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Name(s):______________________________________ Per_______ Bacteria Lab Bacteria are microorganisms that grow everywhere. Bacteria are prokaryotes (Kingdom Monera), which means that they have no true nucleus. They reproduce by binary fission. Most bacteria are benign (benign = good, friendly, kind) or beneficial, and only a few are “bad guys” or pathogens. Agar is a gelatin-like substance with a semi solid surface on which the bacteria can grow while they consume the added nutrients. You have heard of antibiotics and at some point in your life antibiotics were probably prescribed to cure an illness you had. In Part 2 of this investigation, we will use discs that have been impregnated with various antibiotics. These discs will be placed on petri dishes containing bacteria. The purpose of the lab is to determine which of the antibiotics tested would be most beneficial in treatment by observing the effect of two different antibiotics on the growth of the bacteria. The procedure in this lab is similar to the one used in hospitals to determine which antibiotic should be prescribed to treat a patient’s bacterial infection. Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection. Testing for antibiotic sensitivity is often done by the Kirby-Bauer method. Small wafers (discs) containing antibiotics are placed onto a plate upon which bacteria are growing. If the bacteria are sensitive to the antibiotic, a clear ring, or zone of inhibition, is seen around the disc indicating poor growth. Tetracyclines are a family of antibiotics, all products of Streptomyces. Tetracyclines block protein synthesis on isolated ribosomes. It concentrates in certain types of ribosomes possesed only by bacteria, so it is not toxic to the animal. Because of their extremely low toxicity, they were overused in the medical community, and now there is widespread resistance to tetracyclines. However, they are still useful in treatment of some diseases, such as Lyme disease. Streptomycin is a protein synthesis inhibitor binding to bacterial ribosomes to prevent the initiation of protein synthesis. Aminoglycoside usage has been limited because prolonged use has been found to cause kidney damage and injury to the auditory nerves, leading to deafness. Problem: Comparing a variety of surfaces found in a school building, what surface would have the most bacteria on it? Hypothesis: Part 1 Experiment: Materials – cup with water, 2 swabs, Petri dish with nutrient agar, marker Procedure – 1. Use a marker to divide bottom of Petri dish into 2 quadrants. 2. Put your last name on bottom section of Petri dish (part with agar) 3. When you arrive at the location for sample 1, pull out sterile swab and moisten in small amount of water. Thoroughly cover area with moistened swab and then quickly open Petri dish and inoculate dish by streaking a pattern gently across the agar surface without tearing into it. Streak quadrant 1 (1/2 of dish). Quickly reclose Petri dish and dispose of used swab. 4. Repeat step 3 for the other area. 5. Upon return to class, invert Petri dish (agar now on top) and place on tray. Return marker to front table. Shapes/Forms Observations: Record the appearance and number of colonies in the cart below. Area #1 Colony Description # of Colonies Peroxide Test ( + ) Morphology (shape) Colony Description # of Colonies Peroxide Test ( + ) Colony Description # of Colonies Peroxide Test ( + ) Morphology (shape) Area #2 Part 2 Experiment Hypothesis: 1. Each partner should choose one colony to do an antibiotic susceptibility test. 2. Get a new agar plate. One for the 2 of you. 3. Take a sterile swab and very carefully touch the one colony that you want to test and place in broth tube. 4. Swirl swab to mix. Press against sides of tube and then evenly swab ½ of plate. 5. Dispose of swab. 6. Place 2 antibiotic discs to test. Drop these onto plate spaced well apart. Tap gently. 7. Next partner should repeat steps 3-6 8. After one to three days of incubation, observe results without opening the petri dish. 9. Use a ruler to measure the DIAMETER (in millimeters) of the zone of inhibition around each disc and record your observations in the table provided. Bacteria Colony Decription Tetracycline Zone of Inhibition (mm) Strptomycin Zone of Inhibition (mm) You each will be typing your own formal lab report!! This template is provided for you. Use all headings. Name:____________________________________ Title of Experiment: ________________________ Purpose:________________________________________________________ Background: (List the facts necessary to formulate a hypothesis and design the experiment.) _______________________________________________________________ Formulation of Hypothesis I ______________ Formulation of Hypothesis I_______________ Independent Variables (IV):___________________________________________________________ Dependent Variable (DV): the diameter of______________________________________________________ Controlled Variable(s) (Constants) (CV):______________________________________________________ Materials & Equipment: List materials and equipment below. A “material” is something that is used during the lab and is disposed of when the lab is finished . “Equipment” is anything that can be saved to use over. Materials Equipment __________________________ ________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ Procedure Safety (Describe all safety precautions necessary in the experiment): __________________________________ Procedure (Read the narrative description of the procedure provided on an earlier page. Rewrite this procedure in your own words as numbered steps. It should be detailed enough that the entire procedure could be recreated if someone wanted to repeat your experiment.) _______________________________________________________________ Data: Include your data tables Data Analysis: Predictions (If you were to complete an additional trial of this experiment, what other outcomes would you expect?): _______________________________________________________________ Accuracy/Precision (Discuss if the data collected is accurate and precise. NOTE: Accuracy refers to how closely a measured value agrees with the correct value. Precision refers to how closely individual measurements agree with each other. Provide specific instances in which there could have been errors made. Accurate?______________________________________________________ Precise?________________________________________________________ Sources of possible error? _______________________________________________________________ Conclusion: This section should be written in paragraph form. Restate the problem In this lab, an attempt was made to answer the following question/solve the following problem: Restate the hypotheses Support / Refute Hypothesis Which hypotheses were supported?______________________________________________________ Which hypotheses were refuted (rejected)?_____________________________________________________ If any hypotheses were rejected, develop new hypotheses or explanations based on experimental results: How could you improve this experiment for future students to help reduce potential sources of error? (Refer to your list of observations of potential sources of error).