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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
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