MICROTITRE-PLATE COLORIMETRIC ANTIMICROBIAL ASSAY USING BROMOTHYMOL BLUE, BROMOPHENOL RED and METHYL RED AS INDICES OF BACTERIAL GROWTH

The present invention relates to the use of redox dyes, particularly, bromothymol blue, bromophenol red and methyl red, in determining the minimum inhibitory concentrations (MICs) of Vancomycin hydrochloride, Clindamycin phosphate, and Ampicillin sodium against methicillin-sensitive and methicillin-resistant Staphylococcus aureus. The surveillance and clinical isolates were compared to resazurin in microtitre plate-based assay and standard dilution method. The best conditions were tested on Nutrient Broth (NB) with 1 pcnt glucose. Briefly, two-fold serial dilutions of the antibiotic were inoculated with 5x105 CFU/mL of inoculum. Plates were shaken and incubated at 35oC for 24 hours; after which, the dyes were added, and incubation was continued for another five (5) minutes. MIC breakpoints were read and interpreted according to Clinical and Laboratory Standards Institute (CLSI) recommendations. Findings indicate that bromothymol blue, bromophenol red and methyl red are comparative to resazurin using nutrient broth supplemental with 1 pcnt glucose and five minutes incubation time after dye dispensation. The color differentiation clearly indicates bacterial growth. With sensitivity of 100 pcnt and accuracy of 94.305 pcnt of the developed MTR-plate, it is concluded that the developed assay is comparable to TDM and may be used as confirmation for methicillin-resistance in S. aureus. Furthermore, the assay may be used to determine the MICs of different antibiotics against other non-fastidious pathogens. The method is simple, rapid, cheap, convenient, and the options on the dye to be used are increased.