Direct Measurement of Membrane Potential and Resistance in Giant Cells of Escherichia Coli

Felle, Hubert; Stetson, David L.; Long, W. Scott; Slayman, Clifford L.

doi:10.1016/b978-0-12-225402-4.50077-2

Cited by 14 publications

(11 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although direct microelectrode recording of MP has been performed on Escherichia coli that have been artificially enlarged by the manipulation of cell wall synthesis (10), measurement of MP in actively growing bacteria is essentially impossible due to their small size. The distribution of radiolabeled lipophilic cations or charged lipophilic fluorescent dyes across the membrane (11), or the fluxes of potassium (K ϩ ) or rubidium (Rb ϩ ) ions across the membrane, have been used to estimate MP.…”

mentioning

confidence: 99%

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique

Perlmutter²,

et al. 1999

View full text Add to dashboard Cite

Background: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size‐ and MP‐sensitive measurement, and an MP‐independent, size‐sensitive measurement, should provide a better estimate of MP. Methods: Flow cytometry and spectrofluorometry were used to detect red (488 → >600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 → 530 nm) fluorescent. Results: In bacteria incubated with 30 μM dye, aggregate formation increases with the magnitude of the interior‐negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from −50 mV through −120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4°C and washing in glucose‐free medium. Conclusions: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms. Cytometry 35:55–63, 1999. © 1999 Wiley‐Liss, Inc.

show abstract

mentioning

confidence: 99%

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique

Perlmutter²,

et al. 1999

View full text Add to dashboard Cite

show abstract

“…The use of intracellular glass microcapillary electrodes for study of transmembrane potential differences (Osterhout (1925) for giant algal cells, Ling & Gerard (1949) for muscle and nerve), opened up a wide range of preparations for direct study of bioelectric phenomena: in secretion, in energy distribution and in energy conservation, as well as in excitability. Furthermore, over the past 10 years the use of ultrafine microcapillaries (tip diameters of 0-05-0 2 Itm: Brown & Flaming, 1977) has spread to a variety of unexpectedly small cells, including insect optic cells (Jensen & DeVoe, 1983), isolated cultured cells (Sherbet, 1978), small plant cells (Etherton, Keifer & Spanswick, 1977;Bates, Goldsmith & Goldsmith, 1982), fungal spheroplasts (Blatt & Slayman, 1983), and even certain bacteria (Felle, Stetson, Long & Slayman, 1979). At the same time, however, limitations of the microcapillary technique have become glaringly obvious: finite sealing resistances and flooding of cell cytoplasm with spurious salt can easily falsify all of the apparent electrical paramenters of punctured cells (Lassen, 1977;Nelson, Ehrenfeld & Lindemann, 1978;Page, Kelday & Bowling, 1981;Fromm & Schultz, 1981;Blatt & Slayman, 1983).…”

Section: Introductionmentioning

confidence: 99%

Membrane voltage, resistance, and channel switching in isolated mouse fibroblasts (L cells): a patch‐electrode analysis.

Hosoi¹,

Slayman²

1985

The Journal of Physiology

View full text Add to dashboard Cite

SUMMARY1. The whole-cell patch-electrode technique of Fenwick, Marty & Neher (1982) has been applied to single suspension-cultured mouse fibroblasts. Seals in the range of 10-50 GQ were obtained without special cleaning of the cell membranes.2. Rupture of the membrane patch inside the electrode was accompanied by a shift of measured potential into the range -10 to -25 mV, but in most cases with little change in the recorded resistance. The latter fact implied that (i) the absolute resistance of the cell membrane must be in the same range as the seal resistance and (ii) the recorded potential is a poor measure of actual cell membrane potential.3. Steady-state current-voltage curves (range -160 mV to + 80 mV) were generated before and after rupture ofthe membrane patch, and the difference between these gave (zero-current) membrane potentials of -50 to -75 mV, which represents a leak-corrected estimate of the true cell-membrane potential.4. The associated slope conductivity of the cell membrane was 5-15,US/cm2 (assumed smooth-sphere geometry, cells 13-15 ,sm in diameter) and was K+-dominated.5. With 0-1 mm (or more) free Ca2+ filling the patch electrode, membrane potentials in the range -60 to -85 mV were observed following patch rupture, with associated slope conductivities of 200-400 /ZS/cm2, also K+-dominated.6. Similar voltages and conductivities were observed at the peak of pulse-induced 'hyperpolarizing activation' (Nelson, Peacock, & Minna, 1972), and the two phenomena probably reflect the behaviour of Ca2+-activated K+ channels.7. Both the pulse-induced conductance and the Ca2+-activated conductance spontaneously decayed, the latter over periods of 5-15 min following patch rupture.8. Sr2+, Ba2+, and Co2+ could also activate the putative K+ channels, but only Sr2+ really mimicked Ca2+. Co2+ and Ba2+ activated with a delay of several minutes following patch rupture, and deactivated quickly with a small decrease ofconductance and a large decrease of membrane potential. Evidently, Co2+ and Ba2+ affect channel specificity as well as channel opening and closing kinetics.

show abstract

“…[78,79,[131][132][133][134], but cf. [135]) based on bilayer-mediated equilibration according to the Nernst equation [136]. However, we recognise that such cyanine dyes, much as ethidium bromide [120] and other xenobiotics [137,138], are likely to be both influx and efflux substrates for various transporters [139], so such an interpretation should be treated with some caution.…”

Section: Discussionmentioning

confidence: 99%

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Jindal

Thampy

Day

et al. 2018

Preprint

View full text Add to dashboard Cite

Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4h, and reinoculated into fresh media at a concentration (10 5 .mL -1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energisation (using a carbocyanine dye) and DNA distribution. It turned out that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of reinoculation, providing the possibility of a very rapid antibiotic susceptibility test, on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential Urinary Tract Infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

show abstract

Direct Measurement of Membrane Potential and Resistance in Giant Cells of Escherichia Coli

Cited by 14 publications

References 8 publications

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique

Membrane voltage, resistance, and channel switching in isolated mouse fibroblasts (L cells): a patch‐electrode analysis.

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Contact Info

Product

Resources

About