EXPERIMENT Bacteria Identification through Functional Media Motility Testing Hands-On Labs, Inc. Version 42-0248-00-02 Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise. Experiment Summary: You will discuss the purpose of functional media in microbiology. You will describe bacterial motility. You will perform motility testing on Escherichia coli and Staphylococcus epidermidis. www.holscience.com 1 Hands-On Labs, Inc.
Objectives Upon completion of this laboratory, you will be able to: Discuss the purpose of functional media testing in microbiology. Describe forms of bacterial motility. Explain how motility media is used to test for bacterial movement. Perform motility testing on E. coli and S. epidermidis. Relate experimental results to motility properties of microbes. Time Allocation: 3 hours + 48 hours incubation www.holscience.com 2 Hands-On Labs, Inc.
Materials Student Supplied Materials Quantity Item Description 1 Active culture plate-e. coli 1 Active culture plate-s. epidermidis 1 Bleach 1 Camera, digital or smartphone 1 Disposable cup 1 Hand soap 1 Isopropyl alcohol (rubbing) 2 Large paper clip 1 Matches or lighter 1 Permanent marker 1 Roll of paper towels HOL Supplied Materials Quantity Item Description 1 Apron 1 Face mask with ear loops 2 Motility test agar 0.4% -8 ml tubes 2 Pairs of gloves 1 Safety goggles 1 Tea candle 1 Test tube rack, 6 x 21 mm Note: To fully and accurately complete all lab exercises, you will need access to: 1. A computer to upload digital camera images. 2. Basic photo editing software, such as Microsoft Word or PowerPoint, to add labels, leader lines, or text to digital photos. 3. Subject-specific textbook or appropriate reference resources from lecture content or other suggested resources. Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit. www.holscience.com 3 Hands-On Labs, Inc.
Background Functional Media A variety of techniques are employed to determine the identity of an unknown microbe. These identification techniques are not mutually exclusive and can be used in conjunction with one another. Functional media are often used to determine the identity of unknown microbes, and come in several forms. See Figure 1. Three of the primary forms are selective, differential, and enriched. Selective media contain agent(s) that prohibit the growth of some organisms, thus selecting or allowing the growth of other organisms. This is especially helpful when growing mixed cultures containing many different microbes when only one or two specific microbes are of interest. Differential media enable many types of microbes to grow; however, differential media contain an indicator that enables differences between microbes to be visualized. Enriched media are supplemented with essential growth factors for organisms that do not grow well on typical media. In this experiment, you will use differential media to determine motility of two species of bacteria. Motility Figure 1. Functional media. HUANSHENG XU Motility, self-propelled motion, is present in many but not all bacteria. Determining the presence of motility is important for the classification of microbes. Bacteria move by a variety of mechanisms including flagella, axial filaments, and slime secretions. Flagella are rigid structures 15-20 µm long which protrude from the cell surface. See Figure 2. Bacteria cells with a single flagellum are termed monotrichous, and those with a single flagellum at each end are termed amphitrichous. Bacteria with numerous flagella at one end are described as lophotrichous, while those with flagella distributed all over the cell are termed peritrichous. See Figure 2. www.holscience.com 4 Hands-On Labs, Inc.
Figure 2. Bacteria flagella arrangements. Left to right: monotrichous Sebastian Kaulitzki, lophotrichous royaltystockphoto.com, peritrichous Sebastian Kaulitzki More than 50% of the world s population harbor the lophotrichous bacteria Helicobacter pylori in their upper gastrointestinal tract. H. pylori, identified in 1982, has been linked to gastric ulcers. However, over 80% of infected individuals are asymptomatic. H. pylori infections can be controlled with antibiotics. Motility Testing Bacterial motility may be determined using microscopy, functional media, and flagella staining. Functional motility media is a soft gel consisting of 0.4% agar. The gel consistency allows motile bacteria to move throughout the media while preventing diffusion of non-motile species. Motility tests are conducted by stabbing a needle containing a microbe sample into a tube of soft gel. After incubation, the tube is observed for bacterial growth. Growth only along the stab line is a negative test for motility. Turbidity throughout the gel indicates uniform growth and is a positive test for motility. In this experiment you will test E. coli and S. epidermidis for motility using functional motility media. www.holscience.com 5 Hands-On Labs, Inc.
Exercise 1: Motility Testing In this laboratory you will incubate E. coli and S. epidermidis cultures in motility media to determine the motility of each bacteria. Part 1 of 2 1. Clear a work area and gather all materials listed for this experiment. 2. Wash your hands thoroughly with soap and warm water. 3. Put on the safety gloves, face mask, apron, and goggles. 4. Disinfect the work surface by wiping the work surface with a 10% bleach solution. 5. Straighten 2 paperclips and place them in a cup of alcohol. 6. Use the permanent marker to label one motility media tube E. coli. Label the second motility media tube S. epidermidis. 7. Light the candle. 8. Remove the lid from the E. coli motility media tube and use the flame to sterilize the lip. See Figure 3. Figure 3. Sterilizing culture tube opening. 9. Remove a paperclip from the alcohol and shake to dry. 10. Remove the lid from the E. coli culture plate and collect several colonies with the paperclip. See Figure 4. www.holscience.com 6 Hands-On Labs, Inc.
Figure 4. Collecting plated colonies. 11. Stab the paperclip into the motility agar and then carefully remove the paperclip in the same pathway. See Figure 5. Figure 5. Stabbing sample into motility media. www.holscience.com 7 Hands-On Labs, Inc.
12. Flame the opening of the motility media tube a second time before returning the cap. 13. Place the paperclip in undiluted bleach for 1 hour. 14. Repeat steps 8-13 for the S. epidermidis tube. 15. Take a photograph of the tubes. Resize and insert the photograph into Data Table 1 of your Laboratory Report Assistant. Refer to the appendix entitled Resizing an Image for guidance with resizing an image. 16. Place the motility media tubes and your plated cultures in your incubation location. 17. Incubate the motility tubes for 48 hours. 18. Wipe down your work area with a 10% bleach solution. 19. Wash and return items to your kit for future use. 20. Wash your hands thoroughly with soap and warm water. Part 2 of 2 21. Observe the motility media tubes after 48 hours incubation for microbial growth. If no growth is observed, incubate them for an additional 24 hours. 22. Wipe down your work area with a 10% bleach solution. 23. Wash your hands thoroughly with soap and warm water. 24. Put on your goggles, a new pair of gloves, face mask, and apron. 25. Gather the 2 motility media tubes. 26. Observe each tube noting the turbidity of the agar and the definition of the original stab line. A clearly defined stab line and clear agar is a negative result. Note: Compare the level of turbidity to that in the photos you took prior to incubation. 27. Record your observations in Data Table 2 of your Laboratory Report Assistant. 28. Take a photograph of your developed media tubes. Resize and insert the photograph into Data Table 2. 29. Soak the motility media tubes in undiluted bleach for 1 hour before placing in the garbage. 30. Wipe down your work area with a 10% bleach solution. 31. Wash your hands thoroughly with soap and warm water. www.holscience.com 8 Hands-On Labs, Inc.
Questions A. What is functional media? What is the distinction between the three types of functional media? B. What is motility? List three forms of motility in bacteria? C. How does the agar media used for motility testing differ from that used for your plate cultures? D. What can you conclude regarding the motility of E. coli and S. epidermidis based on your results? www.holscience.com 9 Hands-On Labs, Inc.