Nematodes

Examination of bacterial motility

Principle

Bacteria due to their small size and refractive index close to water, are very difficult to visualize in an unstained state. The live examination is however useful to;

• Observe cell activities such as motility and binary fission.
• Observe the natural size and shape of the cell as heat fixation and stains can distort the natural appearance of the bacteria. In this experiment, we will observe the size, shape, and motility of the bacteria. It is essential to differentiate between actual movement and Brownian movement, a vibratory movement due to the bombardment of water molecules in the suspension. The hanging drop method and wet mount preparation make the movement of microorganisms easier to see because they can slow down the movement of water molecules.

Materials and equipment

18-24 h broth culture of Pseudomonas aerogenosa, Bacillus cereus, Proteus Vulgaris, Staphylococcus aureus, Bunsen burner, inoculation loop, inoculation needle, cavity slide, coverslips, glass slides, petroleum jelly, cotton swab, soft agar, microscope.

Procedure

A. Hanging drop method

1. With a cotton swab, apply petroleum jelly around the concavity of the cavity slide.
2. Place a loopful culture in the center of the coverslip.
3. Place the cavity slide with the concave surface facing down over the coverslip so that the culture drop remains in the center of the cavity.
4. Press the slide gently to form a seal between the slide and coverslip.
5. Quickly turn the slide right side up so that the drop continues to adhere to the inner surface of the coverslip.
6. Examine under the microscope. First focus on the drop under low power of the microscope, reduce the light source and continue under the high power of the microscope. 

B. Wet mount

1. With a cotton swab, apply a thin layer of petroleum jelly along the edge of the coverslip.
2. Place a loopful culture in the center of the clean coverslip.
3. Place a clean glass slide over the coverslip and press gently to form a seal between the slide and the coverslip.
4. Quickly turn the slide right side up so that the drop continues to adhere to the inner surface of the coverslip.
5. Examine under the microscope. First focus on the drop under low power of the microscope, reduce the light source and continue under the high power of the microscope.

C. Soft agar method

1. Pour the melted soft agar in a screw cap tube and allow it to solidify.
2. Stab inoculate the soft with the help of an inoculation needle.
3. Incubate the tube in a vertical position at 37°C for 24 h.
4. Examine after the incubation. Growth will be present away from the stab if the organism is motile and along the stab in the organism is non-motile.

Note: Sulfide Indole Motility (SIM) medium can be used, which gives black colored growth due to the production of FeS.