Motility Testing
Cynthia Alonzo, M.S. Version 09-2.01
Read the entire experiment and organize time, materials, and work space before beginning.
Remember to review the safety sections and wear goggles when working with chemicals.
Objectives:
 To learn flagellar structure and arrangements common in microbes, and
 To use direct observation and testing to determine if a given microbe is motile.

Materials
From:
Label or Box/Bag: Qty Item Description:
Student
Provides 1 Microscope
1 Paper Clip 1 10%-bleach Solution 1 Paper towels
From LabPaq 1 Gloves packages - 11 pairs 1 Immersion Oil 1 Slide - Cover Glass - Cover Slip Cube (4)
Culture Media
Bag #2 -
Refrigerate
upon Receipt
Culture Media Bag #2 -
Refrigerate upon
Receipt
2 Agar, 0.4% Motility Test Agar - 8 mL in Glass
Tube
Inoculation
Instruments
Inoculation
Instruments
1 Inoculation Loop, Plastic
Mask Bag Mask Bag 1 Mask with Earloops (11) in Bag 5" x 8"
Slide Box MBK Slide Box MBK 1 Slide-Box-MBK with Blank-Slides (4)
Pre-Lab Preparation: Place saved cultures of E. coli and S. epidermidis (from previous lab) in incubator 12-24 hours prior to the start of the experiment.
Discussion and Review: Many bacteria are capable of motility, the ability to move under their own power. Most motile bacteria propel themselves by special organelles termed flagella. The bacterial flagellum is a noncontractile, semi-rigid, helical tube composed of protein and anchors to the bacterial cytoplasmic membrane and cell well by means of disk-like structures. The rotation of the inner disk causes the flagellum to act much like a propeller.
Bacterial motility constitutes unicellular behavior. In other words, motile bacteria are capable of a behavior called taxis. Taxis is a motile response to an environmental stimulus and functions to keep bacteria in an optimum environment. The arrangement of the flagella about the
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Hands-On Labs, Inc. bacterium is of use in classification and identification. The following flagellar arrangements may be found:
1. monotrichous - a single flagellum at one pole.
2. amphitrichous - single flagella at both poles.
3. lophotrichous - two or more flagella at one or both poles of the cell.

4. peritrichous - completely surrounded by flagella.
One group of bacteria, the spirochetes, has internally-located axial filaments or endoflagella. Axial filaments wrap around the spirochete towards the middle from both ends. They are located above the peptidoglycan cell wall but underneath the outer membrane or sheath. You can not directly observe flagella on most microbes with a standard light microscope in a wet mount.
To detect bacterial motility: we can use any of three methods: direct observation; motility media; and flagella staining, but in this lab we will focus on two methods:
A. Direct observation of motility: With a wet-mount, an actively growing, "young" broth culture can be used for observation of motility. Motile organisms can be seen as they move among each other in separate directions. Direct observation of motility can sometimes be difficult. Cultures, particularly older cultures, may be a mix of both active and inactive members, if the wet mount sample contains a larger percentage of inactive microbes, motility may be difficult to observe. In very active cultures, the opposite problem may exist. When using a microscope, the field of view encompasses a very small area. Fast moving microbes may leave the viewable area before they can be clearly observed.
B. Motility test medium: Semi-solid Motility Test medium may also be used to detect motility. The agar concentration (0.3%) is sufficient to form a soft gel without hindering motility. When a nonmotile organism is stabbed into Motility
Test medium, growth occurs only along the line of inoculation. Growth along the stab line is very sharp and defined. When motile organisms are stabbed into the soft agar, they swim away from the stab line. Growth occurs throughout the tube rather than being concentrated along the line of inoculation. Growth along the stab line appears much more cloud-like as it moves away from the stab. PROCEDURES: Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes may have been incorporated into the sample. Any culture that may contain an
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Hands-On Labs, Inc. unknown organism should be treated as potentially pathogenic. Therefore be certain to wear the gloves and mask provided when handling the cultures to protect yourself from unintended exposure. When you have completed the experiment dispose of the mask and gloves you have been using. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, as with all cultures and materials in your LabPaq, use and store them out of the reach of children, other individuals and pets.
Preparation of bacterial cultures: Use the bacterial cultures from the experiment:
Methyl Red Voges-Proskauer Test. If you are not doing the labs together you will need to make the cultures fresh as described in the experiment Methyl Red Voges-Proskauer
Test.
Part I: Direct Observation of Motility
1. Disinfect your work area.
2. Using the protocol from Experiment 2 make a wet-mount of S. epidermidis.
3. Observe the preparation and record any observations of motility.
4. Repeat Steps 2 and 3 with E. coli.
5. Set aside your cultures for use in Part II.
Part II: Motility Tubes
1. Label each of the motility tubes (0.4% agar) with E. coli and S. epidermidis.
2. Sterilize the inoculating needle (for the purposes of this lab you will use a straightened paper clip) by soaking it in bleach solution.
3. Using aseptic technique, open the motility tube labeled E. coli and the E. coli culture. 4. Insert the inoculating needle directly into the E. coli culture and then straight down the center of the agar tube. Withdraw the inoculating needle out along the same path as the entry being careful not to disturb the agar.
5. Use the aseptic technique to close the tubes.
6. Re-sterilize the inoculation needle by returning it to the bleach solution.
7. Repeat Steps 3 - 6 with S. epidermidis.
8. Incubate the motility tubes at 35o
C - 37o
C for 24 to 48 hours.
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9. After incubation, observe the tubes and record any observations of motility.
10. Soak the motility tubes in a 10%-bleach solution for 1 hour and discard the contents. Clean and rinse the test tubes for future use.
11. You will not be using your E. coli culture for future labs, add bleach to your culture and dispose of it as previously described.
12. Store your S. epidermidis culture in the refrigerator for use in future lab experiments. 13. Disinfect your work area.
Questions:
A. Define the following terms:
 monotrichous
 amphitrichous
 lophotrichous
 peritrichous
B. What are the three commonly used techniques to test motility?
C. Why is semi-solid media used to test for motility?
D. Why might it be difficult to observe motility in your wet mount?
E. Why is it important to use a needle rather than an inoculating loop when inoculating a motility tube?
F. Which of your organisms, if any, were you able to observe motility in your wet mount? G. Which of your organisms, if any, were you able to observe motility in your motility tube?
H. Did your direct and indirect observations of motility show the same results? If they didn’t, why do you think that may have been the case?