Inhibition of Bacterial Growth In Vitro Following ... - Physical Therapy

Inhibition of Bacterial Growth In Vitro Following
Stimulation with High Voltage, Monophasic, Pulsed
Current
Cynthia B Kincaid and Kathleen H Lavoie
PHYS THER. 1989; 69:651-655.
The online version of this article, along with updated information and services, can
be found online at: http://ptjournal.apta.org/content/69/8/651
Collections
e-Letters
E-mail alerts
This article, along with others on similar topics, appears
in the following collection(s):
Electrotherapy
Other Diseases or Conditions
Wound Care
To submit an e-Letter on this article, click here or click on
"Submit a response" in the right-hand menu under
"Responses" in the online version of this article.
Sign up here to receive free e-mail alerts
Downloaded from http://ptjournal.apta.org/
by guest on January 11, 2014

Inhibition of Bacterial Growth In Vitro Following
Stimulation with High Voltage, Monophasic,
Pulsed Current
Low-intensity direct current has been reported to be effective in promoting healing
of infected wounds, and these results have been assumed to apply to stimulation
of wound tissue with monophasic high voltage pulsed current (HVPC). The purpose
of this study was to determine whether HVPC has an inhibitory effect on
growth in vitro of three bacterial species—Staphylococcus aureus, Escherichia coli,
and Pseudomonas aeruginosa—commonly isolated from open wounds. Following
exposure to HVPC, the measured zone of inhibition of bacterial growth was not
significantly different between bacterial species. Inhibition at the anode (positive
pole) occurred secondary to build-up of toxic end products, and inhibition at the
cathode (negative pole) resulted from exposure to HVPC. Duration of exposure
and voltage showed a highly significant linear relationship. Exposure to more
than 250 V of HVPC for at least two hours resulted in some degree of inhibition
of growth in all three bacterial species. [Kincaid CB, Lavoie KH: Inhibition of bacterial
growth in vitro following stimulation with high voltage, monophasic, pulsed
current. Phys Ther 69:651-655, 1989]
Cynthia B Kincaid
Kathleen H Lavoie
Key Words: Bacterial infections, Electric stimulation, Electrotherapy, Wound
healing.
The use of electrotherapy to promote
healing of superficial and deep dermal
wounds has been reported sporadically
in the literature. A historical
review of literature on the use of lowintensity
direct current (LIDC)
revealed that most studies suggest that
LIDC enhances wound healing. 1 - 13
Two reasons cited for this beneficial
effect are the bactericidal effects of
electrical current 14 and the stimulation
of granulation tissue growth by
the use of electrical current. 15
Clinicians have been applying high
voltage pulsed current (HVPC) for
its assumed antibacterial and
wound-healing effects in recent
years. 16 These assumptions are
based almost entirely on results
obtained in LIDC studies. Thurman
and Christian reported a single-case
study involving the successful use of
HVPC for the treatment of a persistent
toe abscess. 16
High voltage pulsed current instruments
produce a waveform markedly
C Kincaid, MS, PT, is Assistant Professor and Associate Director for Clinical Education, Physical
Therapy Program, The University of Michigan-Flint, 1108 Lapeer St, Flint, MI 48502-2186 (USA).
K Lavoie, PhD, is Associate Professor, Department of Biology, The University of Michigan-Flint.
This article was submitted October 24, 1988; was with the authors for revision for eight weeks; and
was accepted March 3, 1989.
different from the waveform generated
by LIDC instruments (Fig. 1).
High voltage pulsed current characteristics
consist of twin-peaked, paired
pulses of high peak and low total current
having a fixed duration of 100 to
200 µsec. Low-intensity direct current
is characterized by a low-intensity,
continuous, unidirectional flow of
current. 15 The actual current used in
this study was delivered in modified
form from a Rich-Mar HV-20* HVPC
device (Fig. 2).
To date, there are no published
reports regarding the effects of HVPC
on bacterial growth. The purpose of
this study was to establish whether
HVPC has an inhibitory effect in vitro
on the growth of bacterial pathogens
commonly found as infectious agents
in wounds.
*Rich-Mar Corp, Rt 2, PO Box 879, Inola, OK 74036-0879.
Physical Therapy/Volume 69, Number 8/August 1989 651/29
Downloaded from http://ptjournal.apta.org/ by guest on January 11, 2014

Method
Organisms Tested
Three bacterial species commonly
isolated from wounds were used as
test organisms. 17 Isolates of Staphylococcus
aureus (gram-positive cocci)
and Escherichia coli and Pseudomonas
aeruginosa (both gram-negative
rods) were obtained from American
Type Culture Collection † stocks.
Procedure and Instrumentation
Our procedure was a slight modification
of the procedure used by Barranco
et al to test the in vitro effect of
weak direct current on S aureus. 14
Sterile disposable plastic petri dishes
were used throughout the experiment.
Stainless-steel wires ‡ (0.035
gauge) used as electrodes were positioned
parallel 50 mm apart and covered
with growth medium containing
the test organisms. With a heated
wire, four holes were melted 3 mm
from the bottom edge of the dish.
The holes allowed parallel placement
of two 15-cm pieces of sterile
stainless-steel wire extending across
the entire width of the dish with the
ends bent to prevent the wires from
rolling and breaking contact with the
medium.
Test organisms were grown in trypticase
soy broth § overnight at 37°C in a
shaking water bath, and enough culture
was added to the test medium to
reach a final concentration of 1 ×10 7
colony-forming units per milliliter, as
determined by standard use of a
hemocytometer.
The test medium selected was
Mueller-Hinton agar, § which is used
in the semiquantitative Kirby-Bauer
technique for determining effectiveness
of antibiotics, because of the
consistency of the widths of zones
indicating inhibition of bacterial
Fig. 1. Comparison of waveforms of
(a) low-intensity direct current (LIDC)
and (b) high voltage pulsed current
(HVPC) stimulation.
growth. 18 A sufficient quantity of
medium inoculated with the test organism
was poured into the prepared
petri dishes to completely cover the
wire electrodes, covered, and allowed
to solidify. Plates were refrigerated for
use within 24 hours of preparation.
For test runs, wires in the petri dishes
were connected by alligator-clip leads
to a HVPC stimulator. The experimental
setup is shown in Figure 3. The
pulse rate was set at 120 pulses per
second (pps) and the interpulse interval
(measured at 50% of peak pulse
amplitude) at 55 µsec. Voltages of
150, 200, 250, and 300 V were applied
to the test organisms for 1, 2, 3, and 4
hours' duration. According to Roberta
A Newton (RA Newton, PhD; unpublished
data), these voltages are within
the normal therapeutic range for
motor effect. Exposure durations were
chosen based on the results of a pilot
study that showed no bactericidal
effect of HVPC at these voltages with
shorter exposure duration. Each petri
dish was incubated at 37°C for 24
hours following exposure to HVPC.
† American Type Culture Collection, 12301 Parklawn Dr, Rockville, MD 20852.
Fig. 2. Modified waveform produced
by high voltage pulsed current stimulator
at settings of 250 V, 120 pps, and 55
µsec pulse pair interval: (a) Waveform as
it leaves instrument; (b) waveform as it
passes through medium in petri dish in
experimental setup.
After incubation, the width of the
zone paralleling the wire electrodes
where no bacterial growth occurred
was measured to the nearest 0.1 mm
using a millimeter ruler. Test results
represent the average of four measurements
per zone of inhibition per
plate.
Subcultures from the zone of inhibition
were checked for sterility by
using an inoculating needle to transfer
medium from the zone into tubes
of sterile nutrient broth and incubating
them at 37°C for 24 hours. Visible
turbidity was used as an indication of
growth. Changes in pH of the
medium were determined by using
pH paper touched to the surface of
the medium.
Determination of the actual current
waveform was made using an oscilloscope.
The pattern was determined
for the output at 250 V from the
HVPC leads and for the electrodes
through a petri dish setup with the
test medium (Fig. 2).
‡ Phoenix Wire Cloth, Inc, 585 Stevenson Hwy, Troy, MI 48083.
§ Difco Laboratories, Inc, 920 Henry St, Detroit, MI 48232.
30/652 Physical Therapy/Volume 69, Number 8/August 1989
Downloaded from http://ptjournal.apta.org/ by guest on January 11, 2014

Controls
Seeded plates with the wire
electrodes in place were incubated
without exposure to HVPC to determine
whether the wires themselves,
the medium, or a combination of
both would be inhibitory to bacterial
growth. Plates were also prepared
with sterile medium (ie, without organisms
added) to determine whether
wire-insertion preparation introduced
contamination.
The presence of potential toxic electrochemical
end products from the
interaction of current, wire, and
medium was determined by sending
current through sterile medium without
organisms. Extreme conditions
(ie, beyond a normal therapeutic
range) of 500 V for either 30 minutes
or 2 hours were used to allow maximum
potential toxin production.
Overlays of medium containing either
S aureus or E coli were poured into
the plates, allowed to solidify, and
then incubated and evaluated as previously
described.
Data Analysis
Significance of zone width, as a function
of the organism, was analyzed by
a two-way analysis of variance
(ANOVA). 20 A regression analysis was
performed using the MIDAS (Michigan
Interactive Data Analysis System)
program on the Michigan Terminal
System.
Results
Inhibition of growth of S aureus, E
coli and P aeruginosa at the cathode
(negative electrode) following exposure
to HVPC is shown in Figure 4.
Each line represents pooled data
from three organisms, three replicates
each, at 300, 250, 200, and 150
V for 1 to 4 hours of exposure. The
ANOVA of the data at 4 hours of
exposure revealed that differences
using different microorganisms
were not significant (F = 2.18;
df = 2,18; p < .10), whereas varying
the voltage resulted in highly significant
differences (F = 152.98;
df= 2,18; p

The current waveform from the
instrument changed greatly as it
flowed through the test medium. The
resistance to flow varied, making it
impossible to measure actual current
flow. Figure 2 shows a reduction in
total current and resistance to flow,as
indicated by the baseline not returning
to zero, as the waveform leaves
the stimulator and passes through the
medium.
Discussion
High voltage pulsed current can be
effective in killing common woundinfecting
bacteria in vitro. All organisms
tested were equally affected by
2 hours of exposure to HVPC above
250 V. The data analysis revealed a
strong positive linear relationship
between the voltage and the duration
of exposure to HVPC.
Exposure to HVPC at the cathode
accounted for most or all killing of
bacterial cells. The increasing pH
observed at the cathode was transient
and probably did not reach levels
extreme enough to directly kill bacterial
cells. No effect on skin pH following
a 30-minute application of HVPC at
100 V was reported by Newton and
Karselis. 20 Such a rise in pH could
have a static effect on growth that,
combined with the lethal effect of
HVPC, would help to keep bacterial
population levels down and enable
body defenses to fightoff the infection.
Effects of HVPC at the anode were
complicated by production of some
toxic electrochemical end products
created by passing current through
the wire. Zones of inhibition around
the cathode were recolonized by
motile bacteria, suggesting that no
permanent change had occurred
there. In contrast, organisms were
unable to recolonize the zone of discoloration
around the anode, suggesting
that lethal end products had accumulated
and persisted. These results
are comparable to those reported by
Barranco et al. 14
Caution must be exercised when
attempting to extrapolate the findings
of in vitro studies to predict results
Fig. A. Width of zone of inhibition at cathode after exposure to high voltage pulse
current at 300, 250, 200, and 150 V versus duration of exposure. Points represent
mean (± range) of pooled data from Escherichia coli, Staphylococcus aureus, and
Pseudomonas aeruginosa.
when applying the same intervention
to infected wounds in human subjects.
Present treatment protocols for
use of HVPC on infected wounds,
however, generally indicate a treatment
duration of 20 to 45 minutes
once or twice a day, with voltage
amplitude adjusted to a subthreshold
level for muscle contraction. Our
study used a much higher voltage
setting for a much longer exposure
duration than those used in current
clinical practice. Human subjects may
not be able to tolerate such high voltage
applications. Alternatively, the
actual current flow through the petri
plates was very low because of resistance
from the test medium. If there
is less resistance to current flow in
human skin, then lower settings might
be bactericidally effective in a clinical
setting.
Future avenues of investigation
include in vivo application of HVPC to
infected wounds as well as the application
of other types of electrical current
to microorganisms in vitro. If
future studies indicate that the exposure
of infected wounds to electrical
current results in decreased infection,
then it may be possible to effectively
treat infected wounds on a home-care
basis with portable electrical stimulators,
thereby making wound management
more cost effective.
Conclusion
Some clinicians use HVPC to inhibit
bacterial growth in infected wounds.
The results of this study indicate that,
although there is inhibition or killing
of bacteria in vitro at the cathode with
the application of HVPC, either the
voltage applied or duration of treatment,
or both, may need to be substantially
increased to achieve healing
of infected wounds. Studies
conducted in vivo are necessary to
32/654 Physical Therapy/Volume 69, Number 8/August 1989
Downloaded from http://ptjournal.apta.org/ by guest on January 11, 2014

determine the effectiveness of HVPC
in wound healing in clinical practice.
Acknowledgments
We thank Mary Ann Cardani for technical
assistance and the laboratory
staff at Hurley Medical Center for providing
clinical isolates used in our
pilot studies. Dr Donald Boys, Department
of Physics, The University of
Michigan-Flint, provided technical
expertise in recording the actual
waveforms generated.
References
1 Assimacopoulos D: Low intensity negative
electric current in the treatment of ulcers of
the leg due to chronic venous insufficiency.
Am J Surg 115:683-687, 1968
2 Assimacopoulos D: Wound healing promotion
by the use of negative electrical current.
Am Surg 34:423-431, 1968
3 Dueland R, Hoffer RE, Scleen WA, et al: The
effects of low voltage current on healing of
thermal third degree burns. Cornell Vet 68:51-
59, 1978
4 Edel H, Freund R: Direct current treatment
of chronic skin ulcer and wound healing by
second intention. Physiotherapy 27:457-464,
1975
5 Gault WR, Gatens PF Jr: Use of low intensity
direct current in management of ischemic skin
ulcers. Phys Ther 56:265-269, 1976
6 Moore AD: Electrostatic discharge for treating
skin lesions. Med Instrum 9:274-275, 1975
7 Tyurlikova LD, Lassi NI: The effect of the
anode of a constant intermittent current on
regenerative process in skeletal muscle. Byulleten'
Éksperimental'noi Biologii i Meditsky
63:71-74, 1967
8 Wolcott LE, Wheeler PC, Hardwicke HM, et
al: Accelerated healing of skin ulcers by electrotherapy:
Preliminary clinical results. South
Med J 62:795-801, 1969
9 Kloth LC, Feedar JA: Acceleration of wound
healing with high voltage, monophasic, pulsed
current. Phys Ther 68:503-508, 1988
10 Akers TK, Gabrielson AL: The effect of high
voltage galvanic stimulation on the rate of
healing of decubitus ulcers. Biomed Sci
Instrum 20:99-100, 1984
11 Rowley BA, McKenna JM, Chase GR, et al:
The influence of electrical current on an
infecting microorganism in wounds. Ann NY
Acad Sci 238:543-551, 1974
12 Rowley BA: Electrical current effects on E
coli growth rates. Proc Soc Exp Biol Med
139:929-934, 1972
13 Carley PJ, Wainapel SF: Electrotherapy for
acceleration of wound healing: Low intensity
direct current. Arch Phys Med Rehabil 66:443-
446, 1985
14 Barranco SD, Spadaro JA, Berger TJ, et al:
In vitro effect of weak indirect current on
Staphylococcus aureus. Clin Orthop 100:250-
255, 1974
15 Nelson RM, Currier DP (eds): Clinical Electrotherapy.
East Norwalk, CT, Appleton &
Lange, 1987, chap 8
16 Thurman BF, Christian EL: Response of a
serious circulatory lesion to electrical stimulation:
A case report. Phys Ther 51:1107-1110,
1971
17 Finegold SM, Martin WJ, Scott EG: Bailey
and Scott's Diagnostic Microbiology. St Louis,
MO, C V Mosby Co, 1978, chap 20
18 Difco Manual: Dehydrated Culture Media
and Reagents for Microbiology, ed 10. Detroit,
MI, Difco Laboratories, 1984, pp 582-585
19 Brownlee KA: Statistical Theory and Methodology
in Science and Engineering, ed 2.
New York, NY, John Wiley & Sons Inc, 1965,
chap 14
20 Newton RA, Karselis TC: Skin pH following
high voltage pulsed galvanic stimulation. Phys
Ther 63:1593-1596, 1983
Physical Therapy/Volume 69, Number 8/August 1989 655/33
Downloaded from http://ptjournal.apta.org/ by guest on January 11, 2014

Inhibition of Bacterial Growth In Vitro Following
Stimulation with High Voltage, Monophasic, Pulsed
Current
Cynthia B Kincaid and Kathleen H Lavoie
PHYS THER. 1989; 69:651-655.
Cited by
This article has been cited by 1 HighWire-hosted articles:
Subscription
Information
http://ptjournal.apta.org/content/69/8/651#otherarticles
http://ptjournal.apta.org/subscriptions/
Permissions and Reprints http://ptjournal.apta.org/site/misc/terms.xhtml
Information for Authors
http://ptjournal.apta.org/site/misc/ifora.xhtml
Downloaded from http://ptjournal.apta.org/ by guest on January 11, 2014