Ng Siang Hang
002206-029 cqn461
Biology Extended Essay
4.1.1
Procedure
I)
Plotting a Standard Curve for Staphylococcus aureus
Firstly, the optical density at 600 nm (OD600) of the Staphylococcus aureus solution cultured by teacher was obtained. The bacteria solution was diluted to 10-3, 10-4 and 10-5, and inoculated onto agar plates. The plates were incubated for 24 hours and then observed. The number of CFU formed on the 10-5 dilution plate was recorded. The total Staphylococcus aureus concentration in the bacteria solution was determined as below: Total
concentration
Total CFU per ml
Dilution factor
Next, the bacteria solution was diluted to 1:2, 1:4, 1:8 and 1:16. OD600 at each dilution was obtained. Staphylococcus aureus concentrations in the dilutions were calculated as follows:
concentration
Total
concentration dilution factor
A standard curve was plotted, with Staphylococcus aureus concentration as the y-axis and OD600 as the x-axis.
II)
Determining the Staphylococcus aureus concentration in the samples [12]
OD600 of Staphylococcus aureus in the samples were obtained by “blanking” OD600 of nutrient broth and Manuka honey. This was done by first preparing blank buffers. Blank buffers are solutions identical to the honey samples (in terms of volume of nutrient broth, volume and concentration of Manuka honey) except that the blanks do not contain Staphylococcus aureus. OD600 of blank buffers were first measured and zeroed. Then, OD600 of the honey samples were taken. The OD600 obtained would be the OD600 of Staphylococcus aureus. Staphylococcus aureus concentrations of the samples were determined by referring the OD600 of Staphylococcus aureus to the standard curve.
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