EXPERIMENT Antibiotic Sensitivity-Kirby Bauer Diffusion Test Author Name Version 42-0238-00-02 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 antibiotics and antimicrobial agents. You will outline how penicillin, novobiocin, and gentamicin work. You will summarize the process of a Kirby-Bauer Diffusion test and then compare Staphylococcus epidermidis sensitivity to three antibiotics. www.holscience.com 1 Hands-On Labs, Inc.
Objectives Upon completion of this laboratory, you will be able to: Describe antibiotics and antimicrobial agents. Outline how penicillin, novobiocin, and gentamicin function as antibiotics. Discuss bactericidal and bacteriostatic agents. Perform a Kirby Bauer Diffusion test with Staphylococcus epidermidis. Measure zones of inhibition. Relate antibiotic resistance to test results for penicillin, novobiocin, and gentamicin. Time Allocation: 3 hours + 48 hours incubation www.holscience.com 2 Hands-On Labs, Inc.
Materials Student Supplied Materials Quantity Item Description 1 Active culture broth-s. epidermidis 1 Bleach 1 Coffee cup or mug 1 Disposable cup 1 Hand soap 1 Isopropyl alcohol (rubbing) 1 Large cooking pot (at least 8 deep) 1 Matches or lighter 1 Oven mitt 1 Permanent Marker 1 Roll of paper towels 1 Source of tap water 1 Stove HOL Supplied Materials Quantity Item Description 1 Antibiotic Disk-Gentamicin in Bag 2 x 3 1 Antibiotic Disk-Novobiacin in Bag 2 x 3 1 Antibiotic Disk-Penicillin in Bag 2 x 3 1 Apron 1 Face mask with ear loops 1 Metric ruler 1 Nutrient agar-18 ml tube 1 Pair of gloves 1 Petri dish, 60 mm 1 Plastic tweezers 1 Safety goggles 1 Sterile swabs (2 per pack) 1 Tea candle 1 Test tube clamp 1 Test tube rack, 6 x 21 mm www.holscience.com 3 Hands-On Labs, Inc.
Note: To fully and accurately complete all lab exercises, you will need access to: 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 4 Hands-On Labs, Inc.
Background Antibiotics Antibiotics are natural substances produced by microbes that destroy or slow the growth of bacteria. Antimicrobial agents are synthetic substances developed in the laboratory that mimic the effects of antibiotics. Generally, however, the term antibiotic is used as the catch-all name and will be used as such throughout this experiment. Antibiotics were discovered in 1928 when Sir Alexander Fleming, a Scottish pharmacologist, noticed a mold growing on agar plates. The mold had killed nearby bacterial colonies. Fleming pursued this discovery and later identified the mold as Penicillium notatum, a fungus that produces the antibiotic penicillin. See Figure 1. Figure 1. Penicillin mold growing on agar plate. catolla Many different organisms produce antibiotics; whether they are used in clinical settings depends on the toxicity and the effectiveness of each agent. The majority of antibiotics used clinically are derived from four main genera. These include two bacteria: Bacillus and Streptomyces, and two fungi: Penicillium and Cephalosporium. Science has continued to expand the search for effective agents. Often, once an antibiotic has been produced from an organism, it can be further manipulated in a laboratory to increase and change its properties regarding toxicity, targets, tissue diffusion, and whether an organism will retain resistance to it. Many types of bacteria have gained resistance to antimicrobials at an astonishing rate. Many times, as soon a new antibiotic is developed, a resistant strain of bacteria soon follows. This is especially true in areas where antibiotic use is either abused or overused. www.holscience.com 5 Hands-On Labs, Inc.
It is estimated that antibiotic resistant bacteria costs the United State approximately 35 billion dollars per year due to increased hospital stays, lost productivity, and premature death. Reducing antibiotic resistant bacteria by just 20% is estimated to lower health care costs by approximately 4 billion dollars per year. In this experiment you will be testing the resistance of Staphylococcus epidermidis to three antibiotics: penicillin, novobiocin, and gentamicin. Penicillin Penicillin is a bactericidal, narrow spectrum drug that targets primarily Gram-positive bacteria. Penicillin disrupts the formation of bacterial cell walls by inhibiting the formation of peptidoglycan cross-links, resulting in cell lysis and ultimately cell death. See Figure 2. Figure 2. Gram-negative and Gram-positive bacterial cell walls. Designua www.holscience.com 6 Hands-On Labs, Inc.
Narrow spectrum antibiotics are active against a select group of bacterial types and are used to treat specific infections when the causative organism is known. Penicillin is considered bactericidal since it leads to cell death. Penicillin is produced by the mold, Penicillium. Novobiocin Novobiocin is a bacteriostatic, narrow-spectrum antibiotic that targets DNA gyrase in some Grampositive bacteria. DNA gyrase is an enzyme necessary for the replication of DNA. Without DNA replication, bacterial cells cease to grow and divide. See Figure 3. Figure 3. DNA replication. Designua Bacteriostatic antibiotics cause an inhibition of growth rather than cell lysis and death. Novobiocin is produced by the bacteria, Streptomyces. Gentamicin Gentamicin is a narrow spectrum, bacteriostatic antibiotic that targets aerobic, Gram-negative bacteria. Gentamicin binds with bacterial ribosomes, cellular structures responsible for protein synthesis. See Figure 4. www.holscience.com 7 Hands-On Labs, Inc.
Figure 4. Protein Synthesis. Alila Medical Media When ribosomes are bound with gentamicin they are no longer able to synthesize protein and the bacterial cell ceases to grow and divide. Gentamicin is synthesized by the Gram-positive bacteria, Micromonospora. Kirby-Bauer Diffusion Test The most common method used in hospital laboratories to assess bacterial antibiotic resistance is the Kirby-Bauer Diffusion test. The Kirby-Bauer Diffusion test is performed using commercially prepared filter paper disks that have been infused with a specified concentration of a particular antibiotic. When placed on an agar containing newly plated bacteria, the antibiotic diffuses away from the disk into the agar, creating a concentration gradient that decreases logarithmically with the distance from the disk. As the bacteria grow, they will be inhibited in regions where the antibiotic concentration is above what the microorganism can handle. Areas where the antibiotic concentration is low will allow for bacterial growth; this growth will lead to the creation of a lawn across the plate, creating a zone around each disk. See Figure 5. Figure 5. Kirby-Bauer Diffusion test plate with zones of inhibition. ggw1962 www.holscience.com 8 Hands-On Labs, Inc.
The diameter of this zone of inhibition is measured in millimeters and compared to a chart that links the size of the zone of inhibition to the dosage that is often administered to a patient. The diameter of the zone is dependent on the sensitivity of a microorganism to a particular antibiotic and the physical properties of the antibiotic itself. Some antibiotics are not as soluble as others and as such will have a different diffusion rate through the agar. Therefore, a very large zone of inhibition does not necessarily mean that the antibiotic is extremely effective. The effective size for a zone of inhibition is calculated through hundreds of assays on many different strains of bacterial isolates correlating this information with many other susceptibility tests. In this method, an organism may be categorized as resistant, susceptible, or intermediate to a panel of antimicrobial agents. An intermediate result is based on when the results can be influenced by other factors. For example, the zone of inhibition may indicate the possibility that an agent would be effective, but the site of infection and the ability of the antibiotic to reach this site is suspect, and may not fully reach inhibitory concentrations. Under this circumstance, it would be recommended that a different antibiotic be selected. www.holscience.com 9 Hands-On Labs, Inc.
Exercise 1: Kirby-Bauer Diffusion Testing In this exercise, you will perform a Kirby Bauer Diffusion test with Staphylococcus epidermidis and three antibiotics: penicillin, novobiocin, and gentamicin. Part 1. Preparation for the Kirby-Bauer Diffusion Test 1. Approximately 1 hour before you begin this exercise, pour 1 agar plate. Refer to the appendix entitled Pouring Agar Plates for guidance with this procedure. Note: Plates may be poured in advance, stored in an airtight bag, and refrigerated for future use. 2. Clear a work area and gather all materials listed for this experiment. 3. Wash 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 divide the agar plate into three equal sections. Label the sections with penicillin, novobiocin, and gentamicin. See Figure 6. Figure 6. Labeled agar plate. 7. Light the candle. 8. Gather 1 sterile swab from the packet. Do not touch the tip to any surface. 9. Remove the lid from the active S. epidermidis broth and flame the lip of the vial. See figure 7. www.holscience.com 10 Hands-On Labs, Inc.
Figure 7. Sterilizing culture lip. 10. Carefully insert the swab into the culture vial and submerge the tip into the broth. 11. Quickly transfer the broth on the swab to the agar plate. 12. Swab the entire surface of the agar including the edges. 13. After swabbing the dish, turn it 90 degrees and repeat the swabbing process. It is not necessary to re-moisten the swab. 14. Place the lid on the plate. Let the plate sit lid-side up until the broth is absorbed by the agar. 15. Flame the lip of the culture vial and replace the lid. 16. Extinguish the candle. 17. Soak the swab in a bleach solution and dispose of it in the garbage. 18. Gather the tweezers and place them in a cup of alcohol for 20 seconds. Remove and shake dry. 19. Using the sterile tweezers, transfer each antibiotic disk to the appropriately labeled section of agar. 20. Gently press each disk onto the agar surface with the tweezers to ensure proper contact. 21. Return the lid to the plate. Let the plate sit lid-side up for 10 minutes to allow the disks to attach to the agar surface. See figure 8. www.holscience.com 11 Hands-On Labs, Inc.
Figure 8. Antibiotic disks applied to plate. 22. Place the plate agar-side up in your incubation location and incubate for 48 hours. 23. Return the culture vial to your incubation location. 24. Wipe down your work area with a 10% bleach solution. 25. Wash and return items to your kit for future use. 26. Wash your hands thoroughly. 27. Based on your knowledge of S. epidermidis and the antibiotics used for this experiment, state a hypothesis regarding the resistance of S. epidermidis to each of the drugs. For example: S. epidermidis will exhibit resistance to penicillin and will be inhibited by novobiocin and gentamicin. Part Two. Collecting Data from the Kirby-Bauer Diffusion Test Check the Kirby-Bauer Diffusion plate for bacterial growth which should appear as a white film covering the agar. If no growth is observed, continue to incubate for another 24 hours. 28. Wipe down your work area with bleach. 29. Wash your hands thoroughly. 30. Put on your goggles, gloves, face mask, and apron. 31. Gather the developed Kirby-Bauer Diffusion plate and the metric ruler. 32. Without removing the lid to the plate, measure the zone of inhibition around each disk by placing the ruler over the center of each disk and measure the diameter of the zone with no bacterial growth. www.holscience.com 12 Hands-On Labs, Inc.
33. Place the agar plate in bleach for 20 minutes before disposing of in the garbage. 34. Clean and return the ruler to your kit. 35. Wipe down your work area with a 10% bleach solution. 36. Wash your hands thoroughly. www.holscience.com 13 Hands-On Labs, Inc.