The Effects of Low-frequency Ultrasound (35 kHz) on Methicillin-resistant Staphylococcus aureus (MRSA) in vitro
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This dual action is designed to fragment tissue and focus ultrasound energy propagated toward the treatment area through the saline jet via the scoop shape. Sterile normal saline was connected to a port on the handpiece and exited the curette as a saline jet, which serves to irrigate and cleanse the wound of tissue fragments, debris, exudate, and other matter as well as to provide a coupling medium for ultrasonic energy transmission directly or through the scoop shape.
The handpiece with its treatment curette probe was mounted vertically on a ring stand (see Figure 2). The curette was submersed to midpoint in the test tube and then treatment was initiated. The test tube was moved vertically up and down in the broth, avoiding contact to the bottom or sides of the tube, and the curette remained submersed throughout the experiment.
LFU treatment of MRSA. Three separate experiments with three replications each were conducted. Bacteria were obtained from clinical stock cultures as described and prepared at concentrations of 106 CFU/mL3 for all experiments except the flow cytometry and ZOI studies; for those, MRSA was prepared at 108 CFU/mL3. LFU was delivered at 35 kHz as described. Control (untreated bacteria) and treated bacterial samples were subcultured and grown overnight at 37˚ C. At 24 hours, bacterial colonies were counted on each plate and CFUs were recorded.
Observational culture changes. All control (untreated) and LFU-treated cultures were visually inspected by a clinical specialist in microbiology. Colonial appearance including size, hemolytic pattern, and odor were recorded per clinical microbiology lab practice.
Scanning electron microscopy. Three samples were prepared from each of the 103 and 104 dilutions prepared from the control (untreated) and 40-kHz, LFU-treated MRSA samples. Control (untreated) and 40-kHz, LFU-treated bacterial samples were adhered to 12-mL cover slips for 1 minute and subsequently rinsed three times in 0.9% isotonic, sterile saline before fixing in 2.5% glutaraldehyde. Fixed samples were transported to the electron microscopy suite in 2.5% glutaraldehyde and then rinsed three times with 0.1 M Millonig’s buffer and subsequently dehydrated in progressively increasing concentrations of ethanol to 100%. Samples were critical point dried, sputter-coated with gold/palladium, and viewed with a Philips 515 scanning electron microscope at 30 kV. Digital images were recorded with a Nikon D80 digital SLR or Polaroid camera. The electron microscopist and primary investigator examined all fields of view for each sample. Digital images were taken of the fields with the greatest number of bacteria present.
Flow cytometry study. Two samples each of 104 dilutions prepared from control (untreated MRSA) and 35-kHz, LFU-treated MRSA (treated for 60 seconds) were submitted to the flow cytometry facility for viability studies. These cells were initially stored at -4˚ C after treatment until transported on ice to the flow cytometry facility (~10 minutes). Cell suspensions were treated with a viability stain (fluorescein diacetate) at 1ug/mL and allowed to incubate for 30 minutes on ice. Viable cells were able to concentrate the fluorescence via the nonspecific esterase activity of the cell and to concentrate this fluorescence in the cell. Nonviable cells were not able to concentrate the fluorescence due to leakiness of the cell wall. Using a BD FACS Canto instrument to detect fluorescence, 10,000 events were analyzed for green fluorescence per cells after gating on the cells using forward and side scatter.