Labs 18-19 Disinfectants and Antibiotics PDF

Labs 19 and 20: Chemical Control of Growth Lab 19 – Disinfectants and Antiseptics Video Labs: https://youtu.be/pLY2t4IskD8 https://youtu.be/qvaY7S1enCA Lab 20 – Antibiotics Video Labs: https://youtu.be/5GHnxYmzXio  Lab 19 – Antiseptics and Disinfectants Purpo...

Labs 19 and 20: Chemical Control of Growth Lab 19 – Disinfectants and Antiseptics Video Labs: https://youtu.be/pLY2t4IskD8 https://youtu.be/qvaY7S1enCA Lab 20 – Antibiotics Video Labs: https://youtu.be/5GHnxYmzXio  Lab 19 – Antiseptics and Disinfectants Purpose – reduce microbial growth by use of Antimicrobial Chemicals Antiseptic – applied to living tissue Disinfectant – applied to non-living surfaces Bacteriostatic Effect – inhibiting growth of microbes Bactericidal Effect – killing microbes Death Reduction Time (DRT) – time to kill 90% of test microbial population Use Dilution Test – bacteria dried onto metal rings, placed in disinfectant 10 min. rings transferred to nutrient agar for growth Antiseptics Antimicrobial Chemicals: o Usually work by disabling proteins, damaging DNA, disrupting cell wall or cell membrane o Always need to know how they effect environment and you!!!! Leave odor, residue, how toxic, how fast do they act, brief or long exposure required, etc…  o 2 organisms: B. Pumilis E. Coli o lab disinfectant and test substance Lab 19 - Activity 1 - Disinfectant 1. Dilute lab disinfectant with sterile water, if necessary to working concentration 2. Transfer 5mL of lab disinfectant to 2 sterile test tubes – label E. coli and B. Pumilis 3. Divide 2 nutrient agar plate into quarters; label as follows: Bacteria added to a. “0”, “D-30s”, “D-4”, and “D-25” and [D for disinfectant!] disinfectant 4. Label 1 plate B. Pumilis and the other E. coli 5. Inoculate “0” sector of both plates using loop with appropriate bacteria. 1. Aseptically transfer 0.5mL of bacteria to each tube 2. Inoculate corresponding plates at each time point. 3. Incubate 37C – evaluate / score growth  Lab 19 – Using Chemicals to Kill Microbes Activity 1 – Disinfectant Results How long does it take disinfectant to kill bacteria? E. coli + Lysol B. Pumilis + Lysol E. coli + Tea tree oil B. pumilis + tea tre oil Lab 19 - Activity 2 - Antiseptic 1. Divide 1 nutrient agar plate in half; label one ½ “before degerming” and the other “after” o 2. Sample lab partners forearm with moist swab. 3. Inoculate “before” sector of plate. 4. “De-germ” partners other forearm with alcohol pad, sample, inoculate “after” plate ½ 5. Incubate 37C – evaluate / score growth Before After “degerming” “degerming” Lab 19 – Using Chemicals to Kill Microbes Activity 2 Antiseptic Results Degerming arm with alcohol Volunteer #1 Volunteer #2 (Dr. T)  Lab 20 – Antibiotic Sensitivity: Kirby Bauer Test Antibiotic: o Molecule produced by one organism that kills or inhibits growth of another organism o Produced by bacteria and fungi o Produced synthetically Antibiotic Mechanism: o Targets biological structures and/or processes Cell wall synthesis: damage it or inhibit cell wall synthesis DNA replication Protein synthesis o These characteristics are unique to prokaryotes or fungal (cell wall) cell types Kirby-Bauer Test: standard test for evaluating antimicrobials by diffusion Antibiotic concentration decreases as diffuses away from antibiotic disk Mueller-Hinton agar: specialized “loose” and “freely” diffusing agar Zone of Inhibition: area of inhibited growth around disk Minimum Inhibitory Concentration (MIC): minimum effective concentration  Lab Manual 16 – Antibiotic Sensitivity: Kirby Bauer Test 2 organisms: Pseudomonas aeruginosa 1. Swab Mueller-Hinton agar plate Staphylococcus aureus Zig Zag Inoculate – 3 directions Loose agar for good and consistent Ab diffusion a. Dip swab into culture and swab plate (3 directions) b. Swab back and forth from one end of plate to other c. Rotate 90 degrees and repeat d. Rotate 45 degrees and repeat e. Let stand 5 min. 2. Place 5 different Ab disks GENTLY on plate Each disk a different ab identified by # on disk a. Dip forceps in alcohol and flame to sterilize Sterilize forceps between Ab disk additions! b. Tap disks gently to ensure full contact to agar c. Label plate for antibiotics used and location of each o Antibiotic diffuses into agar from disk Concentration decreases with diffusion distance!  Lab Manual 16 – Antibiotic Sensitivity: Kirby Bauer Test 2 organisms: Escherichia coli Staphylococcus epidermidis Results: 1. Measure diameter of “Zone of Influence” for each antibiotic a. Measure disk diameter for any with no “Zone of Influence” b. Compare measured diameter to reference table c. Determine if the bacteria is Sensitive (S), Intermediate (I) or Resistant (R) o Antibiotic diffuses into agar from disk Concentration decreases with diffusion distance! o Dosage may be very different to work as medication in human body to treat infection  Lab 20 – Antibiotics Which antibiotics are E. coli sensitive to? Which antibiotics are S. epidermidis sensitive to? Zone of inhibition Antibiotic sensitivity chart p179 “Interactive” Measurement 20 mm 25 mm 15 10 30 mm “Interactive” Measurement 20 mm 25 mm 15 30 mm 10 Learning Objectives Lab 19 Explain how exposure time for chemical disinfectants relates to microbial killing Be able to evaluate the effectiveness of a disinfectant based on data similar to the kind obtained in lab Identify factors that would make a particular bacterial species more resistant to disinfectants State the purpose of degerming skin Use experimental results to evaluate the effectiveness of degerming Lab 20 Explain the terms antibiotic sensitive and antibiotic resistant Explain the purpose of the Kirby-Bauer sensitivity test Be able to interpret results from the Kirby-Bauer antibiotic sensitivity test Explain the zone of inhibition Based on the zone of inhibition, be able to classify an antibiotic’s effectiveness against a given species of bacteria. Note: if this is on the exam, you will have access to a sensitivity chart