Demonstration of Bacterial Motility by Hanging Drop Method

Hanging drop method is useful in determining the size, shape and movement of living microorganisms, especially bacteria without staining them to see their motility due to flagella.  Bacteria, because of their small size and a refractive index that closely approximates that of water, do not lend themselves readily to microscopic examination in a living, unstained state.  However, hanging drop method is useful to: 1) Observe cell activities such as motility and binary fission. 2) Observe the natural sizes and shapes of the cells, since heat fixation and exposure to chemicals during staining cause some degree of distortion.

Aim

To determine the motility of bacteria by hanging drop method

Principle

Bacteria show two types of motility. One is true (actual) motility and the other is Brownian movement.  True motility is due to flagella where as Brownian movement is a vibratory movement of the cells due to their bombardment by water molecules in the suspension.  To determine whether a given organism is motile or not, it is essential to differentiate between true motility and Brownian movement.   Hanging drop preparations make the movement of microorganisms easier to see because they slow down the movement of water molecules in the closed environment of cavity slide.

Requirements

24 hr broth culture of 24 hr old cultures of Escherichia coli,  Proteus, Pseudomonas, Staphylococcus,and Bacillus spp.

Bunsen burner, Inoculating loop, Cavity slides, Coverslips, Petroleum jelly, Cotton swabs, Microscope etc.

Procedure

1.      With a cotton swab, apply petroleum jelly on the four corners of a clean coverslip.

2.      Using sterile techniques, a loopful of the culture is placed on the centre of the coverslip.

3.      Place the depression slide, with the concave surface facing down, over the coverslip so that the depression covers the drop of culture.  Press the slide gently to the coverslip.

4.      Turn the slide right side up so that the drop continues to adhere to the inner surface of the coverslip.

5.      Focus the drop edge under the low power objective (10X) and reduce light source by adjusting the Abb’e condenser. Repeat using high power objective (40X).

Observations

• Escherichia coli – Rod-shaped cells showed swift, directional movement across the field (true motility).

• Proteus spp. – Rod-shaped cells exhibited very rapid, swarming movement across the slide (highly motile).

• Pseudomonas spp. – Slender rod-shaped cells moved actively with clear directional changes (true motility).

• Staphylococcus spp. – Round-shaped cells in clusters showed no movement (non-motile).

• Bacillus spp. – Large rod-shaped cells moved swiftly and directionally across the microscope field (true motility).                                                                                                                                                                                                                                                                              Results

• In the given cultures, Escherichia coli, Proteus spp., Pseudomonas spp., and Bacillus spp. are having true motility, whereas Staphylococcus spp. is non-motile.

Organism	Shape	Motility by hanging drop	Interpretation
Escherichia coli	Rod	Swift, directional movement	Motile
Proteus spp.	Rod	Very rapid, swarming movement	Highly motile
Pseudomonas spp.	Rod	Active, directional movement	Motile
Staphylococcus spp.	Coccus	Vibratory only (Brownian)	Non-motile
Bacillus spp.	Rod	Swift, directional movement	Motile