Continue hourly measurements throughout the deceleration and stationary phases until the cell concentration stabilizes. We will detail what stable means on next page.
Use a serological pipette to sample 1 mL culture. Transfer to a 5 mL centrifuge tube. Note down the sampling time.
2
Disaggregate cell clumps
Vortex the vial 1 minute at maximum speed to disaggregate cell clumps. See Break up clumps and chains if your bacterium needs more aggressive disaggregation than this.
3
Arrest further cell divisions
Transfer to a cold rack to stop cell growth. Let the sample cool for at least 1 minute.
4
Dilute 1:100
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
5
Dilute 1:10 000
Transfer 101 µL of the diluted sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
6
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: staph-epi-harvest or similar.
After completing the measurement, Access will automatically calculate the cell concentration in the culture in the field Cells/mL × Dil.
7
Measure repeat #2 and #3
Repeat steps 4–6 two times more to obtain triplicate repeats. Vortex the sample briefly prior to transferring 101 µL.
Use exactly the same input information as in step 5. When identical sampling time and label is used, Access will treat the data as replicates.
8
Conclude measurements
Stop the measurements when the relative change is <5% two consecutive measurements. We will detail the calculations in Conclude engineering run.
Summary
You tracked the cultures hourly throughout deceleration and stationary phase with triplicate repeats. In the next step, Conclude engineering run, you will decide when to conclude the experiment.
