Microbial physiology and culture (Practical 1 Antibiotics and Agar Coursework

Microbial physiology and culture (Practical 1 Antibiotics and Agar Diffusion) - Coursework Example On the other hand, experiment 2 attempts to investigate the repeatability of the essays using two different methods of antibiotic application to the discs. Lastly, experiment 3 uses gradient plates to qualitatively compare the response of new isolates to a particular antibiotic against a response of a standard strain. Practical 1: Antibiotics and Agar Diffusion Introduction Agar plate diffusion is currently one of the widely used experimental methods to determine the susceptibility of various microorganisms to antimicrobial agents. Generally the principle employed in this method is dependent on the inhibition of the microorganism reproduction on the surface of agar medium by an antimicrobial agent which gradually diffuses into the medium through a filter paper disc. In this regard, the level of susceptibility of the organisms is indicated by the size of the zone of inhibition. Typically when an antibiotic is applied to the agar medium, it moves from its region of high concentration t o the surrounding areas of lower antimicrobial concentration (Cooper, 1993, p.24). Inhibition zone is the clear area of the agar plate where the growth of the microorganisms is prevented by the activities of the microbial agent. The size of inhibition is usually measured and then compared to the standardized measurements in order to determine the level of antibiotic activity on the particular medium. This is primarily because the diameter of the inhibition zone is always a function of the susceptibility of the microorganism and the amount of antibiotics on the medium. Generally there are a number of factors that may affect the antimicrobial activity and consequently determine the size of inhibition zones. Some of the likely factors include agar depth, size of the inoculums and the presence of cations on the medium. The objective of this experiment is to investigate the factors that are responsible for the variation of microbial activity of various antimicrobial drugs on different or ganisms. Methods To investigate the effects of variables such as agar depth, presences of salt and inoculums size on the size of inhibition zones, lawn and seeding methods were used to inoculate the plates. In determining the effect of agar depth on the result of agar plate diffusion, four plates containing 10 cm3, 20 cm3, 20 cm3 and 30 cm3 agar were first swabbed with Eschericia coli 10418 (1:20 dilution). All plates were oriented horizontally except for the third one, which was slanted. Meanwhile, 2 plates each containing 20 cm3 DSTA were inoculated with 100 Â µl of either 1:20 E.coli dilution or undiluted broth culture. 4 discs each containing 20 Â µl 0.5 mg/ml ampicillin were placed in each of the plates. Finally the plates were then incubated overnight at 37Â °C and the variations in the inhibition zone size were recorded. To determine the effect of inoculum size on the antibiotic activity, 0.5mg/ml ampillicin solution and the E.coli dilution were used.2 plates with 20 cm3 D STA were poured into the solution. The first inoculate was then spread uniformly on the entire surface of the plate. On the second plate was also inoculated with a containing broth culture of E.coli was also inoculated. Finally 4 amplicin impregnated papers were placed on each plate before they were incubated. To investigate the effects of potassium or calcium ions on zone sizes, 0.1 or 1.0 ml of 2M KCl or CaCl2