This study was performed to investigate the effects of pulsed electric fields on the membrane permeabilization in two gram-positive and two gram-negative bacterial strains to obtain an understanding of the mechanism of bacterial inactivation by pulsed electric fields.
The experiments were conducted with different electric field strengths, exposure times, and medium pH values (pH 4.0 and 7.0). Additionally, both gram-positive and gram-negative bacteria were investigated to discover possible differences in their reactions to pulsed electric field exposure due to their different cell membrane properties.
The uptake of the fluorescent dye propidium iodide into the cell was measured as it is a sensitive marker of cell membrane integrity. The occurence of this dye in the cell indicates loss of membrane integrity. Propidium iodide uptake was measured before and after pulsed electric field exposure to characterize whether the membrane permeabilization was reversible or not.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
1 Hz
Modulation type:
pulsed
Exposure duration:
not given in this article
|
|
|
Exposure 2:
2 Hz
Modulation type:
pulsed
Exposure duration:
not given in this article
|
|
|
Exposure 3:
Modulation type:
single pulse
Exposure duration:
single pulse of 50 µs
|
|
|
Exposure 4:
Modulation type:
single pulse
Exposure duration:
single pulses of 12,5 - 100 µs
|
|
|
Exposure 5:
Modulation type:
single pulse
Exposure duration:
single pulses of 10 - 400 µs
|
|
cells were stained with PI a) before exposure b) after exposure
| Frequency | 1 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | not given in this article |
| Modulation type | pulsed |
|---|---|
| Pulse width | 2 µs |
| Repetition frequency | 1 Hz |
| Exposure source |
|
|---|---|
| Setup | cylindrical plastic tube with two polished stainless steel electrodes - 0.25 cm apart and with an area of 2.01 cm² - was used as chamber |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| electric field strength | 25 kV/cm | - | measured | - | - |
| Frequency | 2 Hz |
|---|---|
| Type | |
| Waveform | |
| Exposure duration | not given in this article |
| Modulation type | pulsed |
|---|---|
| Pulse width | 2 µs |
| Repetition frequency | 2 Hz |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| electric field strength | 12 kV/cm | - | measured | - | - |
| electric field strength | 19 kV/cm | - | measured | - | - |
| Modulation type | single pulse |
|---|---|
| Pulse width | 50 µs |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| electric field strength | 10 kV/cm | minimum | measured | - | - |
| electric field strength | 25 kV/cm | maximum | measured | - | - |
| Modulation type | single pulse |
|---|---|
| Pulse width | 12.5 µs |
| Additional info |
pulse width: 12.5 - 100 µs |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| electric field strength | 19 kV/cm | - | measured | - | - |
| Modulation type | single pulse |
|---|---|
| Pulse width | 10 µs |
| Additional info |
pulse width: 10 - 400 µs |
| Exposure source |
|
|---|
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| electric field strength | 25 kV/cm | maximum | measured | - | - |
Investiagtions at pH 7.0 showed positive correlation between degree of membrane permeabilization, exposure time, and electric field strength applied in all bacterial strains. In gram-negative bacteria all permeabilized cells died (both reversible and permanent permeabilized cells). In gram-positive bacteria only cells with permanent membrane permeabilization died. The membrane permeabilization was partially reversible in all bacterial strains.
Investigations at pH 4.0 revealed a relationship between loss of cell viability with permanent loss in cell membrane integrity in Listeria monocytogenes, but in Lactobacillus plantarum the degree of permeabilization was higher, and in the gram-negative bacterial strains the permeabilization much lower than the percentage of inactivated cells. The permeabilization in all bacterial strains was permanent.
These results confirm that membrane permeabilization is involved in the mechanism of bacterial inactivation by pulsed electric fields, but the nature of membrane damage and its relationship to cell death depends on the bacterial species and the medium pH value.
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