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Evaluation of five different methods to prepare bacterial extracts for the identification of beta-lactamases by isoelectric focusing

Arstila, T.; Jacoby, G.A.; Huovinen, P.

Journal of Antimicrobial ChemoTherapy 32(6): 809-816

1993


ISSN/ISBN: 0305-7453
PMID: 8144421
DOI: 10.1093/jac/32.6.809
Accession: 008634100

The X-PRESS, osmotic shock, chloroform treatment, lysozyme treatment and ultrasonic disruption methods to release five different plasmid-mediated beta-lactamases from Escherichia coli and one chromosomal beta-lactamase from Enterobacter cloacae were compared. The main activities of TEM-1, SHV-1, OXA-1, OXA-2, PSE-4 and chromosomal P99 beta-lactamases were found at the same isoelectric point irrespective of the method used. However, additional satellite bands were found with TEM-1, OXA-1, OXA-2 and PSE-4 beta-lactamases released by the lysozyme method. In addition, beta-lactamase released by osmotic shock treatment was found to be unstable during storage at -20 degrees C or during the 18 h period of iso-electric focusing at +4 degrees C. Chloroform treatment produced similar band patterns and at least as good an enzyme yield as ultrasonic disintegration and was equally simple and fast to perform.

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