Plasmid-mediated 3-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 ,B-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pl 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of P-lactamases.Analytical isoelectric focusing (IEF) for the detection of P-lactamase enzymes from gramnegative bacteria has been done since the mid1970s (7). This method allows for the characterization and differentiation of many types of Plactamases by visual comparison of the banding patterns and by the isoelectric points of the constituent proteins. Currently, ,-lactamases are characterized by IEF in polyacrylamide gel, a procedure which is a modification of the original work of Matthew et al. in 1975 (7). This experimental procedure requires 18 h of IEF to achieve adequate identification of most ,B-lactamase types. The resulting banding patterns and isoelectric points are consistent, although the origins and chemical composition of the patterns have not been clearly delineated.One alternative to polyacrylamide is agarose, a purified galactan hydrocolloid which was recently introduced as a medium for analytical IEF. Agarose media offer a macroporous matrix of considerable strength at concentrations 51% (4). The macroporous matrix and the low ionic character of agarose minimize the problems of electroendosmosis and molecular sieving associated with polyacrylamide. Agarose is also simpler in gel formulation and is nontoxic, in contrast to the reported toxicity of unpolymerized t Reprint requests should be directed to Antone A. Medeiros, Miriam Hospital, 176 Summit Avenue, Providence, RI 02906.polyacrylamide (10). These factors led us to explore the use of agarose gel IEF for the identification of ,-lactamase enzymes and to compare these results with those established by polyacrylamide gel IEF. MATERIALS AND METHODSBacterial strains. The Escherichia coli and Pseudomonas aeruginosa strains containing plasmid-mediated P-lactamases used in this study were kindly supplied by Margaret Matthew, R. W. Hedges, and George A. Jacoby. E. coli K-12 strains included R6K(TEM-1) (2, 5); RP1(TEM-2) (5); RGN238(OXA-1) (1, 5); R46(OXA-2) (1, 5); R57b(OXA-3) (1, 5); R997(HMS-1) (8); and R1O1O(SHV-1) (8). P. aeruginosa PU21 strains contained plasmids RPL11(PSE-1) (9); R151(PSE-2) (6); Rmsl49(PSE-3) (1...