APuPED MICROBIOLOGY, Sept. 1967, p. 1031-1037 Vol. 15, No. 5Copyright © 1967 American Society for Microbiology Printed in U.S.A.

Inactivation of Microorganisms byElectrohydraulic Shock1

S. E. GILLILAND AND M. L. SPECKDepartment of Food Science, North Carolina State University, Raleigh, North Carolina 27607

Received for publication 17 February 1967

The electrohydraulic shock treatment of microorganisms was accomplished by dis-charging high-voltage electricity (8 to 15 kv) across an electrode gap below the sur-face of aqueous suspensions of the microorganisms. This treatment was effective indestroying Escherichia coli, Streptococcus faecalis, vegetative cells and spores ofBacillus subtilis, and bacteriophage specific for S. cremoris ML1. The presence ofadded protein in bacterial suspensions resulted in reduced bactericidal action. Watersubjected to electrohydraulic treatment retained a certain amount of toxicity whencopper-core electrodes were used to apply the treatment. This was caused by copperliberated from the electrode during electrohydraulic discharge.

Most of the early work involving the use ofelectricity to destroy microorganisms was done inattempts to pasteurize milk or other liquids bypassing an alternating current through the liquid(3, 4, 7, 14). The bactericidal action produced bythese treatments was attributed, for the most part,to heat produced by the flow of current throughthe liquid. The electric current itself was also con-sidered by some to contribute to bacterial death(4). Sandorf (15) studied the effects of highvoltage on the bacterial content of milk. He ob-tained as high as 85% destruction of the bacteriain milk.The killing of microorganisms in aqueous sus-

pensions by submerged high-voltage sparks hasbeen reported in recent years (1, 5, 16; F. Fruen-gel, U.S. Patent 2,931,947, 1960). Brandt et al. (5)found that the microbial population of sewagecould be reduced by as much as 90% with 40discharges. When coliform organisms were sus-pended in water and subjected to submerged high-voltage discharges, 40 discharges reduced thenumber of viable cells from 3.6 X 107 to 2.0 x104 per ml. These results indicated the possibilityof using a submerged high-voltage spark in waterpurification.The submerged high-voltage discharges result

in the formation of tremendously high transientpressure pulses which create shock waves (5, 6).The pressures have been estimated at levels of2,500 to 5,000 atm for discharges at 25 kv (11).

'Contribution from the Department of FoodScience, North Carolina Agricultural ExperimentStation, Raleigh. Published with the approval of theDirector of Research as paper no. 2344 of the JournalSeries.

The pressure pulses are measurable only in microseconds. The application of pressure pulses tomicrobial suspensions has been reported to resultin loss of viability of the organisms being treated(10).

MATERIALS AND METHODS

Test organisms. The organisms employed in thisstudy were Escherichia coli 451B, Streptococcusfaecalis, Bacillus subtilis A (vegetative cells andspores), Micrococcus radiodurans, and a bacteriophagespecific for S. cremoris ML1. All cultures were main-tained on Trypticase Soy Agar (TSA) slants or inTrypticase Soy Broth (TSB; BBL).

Cell crops of E. coli 451B and of S. faecalis wereprepared by growing the cultures in sterile TSB withthe use of a 1% inoculum and an 18- to 20-hr incuba-tion at 35 C. M. radiodurans cells were prepared bygrowing the culture in TGYM (tryptone, glucose,yeast extract, methionine) broth (9) for 24 hr at 37 Con a shaking incubator. The cells were centrifugedfrom the broth at 4,100 X g for 20 min at 2 C and wereresuspended in 1 liter of sterile distilled water fortreatment.

B. subtilis cell crops were prepared for treatmentby spreading 1 ml of a fresh TSB culture on each offive sterile plastic petri dishes (150 X 25 mm) con-taining milk-agar (1.5% agar plus 5% nonfat milksolids). After 16 hr of incubation at 35 C, the cellswere washed from the agar surface with 100 ml ofsterile phosphate buffer (0.0003 M KH2POQ, pH 7.2).Buffered water was used, since unbuffered water wastoxic to the vegetative cells of this organism.

B. subtilis A spores were prepared according to themethod reported by Edwards et al. (6).The stock phage suspension was prepared by inocu-

lating TSB with 1% of a fresh broth culture of S.cremoris ML1, incubating for 3 hr at 32 C, and theninoculating with 1% of the MLI phage and continu-

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GILLILAND AND SPECK

-HIGH VQLTAGE

-GROUND

INSULATION

GROUND

_EPOXY -GLASS(SPARK GAP)

END VIEW -HIGH VOLTAGE

SIDE VIEW

FIG. 1. Electrode structure.

ing the incubation at 32 C until the turbidity disap-peared. For electrohydraulic shock treatment, 1 mlof the stock phage suspension was added to 1 liter ofsterile distilled water.

Equipment description. The electrohydraulic equip-ment used in this study was supplied by General Elec-tric Co., Chemical Systems Branch of Research andDevelopment Center, Schenectady, N.Y. The highvoltage (maximum of 15 kv) was accumulated in aseries of one of four capacitors (6 juf each). The highvoltage from the capacitors was pulsed through anignitron, and the pulse was carried to the electro-hydraulic tank by means of six parallel lengths ofRG-8U coaxial cable, where the submerged high-voltage spark occurred. The high voltage was dis-charged at a rate of one discharge per second.

The design of the electrode system through whichthe high voltage was discharged is shown in Fig. 1.The outer shell of the electrodes was composed of analuminum alloy, and the composition of the center rodor core was copper alloy (ever dure), aluminum alloy,iron, or stainless steel. The insulation material whichseparated the two metal components of the electrodewas epoxy-glass laminated tubing held in place by anepoxy-adhesive. The tip of the electrode was flat, withthe center core protruding slightly (approximately 3mm) beyond the epoxy-glass. The spark gap distancewas determined by the thickness of the epoxy-glassinsulation. The electrode tips were trimmed down on ametal lathe after each experiment, since a certainamount of erosion occurred as a result of the high-voltage discharges. The spark gap sizes varied from0.0625 to 0.25 inch (0.16 to 0.64 cm) for this study.The voltage being used was a guide in determiningwhich gap size to use. The high voltage was fed inthrough the center core of the electrode while theouter shell served as the ground. The electrohydraulic

tank (Fig. 2) in which the high-voltage discharges oc-curred was constructed of stainless steel. The elec-trode was screwed into the tank to a depth of 2.5 inches(6.4 cm) and secured by two lock nuts. The microbialsuspensions (1-liter volume) to be treated were intro-duced into the tank through one of the two plugs in thetank lid. Samples were also removed through one ofthese holes.The time required for the high-voltage pulse to

cause enough ionization at the electrode gap to allowfor dissipation of the high voltage was measured byuse of a Tektronic Type 545B Oscilloscope, equippedwith Type K-Plug-in Unit and a P6015 High VoltageProbe. The probe was connected to the electrical sys-tem at the site of the ignitron. From oscilloscopepatterns, obtained by means of a Tektronic Polaroidoscilloscope camera, the gap ionization times weredetermined. The vertical rise of the oscilloscope signalwas due to the voltage rise, and the horizontal distancetraveled at that voltage level represented the gapionization time. When the discharge occurred, thevoltage dropped.

Preparation of the tank for treatment. The stainless-steel tank and the desired electrode were thoroughlywashed and rinsed with distilled water before assemblyfor each experiment. In most cases, the tank receivedno further sanitation treatment since the microbialpopulations used were so high.

Electrical resistance and temperature of test solutions.The electrical resistance of the liquids to be treated wasmeasured by use of a conductivity cell (cell constant at25 C, Kc = 0.908) and a Leeds and Northrup Porta-ble Electrolytic Resistance Indicator. The specific re-sistance (r) was determined by the formula: r = RIK,where r = specific resistance at temperature t, R =measured resistance at temperature t, and Kc = theconductivity cell constant (0.908 at 25 C). The tem-perature of the bacterial suspension being treated was

FIG. 2. Exploded view of electrohydraulic tank.

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INACTIVATION OF MICROORGANISMS

measured before and immediately after treatmentwhile the suspension was in the electrohydraulic tank.

Enumeration of surviving organisms. Samples weretaken aseptically from the tank and kept in an ice-water bath until examined. Platings were made within20 min after treatment, and the procedures used werethose described in Standard Methods for the Examina-tion ofDairy Products (2) with the exception of mediaused and incubation temperatures.

E. coli strain 451B samples were plated with the useof TSA as the complete medium and minimal salts-agar, containing (NH4)2SO4 as the only nitrogensource, to determine the amount of metabolic injury.The minimal agar was of the same composition as thatdescribed by Straka and Stokes (17). The TSA andminimal agar plates were incubated for 48 hr at 35 C,after which all colonies were counted. The counts ob-tained on TSA were considered as the total numberof survivors, and the counts obtained on the minimalagar were the number of uninjured survivors; the dif-ference in the two counts for each sample representedthe number of metabolically injured cells.M. radiodurans samples were plated on TGYM

agar (9) and were incubated at 37 C for 96 hr.B. subtilis strain A spores were enumerated by the

pour plate method, with the use of fortified nutrientagar (FNA) as described by Edwards et al. (6). Theplates were incubated 48 hr at 45 C before counting.

B. subtilis A vegetative cells and S. faecalis cellswere enumerated by plating with TSA and countingafter 48 hr of incubation at 45 C.

Titers of S. cremoris ML1 bacteriophage were de-termined by use of the limiting dilution technique.Decimal dilutions of the bacteriophage samples weremade in 9 ml of sterile TSB, after which 0.1 mil of an18-hr TSB culture of S. cremoris ML1 was added toeach tube. The tubes were then incubated at 32 C for48 hr, after which the titers of the bacteriophage sam-ples were determined.

RESULTS

Electrode gap ionization time and temperaturechanges. The time required to ionize the electrodegas was dependent on the electrode gap size, thevoltage level, and the electrical resistance of thesolution being treated. An electrode gap size of0.125 inch (0.32 cm) was used for 8- and 10-kvdischarges, and a gap size of 0.25 inch (0.64 cm)was used for 12- and 15-kv discharges. The spe-cific resistances of the aqueous bacterial suspen-sions used in this study were found to be in therange of 104 to 5.3 X 104 ohm-cm. Ionizationtime varied from an instantaneous ionization to atime of greater than 500 ,usec. In most cases, thetime required to ionize the gap was in the rangeof 0 to 300 ,usec.The electrohydraulic shock treatments of bac-

terial suspensions were done at room temperature(22 to 25 C). In 21 experiments in which the tem-peratures were measured (involving total energyinputs of up to 40,000 joules), the temperature of

0- B. subtilis A Spores (IOKv, 24pF)o-B. subtilis A Cells (0OKv,24giF)

00 A-E.coli 451B(lOKv,24pF)A-M.rodiodurons (0OKv,24pF)

\\\\ *~~-S. foecalis (IO Kv, 24pF)*-MLI Bacteriophage (6Kv,24pvF)

10

0. 0.1zwu

wa. 0.01

0.001

0.00004

NUMBER OF DISCHARGES

FIG. 3. Survivor curvesfor various organisms treatedwith electrohydraulic shock (0.125-inch gap copper-coreelectrodes).

the solution being treated never increased by morethan 0.5 C.

Inactivation of various microorganisms. Electro-hydraulic shock treatment was found to be effec-tive in decreasing the viable population of variousmicroorganisms in aqueous suspensions. Thesurvivor curves in Fig. 3 are a representative sam-pling of the results obtained in studying the lethalaction of electrohydraulic shock on a variety ofmicroorganisms. In all the experiments, exceptthe one involving the bacteriophage, the high-voltage level was 10 kv and the capacitance levelwas 244/. In the experiment in which the bacterio-phage was the test organism, the voltage level wasadjusted to 6 kv. The initial concentrations ofmicroorganisms for each experiment were: B.subtilis A spores, 5.5 x 101 per ml; B. subtilis Avegetative cells, 1.4X 108 per ml; E. coli 451B.1.3 X 108 per ml; S. faecalis, 2.7 X 108 per ml;M. radiodurans, 1.0X 107 per ml; and bacterio-phage specific for S. cremoris ML1, 5.0 X 101, perml. Ten discharges resulted in the inactivation ofmore than 95% of the organisms for each speciesstudied. The bacteriophage was the most sucsepti-ble microorganism in this group, and the B. sub-tills spores and M. radiodurans were the mostresistant.

Relationships of energy per discharge to bac-tericidal action. The amount of energy per dis-charge was varied either by changing the voltagelevel or changing the level of capacitance. Theeffect of changing either of these on the energy

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1034~~~~GILLILAND AND SPECK AP.McoIL

100 activation per discharge in most cases increasedo - 8Kv,24p~,F significantly as the capacitance level was in-o0- I0 Kv, 24PiF creased. These results and those in Fig. 4 indicate102Kv,24p F

that, in general, as the electrical energy per dis-10 ~ ~ ~ ~ 2Ky 4iF charge was increased, the amount of bactericidalto ~~~~~-15 Kv, 24p F action per discharge also increased.

Relationship of total energy input to bactericidalaction. The form in which the electrical energy

* ~~~~~~~~wasapplied (i.e., the combination of voltage andcapacitance) had an effect on the total amount of

> ~~~~~~~~~~~~killobtained. In one series of experiments, the5; ~~~~~~~~~~~~totalenergy input, at various levels of energy perD ~~~~~~~~~~~discharge, was held constant at 4,500 joules by

0.1 ~~~~~~~~~varyingthe number of discharges at each energyz0.1 ~~~~~~~~~~level.These experiments were conducted at volt-o ~~~~~~~~~~~~agelevels of 8, 10, 12, and 15 kv at each level of

w capacitance (6, 12, 18, and 24 .if) with the use ofa. ~~~~~~~~~~~copper-core electrodes. E. coli 451B was the test0.01 borganism employed in these experiments. The

total number of discharges used in each case wasselected to give approximately 4,500 joules. InFig. 6, the percentages of survivors obtained areplotted against voltage for each level of capaci-

0.0010

tance. From these results, it can be seen that totalamount of bactericidal action produced by elec-

* ~~~~trohydraulic shock was indeed affected by theform in which the energy was applied. Even

0.00010.00006 III 100 o-6 PF, IOKv0 2 3 4 5 6 7 8 9 10 o-12,uF,lOKv

DISCHARGES *-18pF, I0KVFIG. 4. Effect of voltage level on bactericidal action *06 -241PF, 10Kv

of electrohydraulic shock with Escherichia coli 451B asthe test organism (0.125-inch gap copper-core elec-trode).

can be seen in the equation for computing the ,energy produced by a capacitor discharge: E =CV2, where E = joules, C = microfarad capaci- ; 0.1-otance, and V = kilovolts. Figure 4 shows survivor cr

curves of E. coli 451B obtained from a series of Inexperiments in which the capacitance was held "z .0constant at 244/i and the voltage was varied at 8, w10, 12, or 15 kv. The percentages of survivors w.were plotted against the number of discharges foreach voltage level. The initial bacterial count 0.001for each experiment was in the range of 108 to1.5 X 108 per ml. It is evident from these survivorcurves that the rate of bacterial death increased 0.0001with each increase of the voltage level. 0

Figure 5 shows the survivor curves from a seriesof experiments in which the voltage was held

100

constant at 10 kv, while the level of capacitance 0 2 3 4 5 6 7 8 9 10 11 12 1 3 14 15was varied at 6, 12, 18, or 24 4A. The counts ob- DISCHARGEStamned on the control samples of E. coli 451B in FIG. 5. Effect of capacitance on bactericidal actionthese experiments were again in the range of 108 of electrohydraulic shock with Escherichia coli 45JB asto 1.5 X 108 per ml. The amount of bacterial in- test organism (0.125-inch copper-core electrode).

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TABLE 1. Metabolic injury of Escherichia coli strain451B as a result of electrohydraulic shock

CountsDis- Per-

charges" centgeTotal Uninjured In*rd of injurysurvivors survivors njure

Control 1.3 X 108 1.2 X 10810 1.9 X 105 0.94 X 105 0.96 X 105 5115 6.1 X 103 3.3 X 103 2.8 X 103 4620 1.2 X 103 0.90 X 10I 0.3 X 105 25

a Aluminum electrode, 10 kv, 24 uf, 0.125-inch gap.

o 6pF CAPACITANCEo 12pF CAPACITANCE

0 18pF CAPACITANCE

* 24pF CAPACITANCE

8 10 12 15KI LOVOLTS

FIG. 6. Survivor plots of Escherichia coli 451B afterelectrohydraulic shock treatment at a constant energyinput of 4,500 joules (copper-core electrodes).

though the energy per discharge at 15 kv with 24,uf capacitance (2,700 joules) was much greaterthan at 8 kv with 6 4f capacitance (192 joules),the amount of bacterial death observed after theapplication of 4,500 joules was much greater withthe latter combination. In general, it appearedthat the most efficient production of bactericidalaction by electrohydraulic shock treatment oc-curred at the lower voltage levels for each levelof capacitance. The combination of 8 kv and 64Af capacitance was the most efficient.In one series of experiments, the treated and un-

treated samples of E. coli 451B were plated byuse of both TSA and minimal salts-agar. From thecounts obtained with these two media, the per-centage of the survivors which were injured wasdetermined. Aluminum-core electrodes having a0.125-inch gap were used in the application of theelectrohydraulic treatment at a voltage level of10 kv and a capacitance level of 6 or 24 4uf. Anexample of the results obtained is presented inTable 1. Although metabolic injury decreasedfrom 51 to 25% as the number of discharges in-creased, the amount of death increased.

TABLE 2. Residual bactericidal action of watertreated with electrohydraulic shock

Electrode core Count on Trypti-composition Sample case Soy Agar

Copper Control water 1.6 X 10830 dischargesa 1.8 X 106

Iron Control water 1.3 X 10830 discharges 1.3 X 108

Aluminum Control water 1.3 X 10830 discharges 1.3 X 108

a All discharges were at 10 kv with 24 ,f capaci-tance.

Residual toxicity. When E. coli 451B cells wereadded to electrohydraulic-treated water contain-ing 3 X 10-4 M phosphate buffer (pH 7.2), a por-tion of the cells were killed. The residual toxicityin treated water, however, did not produce asmuch bacterial death as was obtained by directelectrohydraulic treatment of bacterial suspen-sions. Table 2 shows the results from three suchexperiments in which copper-, iron-, and alumi-num-core electrodes (0.125-inch gap) were used.As is evident from these data, the experimentthat involved the copper core electrode was theonly one in which any residual toxicity was noted.The residual toxicity obtained using the coppercore electrode was sufficient to inactivate almost99% of the bacterial population. An analysis forcopper (by use of a Perkin-Elmer 303 Atomic Ab-sorption Spectrophotometer) in the bufferedwater which received electrohydraulic treatmentwith the use of the copper-core electrode revealedthe presence of 5.5 ppm copper. Subsequently,experiments were conducted in which bacterialcells (E. coli 451B) were added to sterile distilledwater and to sterile distilled water containing 5ppm of copper in various forms (CuO, CuC12,CuCl, (CH3COO)2Cu). After an exposure of 20min, the samples were plated on TSA. When com-pared with the control water, all forms of copperexcept cupric oxide were toxic to E. coli 451B.The cupric ions (cupric chloride and cupric ace-

100

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GILLILAND AND SPECK

A-IRON CORE ELECTRODEo - ALUMINUM CORE ELECTRODEo - COPPER CORE ELECTRODE

DISCHARGES

FIG. 7. Survivor curves for Escherichia coli 451Btreated with electrohydraulic shock with the use of elec-trodes made of different metals (discharge voltage, 10kv; capacitance, 24 ,uf; electrode gap, 0.125 inch).

tate) were found to be the most toxic forms ofcopper tested. Thus, the residual toxicity of theelectrohydraulic-treated water, when copper coreelectrodes were used, was caused by the copperions shed into the buffer.

Comparison of copper-, iron-, and aluminum-core electrodes. Experiments were conducted inwhich aqueous suspensions of E. coli 451B (1.1 X108 to 1.5 X 108 per ml) were treated by electro-hydraulic shock with the use of 0.125-inch gapcopper-, iron-, and aluminum-core electrodes.In all of these experiments, the discharge voltagewas 10 kv and the capacitance was 24 jhf. Al-though the rate and amount of kill obtained withthe aluminum-core electrode was greater than thatobtained with the iron-core electrode, both pro-duced an appreciable amount of bactericidalaction (Fig. 7). Greater than 99% kill was ob-tained with all three types of electrodes after 10discharges.

Effect of added protein on bactericidal action.When E. coli 451B cells were suspended in dis-tilled water and in 0.05% bovine serum albuminin distilled water and subjected to electrohy-draulic shock treatment with the use of 0.125-inchgap copper core electrodes, a decreased amount of

death was observed in the suspension containingthe albumin. A discharge voltage of 10 kv with24 1if capacitance was used in both cases. After10 discharges, the aqueous suspension of E. coliwas reduced in population from 1.2 x 108 to4.3 X 104 per ml, whereas the suspension contain-ing the albumin was only reduced from 1.2 X 108to 1.7 X 107 per ml. These results demonstratethe protective effect of added protein in the bac-terial suspension.

DISCUSSION

Many attempts have been made to use elec-tricity in one form or another as a bactericidalagent. Most of these attempts have involved thecontinuous flow of electric current through a thinstream of liquid containing the microorganisms.The killing action of such treatments has, for themost part, been attributed to the heat producedby the current flow. All organisms tested in thepresent study were inactivated by electrohydraulictreatment, although some were less susceptiblethan others. Bacterial spores and M. radioduransexhibited the greatest resistance to electrohy-draulic shock. The bacteriophage, which are oftenmore resistant to heat and chemical bactericides,were found to be much more susceptible to inac-tivation by electrohydraulic shock than were thebacteria.

Water which had been exposed to submergedhigh-voltage discharges was reported by Brandtet al. (5) to retain a certain amount of residualbactericidal activity. The findings in the presentstudy showed that the residual bactericidal actionof water treated with electrohydraulic shock re-sulted from ionic copper released from the copper-core electrodes during the high-voltage discharges.Martin (11) reported that copper atoms in allstates of ionization and excitation could be re-leased from the electrodes used for submergedhigh-voltage discharges, and the present studyconfirms this observation. No residual bacteri-cidal action was noted in water treated with eitheriron- or aluminum-core electrodes.

Brandt et al. (5) noted that the bactericidalaction increased when they increased the amountof electrical energy per discharge. Although ourstudy is in agreement with this, the manner inwhich the energy was applied (the combinationof voltage and capacitance) had an effect on theamount of bactericidal action obtained. Theenergy was utilized more efficiently when appliedat a low voltage (8 kv as opposed to 15 kv) and alow capacitance (6 ,uf as opposed to 24 ,f). Thiswas probably the result of more ionization at theelectrode tip with the lower voltage and capaci-tance.

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The presence of organic matter in the bacterialsuspension during treatment with submergedhigh-voltage discharges lessened the bactericidalactivity, as was also found by Brandt et al. (5).This indicates a nonspecific action by the electro-hydraulic treatment, and may limit the applica-bility of electrohydraulic shock in the destructionof bacteria in many menstrua. The metabolicinjury of E. coli 451B after electrohydraulic treat-ment indicated that the bactericidal nature of thetreatment proceeded in a manner similar to thatcaused by freezing (12), heating (13), and ultra-violet light (8). There were no indications ofdeath caused by mechanical disruption of cellularintegrity.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService Training Grant ES-61 from the Division ofEnvironmental Health Sciences.

LITERATURE CITED

1. ALLEN, M., AND K. SOIKE. 1966. Sterilization byelectrohydraulic treatment. Science 154:155-157.

2. AMERICAN PUBLIC HEALTH ASSOCIATION. 1960.Standard methods for the examination of dairyproducts, 11th ed. American Public HealthAssociation, Inc., New York.

3. ANDERSON, A. K., AND R. FINKELSTEIN. 1919. Astudy of the electropure process of treatingmilk. J. Dairy Sci. 2:374-406.

4. BEATIE, J. M., AND F. C. LEWIs. 1925. The elec-tric current (apart from the heat generated). Abacteriological agent in the sterilization of milkand other fluids. J. Hyg. 24:123-137.

5. BRANDT, B., L. EDEBO, C. G. HEDEN, B. HJORTZ-BERG-NoRDLuND, I. SELIN, AND M. TIGERSCHI-OLD. 1962. The effect of submerged electrical

discharges on bacteria. Tekniskvetenskaplig-forskining. 33:222-229.

6. EDWARDS, J. L., JR., F. F. BUSTA, AND M. L.SPECK. 1965. Thermal inactivation characteris-tics of Bacillus subtilis spores at ultrahigh tem-peratures. Appl. Microbiol. 13:851-857.

7. GELPI, A. J., JR., AND E. D. DEVEREUX. 1930. Ef-fect of the electropure process and of the hold-ing method of treating milk upon bacterialendospores. J. Dairy Sci. 13:368-371.

8. KIMBALL, R. F. 1957. Nongenetic effects of radia-tion on microorganisms. Ann. Rev. Microbiol.11:199-220.

9. KRABBENHOFT, K. L., A. W. ANDERSON, AND P.R. ELLIKER. 1965. Ecology of Micrococcusradiodurans. Appl. Microbiol. 13:1030-1037.

10. LUNDBECK, H., AND 0. SKOLDBERG. 1963. Effectof pressure waves on bacteria suspended in wa-ter. Biotech. Bioeng. 5:167-184.

11. MARTIN, E. A. 1960. Experimental investigationof a high-energy density, high-pressure arcplasma. J. Appl. Phys. 31:255-267.

12. Moss, C. W., AND M. L. SPECK. 1966. Identifica-tion of nutritional components in Trypticaseresponsible for recovery of Escherichia coliinjured by freezing. J. Bacteriol. 91:1098-1104.

13. NELSON, F. E. 1943. Factors which influence thegrowth of heat treated bacteria. J. Bacteriol.45:395-403.

14. PREscoTr, S. C. 1927. The treatment of milk by anelectrical method. Am. J. Public Health 17:221-223.

15. SANDORF, I. J. 1938. Effects of high voltage on thebacterial content of milk. Univ. Nev. Expt.Sta. Bull., vol. 32, no. 2.

16. STONG, C. L. 1957. How to make extremely ener-getic sparks for high-speed photography andother purposes. Sci. Am. 197:148-160.

17. STRAKA, R. P., AND J. L. STOKES. 1959. Metabolicinjury to bacteria at low temperatures. J. Bac-teriol. 78:181-185.

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