EXPERIMENT Biochemical Testing for Microbial Identification Carbohydrate Fermentation Testing Hands-On Labs, Inc. Version 42-0241-00-03 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 and describe carbohydrate fermentation. You will use Durham tubes to test Saccharomyces cerevisiae and Staphylococcus epidermidis for fructose, glucose, and mannitol fermentation. www.holscience.com 1 Hands-On Labs, Inc.
Objectives Upon completion of this laboratory, you will be able to: Discuss the purpose of biochemical testing in microbiology. Explain carbohydrate fermentation. Describe how Durham tubes are used to indicate carbohydrate fermentation. Perform carbohydrate fermentation testing on S. cerevisiae and S. epidermidis. Relate experimental results to metabolic pathways of microbes. Time Allocation: 3 hours + 24 hours incubation www.holscience.com 2 Hands-On Labs, Inc.
Materials Student Supplied Materials Quantity Item Description 1 Active culture broth-s. cerevisiae 1 Active culture broth-s. epidermidis 1 Bleach 1 Camera, digital or smartphone 1 Disposable cup 1 Hand soap 1 Isopropyl alcohol (rubbing) 1 Matches or lighter 1 Permanent marker 1 Roll of paper towels 1 Source of tap water HOL Supplied Materials Quantity Item Description 1 Apron 1 Carbohydrate Fermentation Test: 1 - Fructose powder vial-0.2 g 1 - Glucose powder vial-0.2 g 1 - Mannitol powder-0.2 g 6 Durham test tubes 1 Face mask with ear loops 2 Long thin stem pipets-4.5 ml 2 Pairs of gloves 6 Phenol red broth vials 9 ml 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 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 prevention 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 me dia 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 1. Figure 1. Biochemical testing equipment. borzywoj www.holscience.com 4 Hands-On Labs, Inc.
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 a carbohydrate fermentation test on cultures of S. cerevisiae and S. epidermis. Fermentation Carbohydrates are a group of organic compounds composed of carbon, hydrogen, and oxygen atoms. When carbohydrates are broken down anaerobically (without available oxygen), fermentation occurs. Fermentation is the conversion of pyruvic acid (the by-product of glycolysis), into an acid with or without the production of gas. Common end-products include lactic acid, formic acid, acetic acid, butyric acid, ethyl alcohol, carbon dioxide, and hydrogen. In this laboratory, you will investigate the fermentation of fructose, glucose and mannitol. Fructose is a simple carbohydrate found in fruits and vegetables. Glucose is a simple carbohydrate produced by plant photosynthesis and from the metabolism of glycogen in animals. Mannitol is crystalline alcohol produced by many plants. See Figure 2. Figure 2. Molecular structures of fructose, glucose, and mannitol. adapted from images by lyricsaima Ethyl alcohol, also known as ethanol, is a commercially important product of carbohydrate fermentation. Ethanol is the principal type of alcohol in fermented and distilled beverages. Ethanol is also used as a solvent, an antiseptic, and as a fuel source for automobiles and household heating. See Figure 3. www.holscience.com 5 Hands-On Labs, Inc.
Carbohydrate Fermentation Tests Figure 3. Ethanol formula. Zebor Carbohydrate fermentation tests determine the by-products of fermentation, and thus may be used to differentiate among groups or species of microbes. In these tests, microbes are cultured in a series of broths, each containing an isolated carbohydrate, such as glucose. The acid endproducts of fermentation lower the ph of the broths. Phenol red is commonly used as a ph indicator in carbohydrate fermentation tests. Phenol red turns yellow in acidic solutions. Durham tubes are used to detect the carbon dioxide and hydrogen gases that may be produced by fermentation. These small glass tubes trap gas which is visible as an air bubble. See Figure 4. Figure 4. Gas trapped by a Durham tube. www.holscience.com 6 Hands-On Labs, Inc.
Carbohydrate fermentation results are used to place microbes into the following categories: Fermenter with acid production only Fermenter with acid and gas production Non-fermenter See Figure 5 for an example of each of these categories. Figure 5. Carbohydrate fermentation testing results from left to right: fermenter, fermenter with gas production, non-fermenter. www.holscience.com 7 Hands-On Labs, Inc.
Exercise 1: Carbohydrate Fermentation Testing Part 1 of 2 In this laboratory you will incubate S. cerevisiae and S. epidermidis cultures in fructose, glucose, and mannitol broths to categorize each microbe based on metabolic properties. 1. Clear a work area and gather all materials listed for this experiment. 2. Allow the tubes of phenol red 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 the work surface with a 10% bleach solution. 6. Use the permanent marker to label the 6 phenol red vials: #1 S. cerevisiae Glucose #1 S. epidermidis Glucose #2 S. cerevisiae Fructose #2 S. epidermidis Fructose #3 S. cerevisiae Mannitol #3 S. epidermidis Mannitol 7. See Figure 6 for reference. Figure 6. Labeled phenol red tubes. www.holscience.com 8 Hands-On Labs, Inc.
8. Place the 6 Durham tubes and the 2 dropper pipets in a cup of alcohol. 9. Light the candle. 10. Remove the lid from the S. cerevisiae #1 vial and pass the lip over the flame to sterilize. See Figure 7. Figure 7. Sterilizing vial opening. 11. Carefully transfer half of the glucose powder to the vial. See Figure 8. Figure 8. Adding glucose powder to broth vials. 12. Pass the lip of the vial over the flame a second time to sterilize and return the cap to the vial. 13. Shake the vial until the powder dissolves. www.holscience.com 9 Hands-On Labs, Inc.
14. Repeat steps 10-13 for the 5 remaining phenol red vials. 15. Remove the Durham tubes from the alcohol and allow to dry thoroughly. 16. Insert one Durham tube into each phenol broth vial so that the opening of the Durham tube points toward the bottom of the broth vial. See Figure 9. Figure 9. Durham tube properly positioned in broth vial. Note: If air is initially trapped in the Durham tube, invert the broth vial to allow the air to escape from the Durham tube. Do not proceed until all air is removed from the Durham tube. 17. Remove one pipet from the alcohol and shake to dry. 18. Remove the lid of the active S. cerevisiae culture and flame the lip to sterilize it. 19. Transfer 4 drops of active culture to each of the phenol red tubes labeled S. cerevisiae. 20. Flame the lip of the active S. cerevisiae culture vial and the phelon red tubes before returning the lids. 21. Place the used pipet in undiluted bleach. 22. Repeat steps 17-21 for S. epidermidis. 23. Place the 6 inoculated phenol red broths and 2 active culture broths in your incubation location. 24. Incubate the phenol red broths for 12 hours. www.holscience.com 10 Hands-On Labs, Inc.
25. Wipe down your work area with a 10% bleach solution. 26. Remove the used pipets from the bleach, wrap in paper towels and dispose of them in the garbage. 27. Wash and return all other items to your kit for future use. 28. Wash your hands thoroughly with soap and warm water. Part 2 of 2 29. Observe each of the phenol red broths after 12 hours incubation for color change. If no changes are observed, incubate for an additional 6 hours. Note: Do not incubate the phenol red broths for longer than 24 hours as extended incubation times will result in inaccurate results. 30. Wipe down your work area with a 10% bleach solution. 31. Wash your hands thoroughly with soap and warm water. 32. Put on your goggles, a new pair of gloves, face mask, and apron. 33. Gather the 6 incubated phenol red broths. 34. Observe each broth noting the color of the solution and presence of gas bubbles in the Durham tubes. 35. Record your observations in Data Table 1 of your Laboratory Report Assistant. 36. Categorize each result as: Fermenter with acid production only Fermenter with acid and gas production Non-fermenter (See Figure 10 for reference) Figure 10. Carbohydrate fermentation testing results from left to right: fermenter, fermenter with gas production, non-fermenter. www.holscience.com 11 Hands-On Labs, Inc.
37. Record your results in Data Table 1. 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. Soak the phenol red vials in bleach for 1 hour before placing in the garbage. 40. Wipe down your work area with bleach. 41. Wash your hands thoroughly with soap and warm water. 42. 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. Why are biochemical tests used to identify microbes? B. What is fermentation? What are some common products of carbohydrate fermentation? C. How are Durham tubes used to measure gas production in carbohydrate fermentation tests. D. Do S. cerevisiae and S. epidermidis ferment based on your results? Mention specific endproducts depending on the types of carbohydrates (glucose, fructose, and mannitol). www.holscience.com 12 Hands-On Labs, Inc.