EXPERIMENT. Biochemical Testing for Microbial Identification Methyl Red, Voges- Proskauer, and Catalase Testing

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1 EXPERIMENT Biochemical Testing for Microbial Identification Methyl Red, Voges- Proskauer, and Catalase Testing Hands-On Labs, Inc. Version 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 biochemical testing in microbiology. You will describe the function of methyl red, Voges-Proskauer, and catalase tests. You will perform 3 biochemical tests using Escherichia coli and Staphylococcus epidermidis cultures. 1 Hands-On Labs, Inc.

2 Objectives Upon completion of this laboratory, you will be able to: Discuss the purpose of biochemical testing in microbiology. Describe how the methyl red test detects mixed-acid fermentation. Explain how the Voges-Proskauer test detects acetoin production. Describe how the catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide. Culture microbes in MR-VP broth. Perform biochemical tests on microbes. Relate experimental results to metabolic pathways of microorganisms. Time Allocation: 3 hours hours incubation 2 Hands-On Labs, Inc.

3 Materials Student Supplied Materials Quantity Item Description 1 Active culture broth and plate-e. coli 1 Active culture broth and plate-s. epidermidis 1 Bleach 1 Camera, digital or smartphone 2 Disposable cups 1 Hand soap 1 Hydrogen peroxide 1 Isopropyl alcohol (rubbing) 1 Matches or lighter 1 Pair of scissors 1 Permanent marker 1 Roll of paper towels 1 Source of tap water HOL Supplied Materials Quantity Item Description 1 Apron 2 Blank slides 1 Chem bag-methyl red Voges-Proskauer kit: 1 - Barritt s A reagent-3 ml pipet 1 - Barritt s B reagent-3 ml pipet 1 - Methyl red reagent 0.1%-1 ml pipet 1 Face mask with ear loops 2 Inoculation loops 3 Long thin stem pipettes 2 MR-VP broth-5 ml tubes 3 Pairs of gloves 1 Safety goggles 1 Tea candle 1 Test tube clamp 4 Test tubes, 16 x 125 mm 1 Test tube rack, 6 x 21 mm Note: To fully and accurately complete all lab exercises, you will need access to: 3 Hands-On Labs, Inc.

4 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. 4 Hands-On Labs, Inc.

5 Background Techniques for Identifying Unknown Microorganisms Although approximately 4,000 species of bacteria have been identified, scientists estimate that as many as 3 million more species of bacteria still remain to be identified. Because microorganisms play a key role in the function of ecosystems, understanding these microorganisms can further help with the understanding of how and why ecosystems thrive. Further, the ability to characterize and identify microorganisms is essential for the advancement of human medicine and the avoidance of human epidemics. Food poisoning cases often involve intense work to identify the causative agent. The historic outbreak of the bacteria Listeria moncytogenes, which was associated with cantaloupe in the Western United States, required a coordinated effort from several agencies to identify the genetic variant, or strain, of the microbe. Identification was a key step in determining the point of origin for the infectious bacteria which led to preventative measures against the spread of the disease. Multiple techniques are used to determine the identity of an unknown microbe. These identification techniques are not mutually exclusive and can be used in conjunction with one another. Morphology, staining properties, and selective media provide general information. More specific techniques are required for definitive identification of microbes. One of the most powerful and accurate techniques, and also the most expensive and specialized, is DNA sequencing. A less expensive and often-employed method of microbial identification is a biochemical test. See Figure Hands-On Labs, Inc.

6 Figure 1. Biochemical testing equipment. borzywoj Biochemical tests are designed to identify various metabolic properties of different bacteria species. More importantly, these tests, in conjunction with taxonomy, can lead to the unambiguous identification of an organism. In this laboratory you will perform several common biochemical tests on active cultures of E. coli and S. epidermidis. Methyl Red Test Carbohydrates are a group of organic compounds that are composed of carbon, hydrogen, and oxygen atoms. When carbohydrates are broken down anaerobically (without available oxygen), fermentation occurs. Fermentation is the conversion of sugars to acids, gases, and/or alcohol. The methyl red test is designed to identify microbes that perform mixed-acid fermentation. Mixed-acid fermentation is an anaerobic metabolic process that produces lactic acid, acetic acid, ethanol, carbon dioxide and hydrogen gas. These acidic products can lower the ph of the buffered media used to grow the microbes being tested. Other fermentative organisms produce smaller amounts of less stable acids which are neutralized by the buffering capacity of the media. Acetic acid is the main component of vinegar. Acetic acid has a distinctive sour taste and pungent smell. The global demand for acetic acid is approximately 6.5 million metric tons per year. Food grade vinegar, see Figure 2, is produced by bacteria of the genus Acetobacter in commercial facilities. 6 Hands-On Labs, Inc.

7 Figure 2. A variety of flavored vinegars produced by bacteria. Photology1971 The ph indicator methyl red (p-dimethylaminoaeobenzene-o-carboxylic acid) turns red in ph between 4.4 and 6.0. MR-VP broth contains 0.5% glucose, peptones and buffers. Cultures demonstrating mixed-acid fermentation turn red when methyl red is added to the broth because the mixed-acid fermentation creates acidic by-products that do not neutralize in the buffered solution. Cultures that do not undergo mixed-acid fermentation turn yellow when methyl red is added, indicating a negative test result. Voges-Proskauer Test The Voges-Proskauer test is designed to identify microbes that ferment glucose via the butanediol pathway. Before performing a Voges-Proskauer test, the unidentified cultures are incubated in an MR-VP broth, similar to the process for the methyl red test. However, instead of adding methyl red to the broth, Barritt s reagents are added. Butanediol fermentation produces weaker acids than mixed-acid fermentation. An intermediate of butanediol fermentation is acetoin. Acetoin turns red in the presence of Barritt s reagents indicating a positive result for butanediol fermentation. The methyl red and Voges-Proskauer tests are typically performed together to differentiate bacteria within the family Enterobacteriaceae. The large family of Enterobacteriaceae includes the genus Salmonella. Every year, Salmonella is estimated to cause 1.2 million illnesses in the United States, with 19,000 hospitalizations and 380 deaths. In November 2014, an outbreak of Salmonella in 12 states was linked to bean sprouts. The Centers for Disease Control recommends cooking all sprouts thoroughly to reduce the risk of illness. See Figure Hands-On Labs, Inc.

8 Catalase Testing Figure 3. Raw bean sprouts. Dan loei tam loei Aerobic respiration results in metabolites (by-products) that include oxygen ions and peroxides. These metabolites are chemically reactive and can cause damage to cell structures. Hydrogen peroxide (H 2 O 2 ) is toxic to many microbes. Bacteria that live in oxygenated environments produce the enzyme catalase to neutralize the harmful effects of hydrogen peroxide. Anaerobic microbes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide into oxygen and water. When hydrogen peroxide is added to a colony of catalase-positive microbes (aerobic microbes), oxygen bubbles rapidly form. See Figure 4. A lack of bubbles indicates catalase-negative microorganisms (anaerobic microbes). Figure 4. Hydrogen peroxide, when added to a wound, produces oxygen bubbles when it comes in contact with bacteria that produce the enzyme catalase. schankz 8 Hands-On Labs, Inc.

9 Exercise 1: Methyl Red and Voges-Proskauer Testing In this exercise, you will perform the methyl red and Voges-Proskauer tests on active cultures of E. coli and S. epidermidis. Preparation for the Incubation of Microbes in the MR-VP Broth 1. Clear a work area and gather all materials listed for this experiment. 2. Allow the tubes of MR-VP broth to warm to room temperature (approximately 30 minutes). 3. Wash your hands thoroughly with soap and warm water. 4. Put on the safety gloves, face mask, apron, and goggles. 5. Disinfect the work surface by wiping with a 10% bleach solution. 6. Use the permanent marker to label one MR-VP broth tube E. coli and the other MR-VP tube S. epidermidis. 7. Place 2 dropper pipets into a disposable cup of alcohol. 8. Light the candle. 9. Gather the active E. coli culture vial. 10. Remove the cap and pass the lip of the vial over the flame to sterilize it. See Figure 5. Figure 5. Sterilizing culture lip. 11. Remove the lid of the E. coli MR-VP broth tube and flame the lip of the tube. 12. Remove one dropper pipet from the alcohol and shake to dry. 13. Carefully insert the tip of the pipet into the active E. coli culture and draw up a small amount of broth. 14. Carefully pipet 4 drops of the active culture into the MR-VP tube. 9 Hands-On Labs, Inc.

10 15. Place the pipet into undiluted bleach. 16. Flame the lip of the inoculated MR-VP broth and screw on the cap. 17. Flame the lip of the active culture vial and screw on the cap. 18. Repeat steps 9-17 for S. epidermidis. 19. Extinguish the candle. 20. Place the MR-VP cultures in your incubation location and incubate for 48 hours. 21. Return the stock cultures of E. coli and S. epidermidis to your incubation location to store for future experiments. 22. Remove the pipets from the bleach container, wrap in paper towels, and dispose of in the garbage. 23. Wipe down your work area with a 10% bleach solution. 24. Wash and return items to your kit for future use. 25. Wash your hands thoroughly with soap and warm water. Methyl Red Test 26. Check MR-VP broths after 48 hours for bacterial growth which should appear as turbidity. If no growth is observed, continue to incubate for another 24 hours. 27. Wipe down your work area with a 10% bleach solution. 28. Wash your hands thoroughly with soap and warm water. 29. Put on your goggles, a new pair of gloves, face mask, and apron. 30. Gather the materials required for this experiment. 31. Sterilize the 4 test tubes by rinsing them in alcohol and allowing them to thoroughly dry. 32. Using the permanent marker, label 2 test tubes E. coli and 2 test tubes S. epidermidis. See Figure Hands-On Labs, Inc.

11 Figure 6. Labeled test tubes. 33. Carefully pour half of the incubated MR-VP broth labeled E. coli into each of the corresponding test tubes. Repeat for the broth labeled S. epidermidis. 34. You should have approximately equal amounts of broth in all 4 tubes. See Figure 7. Figure 7. Incubated MR-VP broths divided among test tubes Hands-On Labs, Inc.

12 35. Use scissors to remove the tip of the Methyl Red Reagent pipet over a trash can. Use a wet paper towel to carefully wipe the scissors after cutting the pipet and throw the paper towel into the trash. See Figure 8. Figure 8. Removing the tip of reagent pipet. 36. Pipet 7 drops of methyl red reagent into one of the E. coli and into one of the S. epidermidis test tubes. See Figure 9. Figure 9. Adding reagent to a test tube Hands-On Labs, Inc.

13 37. Immediately observe the color of the broth after adding the drops and record it in Data Table 1 of your Laboratory Report Assistant. If the test is positive, the broth will appear red or pink in color. 38. Take a photograph of your results. Resize and insert the image into Data Table 1. Refer to the appendix entitled Resizing an Image for guidance with resizing an image. 39. Use the 2 remaining test tubes for the Voges-Proskauer test. Voges-Proskauer Test 40. Remove the tip of the Barritt s A Reagent pipet with scissors and add 12 drops to each test tube. Gently mix by swirling the test tube. 41. Remove the tip of the Barritt s B Reagent pipet with scissors and add 4 drops to each test tube. Shake the tubes gently for 30 seconds to expose the contents to oxygen. 42. Place the tubes in the test tube holder and let them sit for 30 minutes. Note: The tubes must not be disturbed for the 30 minute incubation period. 43. After 30 minutes, observe the test tubes for a color change. A positive test is the formation of a red surface layer. See Figure 10. Figure 10. Voges-Proskauer test results. The sample on the right is positive. 44. Record your observations in Data Table 2 of your Laboratory Report Assistant. 45. Take a photograph of your results. Resize and insert the image into Data Table 1. Refer to the appendix entitled Resizing an Image for guidance with resizing an image. 46. Soak the 4 test tubes in bleach for 1 hour. Clean and rinse the test tubes for future use. 47. Wrap the chemical pipets in paper towels and dispose of in the garbage Hands-On Labs, Inc.

14 48. Wipe down your work area with a 10% bleach solution. 49. Wash your hands thoroughly with soap and warm water. Questions A. Why are biochemical tests used to identify microbes? B. What is mixed-acid fermentation? How is methyl red used to determine if a microbe has undergone mixed-acid fermentation? C. Why are the methyl red and Voges-Proskauer tests often performed together? D. How do the experimental results relate to the metabolic pathways used by E. coli and S. epidermidis? 14 Hands-On Labs, Inc.

15 Exercise 2: Catalase Testing In this exercise, you will perform a catalase test using E. coli and S. epidermidis cultures saved from an earlier experiment. 1. Gather the hydrogen peroxide, dropper pipet, inoculation loops, blank microscope slides, incubated E. coli and S. epidermidis culture plates, alcohol, disposable cup and all safety items. 2. Wash your hands thoroughly with soap and warm water. 3. Put on a new pair of gloves, face mask, apron, and goggles. 4. Disinfect the work surface by wiping with a 10% bleach solution. 5. Place the inoculation loops into a cup of alcohol to sterilize. 6. Label one blank slide E. coli and the other slide S. epidermidis with the permanent marker. Draw a dime-sized circle on the center of each slide. 7. Remove one inoculation loop from the alcohol and shake it to dry. 8. Open the lid of the E. coli culture plate and collect several colonies with the inoculation loop. See Figure 11. Figure 11. Collecting colonies from a culture plate. 9. Transfer the collected colonies to the circle on the slide labeled E. coli. See Figure Hands-On Labs, Inc.

16 Figure 12. Transferring colonies to a slide. 10. Pipet one drop of hydrogen peroxide onto the sample and observe it for the formation of oxygen bubbles. The absence of bubbles is a negative test result. 11. Record your observations in Data Table 3 of your Laboratory Report Assistant. 12. Take a photo of the slide. Resize and insert the image into Data Table Repeat steps 7-12 for S. epidermidis. 14. Soak the slides and inoculation loops in undiluted bleach for 20 minutes and dispose of them in the garbage. 15. Return the plated cultures to your incubation location for future experiments. 16. Wipe down your work area with a 10% bleach solution. 17. Wash and return the remaining items to your kit for future use. 18. Wash your hands thoroughly with soap and warm water. 19. When you are finished uploading photos and data into your Laboratory Report Assistant, save your file correctly and zip the file so you can send it to your instructor as a smaller file. Refer to the appendix entitled Saving Correctly and the appendix entitled Zipping Files for guidance with saving the Laboratory Report Assistant correctly and zipping the file Questions A. What is catalase? What types of microbes are more likely to produce catalase? B. Based on the results from your experiment, what can you conclude about the metabolism of E. coli and S. epidermidis? 16 Hands-On Labs, Inc.

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