Hanging Drop Preparation: Introduction, Test Requirements, Procedure, Result-Interpretation, Keynotes, and Bacterial Motility Footages

Introduction of Motility Test for Bacteria/ Hanging Drop Preparation

Bacteria are motile due to having flagellum/flagella. On the flagella, bacteria are motile and non-motile and therefore some bacteria are motile (Vibrio cholerae, E. coli, Proteus species, etc.) whereas some others are non-motile like Klebsiella, Shigella, and Acinetobacter. Motile bacteria generally use flagella as their locomotory organ and they tend to move towards or away from various chemotactic, phototactic, aerotactic, or magnetotactic stimuli. There are various methods to demonstrate bacterial motility and among them, some are hanging drop preparation, simple wet mount, and semi-solid agar (SIM/MIU).

Principle of Hanging Drop Preparation

Organisms are motile due to flagella. The motility tests are used to detect the presence of flagella by bacteria, allowing them to travel in the microscopic field. A drop of the organism in broth is suspended on a clean and grease-free cavity (modified) glass slide, a coverslip is added, and it is observed microscopically for motility.

Requirements for Hanging Drop Preparation

• Fresh broth culture of test bacteria
• Bunsen burner or spirit lamp
• Inoculating/bacteriological loop/ sterile sticks
• Glass slide (with central concavity/paraffin ring/adhesive ring/ applying agar medium pieces-as shown in footage)
• Cover slips (22 by 22 mm)
• Bright-field microscope or phase-contrast microscope

Procedure of Requirements for Hanging Drop Preparation

• Place a loopful of bacterial suspension in the center of a cover slip.
• Place the glass side with stick ring over the coverslip such a manner that coverslip sticks to the ring on the slide. Lift the glass slide immediately and turned around.
• The drop of bacterial suspension will hang on the lower surface of the coverslip.
• Now observe the drop under the low power objective (10X) of the compound microscope.
• Focus the edge of the drop.
• Test organisms tend to accumulate at the edge of the drop.
• After location edge, observe it under high power objective( 40X).

Result-Interpretation of Hanging Drop Preparation

Motility of organisms can be seen. However, it has to be differentiated from the Brownian movement.

1. Darting or shooting: motility:  It is a rapid type of movement e.g. Vibro cholerae and it may move as fast a 200µm per second.
2. Active motility- e.g. Enterobacteriaceae (Except Klebsiella and Shigella)
3. Stately or sluggish movement- Bacillus, Clostridium (oscillatory i.e. pendulum-like movement)
4.  Tumbling motility: e.g. Listeria monocytogenes (Inverted umbrella appearance is seen in soft agar)

Keynotes on Motility of Microbes

1. Enterococcus casseliflavus and E. gallinarum are motile.
2. Listeria organisms are motile at 25°C but not at 35°C, with a characteristic umbrella-shaped growth at the top of the tube. On wet mount, they exhibit tumbling motility.
3. Bacillus species should be motile but lack of motility could indicate Bacillus anthracis.
4. Yersinia enterocolitica is motile at 25°C but non-motile at 35°C.
5. Acinetobacter species are non-motile.
6. Non-fermenting, gram-negative rods, and Enterobacteriaceae vary in their motility.
7. Flagella arrangements are as follows-

• Monotrichous : It is a single flagellum at one pole (also called polar flagellum) and organism having such flagellum is Vibrio cholerae.
• Amphitrichous : It is a arrangement of single flagellum at both poles. e.g. Spirillum species
• Lophotrichous : It is an arrangement of two or more flagella at one or both poles of the cell. e.g. Spirillum undula
• Peritrichous: It is completely surrounded by flagella. e.g. Escherichia coli

Bacterial Motility Footages

Prepared hanging drop ready for microscopy

Hanging drop at a magnification of 100X

Hanging drop at a magnification of 400X

Vibrio cholerae darting motility in hanging drop at a magnification of 400X

Motility of bacteria in hanging drop preparation at a magnification of 800X

Hanging Drop Preparation for Motility testing of Bacteria Step by Step Demonstration

Darting motility test Positive of Vibrio cholerae and Rice water stool of Cholera patient Demonstration

Flagellated Bacteria (Pseudomonas aeruginosa) in Simple Wet Mount Microscopy at various magnifications

Hanging drop for bacterial motility Positive/ Darting motility of Vibrio cholerae from broth culture

Bacillus species and Pseudomonas aeruginosa Motility in Hanging Drop Preparation

Further Reading

• Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
• Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
• Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
• Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
• Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
• Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier
• https://catalog.hardydiagnostics.com/cp_prod/content/hugo/miomedium.ht
• https://www.ncbi.nlm.nih.gov/pubmed/17333741
• https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/SIMMedium.htm