Atlas page 35 & 64

Motility Procedure: Hanging Drop Atlas page 35

Bacteria that possess flagella have the ability to move and thus are motile. Live wet mount/Hanging Drop preparations of a bacterial culture may be viewed under a dark field microscope under oil immersion to determine if an organism is motile.

PROCEDURE: Obtain a clean Hanging-drop slide and a Nutrient Broth. Inoculate the Nutrient Broth and incubate it overnight ONLY.  Remove the Nutrient Broth from the incubator, mix and then using a sterile loop, transfer 10 "loop-fuls" of the broth to the indention in the Hanging-drop slide. Using a toothpick put four small "dabs" of Vaseline in the four corners of a coverslip and place it over the indention in the Hanging-drops slide.  Add immersion oil to the top of the slide and reduce your Iris to very low light.  Focus under oil immersion and look for "tumbling" behavior... NOTE: Vibrating Microbes are NOT motile... You MUST focus within 3 minutes of preparation of the slide as by 5 minutes all the microbes are NON-MOTILE as they are dead - killed from the heat produced by the light source.  Confirm your Positive Hanging-drop Motility with a Stab Motility Test!

You may also prepare the Hanging Drop test by applying a circular "ring" of Vaseline around the edge of depression on the slide using a wooden stick.  Place 5-10 loopfuls of the mixed overnight incubated broth onto a coverslip.  Invert the slide and place it over the coverslip.  Add oil to the top of the coverslip and view it under oil and LOW LIGHT!
Diagram of how to prepare a Hanging Drop Slide for Motility from an "overnight" incubated broth and under oil immersion at LOW LIGHT (iris DOWN)



An alternative method, one that is safer when working with potential pathogens, is the motility stab. A sterile, cool inoculating wire is used to obtain inoculum from a pure culture of the test organism. The needle is stabbed into motility stab media approximately two-thirds of the depth of the media. The wire is then pulled out of the media as close as possible to the location where it entered. The tube is incubated for approximately 1-2 labs and observed for evidence of motility. A non-motile organism will have a clearly defined edge as it grows on the stab line. Motile organisms will be turbid throughout the tube or have fuzzy, diffuse growth at the edges. Some organisms are so motile that the entire tube becomes very turbid (cloudy). Motile and nonmotile bacteria are shown below.

A redish indicator (soluable colorless 2,3,5-triphenyltetrazolium chloride is reduced to an  insoluable pink formazan "haze" by bacteria swimming through it...) may be added to the motility gel to make visualization easier... a fuzzy red tube is growth+motility, a solid red line is growth/nonmotile, and a clear tube indicates no growth...

In the tree photos below... the first is without indicator, the second is with indicator and the third is a plate showing the "rippled" colony growth pattern consistent with motility...

Motility Stab TEST or Exp.
Motility Stab Test with indicator
Motility Plate Observation Procedure

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