Lab Report: Antibiotic Evaluation by the Kirby-Bauer Method

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Lab Report: Antibiotic Evaluation by the Kirby-Bauer Method

Introduction

Chemical antimicrobial agents are chemical compounds capable of either inhibiting the growth of microorganisms or killing them outright. Those which are taken internally to alleviate the symptoms of or promote healing from disease are called chemotherapeutic drugs, and among these is a class of compounds called antibiotics. In order for a chemotherapeutic drug to be classed as an antibiotic, it must be produced by a microorganism such as bacterium or fungus or at least derived from a chemical produced by one. It must also be capable of killing or inhibiting the growth of other microorganisms and of doing so when taken in very small quantities.

To study whether a microbial product qualifies as an effective antibiotic, a standard procedure called the Kirby-Bauer method is employed. This method, which is the procedure recommended by the US Food and Drug Administration, was devised by William Kirby and A.W. Bauer in 1966. In the current protocols involved in the Kirby-Bauer method, Mueller-Hinton standard agar is used as the medium for bacterial culture. The pH of the standard agar is 7.2 to 7.4 and it is poured exclusively to a depth of 4 mm. The medium is heavily inoculated with bacteria and paper disks containing enough of the antibiotic under study to create an optical density of 1 (the McFarland standard) are placed on top of the cultures. By examining the results of incubation in the form of a zone of inhibition around each disk after incubation, it can be determined how effective each antibiotic is against any given bacterium. A minimum inhibitory concentration can then be deduced for the given antibiotic vs. the specific bacterium tested so that appropriate dosage may be determined. Resistant bacteria cultures will show a small or no zone of inhibition if their growth is not sufficiently inhibited for the antibiotic to be a viable candidate in treating infection by that organism. Sensitive cultures, on the other hand, will be appreciably inhibited in their growth or, ideally, eliminated entirely in a relatively large radius around the McFarland standard disk. In this case, the antibiotic under study might be prescribed as a useful counter to illness brought on by that particularly bacterium.

In the experiment discussed here, we tested eight antibiotics against four common opportunistic pathogens, namely Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Of these, the first two are Gram positive and the latter pair are Gram negative. The eight antibiotics tested were: Ampicillin, a beta-lactam antibiotic that inhibits the final stage of bacterial cell wall synthesis by binding to receptors within the cell wall. The result is a porous cell wall and subsequent lysis carried out by the bacteria's own enzymes. It is effective against many bacteria, both Gram positive and Gram negative, and is particularly used in treating infection by E. coli, Salmonella typhosa and Enterococcus faecalis, among others. (DrugBank) This antibiotic is a semi-synthetic derivative of penicillin, which is itself an antibiotic produced by the fungus Penicillium notatum. Bacitracin, a mixture of polypeptides obtained from Bacillus subtilis var Tracy. It inhibits synthesis of the peptidoglycan layer in Gram positive bacteria by preventing the function of a molecule that transports components to synthesis sites. Bacitracin has a low threshold of toxicity when taken orally or injected, but it has found application as a topical ointment in the prevention of wound infection by Staphylococci. (DrugBank) Chloramphenicol, a broad spectrum antibiotic that is produced synthetically but which was originally discovered in a Streptomyces bacterium. It can be employed against several types of infection but most notably has found application in combating typhoid fever cholera. This antibiotic inhibits protein...
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