Control of Microbial Growth Antimicrobial susceptibility Test (Kirby-Bauer Metho

Question

Control of Microbial Growth Antimicrobial susceptibility Test (Kirby-Bauer Method) pp 223-224 (Photographic Atlas) Media: 2 Mueller-Hinton agar plates/group Organisms: make spread plates with NB culture Staphylococcus aureus (24 hr NB broth tube) Escherichia coli (24 hr NB broth tube) Antimicrobials: Chloramphenicol, Ciprofloxacin, Trimethoprim, Penicilin, Streptomycin, Tetracycline Lab 1: Make bacterial lawns with organisms using a sterile cotton swab Apply antimicrobial discs with antibiotic disc dispenser Tape lids to bottoms, and incubate all plates inverted at 37C. Lab 2: Measure each zone of inhibition (mm) and fill in results. Using the Interpretive Chart (Table 7-3), indicate if organism is resistant (R) or susceptible to each antibiotic. Chloramphenicol Ciprofloxican Trimethoprim Penicillin Streptomycin Tetracycline C30 CIPS STX P10 S10 T30 Organism Zone Zone diameter Zone diameter Zone diameter Zone Zone diameter S/R S/R S/R S/R S/R Escherichia coli Staphylococcus aureus S/R S/R S/R S/R S/R S/R S/R 1. How does the antibiotic move from the disk into the agar? 2. Mueller-Hinton agar plates are poured to a standard depth of 4mm. What might be the consequences of pouring the plates thinner (2mm)? 3. Which of the antibiotics shows a difference in susceptibility between the Gram (+) and Gram ) bacteria? Explain.

Explanation / Answer

The moisture in the medium plays a crucial role in disc diffusion, when the antibiotic discs are applied to the agar, moisture from the plate is absorbed, dissolving the antibiotic, and allowing it to diffuse into the agar. Thus, a reduced moisture content will impede the flow of antibiotic and result in smaller zones.

For sensitivity testing the depth of the agar should be 4mm in the center of the plate (approximately 25ml in a 90mm plate). Variation in depth will affect the zone sizes and would affect the distance the antibiotic diffuses from the disk – if the agar is thinner than 4mm, larger zones will appear since the volume is decreased, and the effective antibiotic concentration increased. If the agar is thicker than the 4 mm, smaller zones will appear since the effective antibiotic concentration has been decreased, because the more downward diffusion there is and the less antibiotic available to diffuse outward. Thus, the zone would be smaller. If the agar is intentionally thin, then small modifications to other factors will have a disproportionate effect.

Penicillins, work by interfering with interpeptide linking of peptidoglycan, the strong structural molecule found specifically bacterial cell walls. Cell walls without intact peptidoglycan cross-links are structurally weak, prone to collapse and disintegrate when the bacteria attempts to divide. Since the eukaryotic cells of humans do not have cell walls, our cells are not damaged by penicillins.

The Penicillin antibiotic shows the difference in susceptibility between Gram positive and Gram negative bacteria. Because the Gram-positive bacteria have thick cell walls containing high levels of peptidoglycan, while gram-negative bacteria are characterized by thinner cell walls with low levels of peptidoglycan. The cell walls of gram-negative bacteria are surrounded by a lipopolysaccharide (LPS) layer than prevents antibiotic entry into the cell. Therefore, penicillin is most effective against gram-positive bacteria where DD-transpeptidase activity is highest.

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