Hanging Drop Method

by

What is Hanging Drop Method?

  • The simplest way to observe bacterial motility is a simple wet mount.
  • A more popular method however has been hanging drop technique. This method allows a greater freedom of movement.
  • Freely suspended bacteria show two types of movement, the Brownian movement which is brought about by random collisions and is not true motility. Secondly tee flagellar motility which has short spells of linear movement.

Motility in Bacteria

Motility in bacteria refers to their ability to move or swim actively. The movement of bacteria is important for various physiological processes, such as nutrient acquisition, avoidance of harmful substances, and colonization of different environments. The motility of bacteria can be classified into two main types: flagellar motility and non-flagellar motility.

Flagellar motility

This type of motility is driven by the rotation of flagella, which are long, thin, whip-like appendages that extend from the surface of the bacterial cell. Flagellar motility is further classified into four types, based on the number and arrangement of flagella:

  1. Monotrichous: Bacteria with a single flagellum at one end of the cell, such as Vibrio cholerae.
  2. Lophotrichous: Bacteria with a tuft of flagella at one or both ends of the cell, such as Pseudomonas aeruginosa.
  3. Amphitrichous: Bacteria with a single flagellum at both ends of the cell, such as Spirillum volutans.
  4. Peritrichous: Bacteria with flagella that are distributed over the entire surface of the cell, such as Escherichia coli.

Non-flagellar motility

This type of motility does not involve the use of flagella. Instead, it is driven by other mechanisms, such as gliding, twitching, or swarming.

  1. Gliding motility: Bacteria move by gliding over surfaces, using a variety of mechanisms such as secretion of slime or other surface-associated molecules. Examples of bacteria that use gliding motility include Myxococcus xanthus and Flavobacterium johnsoniae.
  2. Twitching motility: This type of motility involves the extension and retraction of pili, which are hair-like structures that extend from the surface of the bacterial cell. The pili attach to surfaces and pull the bacterial cell forward. Bacteria that use twitching motility include Pseudomonas aeruginosa and Neisseria gonorrhoeae.
  3. Swarming motility: Bacteria move in groups over surfaces by secreting large amounts of slime or surfactants. Swarming motility is seen in several bacterial species, including Proteus mirabilis and Serratia marcescens.

Hanging Drop Method Principle

In the hanging drop method, a small drop of liquid containing cells or microorganisms is suspended from a coverslip or a glass slide inverted over a depression slide. The drop is held in place by surface tension and gravity, and the cells or microorganisms in the drop can freely move and interact with each other.

The hanging drop method is particularly useful for observing the motility of microorganisms such as bacteria and protozoa. Since the drop is suspended in a controlled environment, it allows for better observation of the organisms’ behavior without interference from external factors such as sedimentation.

Requirement of Hanging Drop technique

  1. Glass slides
  2. Microscope
  3. Bunsen burner
  4. Vaseline or Paraffin wax
  5. Cover glass
  6. Saline suspension of given organism

Procedure of Hanging Drop technique

  1. Apply small quantity of Vaseline to the four corners of a cover glass.
  2. Transfer a loopful of suspension (Drop) to the center of the cover glass.
  3. Invert the cavity slide and center the concavity over the drop of culture.
  4. The Vaseline makes the cover glass to adhere on to the slide.
  5. Now carefully turn the slide so that the drop remains suspended in the cavity.
  6. Focus the edge of the hanging drop under low power.
  7. Reduce light and focus under high power.
  8. Bacteria can be seen as transparent objects with low contrast (40 X and 100 X microscope).
  9. Record your observations, either by taking photographs or by making drawings.
  10. Finally, clean the slide and coverslip before storing them properly.

Hanging Drop Method Results

The movement of the cells or microorganisms in the drop can be observed under the microscope, and their motility can be described as either active or passive. Active motility indicates that the cells or microorganisms can move on their own, while passive motility occurs due to the movement of the surrounding fluid.

Tips to Perform Hanging Drop Method

  1. Use a clean slide and coverslip: Make sure that the slide and coverslip you use are clean and free from any debris or dust. Clean them thoroughly before starting the experiment.
  2. Sterilize the loop or needle: If you are using a loop or needle to transfer the sample into the drop, make sure to sterilize it first by passing it through a flame.
  3. Use a small drop: Use a small drop of liquid to ensure that the drop remains suspended in the air. The drop should be no larger than 10-15 microliters.
  4. Apply petroleum jelly: Apply a small amount of petroleum jelly to the edges of the coverslip to seal the hanging drop and prevent it from drying out.
  5. Position the drop correctly: Position the drop in the center of the coverslip, making sure that it is suspended in the air and not touching the slide.
  6. Focus the microscope: Focus the microscope carefully to ensure that the sample is in focus and that you have a clear image of the cells or microorganisms.
  7. Use proper lighting: Adjust the lighting on the microscope to obtain the best image of the cells or microorganisms. It is important to use proper lighting to avoid damaging the sample or altering its behavior.
  8. Be patient: Observe the sample carefully and be patient when observing the behavior of the cells or microorganisms. It can take some time for changes to occur, so be prepared to wait and observe the sample over a period of time.

Hanging Drop Method Uses

The hanging drop method is a useful laboratory technique in microbiology and cell biology. It has various uses, some of which include:

  1. Observation of microbial motility: One of the primary applications of the hanging drop method is to observe the motility of microorganisms, such as bacteria and protozoa. This technique provides a controlled environment that allows for better observation of the organisms’ motility without interference from external factors.
  2. Observation of cell behavior and interactions: The hanging drop method is also useful for studying the behavior and interactions of cells, such as the aggregation or fusion of cells in the formation of multicellular structures. It provides a means of observing the cells in a controlled environment, allowing researchers to study their behavior without interference from external factors.
  3. Drug discovery and testing: The hanging drop method can be used in drug discovery and testing, especially for antimicrobial compounds. The method allows researchers to observe the effects of drugs on microorganisms and cells, providing valuable insights into their mechanisms of action.
  4. Research on infectious diseases: The hanging drop method can be used to study infectious diseases caused by microorganisms. The method allows researchers to observe the behavior and interactions of microorganisms in a controlled environment, providing insights into the pathogenesis of the disease.
  5. Research on tissue engineering: The hanging drop method can be used to study the formation of tissues and organs, which is important in tissue engineering. The method allows researchers to observe the aggregation and fusion of cells, providing insights into the mechanisms of tissue formation.