The Hanging Drop Method was discovered by Robert Koch in 1877. It is a technique used to culture microorganisms, in which a small drop of liquid culture is suspended from the underside of a coverslip. This method is particularly useful for culturing bacteria and other microorganisms that require a small volume of liquid to grow in, and it allows for the observation of microbial growth under a microscope.

Also Read: Temporary Wet Mount (TWM) technique for observation of Living Organisms.

Hanging Drop Method Overview

• The hanging drop method enables the investigation of living microorganisms by fixing a suspension of bacteria in a drop of fluid suspended above a glass slide. The procedure in which microorganisms are suspended in a drop of fluid is known as the hanging drop method. It is a modification of the wet mount method.
• In the wet mount approach, the shape, size, and arrangement of bacteria are easily discernible, but bacterial motility becomes difficult to analyse as the microbial suspension in the hollow slide well is compressed by the coverslip.
• To make the organism visible, light microscopy employs the wet mount method, smear preparation, heat fixing, and stain films.
• Therefore, a hanging drop is a type of wet-mount technique that utilises inoculum of bacteria from liquid broth.
• It is used to assess the motility of various species, such as bacteria, filamentous fungus, and yeasts, because it is a crucial instrument for determining the intrinsic movement of microorganisms. Under a microscope, all bacterial cells, both motile and non-motile, will exhibit Brownian motion, which is characterised by irregular movement.
• In bacteria, self-propulsion is characterised by one of the following mechanisms: flagellar, gliding, corkscrew, or bending motion. Under microscopic examination, several immortalised cells are seen along the fluid’s border in tests of hanging drop motility. In this post, we will explore the definition, technique, considerations, applications, benefits, and drawbacks.
• Before an hanging technique for droplets was employed to test how Nocardia sp. on liquid paraffin droplets. In further research, the technique has been employed extensively to show the morphology of bacterial cells and their motility. After that, in view of the massive drop size that is a result of hanging drop methods, researchers began using the micromanipulation technique in order to gain control over the size of the drop.

Objective of Hanging Drop Method

• To study the motility of microbial cell.

Definition of Hanging Drop Method (Motility test)

• The hanging drop method is the conventional technique for analysing cell motility and morphology by removing living microorganisms from liquid media. The hanging drop method is based on the wet mount preparation approach, as it involves the introduction of living microorganisms into a fluid drop. It employs glass slides with a small central depression, a coverslip, petroleum jelly, microbial solution, and a sterile inoculation loop. In 1878, a chemist named Robert Koch made the initial discovery.
• Previously, the Nocardia sp. on liquid paraffin droplets was examined using the hanging drop technique. The method has been used extensively to illustrate the shape and movement of bacteria as a result of additional research. Later, in order to control the size of the drops produced by the hanging drop method, scientists began employing the micromanipulation technique.

Principle of Hanging Drop Method

The hanging Drop method has been the most common method to study the cell’s movement and morphology, by collecting live microorganisms and removing them from the liquid medium. Hanging drop technique is based by utilizing the principles of preparation using wet mounts, since it involves subjection of living microorganisms to drops of fluid. It uses glass slides with a tiny concave depression in the middle as well as the coverslip, petroleum jelly Microbial suspension, and sterile inoculating loop. It was first identified by a scientist named Robert Koch in the year 1878.

Materials Required for Hanging Drop Method

• Glass slide (glass slide that has depression) or regular glass slide with adhesive , or paraffin rings
• Paraffin wax
• Loop
• Coverslip
• Microscope
• Bunsen burner
• Young broth cultures with mobile bacteria (e.g. Proteus mirabilis)

Quality Control

Validate the technicians’ competency in the hanging-drop method by testing known motile enterococci or Listeria in a broth assay.

Hanging Drop Method Procedure

Hanging drop preparation

1. Apply a paraffin ring or adhesive tape ring to a clean glass slide to create a circular concavity. (This step is unnecessary if a glass slide with a depression is readily available.)
2. Hold a clean coverslip by its borders and use a toothpick to dab vaseline on its corners.
3. Place a loopful of the to-be-tested fresh broth culture in the middle of the prepared coverslip. Use a mild inoculum (not visibly turbid).
4. Turn the prepared glass slide or concavity slide upside down (concavity down) so that the drop of vaseline bonds the coverslip to the slide around the concavity.
5. Turn the slide over so the coverslip is on top and wait one minute for the organisms to “settle.” The droplet may be seen suspended from the coverslip over the concavity.

Microscopic Observation

1. Place the sample in the microscope slide holder and orient it with the unaided eye such that one edge of the drop is beneath the low-magnification objectives.
2. Adjust the goal to its lowest setting using the coarse adjustment, then CLOSING THE DIAPHRAGM.
3. Using the coarse adjustment knob, progressively raise the objective until the edge of the drop is noticed as an uneven line traversing the field of view.
4. Move the slide so that this line (the edge of the drop) intersects the field’s centre.
5. Without raising or lowering the tube, position the high dry objective by swinging it into place (be sure the high dry objective is clean).
6. Observe the slide through the eyepiece and adjust the fine adjustment until the drop’s edge is visible as a thick, often dark line.
7. Focus on the edge of the drop and examine both sides of that line for the extremely little bacteria-containing items. With very small rods or spheres, the cells will appear either dark or somewhat bluish-green and will be composed of tiny structures. Remember that the magnification of the high dry objective is somewhat less than half that of the oil immersion objective.
8. Using the diaphragm lever, adjust the light to enhance the visibility of the cells.
9. Observe the cells, taking note of their shape and clustering, and evaluate if genuine motion can be observed.
10. On the slides of all the organisms, Brownian movement should be obvious, but there should also be evidence of genuine motility.
11. The prepared glass slide must be discarded after washing and soaking in lysol buckets or it must be discarded.

Result and Interpretations of Hanging Drop Method

1. Directional intent motility is a favourable indicator. Positions of mobile organisms fluctuate relative to one another. Brownian movement (random jiggling or shaking owing to molecular bombardment) in which organisms remain in the same relative position to one another should not be confused with genuine motility.
2. Campylobacter and Vibrio cholerae exhibit extremely vigorous movement (darting motility) that appears as small spots darting across the field.

For all organisms that are not able to demonstrate motility, after the initial wet mount Repeat the wet mount following incubation in broth or test via tube method.

• Incubate at 30°C for non-fermenting rods that are Gram-negative (24 hours).
• Incubate enterococci as well as Listeria at 30 degC for 2 hours.
• Other microorganisms could be incubated at temperatures suitable in order to grow, typically 35 degrees Celsius.

When investigating live organisms for the quality of active locomotion, it is crucial to differentiate between real motility, in which the organisms move in different directions and alter their positions on the field, and other forms of active locomotion. Passive drift of the organisms in the same direction in a fluid’s convective current or in a fluid’s convective current. Brownian movement, which is an oscillating motion about a nearly fixed point, is shown by all small things suspended in fluid and is caused by anomalies in their bombardment by water molecules.

Uses of Hanging Drop Method

• The spiral morphology is studied specifically in the hanging drop method because its shape changes during the process of heat fixing.
• Spirochetes, a type of spiral bacteria, require examination in a live state and their shape as well as their arrangement could be observed under a dark-field microscope.
• Mobility or motility in a bacterium can be studied using a hanging drop technique that allows the bacterial cells can freely move within the medium of liquid.
• The cytological changes that take place as cells divide, spore development and the germination of bacteria are the processes that require investigation in a living environment or using the hanging drop method.
• The cytoplasmic inclusions, such as vacuoles, granules and so on. are easily discernible employing this technique.

Notes

• A hanging drop technique is an aseptic technique to examine the samples from liquid culture in lieu of solid medium for culture.
• When using the hanging drop method the microbial suspension is wet-mounted instead of exposing it to techniques like staining, heat-fixing, smearing or smearing, etc.
• It is widely used to study the bacterial shape and arrangement as well as the whether flagella are present.
• The samples collected by the method of hanging drops reveal Brownian motion, in which microscopic particles in the fluid can swim erratically by the kinetic energy held by the molecules of the fluid surrounding.
• The real mobility can be observed through the multi-directional movements of bacteria’s cells over larger distances, rather than the cells that move back and forth. The motility of bacterial cells can be observed with a 10X or 40X objective, which can be seen in the diagram. When using a hanging drop technique using a 10X objective, the first step is to be utilized to concentrate the microscopic image, and then the objective is elevated to 40X for an expanded view of the sample and also to differentiate between immotile and motile cells.
• The petroleum jelly that is applied to the corners of the coverslip functions as a sealant material between the coverlip’s concave depression glass. In addition, it reduces the loss of water and blocks the effects on air flow.
• The use of petroleum jelly in excess could result in false results because it can squeeze toward the middle of the drop, which contains microorganisms. It may also escape from the edges and adhere to the camera’s lens.
• The removal of slides should be done with care since after dipping them in Lysol solution, they must be autoclaved before being reused. The coverslips must be removed and not reused.

Advantages of Hanging Drop Method

• It is an essential instrument for studying bacterial locomotion as well as the shape, size, and organisation of bacteria.
• It does not alter the form and arrangement of the cells.
• In addition, the hanging drop approach offers a better view of bacterial movement than the wet mount method.
• It is also useful for classifying bacteria according to whether they are motile or immobile.
• This method allows for the study of Brownian motion, as the bombardment of water molecules causes the bacterial cells in the field of view to move erratically.
• This approach uses petroleum jelly to bond the coverslip to the hollow slide, which facilitates repeated examination of the tested specimen.

Disadvantages of Hanging Drop Method

• It is dangerous to research harmful microorganisms in a living environment.
• The depression slide is economical, whereas the coverslip is delicate and difficult to handle.

Precautions

• The usage of PPE (Gloves, Mask, Lab coat / Gown, Safety goggles, etc.) is required because you will be handling highly infectious, live microorganisms.
• Use the early culture of the organism, as the organisms are most likely dead in the older cultures.
• Place a drop of sufficient size on the cover glass; it should not be too large or too small and should hang freely in the depression slide’s concavity.
• Observe first with low power goals, then transition to high power and oil immersion objectives for simple observations.
• Adjust the diaphragm accordingly for enhanced contrast, hence minimising observational mistakes.
• Do not confuse the bacterial cell’s passive drifting and Brownian motion with its motility. Observe carefully before classifying creatures as Motile or immobile.

Hanging Drop Method Video

Hanging Drop Method Images

Conclusion

Therefore, we may conclude that the hanging-drop method is the greatest way to analyse the various events of a living organism, the mobility, shape, and arrangement of bacterial cells. The hanging drop method is a type of motility test in which microorganisms from liquid media are placed on a glass slide with a central depression.

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