A Method for Antibiotic Susceptibility Testing: Applicable and Accurate

Background: Emerging antibiotic resistance in pathogenic bacteria has driven the development of new assays for routine antibiotic testing. Objectives: The purpose of this study was to evaluate the effects of different organic solvents in preparing two-fold decreases in serial penicillin concentration coated onto 96-well plates to design a method for antibiotic susceptibility testing. Materials and Methods: Benzyl penicillin was dissolved in each solvent (sterile distilled water, PBS, diethyl alcohol, ethanol, butanol, chloroform, 2-propanol, and acetonitrile). Serial dilutions of each solution were loaded onto a 96-well microtiter plate and incubated at 37°C for 12 h. Next, 200 ?L of sterilized Mueller-Hinton broth was added along with 50 ?L of bacterial suspension at an adjusted concentration equivalent to 0.5 McFarland standards. The prepared plates were incubated at 37?C for 24 h. Optical density (OD) was measured at 540 nm. Results: When comparing the ODs of each sample in 96-well microtiter plates with positive and negative controls, significant antibacterial activity was observed. Most activities ranged from 50 to 200 units of penicillin in samples that were diluted with distilled water, PBS, or isobutyl alcohol as a solvent. Analysis of the results suggested that, when using the aforementioned solvents, the minimum inhibitory concentration of penicillin against a sensitive strain of Staphylococcus aureus was ?50 units of penicillin. Conclusions: The results revealed that the accuracy and feasibility of this method can greatly reduce the waiting period of antibacterial sensitivity tests. Additionally, this method is lowcost and could benefit patients who urgently require proper antibiotic therapy.