Weekly ilab ilab: Bacterial Growth and Controlling Bacterial Growth Scenario/Summary: This week's experiment focuses on bacterial growth and methods to control bacterial growth. This week's experiment will be conducted in two parts: Experiment I: Growth Media and Patterns of Growth and Experiment II: Controlling Microbial Growth. Experiment I: Growth Media and Patterns of Growth. In part I of this week's experiment, you will explore the characteristics of several types of growth media. For example, what are the carbon source and the nitrogen source in nitrate broth? Will a strict aerobe grow in one broth but not another? Experiment II: Controlling Microbial Growth In part II of this week's experiment, you will explore the resources within the VirtualUnknown software and investigate how the components of different media can control bacterial growth. Deliverables: Each week, you will prepare a lab report detailing your experiments for the week. Include all data, observations, and conclusions in the single report. You should be sure to address the questions in the lab steps in the appropriate sections of the lab report. Your lab report will have the following format: Title Introduction: The first section of your lab report will be the introduction. In this paragraph or paragraphs, you are to provide information to the readers so that they can understand the purpose of the experiment. Purpose - a concise statement about the lab's objective. Background - a brief summary of the topic you are investigating. Include any information that would be necessary to understand the stated purpose. Findings - state the major results of the lab exercise. Procedure: This is the second section in your lab report. This section includes information that the reader would need in order to repeat your ilab: Bacterial Growth and Controlling Bacterial Growth Page 1
experimental procedure. Do not include any observations or results in this section. Some questions that you should ask yourself to complete this section include: What chemicals did you use? What equipment? How much of each chemical did you use? How long did you perform a step within the procedure? Observations and Results: This is the third section of your report. In this section, you will communicate what you observed during the experiment. The Results section is typically dominated by calculations, tables, and figures. In your tables, label the axes of any graphs. Discussion: This is the fourth section of your report. In this section, you will explain, analyze, and interpret your observations and results. Additionally, you will draw conclusions based on your existing knowledge. This is also where you demonstrate your understanding of the experiment by construing the significance or meaning of the results. Each week, the lab instructions will detail specific questions that should be addressed in the lab report. Use this section as your guide, NOT the questions listed in the VU Basic Training Manual. Conclusion: This is the last section of your report and is separate from the Discussion section. Within a few sentences, provide a concluding statement about the results of your laboratory. In a scientific publication, this section summarizes the significant aspects and results, and identifies implications for future study. Grading Category Points Introduction Completeness of background 5 Procedure 5 Observations & Results Were the results accurate? 5 Discussion 5 Conclusion 5 Writing Quality Correct grammar, spelling, originality, 5 completeness, etc. Total 30 ilab Steps Step 1: Access the VirtualUnknown Lab Software Access the VirtualUnknown Software, VUMIE 2012, through the ilab link at the top page of this course. See Course Specific Requirement in Syllabus under Course Home for more information about this program. ilab: Bacterial Growth and Controlling Bacterial Growth Page 2
Step 2: Conduct Experiments Part 1 - Bacterial Growth You will conduct two different exercises for this week's experiment. Note: all tests will be performed on the SAME unknown organism. You will only have one case study for this experiment. Conduct Bacterial Growth Test: 1. Click the New Unknown button. A window will open asking you to enter a label and select a subgroup. 2. Type GrowthLab1 in the Enter a Label line. 3. From the Subgroup dropdown menu, select Growth. 4. Click Auto-inoculation allowed and then click OK. 5. Record details of your case study scenario. 6. The Gram Stain window will open. Record results of Gram stain. 7. Include the results in your Lab Report. Conduct "OF Glucose Test": 1. Open the Biochemical Tests Reference Book. (You can find this resource in two way; you can click on the T? button or select the Reference Books from the Help dropdown menu.) 2. Open the OF Glucose Test. Review the steps of the test and determination of test results before you begin your experiment. 3. From the Media dropdown menu, select OF Glucose Broth. The Media dropdown menu is to the far right of the New Unknown button. 4. Enter OFGlucose1 in the Medium Label window to label your sample. Click OK. Two tubes will appear. You will use the sample on the left to inoculate the sample on the right. 5. Turn on Bunsen burner by right clicking on the Bunsen burner and hitting on. You will see the flame of the Bunsen burner. 6. Select Wire from the Tool dropdown menu. Flame the wire in the flame of the Bunsen burner until it glows red. 7. Bring wire over to Inoculum sample. Right click on sample and select Inoculate. ilab: Bacterial Growth and Controlling Bacterial Growth Page 3
8. Check the Traffic Signals in the upper right hand corner of the lab. Check to see that you have successfully inoculated your sample and that no contamination was introduced. 9. Turn off Bunsen burner. 10. Select Pointer tool from Tool menu. 11. Put cursor, with your Pointer tool, over your inoculated sample, drag it, and place it in the 37 degree incubator. You will also see that your inoculum sample will disappear from the workbench. 12. Click the New Day button. Notice that the Virtual Days counter now reads 2. 13. Put cursor over the incubator. Right click and select your sample. Your sample tube will appear on your workspace. 14. Right click on tube and select Record Results. Review the OF Glucose Test Biochemical Test information to determine if the results of the test are positive or negative. 15. When you record the results of the test, your samples will automatically be moved to the biohazard disposal bag. 16. Note the results of this test. In addition, the results from the test are recorded in the Lab Report. Conduct OF Glucose Test with Oil: 1. For this test, you will follow the same steps as the previous test. The only difference is that you will select the OF Glucose with Oil as your medium. 2. Label your medium as follows, OFGlucose Oil. 3. Two tubes are now on your workbench. 4. Following the same steps as above, inoculate your medium. Click Next Day. Note that the Virtual Day counter is now 3. Retrieve your sample from the incubator and record your results. Conduct Indole Production Test: 1. Open the Indole Production Test from the Biochemical Tests Reference Book. Review the steps of this test, including that you will add Kovac s reagent after incubating the inoculated culture for one day. Make note of how you will determine the results of this test. 2. Select Trypton Broth as your medium. Label the medium : IndoleTryptone. Click OK. 3. Inoculate your medium. Place in 37 degree incubator. Click Next Day. ilab: Bacterial Growth and Controlling Bacterial Growth Page 4
4. Retrieve the sample from the incubator. To add Kovac s reagent, select the dropper tool. Note that you can now select a Reagent (this field is now active). Select Kovacs Reagent. 5. Right click on your culture. Remove caps. 6. Place dropper above the tube opening and click your mouse. A window will open that says, Reagent Kovacs successfully added. Click OK. Note any changes in the appearance of your sample. 7. Observe and record the results. Conduct Malonate Utilization Test: 1. Open the Malonate Utilization Test from the Biochemical Tests Reference Book. Review the steps of this test. 2. Select Malonate Broth as your medium. 3. Label sample like this: MalonateTest. Click OK. 4. Inoculate your sample and incubate for 1 day. 5. Retrieve sample from incubator. Observe and record the results. View Lab Report Results: 1. Select Lab Report from the View item on the top menu bar. 2. You can copy and paste the contents of this lab report to a Word document and save. 3. Note that the lab report records the results of each of the five tests that you conducted on your sample. The lab report identifies the number of bacteria that were eliminated after each test. You will want to include this information in your lab report. 4. Also note that you do not know the identity of the bacterium that caused the infection. 5. After you have recorded the information on the lab report, close the document. Determine Identity of Bacterium: 1. Open the Identification Matrix from the View Command. You will find that only possible bacteria that remain after conducting this series of tests will be shown on the Identification Matrix. Make note of the possible bacteria that might cause this infection. 2. From the Unknown command, select Identify. From the drop down menu, select a bacterium that is likely the cause of the agent (based upon the ilab: Bacterial Growth and Controlling Bacterial Growth Page 5
information you have from your tests and by viewing the Identification Matrix). 3. Click OK. Then, click Yes, when the Confirm Identification Window opens. A window will open letting you know if you chose the correct bacterium. 4. Now, open up the Lab report. Under the Identification Information of the lab report, you will see the bacterium that you selected and the bacterium that was assigned. You will also want to record this information in your final lab report. Part II - Controlling Microbial Growth 1. Click on New Unknown button. 2. Type GrowthLab2 in the label field. Select. Controlling Growth from the subgroup dropdown menu. 3. Click Auto-inoculation allowed. Click OK. 4. Record details of your case study scenario. 5. Record gram stain results of the organism. 6. No further tests will be performed. You will answer questions about this topic in your final lab report. See below. Step 3: Write the Lab Report Use the reference areas within the VirtualUnknown Software to answer the questions in the Write lab report section regarding controlling microbial growth. Submit one lab report for both experiments this week. Include the following information for each part: Experiment I: Growth Media and Patterns of Growth and Experiment II: Controlling Microbial Growth Experiment I: Growth Media and Patterns of Growth 1. In the Introduction section, include the information about your case study. 2. In the Observations and Results section, include the following: Gram stain results Describe how the results of the OF Glucose Broth test. How many bacteria were eliminated as causes of the patient s infection by using this test? Describe results from the Indole Utilization test. How many bacteria were eliminated as causes of the patient s infection by using this test? Describe results from Malonate utilization test. How many bacteria were eliminated as causes of the patient s infection by using this test? ilab: Bacterial Growth and Controlling Bacterial Growth Page 6
3. Include the answers to the following questions in the Discussion section and the Conclusion section, as appropriate. Based upon your results, is your bacteria sample a strict aerobe, strict anaerobe, facultative anaerobe, or microaerophile? Why? How important is it to add the Kovac s Reagent in the Indole Utilization test? What is responsible for the color change observed in a positive test? Did the bacteria grow in the tryptone or malonate media? Why or why not? Refer to the resources within the VirtualUnknown software to get details on each media and biochemical test. For the media used in this portion of the experiment, are the media complex or synthetic? Are the media selective, differential, both, or neither? Identify the carbon and nitrogen source for each medium. How many possible bacteria were remaining after conducting these biochemical tests? Which bacteria remained after conducting the four biochemical tests. Using the information in the Identification matrix, what additional tests might help you to identify the bacteria in your case study? NOTE: If you need to refer to the lab report, or the identification matrix for this lab exercise, you can to the upper right hand corner of the lab environment and select GrowthLab1 from the Unknown drop down menu. Experiment II: Controlling Microbial Growth 1. In the Introduction section, include the information about your case study. 2. In the Observations and Results section, include the following: Gram stain results 3. Include the answers to the following questions in the Discussion section and the Conclusion section, as appropriate. Salt is one of the oldest food preservatives. How does salt control microbial growth and reduce spoilage? Which two media include elevated concentrations of NaCl? Would bacteria that have the ability to grow in these two media be more likely to cause food spoilage? Why or why not? Bile salts are natural products of the digestive system that prevent growth of many bacteria. How do bile salts exert antimicrobial activity? What is the concentration of bile salts in bile esculin agar? Why might some bacteria be able to grow on this medium? Nitrate is often used as a preservative in cured meats to prevent anaerobic growth of spoilage bacteria. Why would the inclusion of nitrate inhibit anaerobic growth? ilab: Bacterial Growth and Controlling Bacterial Growth Page 7
Potassium cyanide (KCN) is a potent poison. How might some bacteria be able to grow on media containing potassium cyanide? Step 4: Submit the Lab Report Save your lab report as a Word document titled BIOS242_Week3LabReport_FIRSTNAME_LASTNAME.docx. Submit your lab report into the Week 3 ilab Dropbox. See Syllabus "Due Dates for Assignments & Exams" for due dates. ilab: Bacterial Growth and Controlling Bacterial Growth Page 8