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Disc Diffusion Assay to Determine Antibiotic Sensitivity in Two Different Organisms

The Mechanism of Action Behind Antibiotics Such as Penicillin, Chloramphenicol, and Doxycycline

Disc diffusion assay to determine the antibiotics sensitivity in two different organisms

Maresso (2019) states that antibiotics are drugs that work against bacteria by being either bacteriostatic or bactericidal. All antibiotics usually stop the synthesis of biomolecules essential for bacterial growth. Antibiotics have many types but the main one is Penicillin that is derived from the green mold Penicillium, it prevents the Gram-positive bacteria from forming by binding to proteins in the cytoplasmic membrane which inhibit the synthesis of peptidoglycan in the cell wall resulting in the internal pressures causing the bacterium to swell and burst. Also, it can activate enzymes that break down bonds between molecules in the cell wall which is essential for bacteria survival. Penicillin can treat many diseases like syphilis, meningitis, and gonorrhea. There are many types of penicillin e.g. F, G, and X.

Chloramphenicol and Doxy cycline agents are bacteriostatic which inhibit the growth of bacteria. Scholar (2007) states that chloramphenicol has a large spectrum of activity on gram-positive and gram-negative because it can bind with the 23S rRNA on the 50S subunit, on the bacterial ribosome which interferes with the peptidyl transferase activity that prevents the transfer of amino acids to the growing peptide chains and blocks peptide bonds. Because of this, the bacterial proteinsynthesis gets blocked which impedes cell proliferation. While doxycycline is highly active against intracellular pathogens, gram-positive and gram-negative bacteria. This is because it can reversely and allosterically bind with the 30S and possibly to the 50S subunit of the bacterial ribosome which blocks the attachment of aminoacyl-tRNA to the mRNA-ribosome complex result in inhibition of protein synthesis (G et al., 2009, p .1121-1140).

E.coli is gram-negative, non- pores- forming, rod-bacillus-shaped bacteria with adhesive fimbriae and a cell wall consisting of cytoplasmic, peptidoglycan, and outer membrane. The width of E.coli is about 1.1 to 1.5μm with a height of 2 to 6μm. They can be motile and non-motile. Motile E.coli produces lateral while non-motile has polar flagella. E.coli cells can be arranged as single or in pairs. While S.aureus is agram-positive, non-sporing, round cocci-shaped bacteria with a diameter of about 1μm. They are usually non-motile and non-flagellated with grape-like clusters of cellarrangement (Percival., & Williams, 2014, p. 89-117).

The Kirby-Bauer technique of disc diffusion determines the susceptibility oforganisms e.g. E.coli and S.aureus to antimicrobials. This technique required a lawn of bacteria spread via swab on the surface of the plate, containing an agar medium with paper discs that were filled with antibiotics e.g. chloramphenicol anddoxycycline. Before adding the discs plates were divided into four sections, two foreach antibiotic. Then the plates were incubated overnight at 37C. This allowed thebacteria to grow. From which the zone of inhibition around the disc was measured using a ruler (Parker et al., 2016 p. 605).

Many aseptic techniques were used to prevent contamination during the experiment. Some of those techniques were, disinfecting the tables before and after, using a bunsen burner that created a sterile zone, flaming the mouth of a test tube to prevent the airborne pathogen. Using a sterile swab that spreads the bacteria, and the tweezer to take out the disc. Also, the inoculating loop was run under the bunsen burner for some seconds until it was hot red, which killed the microorganism present and made it sterile. After the experiment waste plates were autoclaved under 121C to kill all the microorganisms (Parker et al., 2016 p. 605).

The aim of the experiment was to see which concentration of antibiotic that works best against the organisms. Also, it was to determine the zone of inhibitions produced by the E.coli and S.aureus organisms.

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