Team:BYU Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.13 Determining E coli concentration with spectrophotometer

Small Phage

March-April

May-June

July-August

September-October

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

2. Plot

VI) Conclusion

The results show us that there is a linear relationship between the optical density reading and bacteria concentration. This means that we can use the optical density reading to determine the concentration of bacteria.