Hanging drop test/preparation

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Hanging drop test is a confirmatory test to identify if an organism is motile or non motile. It can also be used for the presumptive identification of organism on the basis of its characteristic motility like Vibrio cholerae which has a darting type of motility in stool sample of patients with cholera. This test is prepared by suspending a drop of bacterial suspension or watery stool sample over an air-tight chamber prepared in a special depression slide possessing a concave depression in the center or assembled from modelling clay (plasticine) which is a soft, malleable, and nonhardening material.

Materials required:

  • Depression slide or plain glass slide with plasticine ring
  • Coverslip
  • Toothpick
  • Vaseline
  • Bunsen burner
  • Inoculating loop
  • Young broth cultures or rice watery stool sample
  • Microscope

Procedure for Hanging drop test

  1. Take a depression slide. If the depression slide is unavailable, use a clean glass slide and apply plasticine ring, to make circular concavity.
  2. Apply a small amount of Vaseline near each corner of a clean coverslip using a toothpick. (This step is not required if plain glass slide with plasticine ring is used)
  3. Transfer few loopsful of broth culture or watery stool to the center of the coverslip.
  4. Turn the depression slide upside down (concavity down) over the drop on the coverslip so that the vaseline seals the coverslip to the slide around the concavity. If the depression slide is unavailable, turn the prepared glass slide with plasticine ring upside down so that the edge of the coverslip stick on plastice ring with drop at the center.
  5. Invert the slide over so the coverslip is on top and the drop can be observed hanging from the coverslip.
  6. Place the preparation in the stage of the microscope observe the edge of the drop under the low power (10x) and high dry power (40x) objectives.
  7. Observe the cells and note their morphology along with true motility.
  8. Discard the prepared glass slide in a discarding jar with disinfectant solutuion.

Advantages:  This test preserves cell shape and arrangement. The Vaseline-sealed depression also slows down the drying-out process, so the organisms can be observed for longer periods.

Disadvantages: This test is too risky to use with highly pathogenic organisms.

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