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

J Immunol Methods. 1988 Apr 6;108(1-2):153-8. doi: 10.1016/0022-1759(88)90414-0.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriolysis* / drug effects
  • Blood Physiological Phenomena
  • Cell Membrane Permeability* / drug effects
  • Cell Wall / drug effects
  • Cell Wall / physiology*
  • Cephalosporins / metabolism
  • Chromogenic Compounds
  • Escherichia coli / drug effects
  • Escherichia coli / enzymology
  • Escherichia coli / physiology*
  • Humans
  • Melitten / pharmacology
  • Nitrophenylgalactosides / metabolism
  • Polymyxin B / pharmacology
  • Spectrophotometry*
  • Substrate Specificity
  • beta-Galactosidase / metabolism


  • Cephalosporins
  • Chromogenic Compounds
  • Melitten
  • Nitrophenylgalactosides
  • pyridine-2-azo-4-dimethylaniline cephalosporin
  • beta-Galactosidase
  • Polymyxin B