Motility of the bacteria Biology and Biotechnology department

v A large number of bacteria are motile. v Most possess one or more flagella on their surface that allow them to swim. v The pattern of flagellation is an important feature in identification of motile bacteria. v The figure illustrates the commonly observed arrangements of flagella.

v Polar flagella occur at one or both ends of the bacterium (Vibrio cholerae and some species of Pseudomonas). v They may be single or in tufts. v Peritrichous flagella are distributed around the surface of the organism (many Proteus species). v Most motile bacteria move in a straight line for a brief time, then turn and randomly change directions before swimming again.

v The straight line movement is called a run and the turn is called a tumble. v Runs and tumbles are controlled by the clockwise or counterclockwise rotation of the basal body of the flagellum, the motor that is anchored in the cell membrane. v Some bacteria do not tumble, but rather reverse direction when they reverse the rotation of the basal body.

v Many flagellated bacteria can move toward useful chemicals and away from harmful ones. v This ability to control movement in response to chemical stimuli is termed chemotaxis. v Chemotactic bacteria contain receptors in the cell membrane that bind to certain chemicals and cause the basal body to direct either a run or tumble (or forward and reverse directions).

v When the chemical stimulus is an attractant, such as a rich nutrient source, the basal body is made to rotate so that the bacteria swim in straight lines toward the signal for long periods of time. v If the stimulus is a repellant, such as a poison, the basal body reverses direction and causes the bacterium to tumble more often (or reverse direction).

Flagellar Stain : - v Flagella are too thin to be seen by the ordinary light microscope. v Flagella should be amplified (enlarged). Use a stain that is specifically deposited on Flagella thus increasing diameter. v Some flagellar stains employ rosaniline dyes and a mordant, applied to a bacterial suspension fixed in formalin and spread across a glass slide.

v The formalin links to, or “fixes, ” the flagellar and other surface protein of the cells. v The dye and mordant then precipitate around these “fixed” surfaces, enlarging their diameters, and making flagella visible when viewed under the microscope.

v Another method, a ferric-tannate mordant and a silver nitrate solution are applied to a bacterial suspension. v The resulting dark precipitate that forms on the bacteria and their flagella allows them to be easily visualized under the microscope. v This silver-plating technique is also used to stain the very slender spirochetes. v Note: The techniques are somewhat sensitive.

v Hanging Drop Technique: v This method is commonly used to view living organisms for the rapid determination of motility. v The hanging drop is prepared by suspending a fluid sample from a coverslip over a depression well in a specially designed microscope slide.

v Wet mounts can be used for the same purpose, however, wet mounts tend to dehydrate rapidly. v Hanging drops, on the other hand, are sealed within the depression and retain their liquid for longer periods of time. v In both methods, the living specimen is unstained. v For best results, reduce the amount of light passing through the specimen.

Procedure: 1. Place a drop of the bacterial culture (optimally from a young broth culture) in the middle of a cover slip. 2. Place a thin line of petroleum jelly around the edge of the cover slide. 3. Turn the depression slide upside-down (depressed area facing down) and gently touch the cover slide. The jelly holds the cover slip to the slide and also keeps the suspension from drying out.

4. Now flip the entire microscope slide/cover slip combination over. It should look like the diagram below. Positive control: Proteus vulgaris. Negative controle: Staph. Epidermidis.

NOTES: 1. You should be able to differentiate true motility from Brownian motility 2. Brownian movement is usually caused by the activity of water molecules. (characterized by back and forth movement) 3. True motility (the bacterial cells runs and tumble).

v Motility Agar : v Motile bacteria require liquid to move. v Thus bacteria can propel themselves in broth or across the surface of a wet agar plate. v They will not however move when embedded in 1. 5% agar, the minimum concentration v found in most agar media. v Semisolid agar has a reduced agar concentration (0. 4 %) that allows flagellated bacteria to migrate from the site of inoculation.

v Semisolid media are prepared in tubes and are inoculated through most of their length by stabbing v with a needle. v Thus after 48 hours of incubation, growth of a motile organism will be observed as a turbid region extending from the stab. v No motile bacteria will only grow along the stab line. Positive control: Proteus vulgaris. Negative controle: Staph. Epidermidis

Procedure 1. Using aseptic techniques, inoculate the tube by stabbing with the needle to approximately three-quarters of its depth. Be careful to bring the needle into the center of the medium and not to touch the side of the tube. 2. Incubate at room temperature for 48 hours. 3. Examine for growth.

Interpretation : (A) Pattern of growth of a motile organism. The entire medium is turbid with the growth of the organism, which has moved away from the stab line. (B) Pattern of growth of a nonmotile organism. Only the stab line is turbid with growth. Note: Semi solid media with tetrazolium chloride (color indicator)