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Name: Date: Period: Teacher’s Guide: Effect of Tears on Bacterial Growth Objectives: Students will learn: how to perform a controlled experiment how to make serial dilutions how the skin and bodily fluids, such as tears, contain substances that kill microorganisms the skin is the first line of defense, an example of innate immunity lab skills: micropipetting, streak plate, inoculation, incubation of bacteria purpose of a control Before passing out lab: 1. Go over innate vs. adaptive immunity 3. Go over anatomy of bacteria including description of the peptidoglycan cell wall- a large polymer with many covalent bonds (“chain link fence”). Show image below. 4. Mention that penicillin (extracted from mold, Penicillium) acts by preventing the cross-linking in the cell wall of many types of bacteria, which leads to cell lysis. This interference with cell wall synthesis makes penicillin an antibiotic. 5. Go over Ecology standard 10a. Not all bacteria are harmful; they are part of the ecology of the human body, particularly the large intestine. In many cases, bacteria exist in a mutualistic relationship to humans. In the large intestines, for example, bacteria synthesize Vitamins K, and some B vitamins) Introduction: Bacteria can survive in almost every environment on Earth. Why is it, then, that we don’t get sick all the time? There are two kinds of immunity: innate and adaptive. Innate immunity refers to immediate, non-specific defense which does not depend on antigens. Adaptive immunity, on the other hand, entails a specific antigen and results in immunological memory. In this lab, you will explore the effects of tears, which contain the enzyme, lysozyme, on the growth of bacteria. Science Standards: 6a. Students know biodiversity is the sum total of different kinds of organisms and is affected by alterations of habitats 10a. Organisms have a variety of mechanisms to combat disease. As basis for understanding the human immune response: Students know the role of the skin in providing nonspecific defenses against infection. Vocabulary: Lysozyme- enzyme in human tears, egg white,saliva, and other body fluids Bactericidal-anything that kills bacteria Bacteriostatic- anything that limits the growth of bacteria by interfering with protein synthesis, replication, or any aspect of bacterial metabolism Serial dilution- stepwise reduction in concentration- “watering down”- of a particular substance in a neutral agent Inhibition- the suppression of bacterial growth Innate immunity-inborn, immediate, non-antigen based immunity, which does not confer immunological memory; response is the same every time Adaptive immunity- antigen-specific immunity in which exposure results in immunological memory (clones of B lymphocytes, specific antibodies) State the Problem: How do tears affect bacterial growth? Read through this lab and formulate your hypothesis. Hypothesis: _____________________________________________________ Any answer is acceptable Materials: onions E.coli: HB101 K–1lyophilized on LB/agar inoculated filter paper discs saturated with dilutions of tears micropipettes microcentrifuge tubes DI water Incubator Permanent markers for labeling Procedure 1. Slice several onions in an attempt to stimulate tear formation in your own eyes. 2. Swab corner of ears with filter paper. Place soaked filter paper into a tube with 10uL DI water. 3. Label 4 microcentrifuge tubes with the 4 serial dilutions you will make: 1/.1/.01/.001 4. Make 4 serial dilutions of tears (1, .1, .01, .001) Transfer 5uL of tear solution into 50 uL DI water. Make three dilutions total. 5. Label petri dishes so that there are 5 “pie slices” with tear dilutions labeled. 6. Soak filter paper discs into each prospective solution 7. Add inoculated filter paper disc into appropriate quadrant 8. Incubate plates for 48 hours at 37C. OP TIONAL; students may use saliva, which also contains lysozyme Making serial dilutions: 5uL 5uL 5uL Add 5uL from each tube to the adjacent tube to make 10-fold dilutions of tears 45uL water Tube # 1 10 uL tearsremove 5uL and put into next test tube Tube #2 45uL water Tube #3 45uL water Tube #4 #4 4. Observations: In the diagram below, record the 4 tear dilutions, one per “slice.” Indicate approximate number of bacterial colonies in each quadrant. Show “zones of inhibition” in each quadrant. Control (DI water ) No inhibiton No inhibition Pure tears Maximum zone of inhibition expected Dilution = .1 Smaller zone of inhibiton expected Dilution = .001 Smallest zone of inhibition with tears Dilution = .01 The amount of bacterial inhibition going from least to most is as follows: 1. Control (pure DI water) 2. .001 tear solution 3. .01 tear solution 4. .1 tear solution 5. pure tears Analysis Questions: 1. Why was the control in this lab? Answer: The control is the plate of DI water- not containing the variable you are testing. 2. Why is a control group necessary in this lab? Answer: A contol plate is a point of comparison (guide) to help you interpret the experimental results. Without the control, you would not know that it was the tears that caused increasing levels of bacterial death. 3. If you wanted to make 2-fold dilutions of tears (doubling the dilution each time) with a starting quantity of 10uL tears, how would you make the dilutions? Make a labeled drawing below. Possible answer: dilutions showing 1:2, 1:4, 1:8, 1:16 ratios of tears to DI water. 4. Explain how this lab relates to innate or adaptive immunity. Answer: This lab relates to innate immunity, a generalized, all or none immune response; tear response is the similar with every exposure, no buildup of specific white blood cells and antibodies result as with adaptive immunity. 5. Conclusion In 5 paragraphs, answer the following questions: 1. 2. 3. 4. 5. Write a summary of the lab’s purpose, materials, and procedure Re-state your hypothesis and explain whether it was right or wrong. Summarize the lab’s result What does the data indicate about the role of skin in human immunity? What were some avoidable and unavoidable sources of error? How could you extend this lab in an effort to explore the effects of tears on bacterial growth?