Lab 8 Antimicrobial Susceptibility Test PDF

Basic Microbiology Lab: 119 Title: Antibiotic Susceptibility Test Intended Learning Objectives (ILOs): By the end of this lab, the students are expected to: 1. Introduced to the antibiotic resistance and how it occurs. 2. Understand the importance...

Basic Microbiology Lab: 119 Title: Antibiotic Susceptibility Test Intended Learning Objectives (ILOs): By the end of this lab, the students are expected to: 1. Introduced to the antibiotic resistance and how it occurs. 2. Understand the importance of the antibiotic susceptibility test and the definition of MIC and MBC terms. 3. Demonstrate how to perform antimicrobial susceptibility testing using the Kirby-Bauer method. 4. Understand the origin of a zone of inhibition and how it should be measured. 5. Interpret the results of the Kirby-Bauer method and determine if an antibiotic is effective against a particular bacterium. Precaution: 1. All specimens should be treated as infectious. 2. Wear appropriate personal protective equipment for the laboratory procedure. 3. All specimens must be well labelled and avoid any contamination. 4. All samples must be processed in line with SOP designed for the test. 5. Result must be recorded immediately. Background: In clinical sets, choosing the right antibiotic is a crucial step towards the infection treatment and elimination as some microorganisms have developed a resistance against different antibiotics. Antibiotic resistance occurs as result of certain genetic mutation or modification in order for the bacteria to survive the antibiotic effect. Note that not all bacterial mutation beneficial. Antibiotic susceptibility test is used to describe the susceptibility of bacteria to certain antibiotics, i.e. which antibiotic will be most successful in treating a bacterial infection in vivo. This can be approached through calculating the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal 1 Concentration (MBC). MIC can be defined as the minimum concentration of a certain antibiotic that inhibit the bacterial growth while MBC is the minimum concentration of a certain antibiotic to kill all the bacteria. How do antibiotics work? 1. A bactericidal antibiotic kills the bacteria. 2. A bacteriostatic which inhibit the bacterial growth (stops bacteria from multiplying). Basic Mechanisms of Antibiotic Action against Bacterial Cells: 1. Inhibition of Cell Wall Synthesis (most common mechanism) 2. Inhibition of Protein Synthesis (Translation) (second largest class) 3. Alteration of Cell Membranes 4. Inhibition of Nucleic Acid Synthesis 5. Anti-metabolite Activity Kirby-Bauer disc diffusion method: Principle: The Kirby-Bauer disc diffusion method is widely used for testing the sensitivity of bacteria to antimicrobials. Bacterial culture is spread onto the surface of MHA plate (Lawn technique), followed by addition of antibiotic discs to the agar surface and incubate the plates overnight at 37°C. If an organism is susceptible to an antibiotic, a clear zone appears around the disc where the growth of organism has been inhibited, called the zone of inhibition. If resistant, no clear zone of inhibition appears. The diameter of the zone of inhibition surrounding the antibiotic disc is measured to determine whether the micro-organism is Sensitive (S), intermediately sensitive (I) or Resistant (R) to a particular antibiotic. Precautionary note: Ø Don’t forget to swab the plates before adding the antibiotic discs. Ø When you swab your plates, make sure to thoroughly swab the entire plate; do not leave gaps, or you may not see the zones of inhibition well. 2  Ø Tap the centre of each disc once you have placed it on the plate to make sure the disc is in contact with the agar. If not, you may have a false – negative result. Ø Once the discs are all on the surface of the plates, keep them lid side down. Water dropping onto the plate surface will ruin the zones of inhibition. In this practical, you will carry on with your clinical sample and detect the antibiotic susceptibility for both detected organisms using the disc diffusion method. The second part will be about detection of antibiotic resistant mutants and how bacteria can develop resistance to certain antibiotic. PRACTICAL SESSION: PART-A: Kirby-Bauer disc diffusion method. CASE #3 DAY 3: PROCEDURE: Materials: 1. Petri dish containing sterile Mueller-Hinton Agar (MHA). 2. Pure culture in broth 3. Glass Spreader or Sterile cotton Swabs to spread onto the surface of MHA plates 4. Different antibiotic discs. 5. Forceps to place the antibiotic discs or disc dispenser. 6. Bunsen burner to sterilize forceps 7. Ruler to measure zone of inhibition Method: 1. Pick single colony of bacteria and inoculate into nutrient broth. 2. Incubate the broth culture at 37°C for 4-6 hrs in the shaker incubator allowing the bacteria to achieve or exceed the turbidity of the 0.5 McFarland standard. 3. Take a sterile cotton swab and dip it into the bacterial culture test tube to make a lawn of bacteria on each plate (completely cover the plate). 3  4. Allow the agar surfaces to dry for 5 minutes. 5. Pick the antibiotic discs with a sterile forceps or a disc dispenser and place it on the agar plate surface. 6. Invert and incubate the plates overnight at 37°C. 7. Observe the plates for zone of inhibition surrounding the disc. 8. Measure the zone of inhibition using a ruler on the underside of the Petri plate and tabulate your results. Observation: Measure the zone of inhibition using a ruler on the underside of the Petri plate and tabulate results. Results and Interpretations: Compare the results obtained with that of standard (zone size interpretative chart) to determine the sensitivity of each of the organism to each of the antibiotics and record the results. For each drug, indicate on the recording sheet whether the zone size is 4 Sensitive (S), Intermediate (I), Resistant (R), based on the interpretation chart. There are different charts for different organisms Measuring zone of inhibition (in mm or cm) Antibiotics S. aureus P. aeruginosa PART-B: Detection of Antibiotic Resistant mutants PROCEDURE: Materials: 1. 1xConcentration Gradient agar of Streptomycin 2. 1x MHA plate 3. Glass Spreader 4. Micro pipette and a sterile tips. 5. Broth culture of S. aureus Method: 1. Label MHA plate as “control” and the Gradient plate as “Streptomycin” 2. Add 0.1ml of S. aureus culture to both plates. 3. Using the glass Spreader, spread the added culture on the whole agar evenly. 4. Incubate at 37°C for 3-4 days. 5. Observe the results next practical session 5 References: 1. Bharti Arora, D. R. Arora (2015): Practical Microbiology. ISBN: 81-239- 14095-9. Page 1 – 218. 2. Steven Obenauf, Susan Finazzo (2016): Microbiology Fundamentals. A Clinical Approach. Page 1 – 318. 6  Student's Evaluation 1. What are the advantages of antimicrobial susceptibility testing? 2. Which medium is preferred for antimicrobial sensitivity testing? 3. Write four basic mechanisms of antibiotic action against bacterial cells. 4. Define the term MIC and MBC. 5. Explain the MacFarland standard. Instructor’s signature Date: 7