1. Calculate the concentration of the unknown sample. The concentration of the standard is 100μg/ml.

2. Write these formulas: the log of the dilution,… the average of the parallel standards,… and the average of the parallel samples

3. Select the cells with the formulas, click on the tiny square on the right bottom corner of the selected square, and drag it (autofill) into the next lines. Click on the chart wizard

4. Choose the „XY Scatter” chart type Choose the series tab Add data

5. Write the name of the first data series Click on the X values (dilution) Choose the dilution (log) values Click on the Y values (OD values) Choose the standard OD values =Sheet1!$A$17:$A$28 =Sheet1!$C$17:$C$28

6. Do the same procedure with the unknown sample data also

7. Straight line can be fitted on point 5 to 8 of the standard curve, and the on point 3 to point 6 of the sample titration curve

8. Data of the standard curve linear part Data of the sample curve linear part You can use this data to draw the linear parts

9. Right click on the line and choose the „Add Trendline” option

10. Linear trendline Display equation on chart (Options tab)

11. The equation of the standard line Do the same with the sample line

12. sample: y = -0.3778x + 2,8471standard: y = -0.3678x + 3.5265 e.g. OD 1.2  x=4.359OD 1.2  x=6.325 The 10 4.359 =22856 –fold dilution of the sample has equal OD than…. …the 10 6.325 =2113489 –fold diluted standard You can calculate the dilutions of different OD solutions with the equations The sample is 2113489/22856= app. 92x thinner than the 100µg/ml standard 1.08 μg/ml

13. Note This is a demonstrative tutorial example! You can get the result easier! (eg. You need only the equation of the standard trend line compared to an appropriately chosen dilution and OD value of the sample for get the correct result)