The Impact of Noncontact, Nonthermal, Low-Frequency Ultrasound on Bacterial Counts in Experimental and Chronic Wounds

Thomas Serena, MD, FACS; S. Kwon Lee, MS, FACS, FCCWS; Kan Lam, BS, RLAT; Paul Attar, PhD; Patricio Meneses, PhD; and William Ennis, DO

The appropriate skin sample then was attached to a cassette and positioned on the second ring stand. The separation distances, 10 cm for the sham unit and 1 cm for the noncontact ultrasound device, ensured equivalent kinetic energy transfer for both devices as previously described. Three samples each of intact and wounded skin were treated using a 5-minute treatment protocol. This treatment time was selected to simulate treatment time commonly used for smaller wounds in clinical practice. After the treatment, skin samples were embedded in optimal cutting temperature (OCT) and 6-micron frozen sections were created. The sections then were placed on glass using a fluorescence-preserving mounting media and a coverslip was applied. Photographs were taken with a Zeiss fluorescent microscope using a 1.910-second exposure time. The ultimate limit of dye diffusion was determined by applying a long exposure time (>10 seconds) to identify the approximate depth where background illumination was indistinguishable from true fluorescence. Depth measurements were calibrated using the thickness of the epidermis as a standard control of 50 microns (excluding rete pegs).

     In vitro bacteria reduction experiment. During experimental design, it was noted that the saline spray used with noncontact ultrasound might reduce the bacterial count in a petri dish through a simple washout mechanism. Alternative methods for quantifying bacteria reduction in vitro subsequently were explored. It was discovered that bacteria can be trapped using suction to draw the bacteria onto the surface of a sterile 0.2-micron Nuclepore filter. To verify that the integrity of Nuclepore filters would be maintained after extended ultrasound treatment (maximum exposure tested: 10 minutes), aliquots of the fluid that had been sucked through the filter by plating on bacterial growth agar were tested. The Nuclepore filter was applied to the surface of a 150-cc Nalgene analytical filtering unit with a suction unit attached to the canister (see Figure 1). The bacteria then were treated directly on the filter paper itself. For the evaluations of Pseudomonas aeruginosa (ATCC 27317), an inoculum was cultured on nonselective media (trypticase soy agar) and incubated overnight at 37° C. A loopful of the test organism from the agar was transferred into tryptic soy broth and vortexed. This solution was incubated at 37° C for 18 hours and the optical density (OD) was adjusted to 0.16 OD at 625 nm against sterile trypticase soy broth (TSB) as “zero”. Five cc of this solution was transferred to a 495-cc bottle, achieving the final study concentration of 107 colony-forming units (CFU)/mL.

     The noncontact ultrasound unit was attached to a ring stand in a vertical position and the fluid was delivered to the unit through an intravenous catheter of sterile saline. After attaching the noncontact ultrasound or sham units, 100 cc of the bacterial solution was filtered through the treatment membrane. A 5-minute treatment was performed, after which the filter was removed and a strip (0.5 cm x 2.5 cm) from the center of each filter was cut out and stained with live/dead stain. Live-dead percentages were calculated by counting the number of red fluorescent cells (total dead bacteria count) and the number of green fluorescent cells (live bacteria count). Counts were conducted over four microscopic fields for each filter on areas that were exposed to the highest levels of ultrasound (center of the filter). Scanning electromicroscopy was used to provide supporting evidence for the live/dead staining protocol. Similar procedures were carried out with Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), S.


1. Mertz PM, Ovington LG. Wound healing microbiology. Dermatol Clin. 1993;11(4):739–747.
2. Ennis WJ, Meneses P. Wound healing at the local level: the stunned wound. Ostomy Wound Manage. 2000;46(1A suppl):39S–48S.
3. Carignan A, Allard C, Pepin J,Cossette B, Nault V, Valiquette L. Risk of Clostridium difficile infection after perioperative antibacterial prophylaxis before and during an outbreak of infection due to a hypervirulent strain. Clin Infect Dis. 2008;46(12):1838–1843.
4. Costerton JW. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 2001;9(2):50–52.
5. Robson MC, Edstrom LE, Krizek TJ, Groskin MG. The efficacy of systemic antibiotics in the treatment of granulating wounds. J Surg Res. 1974;16(4):299–306.
6. Ennis WJ, Mozen FPN, Massey J, Conner-Kerr T, Meneses P. Ultrasound therapy for recalcitrant diabetic foot ulcers: results of a randomized, double-blind, controlled, multicenter trial. Ostomy Wound Manage. 2005;51(8):24–39.
7. Ennis WJ, Valdes W, Gainer M, Meneses P. Evaluation of clinical effectiveness of MIST ultrasound therapy for the healing of chronic wounds. Adv Skin Wound Care. 2006;19(8):437–446.
8. Kavros SJ, Miller JL, Hanna SW. Treatment of ischemic wounds with noncontact, low-frequency ultrasound: the Mayo clinic experience, 2004-2006. Adv Skin Wound Care. 2007;20(4):221–226.
9. Kavros SJ, Liedl DA, Boon AJ, Miller JL, Hobbs JA, Andrews KL. Expedited wound healing with noncontact, low-frequency ultrasound therapy in chronic wounds: a retrospective analysis. Adv Skin Wound Care. 2008;21(9): 416–423.
10. Johns LD. Nonthermal effects of therapeutic ultrasound: the Frequency Resonance Hypothesis. J Athl Train. 2002;37(3):293–299.
11. Bertuglia S. Mechanisms by which low-intensity ultrasound improve tolerance to ischemia-reperfusion injury. Ultrasound Med Biol. 2007;33(5):663–671.
12. Waldrop K, Serfass A. Clinical effectiveness of noncontact, low-frequency, nonthermal ultrasound in burn care. Ostomy Wound Manage. 2008;54(6):66–69.
13. Young SR, Dyson M. The effect of therapeutic ultrasound on angiogenesis. esis. Ultrasound Med Biol. 1990;16(3):261–269.
14. Unger P. Low-frequency, noncontact, nonthermal ultrasound therapy; a review of the literature. Ostomy Wound Manage. 2008;54(1):57–60.
15. Mukherjee S, Raghuraman H, Chattopadhyay A. Membrane localization and dynamics of Nile Red: effect of cholesterol. Biochim Biophys Acta. 2007;1768(1):59–66.
16. Duc Q, Breetveld M, Middelkoop E, Scheper RJ, Ulrich MM, Gibbs S. A cytotoxic analysis of antiseptic medication on skin substitutes and autograft. Br J Dermatol. 2007;157(1):33–40.
17. Poon VK, Burd A. In vitro cytotoxity of silver: implication for clinical wound care. Burns. 2004;30(2):140–147.
18. Kavros SJ, Schenck EC. Use of noncontact low-frequency ultrasound in the treatment of chronic foot and leg ulcerations: a 51-patient analysis. J Am Podiatr Med Assoc. 2007;97(2):95–101.
19. Whitney J, Phillips L, Aslam R, et al. Guidelines for the treatment of pressure ulcers. Wound Repair Regen. 2006;14(6):663–679.
20. Serena TE, Robson MC, Cooper DM, Ignatius J. Lack of reliability of clinical/visual assessment of chronic wound infection: the incidence of biopsy-proven infection in venous leg ulcers. Wounds. 2006;18(7):197–202.
21. Field FK, Kerstein MD. Overview of wound healing in a moist environment. Am J Surg. 1994;167(1A suppl):2S–6S.

Post new comment

  • Web page addresses and e-mail addresses turn into links automatically.
  • Lines and paragraphs break automatically.
  • Use to create page breaks.

More information about formatting options

Enter the characters shown in the image.