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J Immunol Methods. 1988 Apr 6;108(1-2):153-8.

Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry.

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1
Department of Medicine, UCLA-Center for the Health Sciences, Los Angeles, CA 90024.

Abstract

We developed a dual wavelength spectrophotometric assay that permitted beta-lactamase and beta-galactosidase activities to be measured concurrently in a single sample. We also constructed a target cell, E. coli ML-35p, that was substantially cryptic for its periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless outer membrane (beta-lactamase) or inner membrane (beta-galactosidase) permeabilization occurred. By applying the spectrophotometric assay to whole target cells, we could ascertain the kinetics of inner and outer membrane permeabilization by biological agents, including serum, polymyxin B and mellitin. By monitoring the reactions at an additional wavelength, we could also follow the kinetics of serum-mediated bacteriolysis. These experiments illustrate the principle of multiple wavelength spectrophotometry and provide examples of its use to monitor and dissect the action of biological agents on a gram-negative bacterium.

PMID:
3127470
DOI:
10.1016/0022-1759(88)90414-0
[Indexed for MEDLINE]

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