This document summarizes research demonstrating that high electric fields can kill bacteria and yeasts. The researchers applied direct current pulses up to 25 kV/cm to suspensions of various microorganisms. They found that the electric fields caused death that was not due to electrolysis products or temperature increases. Higher field strengths and longer total pulse times resulted in greater killing of microbes. The sensitivity to electric fields varied between species, with yeasts being more sensitive than bacteria. The research suggests electric fields can kill microbes through a non-thermal mechanism.
Microbial fuel cells could be used to the study growth rates of aerobic microbial species on the basis of voltage produced by them
in the microbial fuel cell assembly. A fresh culture of Rhizobium leguminosarum was added in the anode chamber of a microbial
fuel cell assembly and subsequent voltage produced by it was recorded after every fifteen minutes. The 24 ml/hr of air was
pumped in the anode chamber to maintain the dissolved oxygen level and resistance of 12 ohm was applied across the electrodes.
This process was studied in triplicates and voltage data was recorded. The graph plotted of voltage against time suggested the
growth curve of the species in the microbial fuel cell system. It was found that voltage gradually increased with time ranging from
50 mV to 190 mV with a supply of oxygen in the anode, but it declines gradually to zero in absence of aeration with time and
depletion of nutrients.
The effect of nonthermal plasma needle on staphylococus aureus bacteriaIbrahim Karim
The reduction of the use of antibiotics and dependence on devices and tools that link between physics and
modern therapeutic medicine led to a modern leap in the treatment of patients. In this research was designed nonthermal
plasma needle system at the normal atmosphere pressure and working on argon gas, the system was applied to
gram positive Staphylococcus aureus bacteria isolated from patients with burns from Yarmouk Teaching Hospital,
wounds and Burns Division. The bacteria were exposed to non-thermal plasma generated at different time intervals and
at two values of voltages (4.9, 8). The killing percentage depends on the non-thermal plasma needle system operating
conditions such as gas flow, applied voltage on bacteria and distance between tip needle and isolates (Staphylococcus
aureus) bacteria, the perfect and partial killing percentage increase with the applied voltage increasing. The result
showed various killing percentage were obtained for the bacteria. A perfect killing percentage of these bacteria was
obtained at time (6 min) and the applied voltage were (8 kV) and gas flow (5 l/min).
Effect of temp. on venom of bungarus caeruleusAnju Rana
Common krait is a small snake but its venom is highly poisonous. The venom is neurotoxic and in my research, an effort was done to see the effect of temperatue on venom.
ABSTRACT In the present study, the extracellular synthesis of Silver nanoparticles was done using two different bacterial strains viz. Bacillus flexus and Bacillus pseudomycoides. The silver nanoparticles were confirmed by in color test and characterized by UV-Visible spectroscopy, the λmax observed at 430 nm and 410 nm confirmed the synthesis of AgNPs. FTIR analysis confirms the presence of elemental silver and reveals the dual function of the biological molecule responsible for the reduction and stabilization of AgNPs in the aqueous medium. The XRD showed that silver nanoparticles produced are crystalline in nature with size ranges from 30 to 70 nm. The SEM shows that produced silver nanoparticles are spherical, Pseudo spherical in shape with traces of agglomeration. Further through investigation of Antibiotic Sensitivity/Resistant pattern expressed that out of eighteen virulent enteric bacterial isolates, three isolates showed MAR index equal to 1, which indicates the presence of multiple drug resistance (MDR). MIC values of AgNPs against MDR isolate E7 and K3 was established to be 80 μg/ml whereas, for isolate Sa1 the MIC value was 70 μg/ml. The synergistic effect of antibiotics in conjugation with biologically synthesized AgNPs encourage the susceptibility amongst the tested bacterial cultures; viz. Salmonella followed by Klebsiella and E. coli.
Key-words: Biosynthesis, Synergistic activity, Antibacterial activity, Silver nanoparticles, Multidrug-resistant (MDR)
Microbial fuel cells could be used to the study growth rates of aerobic microbial species on the basis of voltage produced by them
in the microbial fuel cell assembly. A fresh culture of Rhizobium leguminosarum was added in the anode chamber of a microbial
fuel cell assembly and subsequent voltage produced by it was recorded after every fifteen minutes. The 24 ml/hr of air was
pumped in the anode chamber to maintain the dissolved oxygen level and resistance of 12 ohm was applied across the electrodes.
This process was studied in triplicates and voltage data was recorded. The graph plotted of voltage against time suggested the
growth curve of the species in the microbial fuel cell system. It was found that voltage gradually increased with time ranging from
50 mV to 190 mV with a supply of oxygen in the anode, but it declines gradually to zero in absence of aeration with time and
depletion of nutrients.
The effect of nonthermal plasma needle on staphylococus aureus bacteriaIbrahim Karim
The reduction of the use of antibiotics and dependence on devices and tools that link between physics and
modern therapeutic medicine led to a modern leap in the treatment of patients. In this research was designed nonthermal
plasma needle system at the normal atmosphere pressure and working on argon gas, the system was applied to
gram positive Staphylococcus aureus bacteria isolated from patients with burns from Yarmouk Teaching Hospital,
wounds and Burns Division. The bacteria were exposed to non-thermal plasma generated at different time intervals and
at two values of voltages (4.9, 8). The killing percentage depends on the non-thermal plasma needle system operating
conditions such as gas flow, applied voltage on bacteria and distance between tip needle and isolates (Staphylococcus
aureus) bacteria, the perfect and partial killing percentage increase with the applied voltage increasing. The result
showed various killing percentage were obtained for the bacteria. A perfect killing percentage of these bacteria was
obtained at time (6 min) and the applied voltage were (8 kV) and gas flow (5 l/min).
Effect of temp. on venom of bungarus caeruleusAnju Rana
Common krait is a small snake but its venom is highly poisonous. The venom is neurotoxic and in my research, an effort was done to see the effect of temperatue on venom.
ABSTRACT In the present study, the extracellular synthesis of Silver nanoparticles was done using two different bacterial strains viz. Bacillus flexus and Bacillus pseudomycoides. The silver nanoparticles were confirmed by in color test and characterized by UV-Visible spectroscopy, the λmax observed at 430 nm and 410 nm confirmed the synthesis of AgNPs. FTIR analysis confirms the presence of elemental silver and reveals the dual function of the biological molecule responsible for the reduction and stabilization of AgNPs in the aqueous medium. The XRD showed that silver nanoparticles produced are crystalline in nature with size ranges from 30 to 70 nm. The SEM shows that produced silver nanoparticles are spherical, Pseudo spherical in shape with traces of agglomeration. Further through investigation of Antibiotic Sensitivity/Resistant pattern expressed that out of eighteen virulent enteric bacterial isolates, three isolates showed MAR index equal to 1, which indicates the presence of multiple drug resistance (MDR). MIC values of AgNPs against MDR isolate E7 and K3 was established to be 80 μg/ml whereas, for isolate Sa1 the MIC value was 70 μg/ml. The synergistic effect of antibiotics in conjugation with biologically synthesized AgNPs encourage the susceptibility amongst the tested bacterial cultures; viz. Salmonella followed by Klebsiella and E. coli.
Key-words: Biosynthesis, Synergistic activity, Antibacterial activity, Silver nanoparticles, Multidrug-resistant (MDR)
Combined application of sub-toxic level of silver nanoparticles with low powe...Nanomedicine Journal (NMJ)
Objective(s):
Electromagnetic radiations which have lethal effects on the living cells are currently also considered as a disinfective physical agent.
Materials and Methods:
In this investigation, silver nanoparticles were applied to enhance the lethal action of low powers (100 and 180 W) of 2450 MHz electromagnetic radiation especially against Escherichia coli ATCC 8739. Silver nanoparticles were biologically prepared and used for next experiments. Sterile normal saline solution was prepared and supplemented by silver nanoparticles to reach the sub-inhibitory concentration (6.25 μg/mL). Such diluted silver colloid as well as free-silver nanoparticles solution was inoculated along with test microorganisms, particularly E. coli. These suspensions were separately treated by 2450 MHz electromagnetic radiation for different time intervals in a microwave oven operated at low powers (100 W and 180 W). The viable counts of bacteria before and after each radiation time were determined by colony-forming unit (CFU) method.
Results:
Results showed that the addition of silver nanoparticles significantly decreased the required radiation time to kill vegetative forms of microorganisms. However, these nanoparticles had no combined effect with low power electromagnetic radiation when used against Bacillus subtilis spores.
Conclusion:
The cumulative effect of silver nanoparticles and low powers electromagnetic radiation may be useful in medical centers to reduce contamination in polluted derange and liquid wastes materials and some devices.
Keywords
Electromagnetic Radiation; Silver nanoparticles; disinfection process
Radial immunodiffusion (RID) or Mancini method, Mancini immunodiffusion or single radial immunodiffusion (SRID) assay is an immunodiffusion technique used in immunology to determine the quantity or concentration of an antigen in a sample
Effect of cold atmospheric pressure plasma needle on DNAiosrjce
Non-thermal argon plasma needle at atmospheric pressure was constructed. The experimental setup
was based on a simple and low cost electric components that generate a sufficiently high electric field at the
electrodes to ionize various gases which flow at atmospheric pressure. A high AC power supply was used with
1.1Kv and 19.57KHz. Non-thermal atmospheric pressure plasma has much potential for medical applications.
The atmospheric pressure plasma needle contains almost equal amounts of positive and negative charges. In
this research we focused on the possible biomedical application of the atmospheric pressure plasma needle,
where we used plasma ignited in Argon (Ar) gas flow to cause strand breaks in DNA. However, the mechanism
by which plasma destroys DNA is not yet understood. In this study we investigated the effects of exposure time
and distance between the plasma source and the DNA sample. In this study we found that plasma does indeed
alter DNA. We came to this conclusion through qualitative interpretations using a standard gel electrophoresis
technique on DNA samples treated with the atmospheric pressure plasma needle.
The current research aimed at fabricating plant extract mediated biosynthesized silver nanoparticles (AgNPs) utilizing thorn extract of Bombax ceiba (TEBC). The synthesized AgNPs was characterized by UV spectroscopy where the surface plasmonic resonance peak (SPR) was located at 222 nm. The scanning electron microscopy (SEM) studies demonstrated that the morphology of fabricated nanomaterials was primarily cylindrical of average size of 20-30 nm with some spindles of size >50 nm. The anti-microbial evaluation against Staphylococcus aureus revealed that AgNPs exhibited notable activity with ZOI of 27.2 mm at MIC of 25 μg/mL. The outcome of this research evidently signified that the biofabricated AgNPs using TEBC may be a new greener approach or technology to formulate anti-bacterial nanodrugs in future.
High voltage pulse technique or High intensity pulsed electric field processing involves the application of electric pulses of high voltage
20-80 kV/cm to the food placed between two electrodes.
The applied electric field create a pores on the cell membrane, thus the phenomenon known as Electroporation or electropermeabilization.
The effect of Electroporation can be divided into four steps:
An increase in the transmembrane potential
Pore formation
Evolution of the number and size of the pores
Pore resealing
Combined application of sub-toxic level of silver nanoparticles with low powe...Nanomedicine Journal (NMJ)
Objective(s):
Electromagnetic radiations which have lethal effects on the living cells are currently also considered as a disinfective physical agent.
Materials and Methods:
In this investigation, silver nanoparticles were applied to enhance the lethal action of low powers (100 and 180 W) of 2450 MHz electromagnetic radiation especially against Escherichia coli ATCC 8739. Silver nanoparticles were biologically prepared and used for next experiments. Sterile normal saline solution was prepared and supplemented by silver nanoparticles to reach the sub-inhibitory concentration (6.25 μg/mL). Such diluted silver colloid as well as free-silver nanoparticles solution was inoculated along with test microorganisms, particularly E. coli. These suspensions were separately treated by 2450 MHz electromagnetic radiation for different time intervals in a microwave oven operated at low powers (100 W and 180 W). The viable counts of bacteria before and after each radiation time were determined by colony-forming unit (CFU) method.
Results:
Results showed that the addition of silver nanoparticles significantly decreased the required radiation time to kill vegetative forms of microorganisms. However, these nanoparticles had no combined effect with low power electromagnetic radiation when used against Bacillus subtilis spores.
Conclusion:
The cumulative effect of silver nanoparticles and low powers electromagnetic radiation may be useful in medical centers to reduce contamination in polluted derange and liquid wastes materials and some devices.
Keywords
Electromagnetic Radiation; Silver nanoparticles; disinfection process
Radial immunodiffusion (RID) or Mancini method, Mancini immunodiffusion or single radial immunodiffusion (SRID) assay is an immunodiffusion technique used in immunology to determine the quantity or concentration of an antigen in a sample
Effect of cold atmospheric pressure plasma needle on DNAiosrjce
Non-thermal argon plasma needle at atmospheric pressure was constructed. The experimental setup
was based on a simple and low cost electric components that generate a sufficiently high electric field at the
electrodes to ionize various gases which flow at atmospheric pressure. A high AC power supply was used with
1.1Kv and 19.57KHz. Non-thermal atmospheric pressure plasma has much potential for medical applications.
The atmospheric pressure plasma needle contains almost equal amounts of positive and negative charges. In
this research we focused on the possible biomedical application of the atmospheric pressure plasma needle,
where we used plasma ignited in Argon (Ar) gas flow to cause strand breaks in DNA. However, the mechanism
by which plasma destroys DNA is not yet understood. In this study we investigated the effects of exposure time
and distance between the plasma source and the DNA sample. In this study we found that plasma does indeed
alter DNA. We came to this conclusion through qualitative interpretations using a standard gel electrophoresis
technique on DNA samples treated with the atmospheric pressure plasma needle.
The current research aimed at fabricating plant extract mediated biosynthesized silver nanoparticles (AgNPs) utilizing thorn extract of Bombax ceiba (TEBC). The synthesized AgNPs was characterized by UV spectroscopy where the surface plasmonic resonance peak (SPR) was located at 222 nm. The scanning electron microscopy (SEM) studies demonstrated that the morphology of fabricated nanomaterials was primarily cylindrical of average size of 20-30 nm with some spindles of size >50 nm. The anti-microbial evaluation against Staphylococcus aureus revealed that AgNPs exhibited notable activity with ZOI of 27.2 mm at MIC of 25 μg/mL. The outcome of this research evidently signified that the biofabricated AgNPs using TEBC may be a new greener approach or technology to formulate anti-bacterial nanodrugs in future.
High voltage pulse technique or High intensity pulsed electric field processing involves the application of electric pulses of high voltage
20-80 kV/cm to the food placed between two electrodes.
The applied electric field create a pores on the cell membrane, thus the phenomenon known as Electroporation or electropermeabilization.
The effect of Electroporation can be divided into four steps:
An increase in the transmembrane potential
Pore formation
Evolution of the number and size of the pores
Pore resealing
Electroporation-A simple notable technique in our science field.Technique which is a magic of electric voltage.Technique that works on the mechanism of transient aqueous pore model.
Control of metabolic activities of E.coli and S. aureus bacteria by Electric ...researchinventy
Low electric currents generated using conductive electrodes have been used to increase the efficacy of antibiotics against bacterial biofilms, a phenomenon termed “the bioelectric effect” that formed metal ions and free radicals which can inhibit the growth of planktonic Staphylococcus aureus (S.aureus) and Escherichia Coli (E.Coli) the effect is amplitude and frequency dependent, the aim of present study to define the parameters that are most effective against bacterial growth also to investigate the comparative study through inactivation of metabolic activities, growth rate, morphology, bacterial conductivity and antibiotic sensitivity between gram negative E.Coli and gram positive S.aureus bacteria by extremely low frequency electric field (ELF-EF). In this work, the frequency of electric impulses that interfere with the bioelectric signals generated during E.Coli and S.aureus cellular division is investigated in order to compare cell viability, number of colony forming units (CFU) and growth rate (optical density at 600nm) bacterial conductivity and antibiotic susceptibility. Also morphological cellular structure was investigated by transmission electron microscope (TEM). The results revealed that a highly significant inhibition effect occurred when S.aureus and E.Coli was exposed to resonance of 0.8, 0.5 Hz square amplitude modulated waves (QAMW) respectively for 2hours exposure .Moreover, exposed cells became more sensitive to the tested antibiotics compared to control. Significant ultra-structural changes occurred as observed by TEM which indicated morphological changes. It will be concluded that, the use of 0.8, 0.5 Hz QAMW in controlling the biological activity of S.aureus and E.coli respectively seems to be a new and promising medical activity
Control of metabolic activities of E.coli and S. aureus bacteria by Electric ...inventy
Low electric currents generated using conductive electrodes have been used to increase the efficacy of antibiotics against bacterial biofilms, a phenomenon termed “the bioelectric effect” that formed metal ions and free radicals which can inhibit the growth of planktonic Staphylococcus aureus (S.aureus) and Escherichia Coli (E.Coli) the effect is amplitude and frequency dependent, the aim of present study to define the parameters that are most effective against bacterial growth also to investigate the comparative study through inactivation of metabolic activities, growth rate, morphology, bacterial conductivity and antibiotic sensitivity between gram negative E.Coli and gram positive S.aureus bacteria by extremely low frequency electric field (ELF-EF). In this work, the frequency of electric impulses that interfere with the bioelectric signals generated during E.Coli and S.aureus cellular division is investigated in order to compare cell viability, number of colony forming units (CFU) and growth rate (optical density at 600nm) bacterial conductivity and antibiotic susceptibility. Also morphological cellular structure was investigated by transmission electron microscope (TEM). The results revealed that a highly significant inhibition effect occurred when S.aureus and E.Coli was exposed to resonance of 0.8, 0.5 Hz square amplitude modulated waves (QAMW) respectively for 2hours exposure .Moreover, exposed cells became more sensitive to the tested antibiotics compared to control. Significant ultra-structural changes occurred as observed by TEM which indicated morphological changes. It will be concluded that, the use of 0.8, 0.5 Hz QAMW in controlling the biological activity of S.aureus and E.coli respectively seems to be a new and promising medical activity.
It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper.
It gives sharper bands.
Provides a good background for staining glycoproteins.
ADVANTAGE:
No tailing of proteins or hydrophilic materials.
Available in wide range of particle size and layer thickness.
Give sharp bands and offer good resolution.
High voltage can be applied which will enhance the resolution.
Effects of electric field on histopathological study, electrical properties a...inventy
The present work was undertaken in order to investigate the effects of electric field (EF) of strength 50Hz-3KV/m on the histopathology, dielectric properties and liver function tests in albino rats. Fifty male albino rats were equally divided into three groups namely A, B, and C. Animals of group A used as control group which didn't receive any treatment . Animals of group B was divided into two subgroups namely B1 and B2 which were discretely exposed to 50HZ, 3KV/m electric field for a period of 15 day (8 hours/day, 5day/week). Group B2 animals were left to survive and housed at normal environmental conditions similar to control group A for a period of 15 day post exposed. Animals of group C are divided into two subgroups namely C1 and C2 were discretely exposed to the electric field for a period of 30 day (8 hours/day, 5day/week). Group C2 animals were left to survive and housed at normal environmental conditions similar to control group A for a period of 15 day post exposed. At the end of this period, blood and tissues samples were collected from all groups for experimental investigations. The dielectric constant (έ), electrical conductivity (σ) was measured in frequency range 42Hz-5MHz to investigate any changes in liver structure through studding histopathological examination. Also, the liver function was studied through analysis of glutamic oxaloacetic transaminase (GOT), glutamic pyruvie transaminase (GPT) and total protein (TP) after exposure to electric field this biochemical parameters have been evaluated in the blood serum of rats. The obtained results show high significant changes in the value of έ and σ of liver tissues for all groups exposed to EF as compared with control group. The levels of GOT and GPT were increased up to four times their values during the period of exposure to EF. These variations were recovered during two week after stopping exposure but they did not return to its original control values before exposure. On microscopic level; liver histological observations in liver cells which revealed some alterations including hepatic tissue with two portal tracts showing mild florous expansion and a dilated central vein, also ghosts of hepatocytes denoting necrotic changes also shows hepatic tissue with dilated central veins engorged with blood and splitting out to adjacent hepatocytes.
Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.
Similar to 1967 sale effect of high electric fields on microorganisms_killing bacteria and yeasts (20)
More information can be found on:
- www.purepulse.eu
- http://www.pinterest.com/toptechtalks/purepulse-pef-20/
- http://en.topwiki.nl/index.php/PurePulse_-_PEF_2.0
More information can be found on:
- www.purepulse.eu
- http://www.pinterest.com/toptechtalks/purepulse-pef-20/
- http://en.topwiki.nl/index.php/PurePulse_-_PEF_2.0
Lunchlezing op het ministerie van EZ te Den Haag over Disruptieve Innovatie, de transitie naar de 6e cyclus van kondratief, voedselinnovaties en voedseltechnologie. De grote PLOF's
see more on:
wdeheij.blogspot.com
follow @deheij on twitter
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www.topwiki.nl
www.top-bv.nl
www.toptechnologytalks.nl
www.foodforfood.info
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1967 sale effect of high electric fields on microorganisms_killing bacteria and yeasts
1. BIOCHI1VflCA ET BIOPHYSICA ACTA 781
BBA 25 876
EFFECTS OF HIGH ELECTRIC FIELDS ON MICROORGANISMS
I. KILLING OF BACTERIA AND YEASTS
A. J. H. SALE AND W. A. HAMILTON*
Unilever Research Laboratory, ColworthHouse, Sharnbrook, Bedford (GreatBritain)
(Received July 7th, 1967)
SUM~C[ARY
A lethal effect of high electric fields on a number of species of vegetative bacteria
and yeasts has been demonstrated. Fields up to 25 kV/cm have been applied as a
series of direct current pulses to suspensions of the organisms. Death of the organisms
was not due to tile products of electrolysis; the temperature rise of the suspension
was small and did not cause the lethal effect. The degree of kill of a population was
determined by the product of the pulse length and number of pulses, and by tile
field strength in the suspension. The various species differed in their sensitivity to
the electric field, the yeasts being more sensitive than the vegetative bacteria.
INTtiODUCTION
Most of the investigations into tile bactericidal action of electric fields have
been carried out at radio frequencies. In I949 BUI~TON~ reviewed the literature, in
whiclh there are accounts both for and against the existence of bactericidal effects.
There is some uncertainty concerning the heating effects, but it seems that a non-
thermal bactericidal effect might occur if high enough fields are applied. Amongst
the more recent literature the highest field strength appears to be 2 kV/cm reported
by INGRAMAND PAGE 2, who found no lethal effect.
Few reports of the effects of direct current (d.c.) pulses have appeared.
GOSSLING3 claimed that, if the power density is high enough, micro-organisms may
have mutations induced and may be destroyed. FEDOROVAND ROGOV4 reported a
bactericidal effect in milk, attributing the effect to impact and cavitation. Inactivation
of micro-organisms is also reported by DOEVENSPECK5.
We have extended these studies to high field strengths using d.c. pulses up to
25 kV/cm to try to find out whether a non-thermal bactericidal effect exists. In
particular we have attempted to establish which of a number of parameters controlled
the lethal effect on micro-organisms.
If a bactericidal effect due specifically to very high electric fields is to be demon-
strated, the fields must be applied in very short pulses with long intervals between
pulses, to minimise the temperature rise.
* Present address: Department of Biological Chemistry, University of Aberdeen, Scotland.
Biochim. Biophys. Acta, 148 (1967) 781-788
2. 782 A.J. lifo SALE; W. A. ;frA2vflLTON
METHODS AND MATERIALS
Aj)~ara~us
A pulse generator was built to provide approximately rectangular d.c. pulses
into a resistive toad; the design followed conventional practice with no notable novel
features. The pulse voltage was continuously adjustable up to io kV and the pulse
length could be varied from 2 to 20 ~sec in steps of 2/zsec. The pulse repetition rate
was one per second and the number of pulses per treatment was varied as required.
The pulses were monitored by an oscilioscope and could be photographed. Fig. z
illustrates the shapes of the voltage pulse applied to the treatment cell and the current
pulse passing through it.
The treatment cell, which forms the load, is illustrated in Fig. 2. The sample
was bounded by the carbon electrodes, the poiythene spacer and air, so that apart
from the meniscus and any crevices, the whole of the sample received uniform treat-
ment. By using a variety of spacers the electrode area and thickness of sample could
be varied, and hence the electrical resistance and electric field. The field strength
was limited to less than 3o kV/cm by the electrical breakdown of the air above the
sample. Provision was made for temperature control by the circulation of water
through the brass blocks that support the carbon electrodes. The water was at room
temperature (approx. 2o°) unless otherwise stated.
Fig. I. Voltage and current pulse shapes 20 #sec).
Biochim. Biophys. Acta, 148 ~i967) 781-788
4. 784 A.j.H. SALE, W. A. HA2VflLTON
RESULTS
Lethal effect of d.c. pulses
In preliminary experiments d.c. pulses -wereapplied to suspensions of the various
vegetative bacteria and yeasts listed under organisms. There was a tethai effect when
the pulse amplitude was high enough. E. coli was chosen as the test organism, for
a study of the parameters likely to influence the kill. The sensitivity of the cel!s to
d.c. pulses was not influenced by the stage of growth at which the ceils were harvested,
by the presence or absence of oxygen during growth, nor by varying the pH of the
suspending medium from 4 to 9- Therefore approximately neutra! NaC1 solutions were
used as the suspending media for the study of the effects of the electrical conditions
on the degree of kill, which was measured in terms of percentage survival of the
population.
Insig~cifica~ce of electrolysis
Electrolysis occurs under d.c. conditions and this was visible as a burst of gas
generated at the electrode surface by each pulse; simultaneously a marked disturbance
of the liquid could be seen. The possibility that the products of electrolysis might be
causing the lethal effect had to be examined. If the pulse treatment was carried out
with the organisms suspended in a gel, one would expect that the gases produced
by electrolysis would only contact the gel surfaces adjacent to the electrodes and
not penetrate the gel. So examination of the organisms in the gel should reveal
whether electrolysis was responsible for the lethal effect. First it was necessary to
show that the products of electroiysis did not penetrate the gel. A molten nutrient
agar containing Io % rezazurin was put into the treatment cell and allowed to set.
After a number of pulses the gel was removed. Only the surface next to the cathode
had become pink, so the nascent hydrogen had not diffused into the agar. Next a
suspension of E. coZiin o.1% peptone water was mixed with molten agar at 48 ° and
put in the treatment cell and allowed to set. After a number of pulses the gel was
removed and thin slices cut so that. viewing across the agar. one edge had been
adjacent to the cathode and the other to the anode. The slices were placed on a
microscope slide and under a cover slip, and incubated in moist conditions at 37 °
for 4 h. The agar was then examined for microcolonies and compared with similar
microcultures of untreated suspensions Live organisms would give rise to micro-
colonies whereas dead ones would remain as single organisms. There were many
colonies throughout the untreated suspension. In the treated preparation there were
few colonies but many single orgamsms, which could be seen uniformly distributed
throughout the agar. Therefore the kill occurred even where the products of elec-
trolysis were absent.
Insig~ifiea~ee of heating
In many experiments it would have been possible for the amount of energy
supplied in pulses to the suspensions in the treatment cell to have markedly heated
the liquid if there had not been any cooling by the thermal capacity of the cell and
the cooling water. It was not practicable to have a thermoeouple present during the
treatment and when one was introduced into the suspension afterwards no significant
rise was registered.
l?iochim, Biophys. AclG 148 (i967) 781-788
5. KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 785
However, it was possible to obtain the temperature rise of the suspension itself
during an actual treatment from the change of its electrical resistance. The suspending
medium was an NaCl solution, of which the resistance changes by 2.4 % per degree.
The resistance change was found from the amplitudes of the voltage and current
pulses of the treatment. Although the accuracy was limited, a temperature change
of 5° was readily detected. The maximum temperature rise found in this way was IO°,
which appears among the examples of a number of treatments of IO pulses of 2o/,see
quoted in Table I.
In the table the energy input for each treatment has also been calculated. The
energies expressed in cal/cm~ would be numerically equal to the temperature rises
in degrees that could have occurred if there had not been any cooling, but the actual
temperature rises were found to be much less. The energy inputs for very many con-
ditions of voltage, current, conductivity, number of pulses and pulse length have
been gathered together in Fig. 3, where the percent survival has been plotted against
the energy input. There was no correlation between the energy and the degree of kill.
Effect of time of treatment
When the number of pulses was increased, while keeping the other conditions
constant, the degree of kill increased rapidly at first and then more slowly. The effect
of altering the pulse length was that when the length was shortened more pulses
were required to achieve the same degree of kill. If these two effects were combined,
it was found that the degree of kill appeared related to the product of the number
of pulses and the pulse length, that is, to the total time for which the voltage is
actually applied to the cell. This is illustrated by the example in Fig. 4, which shows
TABLE I
EFFECT OF VARYING THE ELECTRIC FIELD AND CONDUCTIVITY OF THE 1VIEDIU1ViON CURRENT DENSITY,
ENERGY, TEMPERATURE RISE AND DEGREE OF KILL OF E. coli
Treatment consisted of I0 pulses of 20 #sec at room temp. (approx. 20°).
Conductivity Electric field Current density Energy Temperature Survivors
(mr2-1 ) (k V / cm ) (A /cm ~) (cal/cm3) rise (% )
0.8 lO. 5 8 4 <5 21
0.8 21 17 17 <5 <i
1.6 lO. 5 17 8 o 4°
1.6 21 34 34 5 i
3.2 lO.5 34 17 <5 22
1.6 5.3 8 2 o 94
3 .2 4.9 16 4 o ioo
3 .2 18.5 59 52 io <I
6.4 4-9 31 7 <5 ioo
6.4 9.5 61 27 >5 < IO 44
0.9 18. 5 18 I5 <5 2
1.2 14. 5 17 12 <5 4
1.6 11. 5 18 io <5 17
1.7 11.4 19 Ii <5 25
2.0 io 20 9 <5 5 °
2.6 7-5 19 7 o 80
2.6 15 39 28 5 3
2.6 19.5 51 47 >5 <io 2
3 .2 14.5 46 32 8
Biochim. Biophys. Acta, 148 (1967) 781-788
7. KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 787
shown by crosses in the same figure are the results of experiments carried out with
the water circulated through the treatment cell at 4°° , so that the temperature of
the suspension was 4o0 instead of 2o° at the start of the treatment; the relationship
between the degree of kill and electric field was not significantly altered.
Zl°eio
S ~8 25
kV/cm
Fig. 7. Relationship between degree of kill and electric field for various organisms, io pulses of
20/~sec. S.C., Saccharomyces cerevisiae; C.U., Candida utilis; E.C., Escherichia coli; M.P., motile
pseudomonad; C.W., Clostridium welchii ; M.L., Micrococcus lysodeikticus.
Sensitivity of the various @cries
A few organisms, other than E. coli, were also treated with IO pulses of 2o ~sec
at 20 ° to find out how the degree of kill was related to electric field. The species
were M. lysodeikticus, C. welchii, B. megaterium, S. cerevisiae, C. utilis and the motile
pseudomonad, and tile results are shown in Fig. 7. In all cases the relationships were
similar but the species differed in their sensitivity to the electric field.
DISCUSSION
In the gel experiment the organisms were killed whether they were in contact
with the products of electrolysis or not, so the products did not cause the kill. Further
evidence is provided by the kill being independent of current density, because the
rate of generation of the products is proportional to current density.
There is evidence that the cooling of the suspension was remarkably effective;
although the energy inputs were as high as 5° cal/cm3 (which would raise the tempera-
ture by 5o° in the absence of cooling), the greatest temperature rise that was recorded
was IO°. This rapid cooling, which occurred in the IO see of the IO pulse treatments,
may be accounted for, not by conduction, but by intense mixing caused by the bursts
of gas generated at the electrodes by each pulse. The small actual temperature rises
were insufficient for temperatures lethal to E. coli to be reached, and the lack of
correlation between the degree of kill and energy input is evidence for the non-thermal
nature of the kill. Further evidence is provided by the lack of any increase of kill
when the starting temperature of treatments was raised from 2o ° to 4o°, and also
provided by the shape of the survivors vs. total time curve (Fig. 3)- If the kill had
been thermal the survival would have been expected to remain at IOO% until enough
Biochim. Biophys. dcta, 148 (1967) 781-788
8. 788 A. ~. H. SALE, ~,V A. HAMILTON
time had elapsed for the suspension to have reached a lethai temperature and ther.
fallen rapidly; but the survival fell immediately and rapidly before leveliing off.
The evidence therefore suggests that the kill was non-thermal, in that it was
not due to heating of the suspension as a whole. The data of Figs. 6 and 7 show that,
for a given time of treatment, the kill was determined by the electric field in the
suspension. This suggests that when an electrical potential in or around the organism
reached a sufficient magnitude, irreversible damage was done. The nature of the
damage to the organism will be discussed in the subsequent paper.
ACKNOWLEDGEMENTS
We wish to thank Mrs, C. A. FOULCER, L. STUTTARD and W. L. Km~~ for their
technical assistance.
REFERENCES
I H. BURTOn-,NaEional Inslitute for Resec~ch in Dairying paper no. zo4z Reading (I949).
2 M. INGRAMANDL. J. PAGE,Proc. Soc. Appl. Bact., 16 (1953) 69.
3 B. S. GOSSLING,Brit. Pat. 845 743-
4 N. E. FEDOROVAND I. A. ROGOV,via Dairy Sci. Abstr., 23 (1963) 312.
5 DOEVENSPECX,Fleischwirtschctfl, 13 (196I) 986.
Biochim. Biophys. Acta, I48 (1967) 781-788