Introduction - Central New Mexico Community College | CNM



Unit 14: Chemical Agents That Inhibit Bacterial GrowthBy Karen Bentz, Patricia G. Wilber, Heather Fitzgerald, and Andrea Peterson, 2022Creative Commons Attribution-NonCommercial 4.0 International License.IntroductionThere 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 a patient (humans, pets, livestock, etc.) to inhibit the growth of bacteria (bacteriostatic) or kill bacteria (bactericidal). Bacteriostatic antibiotics can very effective if a person has an immune system strong enough 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 have often evolved resistance to the different antibiotics produced by soil microorganisms while normal flora on or inside the human body (and other non-human patients) are generally susceptible, UNLESS (drum roll) they are over exposed to these drugs. The result of overexposure is antibiotic resistance.Because of the problem of antibiotic resistance, antibiotic resistance tests can be performed to ensure antibiotics prescribed for any given infection are truly effective. The Kirby-Bauer method is widely used for this purpose. The effects can be determined by measuring the diameter of a zone of inhibition around a given antibiotic. The measurement of the zone is then compared to standards to determine if the bacteria is sensitive to the antibiotic (killed or inhibited by it), or resistant to it (unaffected). Many cleaning agents have words such as “germicide”, “antiseptic” or “disinfectant”, on their labels. If a term ends in “cide”, the agent should kill the specified organism. “Antiseptic” means that the agent prevents bacterial growth and is safe to use on living tissue, but does not necessarily kill the organism. Mouthwash and betadine are antiseptics. Disinfectants are harsher and generally kill microorganisms. They are not safe to use on living tissues and are used to clean inanimate surfaces such as floors and hospital beds. Bleach and Lysol are disinfectants. When purchasing household cleaners, you might want to read the label to see if active ingredient is a disinfectant or not.DAY 1Kirby-Bauer Method for Testing Bacterial Sensitivity to AntibioticsMaterials (per group)1 Mueller-Hinton agar plate1 swabvarious antibiotic discs1 tweezersBacteriaEscherichia coli (Ec)Pseudomonas aeruginosa (Pa)Staphylococcus aureus (Sa)Figure 14-1. Lawn on a Mueller-Hinton Plate with 8 different antibiotics.Figure created by Karen BentzProcedure for Kirby-Bauer Inoculation Choose one bacteria or use the bacteria designated by your instructor. Label the bottom of your Mueller-Hinton plate with the name of the bacteria that you will be usingWet 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.Sterilize a pair of tweezers and use them 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. Place your plates upside down for incubation.Disinfectant and Antiseptic Effects on Bacterial GrowthMaterials (per person)1 T-soy plate1 swab2 paper discs1 tweezers2 cleaning solutions of your choiceBacteriaEscherichia coli (Ec)Pseudomonas aeruginosa (Pa)Staphylococcus aureus (Sa)Figure 14-2. SOME possibilities. Figure created by Karen Bentz and Patricia G. WilberProcedure for Antiseptic/Disinfectant InoculationThere are various ways you can approach this exercise. Your instructor will show you some options. For example, you could make a lawn of one species and use two discs as shown, or you could make two lawns (divide the plate in half) and use four discs.Create your own chemical discs by holding a paper disc with sterilized tweezers and soaking the disc in the chemical of your choice.Still holding your paper disc with a tweezers, blot off the excess chemical with a Kim-wipe and place the disc on your bacterial lawn. Label your plate, properly, and indicate which chemical is on which disc on the label on the plate (don’t try to label the disc!).Repeat the process of preparing a chemical disc using your additional chemicals, and place them appropriately on your plate.Invert plates and put into the rack for incubation.DAY 2: Results and InterpretationKirby-BauerExamine your plate for zones of inhibition, which will be clear areas of no growth around the disc. Measure the diameter of the zone in millimeters (mm). Compare your zone of inhibition measurement to a standard (Table 14-1)Record your results in Table 14-2 by recording the measurements, then circle the species that are SENSITIVE (killed) by the antibiotic.Collect results for the other two bacteria from other student groups. Table 14-1: Antibiotic zone of inhibition diameter Standards (mm)DrugResistantIntermediateSensitiveAmpicillin (AM)≤ 11 mm12-13 mm≥ 14 mmCephalothin (CF)≤ 1415 - 17≥ 18Erythromycin (E)≤ 1314 - 22≥ 23Gentamycin (GM)≤ 1213 - 14≥ 15Penicillin (P)≤ 28None≥ 29Streptomycin (S)≤ 1112 - 14≥ 15Tetracycline (TE)≤ 1415 - 18≥ 19Bactrim (SXT)≤ 1011 - 15≥ 16Table 14-2: Kirby-Bauer results. Enter your zones of inhibition that you measures and circle those that indicate sensitivity.AntibioticPseudomonasE. coliStaph. aureus Ampicillin (AM) Cephalothin (CF) Erythromycin (E) Gentamycin (GM) Penicillin (P) Streptomycin (S) Tetracycline (TE) Bactrim (SXT)Insert Photos of Results Here:Antiseptics/DisinfectantsYou will not measure a zone of inhibition for the chemicals you tested. Instead record (Table 14-3) a chemical as inhibiting growth if there is a zone of inhibition greater than 10mm.Look at two plates for each species and determine which products inhibited growth of the bacteria and which did not for each plate observed. Table 14-3: Effectiveness of Various ChemicalsBacteriaProducts thatInhibited GrowthProducts thatDid Not Inhibit GrowthPseudomonas aeruginosaPseudomonas aeruginosaEscherichia coliEscherichia coliStaphylococcus aureusStaphylococcus aureus Post-Lab QuestionsName: _____________________Look at the label on TWO products that inhibited bacterial growth and write down the name of the active ingredient in each. #1___________________________________#2___________________________________What was the most surprising thing you saw in the results of the products the class tested?What is the active ingredient in mouthwash? ___________________________________Look it up if you are not familiar with the ingredient. Is mouthwash antibacterial or bacteriostatic?Why would you not want to use a mouthwash that said “disinfectant” on the label?Which species is resistant to the most antibiotics? _____________________________ (correct scientific name and format, please)Why might this be so?Which species is sensitive to the most antibiotics?Why might this be so?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.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? ................
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