BIOCHI1VflCA ET BIOPHYSICA ACTA 781
BBA 25 876
EFFECTS OF HIGH ELECTRIC FIELDS ON MICROORGANISMS
I. KILLING OF BACTERIA AN...
782 A.J. lifo SALE; W. A. ;frA2vflLTON
METHODS AND MATERIALS
Aj)~ara~us
A pulse generator was built to provide approximate...
KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 783
Electrical conductivities of media were measured with a Wayne Kerr Univer...
784 A.j.H. SALE, W. A. HA2VflLTON
RESULTS
Lethal effect of d.c. pulses
In preliminary experiments d.c. pulses -wereapplied...
KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 785
However, it was possible to obtain the temperature rise of the suspension...
786 a. j, H~ SALE, W, A, HAI~fILTON
that the percent survival fell rapidly from the start of the treatment and then tended...
KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 787
shown by crosses in the same figure are the results of experiments carrie...
788 A. ~. H. SALE, ~,V A. HAMILTON
time had elapsed for the suspension to have reached a lethai temperature and ther.
fall...
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1967 sale effect of high electric fields on microorganisms_killing bacteria and yeasts

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1967 sale effect of high electric fields on microorganisms_killing bacteria and yeasts

  1. 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. 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
  3. 3. KILLING OF MICROORGANISMS BY ELECTRIC FIELDS 783 Electrical conductivities of media were measured with a Wayne Kerr Universal Bridge, B22I, operating at 1592 cycles/sec; the conductivity cell had platinum black electrodes. Cur'boM electrodes / "Brass i] Co0 ant ' , I- ] I connections I' - ..... -~_~i~f Polyt henei ~TI -- - ©/j spacer4e Fig. 2. Treatment cell Organisms The following species were used: Escherichia eoli, Staphylococcus aureus, Mioro- coccus lysodeiktieus, Sarcina lutea, Bacillus subtilis, B. cereus, B. megaterium, Clos- tridium welchii, a Gram-negative oxidase-positive motile rod, which was isolated as a culture contaminent and classified as a pseudomonad, and two yeasts Saecharomyces cerevisiae and Candida utilis. Organisms other than Cl. welchii were grown for 16 h at 30 ° in a yeast glucose broth: 0.5 % glucose, 0.5 % peptone, 0.5 % Na2HPO4, 0.5 % yeast extract, 0.5 % lab lemco (pH 7-3)- 5oo-ml erlenmeyer flasks containing IOO ml medium were used as culture vessels. The cultures were aerated by mounting the flasks on a reciprocal shaker. The cells were harvested by centrifugation, washed once and resuspended in NaCI solutions of concentration appropriate to the experiment. For viable counting, suspensions were serially diluted in o.i % peptone and I-ml volumes of the dilutions plated out in yeast glucose broth plus 1.5 % agar. After setting, the agar plates were incubated at 37 ° for 24 or 48 h. A cell was considered to have been killed when it did not give rise to a colony under these conditions. Cl. welchii were grown in a reinforced clostridial medium: I.O % peptone, o.I % glucose, I.O % lab lemco, o.15 % yeast extract, 0.5 % sodium acetate, o.i % soluble starch, 0.05 % L-cysteine, i % ascorbic acid (pH 7.4)- IOO ml flat bottles were filled to the neck with medium, and after steaming to drive off dissolved oxygen and cooling, they were inoculated and incubated for I6 h at 3o°. Viable counts were estimated by the most probable number technique, using the same medium and 5 tubes per dilution. Bioohim. Biophys. _dcta, 148 (i967) 781-78 8
  4. 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. 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
  6. 6. 786 a. j, H~ SALE, W, A, HAI~fILTON that the percent survival fell rapidly from the start of the treatment and then tended towards a constant value. Effect of electricfield In the series of experiments quoted in the table the conductivity of the sus- pending medium was varied so that ~he electric field and current density could be varied independently, while keeping to zo pulses of 2o ffsec. Examination of the current density and the survival revealed no correlation between the two, as Fig. 5 shows. However, by plotting the survival against electric fieid it was revealed that the eIectric field controlled the degree of kill, as shown by the circles in Fig. 6. Aiso I0C • o., °o° o.o ; o o ° "'2. .° 6 °. • , IC •• IO- ZoV © " 6!"> '~s 0 1; "2'0 " 30 ' 40 ' 5b 6C ) 200 400 660 8,00 IOOC cal /cm 3 Jsec Fig. 3- Effect of the energy input oi a treatment on the degree of kill of E. coli, for a range of conditions: electric field, 5-21 kV cm; pulse length, 4-2o .~sec; number of pulses, 1-99; media, 0.05-0. 4 % NaCl; conductivity, 0.8-6. 4 m~ -i. Fig. 4- Illustration that the degree of kill fell rapidly at first and then tended to a constant value as the total time of actual voltage application increased. Electric field 14 kV ,cm; medium o.i % NaC1; O, IO-#sec pulses; X, 2o-#sec pulses. • ~ 1 0 0 ~ o o 10 10 k c Sn S 15 210 310 " 4; 5; 60 0 ; 10 1; "*2;* 25 A/cm 2 kV/cm Fig. 5. Effect of the current density on the degree of kill. Various conductivities from o.o 5 to 0. 4 % were used to obtain independent variation of the electric field and current density, zo pulses of 20 #see. Fig. 6. Relationship between the degree of kill of E. coIi and electric field, also showing a lack of temperature effect. IO pulses of 20 #see. O, room temperature (2o°); x, elevated tempera.- ture (40°). Biochim. Biophys. Acta, 148 (z967) 78z-788
  7. 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. 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

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