Determine concentration after incubation
Now, you will determine the concentration after overnight incubation. Cell concentration is typically in a ball-park range of 1 × 1010 cells/mL, and therefore a dilution of 1:10,000 is usually suitable. This dilution is obtained by two sequential 1:100 dilutions. Below we demonstrate this step by step using the Access interface. You can also use BactoBox® as a standalone device and do the calculations manually.
Retrieve and disaggregate a sample of the overnight culture
Use a sterile serological pipette to transfer 10 mL of the overnight culture to a sterile vial.
Vortex thoroughly to disperse any cell clumps. For an E. coli culture it is usually sufficient to vortex 30 seconds at max speed.
Proper vortexing is important for reliable results!
Ensure that you see a proper vortex forming. Poor vortexing can cause inadequate disaggregation, leading to low precision in replicates and non-linearity in dilution series.
Different pedestals exists for different sizes of vials. In the video, we show a multipurpose pedestal that works for many types of vials. Ensure that your pedestal is compatible with the given vial.
Dilute 1:100
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
Dilute 1:10 000
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
Measure the diluted sample on BactoBox®
Transfer the tubing kit to the 1:10,000 vial.
Press Measure in Access to start a new measurement.
Dilution: 10 000
Label: Overnight_culture
After completing the measurement, Access will automatically calculate the bacterial concentration in the shake flask. To view the full table, drag the panel sideways.
Summary
Now you know the bacterial concentration of your overnight culture. With cells from an agar plate as input material, you now have a liquid starter culture with a well-defined cell concentration.
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