It
is the
tension 
between creativity and skepticism that has produced the stunning
and unexpected
findings of science. —Carl Sagan
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Appendix:
1. Prepare an over-night bacteria
culture s Pipette 20.0ml of LB liquid medium from the large flask (containing LB) to the 3 empty flasks s
Joint the bulb to the pipette. s Obtain 10.0mL of LB liquid. s Flame the large beaker containing LB before and after. s Release LB liquid to the empty flask. s Flame the flask before and after. s Obtain another 10.0mL for the same flask to accumulate 20.0mL s Repeat the same procedure for all 3 flasks. s Obtain inoculating loop (disposable). The inoculating loop does not need to be flamed. s Inoculate a single E. Coli colony from the agar plate to the 3 flasks. s Place the 3 flasks inside the shaking incubator for 24h at 37°C
2. Standardize the concentration of bacteria to 0.01 s
Put 1000μL
of LB into the control
couette s
Put 900μL
of LB and 100 μL
of overnight bacteria culture into
the testing couette s
Insert both couette into the specphotoment s Standardize the concentration of bacteria to 0.02 s Use the formula CiVi = CfVf
3. Determination of E. coli concentration in LB that is suitable for plate reader One of the E. coli
colonies grown on solid nutrient agar plate was inoculated in liquid LB medium
in an Erlenmeyer flask to obtain a pure culture. Aseptic technique and sterile
materials and equipment were used to minimize possibilities of contamination.
The flask was then incubated for 24hrs at 37ºC in the shaking incubator. After
which the bacteria concentration was standardized to 0.01 OD600 using
a specphotometer (see appendix 2). Then the sample was diluted using 10-fold
dilution to a final concentration of 0.01X 10-7. The dilution was
created by adding 200uL of the liquid culture in the first lane of the 96-well
plate, and 180uL of LB to lane 2 - lane 8. 20uL was from the fist lane was
transferred to the second lane using pipette and mix well. The transfer was
continued to the 8th lane and 20uL was taken out from it to ensure even volume.
The first lane was labeled 0 for a 100 dilution factor (sample with
original concentration), the second lane was labeled 10-1, and so
forth. The process was repeated for another plate. Both plates were incubated
for 24hrs. When the plates were read by the plate reader, the OD600
readings showed that the original concentration was within the concentration
limit of the plate reader; therefore 0.01 OD600 was used as the
bacteria concentration in this experiment. The different concentrations were
plated out on agar plates to examine the viability of the bacteria and the
number of colonies for each concentration.
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Enhancement of Antibiotic Action with an Application of
Ultrasound 29/04/2007 |
