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Procedures to determine MIC and MBC
Antibiotic sensitivity testing is carried out to determine the exact antibiotic agent used for a bacterium. This can be carried out by two different procedures; the disc diffusion test and the dilution test. Disc diffusion tests are used to determine vulnerability of bacteria to antibiotics. In this method, discs infused with known concentrations of antibiotics and inoculated with a tested bacterium are placed on an agar plate. The place is then incubated at 37oC for 18-24 hours. Diffusion will take place at this point. The concentration of antibiotics is high near the antibiotic and decreases with distance. Vulnerability of the antibiotic is determined by measuring the zone of growth inhibition around the disc. There are different types of disc diffusion methods that can be performed. These methods are Kirby-Bauer disc diffusion method, Stokes disc diffusion method and Primary disc diffusion method. However, these methods do not determine the MIC and MBC.

Unlike the disc diffusion method, the dilution methods do determine the MIC and MBC of an antimicrobial agent. The MIC estimation is important and useful since it regulates the therapeutic dose of the antibiotic and it tests antimicrobial sensitivity of slow growing bacteria. There are three methods to the dilution method including the broth dilution method, agar dilution method and Epsilometer test or the E-test.

The broth dilution test is quantitative since it determines the MIC of antimicrobial agents that inhibit the growth of organisms. In this method, the antimicrobial agent is successively diluted in Mueller-Hinton broth by doubling the dilutions in tubes with a standard suspension of the tested organism added to each tube. It is mixed gently and incubated at 37oC for 16-18 hours. The MIC is the observed and noted of the lowest concentration of the drug at which there is no growth. The advantage of this method is that it is a simple procedure for testing a small number of isolates and the MBC can be determined from the same tubes. To determine the MBC, one must subculture the tubes that show no growth on nutrient agar plates. It is then incubated overnight. The tube that shows the lowest concentration of the drug that failed to show any growth on the agar plate is the MBC of that organism. This is the only method that determines MBC.

The agar dilution method is also quantitative since it determines the MIC of antimicrobial agent against the tested organism. This method is important and useful because it tests organisms from infections and it verifies equivocal results of the disc diffusion method. Dilution of the antibiotic are made in the Mueller-Hinton agar (note that it is an agar not a broth) that has been made in distilled water added to the agar plate that has been melted and cooled to not more than 60oC. It is then poured onto Petri dishes. The tested organism is inoculated and incubated overnight. The plates are then observed for the presence or absence of the growth of the bacterium. The lowest concentration at which the bacterium is completely inhibited is the MIC. The advantage to this method is that a number of organisms can be tested on each plate.

The E-test is an automated system that measures the MIC of the bacterial isolate. In this method, an absorbent plastic strip with a antibiotic powerless on one side and MIC interpretative scale equivalent to 15 twofold MIC dilutions on the other side is placed on an agar plate. The organism is then inoculated with the MIC scale facing toward the opening side of the agar plate. It is then incubated overnight. After incubation, an oval-shaped zone of growth inhibition is observed around the strip. The MIC is read from the scale at the intersection of the zone with the strip. The E-test is very useful since it interprets the MIC of the antibiotic easily.