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Bacterial Motility

Carolina Labsheets™

In this lab, students perform two exercises that investigate microbial motility. One is on a microscopic level, using the “hanging drop” slide preparation method to directly observe motile cells. The second exercise is on a macroscopic level and involves inoculating motility test media. The MIO media used in this exercise (825862) also screens for indole and ornithine decarboxylase activity.

This activity assumes that students have previously worked with bacteria and are familiar with sterile technique. If this is not the case, we recommend that students complete the activities described in our “Introduction to Sterile Technique” LabSheet before beginning this activity.

Student Lab Sheet

Needed Materials*

Bacillus cereus, broth culture (154872)

Enterobacter areogenes, broth culture (155031)

Micrococcus roseus, tube culture (155160)

Motility Indole Ornithine (MIO) Media (825862)

Bunsen burner

inoculating loop and inoculating needle

concavity slide (632250)


petroleum jelly or paraffin

wax test tube racks

disinfectant (e.g., 70% ethanol, Lysol®, bleach solution)

microscope with 400× total magnification


Optional Materials
Slides 294198 (Polar Amphitrichous [Spirillum volutans], w.m.) and 294204 (Peritrichous [Proteus vulgaris], w.m.) are stained to show bacterial flagella.

You can substitute 825902 (Motility Test Medium Agar without TTC) or 825903 (Motility Test Medium Agar with TTC) for 825862 MIO media. Note: Neither of these substitutes is suitable for indole testing.

TTC (triphenyl tetrazolium chloride) is reduced by bacterial dehydrogenases, changing from clear to red. If the red color is restricted to the area of the stab, the bacterium is nonmotile. If the bacterium is motile, the red color spreads out from the stab. If the bacterium is highly motile, the entire tube of medium could become red.


Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.

This activity requires that students work with bacterial cultures and open flames. Have students use sterile technique at all times and wear appropriate safety equipment (such as safety glasses or goggles, gloves, and aprons).

Have them wipe down all work surfaces with disinfectant before and after the lab, and wash their hands after entering and before exiting the lab. Disinfect cultures and any contaminated items remaining at the end of the lab by autoclaving them or by flooding them with disinfectant overnight before proper disposal.


Students can work individually, if desired, but students working in pairs will have the chance to both inoculate their media and share the duties in preparing the hanging drop slide. Both can share a microscope to observe and record their findings.

Optimal incubation temperatures are 30°C for Enterobacter areogenes and 25°C for Micrococcus roseus. Both can be incubated at room temperature, but it might take longer to obtain results.

Inoculating Media

Each group will need the following:
     test tube rack
     Enterobacter areogenes culture
     Micrococcus roseus culture
     2 tubes of motility media
     inoculating needle
     Bunsen burner

“Hanging Drop” Slide Preparation

Each group will need the following:
     test tube rack
     Bacillus cereus broth culture
     concavity slide
     petroleum jelly or paraffin wax
     inoculating loop
     Bunsen burner

Brownian motion can sometimes be mistaken for motility. If a cell is truly motile, it will move over a distance and not simply vibrate back and forth. Bacterial cells are most motile during the log phase of growth and may lose their motility as the culture ages.

Optional: Other bacterial cultures can be tested in addition to or instead of the ones recommended above. Note that the hanging drop method is not safe for use with bacterial cultures that we list as pathogens. MIO medium also can be used to test for indole production; see our “Bacteria: The Indole Test” LabSheet for details.

Answer Key to Questions Asked on the Student LabSheet

  1. What is bacterial motility?
    Bacterial motility is the ability of a bacterium to move by propelling itself spontaneously.
  2. Your culture tube contains a motility medium. What is its purpose?
    The motility medium indicates whether the bacteria have the ability to grow or migrate from a point of inoculation.
  3. How can you tell the difference between a negative and a positive motility test using this medium?
    A positive test is indicated by the spreading of cloudy, diffuse growth in the media from the point of inoculation. This is not the case with a negative test result.
  4. What anatomical feature allows most bacteria to become motile?
    Possession of one or more flagella allows a bacterium to be motile.
  5. List four flagellum (or flagella) arrangements and draw a simple picture illustrating each.
    The arrangements are (1) monotrichous, (2) lophotrichous, (3) amphitrichous, and (4) peritrichous. Students’ illustrations will vary, but should accurately depict the listed arrangements.

  6. Using a textbook or other resource, describe the flagella arrangement (if any) of:
  • Enterobacter areogenes
    Enterobacter is peritrichous, having flagella over the entire surface of each bacterium.
  • Micrococcus roseus
    Micrococcus is nonmotile and lacks flagella.
  • Bacillus cereus
    Bacillus is peritrichous, having flagella over the entire surface of each bacterium.
  1. Describe the type of motility of Bacillus cereus under the low-power objective and high-power objective.
    Under the low-power objective, you can see the rod-shaped bacteria slowly move in a twitching, erratic manner. Some move generally in one direction, while others change course nearly constantly. Under a highpower objective, these movements are exaggerated. The twitching and vibrating movements are more apparent, and some bacteria can be observed moving in a spinning motion.
  2. Design a test where one can manipulate the environment or add a dependent variable causing a change in motility.
    Student’s answers will vary; they might choose to make the environment more acidic or more basic, alter its temperature, light availability, or other factors.
  3. Which of the inoculated media tested positive for motility? How can you tell?
    The media inoculated with Enterobacter tested positive, exhibiting a spreading of cloudy, diffuse growth from the point of inoculation.
  4. Using a microbiology textbook or other resource(s) describe any other bacteria that may test negative for motility.
    Answers will vary, but might include Corynebacterium, Lactobacillus, Sarcina, and Streptococcus



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