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

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A Newton (RA Newton, PhD; unpublished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
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