BISC209: Motility

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. Three different ways to examine motility are available to you. You can choose which one or ones you wish to perform.

1. Flagella can be demonstrated by special staining techniques 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 refer to: Special Stains: Endospore, Acid fast, Capsule, and Flagella.

2. The demonstration of motility can also be examined using special media. These tests rely 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) It is useful not only to determine motility but also tests 2 metabolic pathways. SIM medium contains nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which contains amino acids, including tryptophan. If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result. The sulfur and motility test results should be determined before you perform the indole test. Some bacteria possess the ability to produce the enzyme tryptophanase, which hydrolyzes tryptophan. The end products of this hydrolyzation are indole, pyruvic acid, and ammonia, by way of deamination. The Kovac’s reagent that you add to the SIM medium to test for indole contains hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol. DMABA reacts with indole to produce a red quinoidal compound. If the reagent turns red, the indole test is positive.

The protocol for the Motility Test is described below.

Inoculating the SIM tubes involves a technique you have not yet practiced. It is very similar to inoculations using the loop, but you will use an inoculating needle (the wire extending from the handle will not have a loop on the end) and after picking up a visible amount of your isolate on the tip of the needle you will stab it deeply into the center of the medium in the SIM tube.

• Using your best aseptic technique, remove an inoculum from your unknown culture with an inoculating needle and inoculate the SIM tube by stabbing down the center of the tube, stopping just before the bottom or most of the length of the needle. Withdraw the needle (or loop) through the same inoculation channel.
• Inoculate a second tube with a known motile control culture, such as E. coli or Proteus sp.
• Incubate both tubes at 37˚C for 24-48 hours or longer if appropriate for your isolate.
• Examine the inoculated SIM tubes for growth. If growth is restricted to the line of inoculation and is well demarcated, with no growth in the rest of the agar, the organism is considered non-motile. Growth that occurs throughout the medium indicates a motile organism.

3. A suspension of an isolate in either a young broth culture on the or a small amount of growth on solid medium transfered into liquid medium is another way to observe motility. The presence of directed movement rather than Brownian motion in living cultures 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 broth culture 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.

Links to Labs in the Soil Microbes Project

Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9