Hanging Drop Method
- Hanging drop technique allows for the analysis of living microorganisms. This involves fixing the Microbial suspension into a drop of liquid over the slide of glass.
- Thus, hanging drop is the method where microorganisms are suspended in the drop of fluid.
- It’s a modified version of the wet mount method.
- The size, shape and arrangement of bacteria is easy to identify in the wet mount method, however the motility of bacteria becomes difficult to determine because the microbial suspension inside the cavity slide’s well is compressed by the coverlip.
- Light microscopy allows the use of wet mount methods as well as smear preparation, stain and heat fixing films to make the living thing visible to us.
- A hanging drop is a type of wet-mount procedure that makes use of bacteria that have been inoculated into the broth liquid. It’s a crucial instrument for determining the movement of the microorganisms. That’s why it is it is utilized to determine the movement of various organisms such as filamentous fungi, bacteria and yeasts.
- All bacterial cells display erratic movement, including non-motile and motile forms under the microscope. This we refer to the Brownian motion.
- However, the self-propulsion of bacteria is detected by one of the following mechanisms, such as flagellar, gliding or corkscrew and bending motions.
- In hanging drop motility tests there are many immortalized cells at the edges of the fluid under microscopic scrutiny.
- 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.
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
- Bunsen burner
- Young broth cultures with mobile bacteria (e.g. Proteus mirabilis)
Procedure of Hanging Drop Method
- Clean a glass slide, and then apply an adhesive-tape ring to create circular concavities. (This procedure is not required when a glass slide that has depression is in the market).
- Place a clean coverslip on its edges, and then carefully apply vaseline along its corners using the toothpick.
- Put a loopful of freshly prepared broth to test in the middle of the coverlip. Make sure to use a thin inoculum (not visible as in any way turbid).
- The glass slide that you have prepared or concavity slide upside-down (concavity downwards) over the drops on the coverslip to ensure that the vaseline is sealed the slideslip within the concavity.
- The slide should be turned over so that it is on the top. let the organisms “settle” for a minute. The drop is visible hanging across the concavity.
- Place the sample inside the slide holder of the microscope and align it with the naked eye to ensure that an part of it lies under the objectives with low power.
- Move the objective towards its lowest setting with the coarse adjustment, and close the DIAPHRAGM.
- Examine the eyepiece through the lens and slowly raise the objective by using the knob for coarse adjustment until the end of the drop is visible in an irregular line, which crosses the field.
- Slide the slide so the lines (the edges of the slide) run through the center of the field.
- Without lifting or lower the tubing slide the high dry object to position (be sure that the high dry object is clean).
- Examine the slide with the eyepiece, and then adjust the fine adjustment until the edges of the drop is perceived as a long generally dark line.
- Then, focus on the edge of the drop and then look on every side to find tiny things which are bacteria. The cells appear as greenish or dark very tiny particles or rods. Keep in mind that the high dry objective magnifies just a bit less than half the size of the oil objective that is used for immersion.
- Modify the light by via the diaphragm lever, to enhance the visibility that the cells have.
- Examine the cells, noting their form and structure and decide the degree to which true motility can be seen.
- Brownian motion should be evident on the slides of all organisms, however, they should be evidence of true mobility.
- Clean the slide after washing and then soak it in lysol buckets , throw away the glass slide you have prepared.
Result and Interpretations of Hanging Drop Method
- Directional molecular purpose is a favorable test. Motile organisms move in relation to each other. Brownian movements (random shaking or jiggling caused by molecular bombardment) which is when organisms are in the same position in relation to each other cannot be taken for genuine molecular motility.
- Vibrio cholerae and Campylobacter display an extremely dynamic motion (darting motility) that is visible as tiny dots that dart into or out.
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 examining living organisms to determine the characteristic of active locomotion, it’s important to differentiate true mobility, in which the organisms move in different directions and shifts their position within the field. This is different from or passive drifting of organisms moving in the same direction within convectional flow within the fluid, or Brownian movement that is an oscillatory move around the same point by all tiny bodies that are suspended in fluids because of irregularities in the bombardments they receive from the 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.
- 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 to analyze the motility of bacteria as well as the shape, size , and configuration of bacteria.
- It doesn’t change the shape or arrangement of cells.
- In addition hanging drop, this method offers a clearer image of the motility of bacteria in comparison to the wet-mount method.
- It also aids in the classification of bacteria, determining if they are motile or not.
- The Brownian movement is a study method, which triggers erratic motion of bacteria in the view field because of the explosive bombardment by water molecules.
- The petroleum jelly that is used in this technique is used to seal the coverslip onto the slide, which aids in the examination of the specimen tested for several times.
Disadvantages of Hanging Drop Method
- It’s a risky research of pathogenic bacteria in an environment that is living.
- The slide for depression is affordable and the coverslip is a bit fragile to work with.
We can therefore conclusively say that hanging drops are the ideal method to examine the various actions of the organism when it is in its living condition including the movement in shape, form and arrangement of bacteria’s cells. Methods for hanging drops is one kind of motility test. It is done by taking microorganisms of liquid media and placing them on the glass slide that has an indentation in the middle.