After removing irrelevant cultures, it's time to measure the concentration of the remaining overnight cultures. Begin with the culture having the lowest turbidity, which in this case is the vial -8 . We will use a 1:1 000 dilution in two sequential dilution steps: An initial 1:100 dilution followed by a additional 1:10 dilution.
1
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.
2
Dilute 1:100
Transfer 101 µL of your sample to 10 mL of diluent. Vortex 10 seconds at maximum speed.
3
Dilute 1:1 000
Remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.
Transfer 1 mL of the 1:100 dilution to the this vial. Vortex 10 seconds at maximum speed.
4
Measure on the diluted sample
Transfer the tubing kit to the 1:1 000 vial.
Press Measure in Access to start a new measurement.
Dilution: 1 000
Label: overnight_-8
Pro tip: 1-step 1:1 001 dilution.
The described 2-step procedure for a 1:1,000 dilution is reliable with high precision. However, it demands a longer sample preparation time. If you have good pipetting skills, you can save time, sample and diluent by doing a 1-step dilution. Simply transfer 10 µL of sample to 10 mL of diluent to get a 1:1 001 dilution.
Summary
Of the remaining vials, the culture with the lowest turbidity has a concentration of 1.4 × 109 cells/mL. Next, we will Check remaining cultures to identify the culture that has just reached onset of stationary phase.
