Motility test by using hanging drop method (explained)

Principle 

The motility test by hanging drop method is a laboratory technique used to observe the motility of bacteria. The principle behind the hanging drop method is based on the fact that the movement of bacteria can be observed by placing a small drop of bacterial culture on a microscope slide and suspending it over a depression in the slide. The hanging drop is then observed under a microscope to observe the motility of the bacterial cells.

The principle of the hanging drop method is based on the physical properties of bacteria and the microscope. Bacteria are small, single-celled organisms that are too small to be seen with the naked eye. In order to observe bacteria under a microscope, they must be stained or suspended in a solution that makes them visible. However, staining or suspending bacteria can also affect their motility and behavior.

The hanging drop method overcomes this problem by suspending bacteria in a small drop of fluid, usually a saline solution, which is placed on a cover slip. The cover slip is then inverted and placed over a depression in the microscope slide. The fluid drop is held in place by surface tension between the cover slip and the slide, forming a “hanging drop”.

Because the bacteria are suspended in a drop of fluid, they are free to move about and exhibit their natural motility. The microscope is then used to observe the hanging drop and the movement of the bacterial cells. By observing the pattern of movement of the bacterial cells, one can determine whether the bacteria are motile or non-motile, and the direction and speed of their movement.

Purpose of the test

The purpose of the motility test by hanging drop method is to determine the presence or absence of motility in a bacterial culture. The test helps to differentiate between motile and non-motile bacteria, which is a crucial step in the identification of bacterial species.

Materials and Methods

Materials

Microscope slide: A clean, dry microscope slide is needed to prepare the hanging drop. The slide should be free from any dirt, dust, or debris that could interfere with the observation of bacterial motility.

Cover slip: A clean, dry cover slip is needed to prepare the hanging drop. The cover slip should be free from any dirt, dust, or debris that could interfere with the observation of bacterial motility.

Inoculating loop: A sterile inoculating loop is needed to transfer the bacterial culture to the hanging drop. The loop should be sterilized by heating it until it is red hot, and then allowing it to cool before use.

Sterile saline solution: A sterile saline solution is needed to prepare the hanging drop. The saline solution should be sterile to avoid contamination of the bacterial culture.

Bacterial culture: A pure bacterial culture is needed to perform the motility test. The culture should be free from any contamination or other bacteria that could interfere with the observation of bacterial motility.

Preparation of the Hanging Drop:

• Apply petroleum jelly with a cotton swab all the way around the concavity of the depression slide to make a hanging-drop preparation. 
• After that, centre a loopful of the culture using sterile procedures on a clean coverslip. 
• With the concave surface pointing downward, place the depression slide over the coverslip to cover the culture drop. 
• Gently press the slide to seal it to the coverslip. Turn the slide right side up quickly to ensure that the drop sticks to the coverslip’s inside surface. 
• Start by focusing on the drop culture with the low-power objective, then dim the light source by adjusting the Abbé condenser to study the preparation under a microscope. 
• Repeat the inspection using the high-power objective. 
• Finally, note down observations in your record

Observation and Interpretation:

Observation and Interpretation:

After the hanging drop has been prepared, it should be examined under a microscope to observe bacterial motility. The hanging drop method allows for the observation of live bacteria in a natural state, which can provide valuable information about the bacteria’s motility.

Examination of the hanging drop under a microscope:

The hanging drop should be examined under a microscope immediately after preparation to observe bacterial motility. A microscope with a high magnification and good resolution should be used to observe the bacteria. The hanging drop should be viewed under both bright field and dark field microscopy to enhance the visibility of the bacteria.

Interpretation of motility:

The interpretation of motility in the hanging drop method is based on the observation of the movement of bacteria within the bacterial suspension.

Positive motility

Positive motility is observed when bacteria move actively in the bacterial suspension. The movement can be seen as a rapid, continuous, and smooth motion of the bacteria. The bacteria may exhibit a variety of motility patterns such as tumbling, darting, or swarming. The presence of positive motility can indicate the presence of motile bacterial species.

Negative motility

Negative motility is observed when bacteria do not show any movement or appear to be stationary in the bacterial suspension. The bacteria may be immobile, or they may exhibit Brownian motion, which is a random movement caused by the bombardment of water molecules. The absence of motility can indicate the presence of non-motile bacterial species.

In some cases, the hanging drop method may not be sufficient to detect bacterial motility. In these cases, other tests such as the flagella staining or the motility agar test may be used to confirm the presence or absence of bacterial motility.

Note: It is important to observe the hanging drop immediately after preparation, as bacterial motility can decrease over time.

Discussion

The hanging drop method is a simple and reliable technique used to observe bacterial motility. The discussion of the hanging drop method should include the significance of the motility test in bacterial identification, a comparison of the hanging drop method to other motility tests, the limitations and sources of error in the hanging drop method, and possible improvements to the method.

Significance of motility test in bacterial identification:

Motility is an important characteristic used in the identification of bacterial species. The presence or absence of motility can help to distinguish between different bacterial species. The hanging drop method provides a quick and easy way to observe bacterial motility and can be used as a primary test to identify motile bacterial species.

Comparison of the hanging drop method to other motility tests:

The hanging drop method is one of several methods used to test for bacterial motility. Other methods include the motility agar test, flagella staining, and wet mount preparation. The motility agar test is a simple and reliable method that involves the observation of bacterial movement on an agar plate. Flagella staining involves staining the flagella of bacteria to make them visible under a microscope. Wet mount preparation involves placing a small amount of bacterial suspension on a microscope slide and observing the movement of bacteria.

Compared to other motility tests, the hanging drop method provides a quick and easy way to observe bacterial motility. It allows for the observation of live bacteria in a natural state, which can provide valuable information about the bacteria’s motility patterns.

Limitations and sources of error in the hanging drop method:

One of the main limitations of the hanging drop method is the need for a specialized microscope stage that can hold the hanging drop. The hanging drop can also dry out quickly, which can affect the accuracy of the results. In addition, the hanging drop method may not be sufficient to detect bacterial motility in some cases, and other tests may be needed to confirm the presence or absence of motility.

Sources of error in the hanging drop method include contamination of the bacterial culture, incorrect preparation of the saline solution, and inaccurate observation of bacterial motility. The hanging drop method requires skill and practice to ensure accurate results.

Possible improvements to the method:

One possible improvement to the hanging drop method is the use of automated microscopy. Automated microscopy can provide more accurate and consistent results than manual microscopy. Another possible improvement is the use of a digital camera to capture images of bacterial motility, which can be analyzed using software to provide more precise measurements of bacterial movement.

Implications for further research

Further research can be conducted to explore the potential use of the hanging drop method in the identification and classification of bacterial species. This can include the use of automated microscopy and digital imaging to improve the accuracy and consistency of the test results.

Another area of research could be the development of new techniques to improve the hanging drop method, such as the use of different types of bacterial cultures or the modification of the saline solution to improve the viability of the bacterial cells.

Conclusion

In summary, the hanging drop method is a reliable and useful technique for observing bacterial motility. It is a simple and quick test that can provide valuable information about bacterial species. By preparing the hanging drop, observing the motility, and recording the results, one can easily identify the motility pattern of the bacterial species being tested.

Frequently Asked Questions 

References 

• Tortora, G. J., Funke, B. R., & Case, C. L. (2021). Microbiology: An introduction. Pearson Education Limited.
• Willey, J. M., Sandman, K. M., Wood, D. H., & Prescott, L. M. (2019). Prescott’s microbiology (11th ed.). McGraw Hill.
• Wang S, Wang X, Boone J, Wie J, Yip KP, Zhang J, Wang L, Liu R. Application of Hanging Drop Technique for Kidney Tissue Culture. Kidney Blood Press Res. 2017;42(2):220-231. doi: 10.1159/000476018. Epub 2017 May 4. PMID: 28478441; PMCID: PMC6050513.
• https://microbiologynote.com/hanging-drop-method/