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5.13 Determining E coli Concentration With Spectrophotometer
I) Purpose
- To determine if OD600 can be used to determine E coli concentration
- According to reference The number of cells in a bacterial culture can be estimated by reading the absorbance at 600nm (OD600). For E. coli cells, an OD600 of 1.0 ≈ 5E8 cells/ml
II) Expected Outcome
- linear relationship between absorption and 1/dilution factor
III) Reagants Used
- E. Coli BL21 overnight from 5.12; LB prepared by TA in 100mL flask
IV) Actual Procedure
1. Dilution series
- Label 6 tubes 1 through 6, to each of the test tubes add 2mL of LB.
- 5mL of E coli overnight (21 hours) was to test tube 1. The content was then pipetted up and down to mix well.
- Performing dilution series by transforming 5mL from tube n to tube n+1 in order of n=1,2,3,4,5. The dilution factor is thus 7/5
2. Measurements using spectrophotometer
- Spectrophotometer was warmed up and set to take measurements at 600nm.
- Pure LB was used to as blank
- Before each measurement, the cuvette was rinsed with 1mL of distilled water and 1mL of E coli solution to be measured.
- 1mL of E coli solution at various concentration was pipetted into each cuvette for measurement.
V) Results
1. Raw data
Data From Spectrophotometer Reading
| Test Tube
| Concentration with Respect to Overnight
| Absorption at 600nm
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| 0
| 1
| 0.623
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| 1
| 0.714
| 0.451
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| 2
| 0.510
| 0.313
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| 3
| 0.364
| 0.288
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| 4
| 0.260
| 0.182
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| 5
| 0.186
| 0.098
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| 6
| 0.133
| 0.137
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2. Plot
3. Linear Regression Result
ADD
VI) Conclusion
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