Hanging drop preparation is a special type of wet mount in which a drop of medium containing the organisms is placed on a microscope slide, often used in dark illumination to observe the motility of bacteria.
In this method, a drop of culture is placed on a coverslip that is encircled with petroleum jelly (or any other sticky material). The coverslip and drop are then inverted over the well of a depression slide. The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation. This preparation gives good views of microbial motility.
- Glass slides (glass slide with depression) or normal glass slides with adhesive or paraffin ring
- Paraffin wax
- Bunsen burner
- Young broth culture of motile bacteria (e.g. Proteus mirabilis)
Validate the competence of technologists in the hanging-drop method by testing known motile enterococci or Listeria in the broth assay.
- Take a clean glass slide and apply a paraffin ring, adhesive-tape ring to make circular concavity. (This step is not needed if a glass slide with depression is available).
- Hold a clean coverslip by its edges and carefully dab vaseline on its corners using a toothpick.
- Place a loopful of the fresh broth culture to be tested in the center of the prepared coverslip. Use a light inoculum (not visibly turbid).
- Turn the prepared glass slide or concavity slide upside down (concavity down) over the drop on the coverslip so that the vaseline seals the coverslip to the slide around the concavity.
- Turn the slide over so the coverslip is on top and allow organisms to “settle” for a minute. The drop can be observed hanging from the coverslip over the concavity.
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- Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.
- Turn the objective to its lowest position using the coarse adjustment and CLOSE THE DIAPHRAGM.
- Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the edge of the drop is observed as an irregular line crossing the field.
- Move the slide to make that line (the edge of the drop) pass through the center of the field.
- Without raising or lowering the tube, swing the high dry objective into position (be sure the high dry objective is clean).
- Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can be seen as a thick, usually dark line.
- Focus on the edge of the drop carefully and look at each side of that line for very small objects that are the bacteria. The cells will look either dark or slightly greenish, with very small rods or spheres. Remember the high dry objective magnifies a little less than half as much as the oil immersion objective.
- Adjust the light using the diaphragm lever to maximize the visibility of the cells.
- Observe the cells noting their morphology and grouping and determine whether true motility can be observed.
- Brownian movement should be visible on slides of all the organisms, but there should also show true motility.
- Wash the depression slide and after soaking in lysol buckets or discard the prepared glass slide.
Result and Interpretations
- Directional purposeful motility is a positive test. Motile organisms change position with respect to one another. Brownian movement (random jiggling or shaking due to molecular bombardment), where the organisms remain in the same relative position with respect to each other, should not be mistaken for true motility.
- Campylobacter and Vibrio cholerae display very active motility (darting motility) which appears as tiny dots darting in and out of the field.
For all organisms negative for motility by initial wet mount, repeat the wet mount after incubation in broth, or test by tube method.
- Incubate at 30°C for nonfermenting, Gram-negative rods (24 h).
- Incubate enterococci and Listeria at 30°C for 2 h.
- Other organisms may be incubated at temperatures optimal for their growth, usually 35°C.
While examining living organism for the property of active locomotion, it is essential to distinguish true motility, whereby the organism move in different directions and change their positions in the field, from either
Passive drifting of the organisms in the same direction in a convectional current in the fluid or
Brownian movement, which is an oscillatory movement about a nearly fixed point possessed by all small bodies suspended in fluid and due to irregularities in their bombardments by molecules of water.
Hanging drop preparation is used to study the motility of bacteria;
- Presumptive diagnosis of Vibrio Cholerae from rice watery stools. Click to view the video of Hanging drop preparation
- Listeria monocytogenes shows tumbling motility, particularly in hanging drop preparations prepared from overnight broth cultures.