Fifty antibacterial agents, mainly antibiotics, were examined by a high voltage electrophoretic technique which determined their migration distances in agar and agarose gels at both pH 6·0 and 8·0. Comparison of the different migration distances in the two gels formed the basis for identification. Bio‐autography, using Bacillus cereus var. mycoides or Micrococcus luteus, was used to visualize the antibiotics. The method was satisfactory for identifying many antibiotics in animal tissues and animal feeding stuffs, and was capable of distinguishing antibiotic activity from that of naturally occurring inhibitors often present in those materials. It has also been used to identify antibiotics in urine and pharmaceutical preparations, and a wider application in medical microbiology is indicated.
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
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.