Motility: Introduction, Types, Detection Methods and Keynotes

Introduction of Motility

Flagellar motility is due to rotation due to in the anti-clock direction for changing of direction, the agar tumbles in a clockwise direction then again starts motility in an anticlockwise direction.

Types

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

Detection Methods

Wet mount- Motility of organism can be seen. However, it has to be differentiated from the Brownian movement.
Demonstration of flagella by staining: Silver impregnation cells or structures too thin to be visible under an ordinary microscope may be rendered visible if they are thickened by the impregnation of silver on their surface. These are used for the demonstration of spirochetes as well – Ryu’s Stain
Hanging Drop: The organism is to be looked at the periphery of the drop because organisms are generally aerobic.

In semi-solid agar

In biochemical media like SIM (Sulfide, Indole, Motility), MIU (Motility, Indole, Urea), and MIO (Motility, Indole, Ornithine)
U- Tube method
Cragie’s tube method

To see the motility of an anaerobic organism

Hanging drop method- Seal around by wax or paraffin
Capillary tube method- culture the organism in RCM and take it in a capillary and seal both ends of the tube and fix in a slide and observe under the microscope.

Principle

Organisms are motile due to flagella and motility tests use to detect the presence of ﬂagella by bacteria, allowing them to travel in and out of the microscopic ﬁeld or beyond their initial inoculation in agar. For the wet preparation, a drop of the organism in broth is suspended on a clean and grease-free glass slide, a coverslip is added, and the culture is observed microscopically for motility. Occasionally the organism is incubated in the broth prior to examination whereas, in the tube test, semisolid motility medium like SIM (Sulfide, Indole, Motility) or MIU (Motility, Indole, Urea) or MIO (Motility, Indole, Ornithine) is inoculated in a straight line down through the center of a tube. Motile organisms will migrate out from the line of inoculation, causing visible turbidity throughout the tube. Non-motile organisms will grow only along the line of inoculation.

Requirements Test

Organisms tested-Campylobacter; Legionella; enterococci; Enterobacteriaceae; Listeria; Bacillus; other gram-positive rods; non-glucose-fermenting, gram-negative rods; and any other organism where motility is useful for identiﬁcation
Broth media like TSB or BHI or Nitrate broth
A semi-solid medium like SIM, MIU or MIO
22- by 22-mm coverslips and microscope slides
Bright-ﬁeld microscope or phase-contrast
Sterile inoculating needle or sticks
Control strains

Positive Control (PC):Escherichia coli 25922

Negative Control (NC): Klebsiella pneumoniae 13883

Procedure

Wet mount preparation

For Inoculum preparation, use fresh growth from an agar plate and suspend isolated colonies in broth. Use a light inoculum i.e. not visibly turbid.
It is acceptable to suspend the organism in a small amount of medium for an initial wet mount but follow with incubation of a larger amount of broth media if the result is negative.
Choosing the medium uses any broth which does not contain carbohydrates and will support the growth of the organisms like BHI, TSB, and nitrate broth.
Always use a broth for Bacillus spp.
Use 0.5 ml of BHI or TSB for enterococci.
Normal Saline can be used for gram-negative rods.
Use warm sterile tap water for Legionella.

Examination

Wear gloves and place a small drop of fresh liquid on the center of a microscope slide; add a coverslip. Allow organisms to settle for a minute.
Focus on 10x objective and observe under high power (40X). For a light microscope, decrease the light by closing the diaphragm. Preferably, using a phase-contrast microscope.
For all organisms negative for motility by initial wet mount, repeat the wet mount after incubation in broth, or test by tube method below.
Incubate at 30°C for non-fermenting, gram-negative rods for 24 hours.
Incubate enterococci and Listeria at 30°C for 2 hours
Other organisms may be incubated at temperatures optimal for their growth, usually 35°C.

Keynotes

Enterococcus casseliﬂavus and E. gallinarum are motile.
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.
Bacillus spp. should be motile but lack of motility could indicate Bacillus anthracis.
Yersinia enterocolitica is motile at 25°C but non-motile at 35°C.
Acinetobacter species are non-motile.
Non-fermenting, gram-negative rods, and Enterobacteriaceae vary in their motility.

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