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Unit 14 Unit 14: Chemical Agents That Inhibit Bacterial Growth By Karen Bentz, Patricia G. Wilber and Heather Fitzgerald. Copyright Central New Mexico Community College, 2015 Introduction There are many chemicals that will kill or inhibit the growth of microorganisms. An antibiotic generally refers to a chemical that can be used on or inside the human body to reversibly inhibit the growth of bacteria (bacteriostatic) or irreversibly kill bacteria (bactericidal). Antibiotics that only inhibit bacterial growth may still be very effective if a person has a strong enough immune system to eliminate the bacteria that have stopped growing. Antibiotics are most often produced by soil fungi (Penicillium) and soil bacteria (Streptomycetes). Bacterial species that normally reside in the soil will have developed natural resistance to the different antibiotics produced by soil microorganisms. Although bacterial species that normally reside on or inside the human body (normal flora) may not have been resistant to the antibiotics produced by soil microorganisms, the continued use of prescription antibiotics can promote the development of resistance among our normal flora. An even bigger concern is the sharing of genes that encode for antibiotic resistance between bacteria residing in soil, bacteria that are part of our normal flora, and bacteria that are disease causing pathogens. Antibiotics generally work best against specific types of bacteria. For instance, penicillin is most effective against Gram(+) bacteria, as it does not easily penetrate the Gram(-) outer lipid membrane. When treating an infected patient, it is important to first determine which antibiotics the bacterial pathogen is susceptible to. However, it is also important to understand that the susceptibility of a bacteria to an antibiotic is only one factor that is used in selecting the most appropriate antibiotic. There are many other factors that are not measured by the clinical laboratory tests which are just as important. The other factors that impact choice of antibiotic include: (1) the site of infection and which drugs can penetrate to that site most effectively; (2) the risk-benefit related to toxicity of the drugs being considered; (3) known patient allergies or previous adverse events associated with specific antibiotics; (4) current medications being taken by the patient and potential adverse interactions; (5) and age of the patient and counter-indications for pediatric or geriatric populations. It is also important to understand that the susceptibility of a bacterial pathogen to an antibiotic is only one factor that determines success in treating an infection. Other factors that are just as important include: (1) the strength of patient’s immune system; (2) variability among patients related to penetration of antibiotics to site of infection; and (3) the need for surgical intervention to cure an infection. All the antibiotics in the world will not cure an infection associated with an abscess, unless that abscess is first opened and drained. Similarly, all the antibiotics in the world will not effectively treat an infected artificial joint or heart valve, unless the infected joint/valve is first removed by surgery. Although there are many factors working together to Unit 14 Page 1 Unit 14 cure a bacterial infection, the goal of the clinical microbiology lab is to help the health-care providers select the most appropriate antibiotic for the patient. The Kirby-Bauer method is widely used for testing the effects of various antibiotics on bacterial growth. A bacterial lawn is first created on a large petri plate containing a special agar medium. Paper discs containing specified amounts of an antibiotic are then put on top of the bacteria. After incubation if the antibiotic has inhibited the growth of the bacteria there will be a clear zone around the paper disc. The effect of the antibiotic on bacterial growth can be determined by measuring the diameter of the circle of inhibition with a metric ruler. This diameter is called the zone of inhibition. The measurement of the zone is then compared to standards to determine if the bacteria is sensitive to the antibiotic, or resistant to it. Although the KirbyBauer allows healthcare professionals to determine which antibiotics a pathogen is sensitive to or resistant to, the test is not quantitative. The results of the Kirby-Bauer only provide a list of drugs which the healthcare professionals can take to the next step to select the most appropriate for that particular patient (see above for other factors involved in decision). Keep in mind that all living organisms, including bacteria and humans, show genetic variation. Genetic variation is the reason life is able to survive in a constantly changing environment. The genetic variation in bacteria means that some bacteria will have resistance to a particular antibiotic. Treatment with that particular antibiotic will select for those bacteria that are resistant, and they will proliferate. Likewise, in the human population there will be individuals with genetic variations that make them more susceptible to bacterial infection. Because of human and bacterial genetic variation, antibiotics are never 100% effective in treating bacterial infections in humans. Many cleaning agents have words such as “sanitize”, “germicide”, and “antiseptic” in their product labeling, but what do these terms really mean as far as microbial growth is concerned? If a term ends in “cide”, such as viracide, then the agent should kill the specified organism. The term antiseptic means that the agent prevents bacterial growth, but does not necessarily kill the organism. The term sanitation refers to any cleaning technique that removes microorganisms from an object Chemicals used to inhibit microbial growth outside of the human body are termed disinfectants or antiseptics. Disinfectants do more to actually kill microorganisms, while antiseptics merely prevent their growth. Disinfectants are used to clean inanimate surfaces such as floors and hospital beds. Antiseptics such as mouthwash are safe to use on human skin, but do little to actually kill microorganisms. When purchasing household cleaners, you might want to read the label to see what the active ingredient is, to see if it will kill microorganisms or just keep them from growing. Unit 14 Page 2 Unit 14 DAY 1: Inoculation I. Kirby-Bauer Method for Testing Bacterial Sensitivity to Antibiotics Materials (per group) Lawn of assigned bacteria 1 Mueller-Hinton agar plate Antibiotic disc 1 swab various antibiotic discs 1 tweezers Bacteria o Escherichia coli (Ec) o Pseudomonas aeruginosa (Pa) o Staphylococcus aureus (Sa) Procedure for Kirby-Bauer Inoculation 1. For the bacterial lawn, choose one bacteria or use the bacteria designated by your instructor. 2. Label the bottom of your Mueller-Hinton plate with the name of the bacteria that you will be using 3. Wet the swab in water and use it to pick up bacteria. Create a solid lawn of bacteria on your plate. Do not leave any gaps, or you may not see the zones of inhibition correctly. 4. Sterilize a pair of tweezers and use it to place one of each type of antibiotic disc onto the plate. Make sure the discs are spaced as far apart as possible on the plate. Tap each disc with the tweezers once you have placed it on the plate to make sure the disc sticks to the plate. 5. Place your plates upside down for incubation. Inverting your plate prevents water from dripping onto the plate and ruining the zones of inhibition. Unit 14 Page 3 Unit 14 II. Disinfectant and Antiseptic Effects on Bacterial Growth Materials (per person) 1 T-soy plate 1 swab 1 tube of sterile water 2 paper discs 1 tweezers 2 cleaning solutions of your choice Bacteria o Escherichia coli (Ec) o Pseudomonas aeruginosa (Pa) o Staphylococcus aureus (Sa) Lawn of your choice of bacteria Discs soaked in different chemicals Procedure for Antiseptic/Disinfectant Inoculation 1. Choose one of the bacteria to use and create a lawn of bacteria on the petri plate. 2. On the bottom of the plate, draw a line down the middle of the plate and write the chemicals you are going to test on either side of the line. 3. Create your own chemical discs by holding a paper disc with the tweezers and soaking the disc in the chemical of your choice. 4. Still holding your paper disc with a tweezers, blot off the excess chemical with a Kimwipe and place the disc on your bacterial lawn. 5. Repeat the process of preparing a chemical disc using your second chemical, and put this disc on the other side of your plate. 6. Invert plates and put into rack for incubation. Unit 14 Page 4 Unit 14 DAY 2: Results and Interpretation I. Kirby-Bauer 1. Examine your plate for zones of inhibition, which will be clear areas of no growth around the disc. 2. Measure the diameter of the zone in millimeters (mm). 3. Compare your zone of inhibition measurement to a standard (Table 14.1) 4. Record your results in Table 14.2 by putting a check by each antibiotic that the bacteria is sensitive to. 5. Collect results for the other two bacteria from student groups that tested a different bacteria then you did. Table 14-1: Antibiotic Zone Diameter Standards (mm) Drug Resistant Intermediate Sensitive Ampicillin (AM) ≤ 11 mm 12-13 mm ≥ 14 mm Cephalothin (CF) ≤ 14 15 - 17 ≥ 18 Erythromycin (E) ≤ 13 14 - 22 ≥ 23 Gentamycin (GM) ≤ 12 13 - 14 ≥ 15 Penicillin (P) ≤ 28 Streptomycin (S) ≤ 11 12 - 14 ≥ 15 Tetracycline (TE) ≤ 14 15 - 18 ≥ 19 Bactrim (SXT) ≤ 10 11 - 15 ≥ 16 ≥ 29 Unit 14 Page 5 Unit 14 Table 14-2: Kirby-Bauer Results Showing Antibiotic Sensitivity (Put a check in the boxes by the antibiotic that the bacteria is SENSITIVE to) Antibiotic Pseudomonas Ampicillin (AM) Cephalothin (CF) Erythromycin (E) Gentamycin (GM) Penicillin (P) Streptomycin (S) Tetracycline (TE) Bactrim (SXT) Insert Photos of Results Here: Unit 14 Page 6 E. coli Staph. aureus Unit 14 II. Antiseptics/Disinfectants 1. You will not measure a zone of inhibition for the chemical you tested. Instead record (Table 3) a chemical as effective if there is some inhibition of growth around the disc. 2. Look at other student’s results to determine which cleaners inhibit growth of the bacteria that you did not test. Table 14- 3: Effectiveness of Various Chemicals Bacteria Chemicals that Inhibited Growth Chemicals that Did Not Inhibit Growth Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Post-Activity Questions 1. Which bacteria is resistant to the most antibiotics? Why might this be so? 2. Which antibiotic provided the largest zone of inhibition on the plate you inoculated? Is this antibiotic the most potent among those that you tested? Defend your answer Unit 14 Page 7 Unit 14 3. You are caring for a patient that is severely immunocompromised and has a serious infection. The clinical microbiology lab has performed the Kirby-Bauer test and followed that with a test to measure potency of those drugs the bacteria is sensitive to. The clinical microbiology lab reports that the most potent drug is a bacteriostatic drug. Is this the drug that should be selected to treat the infection? Defend your answer. 4. Which, if any, of the cleaning products that you tested in lab inhibited growth of bacteria? Look at the label on the cleaning product and record its active ingredient. 5. Why would you not want to use a mouthwash that said “disinfectant” on the label? 6. Do you think long term exposure to household cleaning agents could be detrimental to human health? Of those cleaners you tested, which do you think has/have the potential to adversely affect human health? Why do you think this is so? The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Mike Jillson and Alex Silage for IT support, and our dean John Cornish . Unit 14 Page 8