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Antimicrobial Agents and Chemotherapy, April 2000, p. 827-834, Vol. 44, No. 4
Department of Medicine, McMaster University,
Hamilton, Ontario, Canada,1 and
Howard M. Shapiro, M.D., P.C., West Newton,
Massachusetts2
Received 11 June 1999/Returned for modification 13 October
1999/Accepted 27 December 1999
Although flow cytometry has been used to study antibiotic effects
on bacterial membrane potential (MP) and membrane permeability, flow
cytometric results are not always well correlated to changes in
bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of
antibiotic-treated and untreated Staphylococcus aureus and
Micrococcus luteus cells. MP was calculated from the ratio
of red and green fluorescence of diethyloxacarbocyanine
[DiOC2(3)]. A normalized permeability parameter was
calculated from the ratio of far red fluorescence of the nucleic acid
dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial
counts were calculated by the addition of polystyrene beads to the
sample at a known concentration. Amoxicillin increased permeability
within 45 min. At concentrations of <1 µg/ml, some organisms showed
increased permeability but normal MP; this population disappeared after
4 h, while bacterial counts increased. At amoxicillin concentrations above 1 µg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused
smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 µg/ml did not change permeability, while a
tetracycline concentration of 4 µg/ml permeabilized 50% of the bacteria; 4 µg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on
permeability; chloramphenicol did not change either permeability or MP.
Erythromycin pretreatment of bacteria prevented streptomycin and
amoxicillin effects. Flow cytometry provides a sensitive means of
monitoring the dynamic cellular events that occur in bacteria exposed
to antibacterial agents; however, it is probably simplistic to expect
that changes in a single cellular parameter will suffice to determine
the sensitivities of all species to all drugs.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multiparameter Flow Cytometric Analysis
of Antibiotic Effects on Membrane Potential, Membrane Permeability, and
Bacterial Counts of Staphylococcus aureus and
Micrococcus luteus

*
Corresponding author. Mailing address: 283 Highland
Ave., West Newton, MA 02465-2513. Phone: (617) 965-6044 or (617)
576-0660. Fax: (617) 244-7110. E-mail: hms{at}shapirolab.com.
Present address: University of California, Los Angeles, Los
Angeles, CA 90095.
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