Examination of Motility by Semisolid agar method

Aim

To examine motility of a given organism using semisolid agar method.

Principle

Bacteria have special structures which allow directed movement or motility. Flagella are the most important organelles of motility. They allow bacteria to move towards favourable environments and away from unfavourable environments. There are different ways to examine motility. Hanging drop technique allows cells to be observed in a wet mount. However, this determination can be difficult because bacteria are small enough to be bounced around by water molecules. This random movement called Brownian motion can be confused with self- propelled motility.

Motility examination is done using semi-solid soft agar in a test tube (without a slanted surface) based on the ability of bacteria to move through semi solid medium. The growth of motile bacteria in such a tube will produce turbidity throughout the solid medium whereas the non-motile organism will grow only along the line of inoculation. Cells are stab-inoculated into the agar (the top surface is not inoculated). Non-motile bacteria will only grow where they are inoculated. Motile bacteria will grow along the stab and will also swim out away from the stabbed area. Thus, a positive result is indicated by diffuse (cloudy growth), especially at the top and bottom the stab. A negative result is indicated by growth in a distinct zone directly along the stab.

This test only works with facultative anaerobic organisms. Obligate aerobes will grow on the top of the agar but not in the stab line.

Procedure

Inoculating the soft agar deep tube is very similar to inoculation using an inoculation loop except that the inoculating needle is flat without a loop at its end.

1.      A semisolid agar (Nutrient broth with 0.5% agar) deep tube was prepared and sterilized.

2.      A visible amount of culture was taken on the tip of a flame sterilized inoculating needle. It was stabbed deeply into the centre of the medium in the tube (about 2/3^rd way into the agar), stopping just before the bottom of the tube. The needle was withdrawn through the same inoculation path, with minimum disturbance of the medium.

3.      The stab should be as straight as possible. This is important since the amount of growth away from the stab will be evaluated for motility. A messy stab will be difficult to evaluate

4.      Incubated at 37^oC for 24-48 hours.

5.      Observed the result by holding the tube against a light source.

Observation

Motility detection is possible due to semi-solid nature (low concentration of agar) of soft agar. Growth radiating out from the central stab inoculation line indicates that the test organism is motile. Motile organisms such as E. coli will exhibit growth radiating from the stab inoculation line. Non-motile organisms such as Staphylococcus aureus, will exhibit growth only along the stab inoculation line.

 

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Motility determination - Hanging Drop technique

Aim

To perform hanging drop technique to determine whether the given bacteria is motile or not

Principle

Hanging drop preparation is a wet mount preparation in which a drop of medium containing the organisms is placed on a microscope slide to observe the motility of bacteria.

In this method a drop of culture is placed on a coverslip that is encircled with petroleum jelly. The coverslip and drop are then inverted over a cavity slide. The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation. This preparation allows easy observation of microbial motility under a microscope.  The hanging drop preparation under microscope shows microorganism moving which may be either

• The organisms moving in different directions and changing their positions in the field
• Passive drifting of the organisms in the same direction due to convectional current in the fluid
• Brownian movement, which is an oscillatory movement possessed by all small particles suspended in fluid and due to bombardments with molecules of water.

The passive drifting and Brownian movement should not be interpreted as motility.  Brownian movements and passive drifting off are shown by all the organisms (both motile & non-motile) and are visible in the Hanging drop preparation.  A precise and accurate examination is required to differentiate the Brownian movement with true motility of bacterial cell.

Materials required

1.      Cultures - 24 hour nutrient broth culture of Escherichia coli, Bacillus cereus

2.      Reagents - Paraffin wax

3.      Equipments - bunsen burner, inoculating needle, Coverslip, Cavity slide, microscope

Procedure

1.      Vaseline or paraffin wax was applied on the edges of a clean coverslip using a toothpick.

2.      A loopful of the broth culture was placed in the center of the coverslip.

3.      A clean cavity slide was turned upside down over the coverslip so that the vaseline seals the coverslip to the slide around the cavity.

4.      The preparation was turned over so that the coverslip comes on top and the drop hangs down from the coverslip over the cavity.

5.      The preparation was observed under microscope and the edge of the drop was focused which will be seen as a thick, dark line.

6.      The edge of the drop was carefully observed for bacteria and their movements. 

Observation

Brownian movement was easily visible and there were bacterial cells showing true motility.

Result

The given bacterial culture contains motile bacteria.