BIO 113-001 Name __________________________ Date ________ General Biology Lab ANTIBIOTIC EFFECTIVENESS PURPOSE The purpose of the Kirby- Bauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds in order to assist a physician in selecting treatment options for his or her patients. The pathogenic organism is ideally grown on Mueller- Hinton agar in the presence of various antimicrobial impregnated filter paper disks. The presence or absence of growth around the disks is an indirect measure of the ability of that compound to inhibit that organism. BACKGROUND Determination of bacterial resistance to antimicrobials is an important part of the management of infections in patients. The disk diffusion method of Kirby and Bauer has been standardized and is a viable alternative to broth dilution methods for laboratories without the resources to utilize the newer automated methods for broth microdilution testing. When a 6- mm filter paper disk impregnated with a known concentration of an antimicrobial compound is placed on a Mueller- Hinton (MH) agar plate, water is immediately absorbed into the disk from the agar. The antimicrobial begins to diffuse into the surrounding agar. The rate of diffusion through the agar is not as rapid as the rate of extraction of the antimicrobial out of the disk, therefore the concentration of antimicrobial is highest closest to the disk and a logarithmic reduction in concentration occurs as the distance from the disk increases. The rate of diffusion of the antimicrobial through the agar is dependent on the diffusion and solubility properties of the drug in MH agar and the molecular weight of the antimicrobial compound. Larger molecules will diffuse at a slower rate than lower molecular weight compounds. In combination, these factors result in each antimicrobial having a unique breakpoint zone size indicating susceptibility to that antimicrobial compound. The organism will grow on the agar plate while the antibiotic “works” to inhibit (slow down) the growth. If the bacteria is susceptible (sensitive) to a specific antibiotic, there will be no growth around the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. The measurement is compared to the criteria set by the Clinical and Laboratory Standards Institute (CLSI). Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I), or Susceptible (S) as shown in Table 1. PROTOCOL 1. Label the BOTTOM of four plates with your group initials, date, and bacterial species. The outline of your plates should look similar to the drawing below. (**Note: You do not have to label each section with the antibiotic name, as the antibiotic disk are labeled with the abbreviated antibiotic name. 1 2. 3. 4. 5. 6. 7. 8. Dip a sterile cotton swab into the bacterial culture, then spread the culture evenly over the entire surface of the agar. Be careful not to destroy the agar. Allow any extra liquid present to dry. Rotate the plate until the entire surface is covered. Resuspend the same cotton swab back into the same bacterial culture, and spread the culture evenly over the entire surface. Dip the tweezer in ethanol and then flame-sterilize them. Next, gently pick up a disk containing the first antibiotic you wish to test. Gently place the disk on top of the agar, in the center of the appropriate section on the plate, and lightly press it down with the tweezers. Between each disk, sterilize the tweezers by dipping them in ethanol and flaming sterilization. Place the disks containing the antibiotic in the appropriate section. Take care not to move the disk around too much, as this could skew the bacterial growth in the surrounding area. Repeat steps 2-5 for EACH bacterial species. Every time you switch to a new bacterial species, use a NEW sterile cotton swab. Incubate the plates inverted (flipped upside down – agar side should be facing you) overnight or until cells have grown out completely. Measure the area of inhibited bacterial growth with a ruler. Compare your measurements to the guidelines chart below to determine if the different bacterial species are susceptible (sensitive) to, intermediately sensitive to, or resistant to the antibiotic. Table 1. Guidelines Chart Antibiotics Ampicillin Chloramphenicol Penicillin Streptomycin Tetracycline Disk Concentration 10 µg 30 µg 10 U 10 µg 30 µg Zone Diameter Measured (mm) Susceptible (S) Intermediate (I) Resistant (R) 14 or more 12-13 11 or less 18 or more 13-17 12 or less 15 or more 13-14 12 or less 21 or more 15-20 14 or less 19 or more 15-18 14 or less 2 RESULTS 1. 2. TAKE PHOTOS OF YOUR PLATES. THIS LAB WILL BE USED IN YOUR FINAL LAB REPORT. Record your results in the table below following overnight incubation at 37oC. Measure in centimeters and then convert the units to mm. Convert cm to mm by multiplying by 10. Escherichia coli (E. coli) Antibiotics Ampicillin, 10 mcg Chloramphenicol, 30 mcg Penicillin, 10 units Streptomycin, 10 mcg Tetracycline, 30 mcg Zone of Inhibition (mm) Interpretation (R, I, S) Bacillus subtilis (B. subtilis) Antibiotics Ampicillin, 10 mcg Chloramphenicol, 30 mcg Penicillin, 10 units Streptomycin, 10 mcg Tetracycline, 30 mcg Zone of Inhibition (mm) Interpretation (R, I, S) TAKE PHOTOS OF YOUR PLATES. THIS LAB WILL BE USED IN YOUR FINAL LAB REPORT. 2. Give a brief description of the basic mechanism of action of each antibiotic. Antibiotic Mechanism of Action Ampicillin Chloramphenicol Penicillin Prevents formation of the cell wall Streptomycin Tetracycline 3
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