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. 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 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 tubes) It is useful not only to determine motility but also tests for these two other metabolic pathways. Motility should be assessed first.
SIM medium contains nutrients, iron, and sodium thiosulfate. Sodium thiosulfate and ferrous ammonium sulfate are indicators of hydrogen sulfide production. The ferrous ammonium sulfate reacts with H₂S gas to produce ferrous sulfide, a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result. The sulfur test shoud be determined before you perform the indole test.
Casein peptone contains amino acids, including tryptophan. If tryptophan is attacked by microorganisms possessing the enzyme tryptophanase the trytophan is cleaved to produce three end products. One of these end products is indole, produced in aerobic conditions, and skatole, produced in anaerobic conditions. Amyl alcohol in Kovacs reagent acts as a solvent for indole which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. Skatole will also give a positive indole reaction. Organisms which do not produce the enzyme produce no color change in the medium when Kovacs is added.

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.
  This test is also described in Enzyme tests
  
  Motility and Hydrogen Sulfide Production/Indole Formation from SIM medium
  Procedure
  • Using aseptic technique, transfer a small amount of bacteria to be tested from a pure colony with an inoculating needle and make a stab inoculation into SIM agar tubes.
  • Inoculate a positive control organism using the same technique.
  • Incubate at 37C for 24 to 48 hours.
    
  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.
  • A blackening of the medium, either along the stab line or throughout the medium, indicates the production of hydrogen sulfide by the microorganisms.
  • To detect indole production, add three or four drops of Kovacs’ reagent and observe for a red color(positive reaction).

  
  Control Organisms:
  

  Organism	ATCC	Motility	H2S	Indole
  Escherichia coli	25922	+	-	+
  Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium	13311	+	+	-
  Shigella flexneri	9199	-	-	-

  
  

  Reagents Kovac's reagent

  
  
  

3. 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 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 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.

Links to Labs in the Soil Microbes Project

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