It
is the
tension 
between creativity and skepticism that has produced the stunning
and unexpected
findings of science. —Carl Sagan
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Procedures:
E.
coli
liquid culture with concentration of 0.01 was pipetted to 96-well plates, each
well has a content of 200uL. There plates were exposed to 42kHz ultrasound at
five different time lengths: 10min, 20min, 30min, 40min, 50min and 60min.
Another plate was not sonicated, which acted as a control. After the sonication,
all the plates were incubated for 24hrs. The viability of E. coli was
based on the values obtained from the plate reader and was compared with the
control, which had no bacteria.
Figure 10. 96-well plate. It is used to grow
bacteria and to ensure 2. Test for MIC of spectinomycin on E.
Coli DH5α In order to find the MIC of spectinomycin, a 2-fold serial
dilution with broth was used to dilute spectinomycin concentration covering a
range of 256μg/mL to 1μg/mL. The dilution was created by adding
spectinomycin of concentration of 256μg/mL to the first lane and adding 100μL
of E. coli of concentration of 0.01
from lane 2 to lane 12 in the round bottom 96-well plate. Then 100μL of
spectinomycin was transferred from the first lane to the second lane and mix
well. This transfer was continued to the 12th lane, in which the concentration
was reduced to 1μg/mL. This process was repeated for one more plate, which
acted as the control. Note that the actual E.
coli concentration was 0.005, due to 100μL of bacteria of concentration
0.01 and 100μL of spectinomycin being added to the initial lane of the
serial dilution. The two 96-well plates were then incubated for 24h and OD
levels read by the plate reader. The lowest concentration of spectinomycin
resulting in almost no growth of the bacteria was identified as the MIC, and the
concentration of spectinomycin that was one deviation below the MIC was the one
used in the synergy test.
3. Test
for synergy (combination of ultrasound and
spectinomycin) After the two preliminary tests were done for the effects E.
coli generated by ultrasound and the MIC of spectinomycin, the next step was
to test if the ultrasound increased the effectiveness of spectinomycin in
killing E. coli. Five 96-well plates
were set up for E. coli with a concentration of 0.005 with added spectinomycin of
concentration one deviation below the MIC. Then the plates were sonicated with
ultrasound with a frequency of 42kHz over different time length: 15min, 30min,
45min, and 60min. Another plate was set up without sonication as the control.
The plates were then incubated for 24h and their OD levels read by the plate
reader. The viability of E. coli was
based on the values obtained from the plate reader and were compared with the
control (no sonication).
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Enhancement of Antibiotic Action with an Application of
Ultrasound 01/05/2007 |
