SOP on Motility Test by Hanging Drop Method
1.0 Objective
To lay down the procedure for performing the Motility Test by Hanging Drop Method for determination of motility of microorganisms in the Microbiology Laboratory.
2.0 Scope
This SOP is applicable to the Motility Test by Hanging Drop Method performed in the Microbiology Laboratory.
3.0 Responsibility
Microbiologist or above of Microbiology Laboratory:
- Preparation and execution of the SOP.
- Recording and reporting of results.
Head – Microbiology Section / Nominee:
- Review and checking of the SOP.
- Ensuring proper implementation.
4.0 Accountability
Head – Quality Control / Nominee:
- Ensuring compliance with this SOP.
5.0 Procedure
5.1 Preparation of Bacterial Suspension
5.1.1 Select a fresh, well-isolated microbial colony from a suitable culture medium.
5.1.2 Prepare a light suspension of the microbial colony in sterile normal saline or sterile broth to obtain a slightly turbid suspension.
5.2 Preparation of Hanging Drop Slide
5.2.1 Take a clean, grease-free cover slip.
5.2.2 Apply a thin layer of grease or petroleum jelly (Vaseline) around the rim of the cavity (depression) of a clean hanging-drop slide.
5.3 Transfer of Culture
5.3.1 Using a sterile inoculating loop, aseptically transfer one small drop of the prepared microbial suspension to the center of the clean cover slip.
5.4 Mounting the Preparation
5.4.1 Invert the cavity slide carefully and place the depression directly over the drop on the cover slip.
5.4.2 Press gently so that the grease forms a firm seal between the slide and cover slip.
5.4.3 Quickly and carefully invert the entire assembly so that the drop hangs freely in the depression of the slide.
5.4.4 Ensure that the drop does not touch the bottom of the cavity.
5.5 Microscopic Examination
5.5.1 Place the prepared slide on the microscope stage.
5.5.2 Using the low power objective (10X), focus on the edge of the hanging drop.
5.5.3 Reduce the light intensity for better contrast. The edge of the drop will appear as a bright wavy line against a gray background.
5.5.4 Switch to the high dry objective (45X) and refocus carefully on the edge of the drop.
5.5.5 Observe the bacteria for movement.
5.6 Interpretation of Results
5.6.1 True motility is characterized by directional, purposeful movement of bacteria across the field.
5.6.2 Brownian movement appears as random vibration without actual displacement of the organism.
5.6.3 Carefully differentiate true motility from Brownian movement before recording results.
6.0 Precautions
6.1 Use fresh (18–24 hour) cultures for accurate results.
6.2 Avoid air bubbles while preparing the hanging drop.
6.3 Do not allow the drop to fall or touch the bottom of the depression.
6.4 Handle slides and cover slips carefully to prevent contamination or breakage.
6.5 Perform all aseptic procedures under appropriate laboratory conditions.
7.0 Documentation
7.1 Record the following details in the appropriate laboratory record:
- Name of organism
- Date of test
- Method used (Hanging Drop Method)
- Observation (Motile / Non-motile)
- Analyst signature
- Reviewer signature