EXPERIMENT. Environmental Influences on Microbial Growth Salt Tolerance Testing

Transcription

1 EXPERIMENT Environmental Influences on Microbial Growth Salt Tolerance 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 describe the environmental conditions affecting microbial growth. You will discuss osmolarity and define the terms halotolerant and halophile. You will perform salt tolerance testing on Saccharomyces cerevisiae and Staphylococcus epidermidis and relate your results to conditions where these microbes occur. 1 Hands-On Labs, Inc.

2 Objectives Upon completion of this laboratory, you will be able to: Identify environmental conditions affecting microbial growth. Discuss how osmolarity influences microbial growth. Define isotonic, halotolerant, and halophile. Perform salt tolerance testing on E. coli and S. epidermidis. Relate experimental results to environments where microbes occur. Time Allocation: 3 hours + 48 hours incubation 2 Hands-On Labs, Inc.

3 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 HOL Supplied Materials Quantity Item Description 1 Apron 1 Face mask with ear loops 2 Long thin stem pipettes 2 Nutrient broth with 1% NaCl-10 ml tubes 2 Nutrient broth with 7% NaCl-10 ml tubes 2 Nutrient broth with 15% NaCl-10 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. 3 Hands-On Labs, Inc.

4 Background Microbial Environments There are 10 million species of microbes on Earth living in diverse habitats. Microbes exist in fresh and salt water, above and below ground, and on and within other organisms. Microbes even thrive in extreme environments such as glaciers, hydrothermal vents, alkali pools, and even inside nuclear reactors. This diversity of habitats demonstrates the adaptability of microbes. See Figure 1. Figure 1. Edge of Grand Prismatic Spring in Yellowstone National Park. The bright colors are produced by a variety of bacteria living in the pool. Krzysztof Wiktor Numerous factors such as temperature, oxygen availability and ph influence where microbes are found in the environment. Temperature limits the distribution of many organisms. Enzymes, catalysts for metabolic reactions, typically function within a narrow temperature range. Oxygen availability also affects microbial distribution. Although oxygen is required for metabolism in many microbes, it is toxic to other species. The ph of the environment also influences microbial growth. While most microbes thrive in neutral conditions, some species can only metabolize and grow in either very acidic or very alkaline environments. In this laboratory you will investigate the importance of the fourth environmental variable determining the distribution of microbes: osmolarity. 4 Hands-On Labs, Inc.

5 The microbe Pyrolobus fumarii is capable of growing at temperatures as high as 113 C. At the other extreme, the bacteria Pseudomonas, Vibrio, and Flavobacterium are found in arctic regions with temperatures as low as -20 C. Osmolarity Osmolarity refers to the concentration of solutes within a solution. Typically, microbes contain a high number of solutes within their cytoplasm which makes them susceptible to osmotic changes. For example, if a microorganism is placed into a solution with high osmolarity (hypertonic), such as a very salty solution, water from within the cytoplasm will move out of the cell. With the loss of water the stability of the cell membrane and other structures is reduced and the cell will shrink (crenate) eventually leading to cell death. Conversely, if a microorganism is placed in a solution with low osmolarity (hypotonic), such as distilled water, water will move into the bacterium. See Figure 2. If the cell wall is unable to mediate the influx of water, the cell will burst. Figure 2. Tonicity of 5 Hands-On Labs, Inc.

6 Most microorganisms prefer to grow under isotonic (equal) or slightly hypotonic environmental conditions but there are some that either tolerate or require hypertonic solutions. Halotolerant microbes are able to survive at high salt concentrations but do not require these conditions for growth. Other microorganisms are considered halophiles because they require a high salt concentration for growth. Bacteria such as these can be found in lakes like the Great Salt Lake in the United States where NaCl levels can range from 5% to 27%. See Figure 3. Figure 3. Great Salt Lake. The pink colored water is caused by halophilic Broder Van Dyke 6 Hands-On Labs, Inc.

7 Exercise 1: Salt Tolerance Testing In this laboratory, you will incubate S. cerevisiae and S. epidermidis culture broths containing differing salt concentrations to determine an approximate osmolarity range for each microbe. Part 1. Inoculating Saline Broths 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 it with a 10% bleach solution. 5. Using the permanent marker, label each one of the 1% NaCl, 7% NaCl, and 15% NaCl broth tubes as S. cerevisiae and the other as S. epidermidis. See Figure 4. Figure 4. Labeled NaCl broth tubes. 6. Place the stems of the pipets into a cup of alcohol to sterilize. 7. Light the candle. 8. Remove one pipet and shake it briskly until dry. 9. Remove the lid from the S. cerevisiae 1% NaCl tube and pass the opening over the candle flame to sterilize it. See Figure Hands-On Labs, Inc.

8 Figure 5. Sterilizing culture tube opening. 10. Remove the lid from the active culture vial of S. cerevisiae and flame the opening to sterilize it. 11. Pipet 2 drops of active S. cerevisiae culture broth into the 1% NaCl tube. 12. Pass the opening of the newly inoculated 1% NaCl tube over the flame before affixing the cap. 13. Repeat steps 9-13 for the 7% and 15% NaCl tubes. 14. Place the used pipet into a container of undiluted bleach for 20 minutes. 15. Perform steps 8-14 for the S. epidermidis NaCl tubes and active S. epidermidis culture. 16. Take a photograph of your inoculated NaCl broths. Resize and insert the image into Data Table 1 of your Laboratory Report Assistant. Refer to the appendix entitled Resizing an Image for guidance with resizing an image. 17. Place the NaCl broths in your incubation location for 48 hours. 18. Return the active S. cerevisiae and S. epidermidis culture vials to your incubation location for use in future experiments. 19. Dispose of the used and bleached pipets in the garbage. 20. Wipe down your work area with a 10% bleach solution. 21. Wash and return the other items to your kit for future use. 22. Wash your hands thoroughly with soap and warm water. Part 2. Observations of Cultures 23. Observe the NaCl broth tubes after 48 hours for growth, which will appear as turbidity. If no growth is observed, incubate for an additional 24 hours. 24. Wipe down your work area with a 10% bleach solution. 25. Wash your hands thoroughly with soap and warm water. 8 Hands-On Labs, Inc.

9 26. Put on your goggles, a new pair of gloves, face mask, and apron. 27. Gather the six NaCl broth tubes. 28. Observe each tube noting the turbidity of the broth. A clear broth is a negative test result. Note: Compare the level of turbidity in the incubated tubes to that in the photos you took prior to incubation. 29. Record your observations in Data Table 2 of your Laboratory Report Assistant. 30. Take a photograph of your incubated media tubes. Resize and insert the image into Data Table Soak the NaCl broth tubes in undiluted bleach for one hour before placing in the garbage. 32. Wipe down your work area with a 10% bleach solution. 33. Wash your hands thoroughly with soap and warm water. 34. 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. Identify three environmental influences on microbial growth. How does each affect microbial distribution? B. What is a halotolerant microbe? How do halotolerant microbes differ from halophiles? C. Based on your experimental results, categorize each microbe as preferring isotonic environments, being halotolerant, or as a halophile. D. S. cerevisiae occurs in nature on ripe fruit. S. epidermidis naturally occurs on human skin. How do your experimental results relate to the natural environment of each microbe? 9 Hands-On Labs, Inc.