Antibiotic Identification by High Voltage Electrophoresis Bioautography

Abstract

Abstract The high voltage electrophoresis bioautography method is applicable to meat, milk, and animal feeds. Meat is freeze-dried, powdered, and extracted with acetonitrile-water (9 + 1), and the extract is concentrated by evaporation at room temperature. Milk is examined directly or following acetonitrile-water extraction. Feed is extracted with acetonitrile- water. Samples or extracts are applied to preliminary assay plates of antibiotic medium No. 1 at pH 6 and 8, seeded with Micrococcus luteus (ATCC 9341), M. luteus DHSR (ATCC 9341A), Bacillus cereus (ATCC 11778), or B. cereus K250 TR (NCIB 11183), and nutrient agar at pH 7 seeded with B. subtilis BGA. Inactivation of penicillinase indicates beta-Iactam antibiotics. Addition of trimethoprim increases sensitivity to sulfonamides. After 18-24 h incubation at 30°C, plates yielding clear inhibition zones guide selection of conditions for subsequent electrophoresis bioautography. Extracts are applied (5-100 |xL) to 10 mm diameter wells on electrophoresis plates 60 cm long and 40 cm wide, with a gel depth of 1.6 mm. The support medium is 1% agar and 1 % agarose in Tris/succinic acid buffers pH 6 and pH 8. A potential of 1500 V is applied for 1.5 h at 15°C. Following electrophoresis, the migrated antibiotics are visualized by over-layering with antibiotic medium No. 1, pH 6 or 8, seeded with M. luteus or B. cereus spore suspension; plates are incubated for 18-24 h at 30°C. Identification is based on results of preliminary screening together with electrophoretic migration distances and inhibition zone appearances compared with standards