging drop slide is a quick way to examine motility.
Put 4 tiny drops of oil on each corner of a clean dry coverslip.
Aseptically transfer a loop full of culture suspended in a liquid to the center of a coverslip.
Locate a depression slide. (This slide will be thicker than a slide used to make a smear and will have 1 or 2 bowl-like depressions.)
Turn the depression slide upside down so that the bowl is over the drop on the coverslip. Touch the coverslip gently and the oil and coverslip will adhere lightly to the slide.
Flip the slide over and examine the drop using the compound scope at 450x magnification with the diaphragm on the microscope adjusted (most of the source of light blocked) to enhance the contrast of the transparent organisms.
- Non motile organisms will shake back and forth in the drop, true motility is shown when an organisms makes progress through the field of view.
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