To study bacterial motility by using the hanging drop technique.
Broth cultures (24 hrs) of Proteus vulgaris and Escherichia coli., vaseline or petroleum jelly, Bunsen burner, inoculating loop, depression (cavity) slide, coverslip, etc.
Living microorganisms can be studied to determine the natural size and shape of cells, cellular arrangement, motility, reactions to various chemicals, and response to environmental factors. Wet mount and hanging drop techniques are commonly used for direct examination of living microorganisms (e.g. bacteria, protozoa, fungi). The hanging drop technique is routinely used for the study of bacterial motility.
Advantages of the Hanging Drop Technique:
- It is easy to study the motility of bacteria and is less time-consuming.
- The hanging drop is surrounded by an air space hence, the capacity of aeration is increased.
- This technique is also used for observation of the size, and shape of microorganisms in a living state.
Disadvantages of the Hanging Drop Technique:
- A hanging drop may be evaporated in a short time.
- In this technique, three different refractive indices are involved in the light path. Hence, good microscopy is not possible with this preparation.
Flagella are responsible for the motility of bacteria and this is called an ‘organ of locomotion’. The number and arrangement of flagella are characteristic of each bacteria. Flagella may be arranged on bacterial body as monotrichous, lophotrichous, amphitrichous or peritrichous. The flagella have three basic parts filament, hook, and the basal body.
Clean and flame a hanging drop slide (depression slide) and place it on the table. Using a fine needle, make a thin layer of petroleum jelly on the periphery of the cavity of the slide [Fig. 1 (a)]. Clean a cover slip and apply petroleum jelly on each of the four corners of the coverslip, using a fine needle or match stick. Transfer a loopful of the culture in the center of the coverslip [Fig. 1 (b)]. Invert the cavity slide over the coverslip containing the culture drop in such a manner that the drop comes into the center of the cavity. Allow the coverslip to adhere to the jelly and gently turn the slide so that the drops of culture hang in the cavity [Fig. 1 (c)]. Place the slide on the microscope stage and carefully focus the edge of the drop so that it appears across the center of the field. Use high power objective and adjust the amount and intensity of light to obtain a sharp view [Fig. 1 (d)].
Observations and Results:
Observe the behavior of microorganisms on the inner side of the drop edge and record the size, shape, and motility of the bacteria. Check whether the motility of organisms is true motility or Brownian movement (vibrating or dancing movement of bacteria in suspension due to bombardment by the molecules of water).
The organisms change their position and move from one place to another (true motility). Hence, the given culture is motile.
- The density of the culture broth should be neither too high nor too low.
- Carboxymethyl cellulose (2%) may be used to slow down the speed of more actively motile organisms.
- The depression slide is inverted over the coverslip in such a way that the suspension does not touch or spread the surface of the concavity.
- For observation of the motility of pathogenic organisms, generally semi-solid agar technique is used.
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