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Antibiotic Evaluations on Gram-Negative and Gram-Positive Bacteria by Using Kirby-Bauer Experiment

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Lab Report 1: Antibiotic Evaluations on Gram-negative and Gram-positive bacteria by using Kirby-Bauer Experiment


Microorganisms are abundant and exist virtually in every corner of the Earth surface. Some microbes are vital for human survival, but some pathogens pose a threat to public health. To combat with these pathogenic microorganisms, antimicrobial agents are developed to either inhibiting (static) or killing (cidal) pathogens including bacteria and yeast. One class of common chemical agents is antibiotics which were first used by Selman Waksman who discovered more than twenty antibiotics at the time [1].

However, pathogens have the ability to mutate at a rapid rate and develop multiple mechanisms to escape the effect of antibiotics by alternating their targeted proteins, degrade their effects or compensate one pathway for another inhibited by active components of antibiotics. This behavior is called antibiotic resistance and its level is unique to each of the pathogens and between populations. Due to the popularity of antibiotics, antibiotic resistances have become common problems during treatment of the patients. Therefore, it is important to measure the resistance of each pathogen and its sensitivity to antibiotics.

William Kirby and A.W. Bauer devised Kirby Bauer experiments by using Mueller-Hinton broth in agar as a medium for bacteria [2]. Each antibiotic is loaded on a paper disc and diffuses into the medium when placed on top. There are twelve antibiotics are tested in the experiment including Amoxicillin, Azithromycin, Cephalothin, Ciprofloxacin, Gentamicin, Penicillin G, Piperacillin, Polymyxin B, Rifampin, Sulfadiazine, Tetracycline and Vancomycin. Since the concentrations of antibiotics are higher closer to the disk, the susceptibility of each bacterium is determined from the zone around the disk where no growth occurs. This zone around the paper disk is called zone of inhibition which is measured in

diameter of the region surrounding each antibiotic disk that lacks growth. Resistant bacteria will show little to no zone of inhibition around paper disks.

In this experiment, Gram-negative isolate B of bacteria in genus Acinetobacter from skin and Gram-positive isolate D of bacteria in Bacillus genus from soil were examined to determine the amount of resistance to different antibiotics. During the experiment, the amount of concentration of all antibiotic are kept constant to precisely determine the sensitivity of the bacteria to antibiotics. The bacteria in the subject of the experiment are at the log phase when they are at maximum activities and perform DNA replication, translation and cell wall synthesis [3]. This allows the examination of the maximum resistance potentials of the bacteria to antibiotics.

However, this experiment will not be able to provide any mechanisms behind the behaviors of the bacteria to antibiotic. There is no direct evidence of how or



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