Motility test (hanging drop and semisolid agar)

Principle

• Flagellated bacteria move actively through liquid or soft agar.
• Hanging drop allows direct viewing of living organisms; semisolid agar shows spreading growth away from a stab line.

Specimen

• Young broth culture (a few hours) for the hanging drop; pure colony for the semisolid agar stab.

Reagents and equipment

• Cavity (depression) slide, coverslip, petroleum jelly, microscope with reduced light or phase contrast.
• Motility test medium with about 0.4% agar (typical range 0.3–0.5%), often with triphenyltetrazolium chloride (TTC), which turns growth red.

Procedure

• Hanging drop: ring the coverslip with petroleum jelly, place a drop of culture in the center, invert the cavity slide onto it and flip over so the drop hangs.
• Focus on the drop edge with the 10× then 40× objective using low light.
• Semisolid agar: stab straight down to about 1–2 cm from the bottom with a straight needle and withdraw along the same line.
• Incubate at 35–37 °C for 18–24 h; also at 22–25 °C for organisms such as Listeria and Yersinia enterocolitica.

Results

• True motility: organisms change position and cross the field in a directional way.
• Brownian movement: vibration in place without progression, which is not motility.
• Semisolid agar: diffuse cloudiness (or red color with TTC) spreading from the stab = motile; growth confined to the stab line = non-motile.
• Listeria shows umbrella-shaped growth in soft agar at room temperature and tumbling motility in wet mounts; Vibrio and Campylobacter show darting motility.

Quality control and pitfalls

• Controls: a motile organism (e.g. E. coli or Proteus) and a non-motile one (e.g. Klebsiella pneumoniae).
• Old cultures or wrong temperature can give false negatives; water currents can mimic motility in wet preps.
• A crooked stab can give false positives.

Clinical use

• Differentiates Enterobacterales (Klebsiella and Shigella are non-motile) and identifies Listeria, Vibrio and other genera.