Track deceleration phase

In the section Plan experimental timeline you did the calculations needed to get a culture in the deceleration phase. At this point, the concentration is usually 1×10⁹–1×10¹⁰ cells/mL and dilutions of 1:10,000 should be sufficient to get within the BactoBox® working range.

Use triplicate repeats to get a good assessment of mean and standard deviation. This is important to know if the concentration is truly changing or if differences in concentration is simply due to inherent variability. We use a cold rack for arresting further cell divisions as otherwise the concentration may drift during repeat measurements.

Stop further cell divisions by quick cooling

Arrest further growth by transferring the sample to an cold rack.

We want precise measurements with small error bars for each measurement method. If the cells are still dividing post-sampling, this will lead to drift in concentrations and pronounced variability.

The below video demonstrates the sample workup.

Step by step

1

Retrieve a sample

Use a serological pipette to sample 1 mL culture.

Transfer to a 5 mL centrifuge tube and 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.

Pro tip: Add information on incubation time to measurements

The Access column calcHPI (calculated hours post inoculation) is a straightforward way to append incubation time to your measurements. If your cultures were inoculated 24 hours before the first measurement, then add the number 24 to the HPI cells for each label. This will automatically offset the measurements with 24 hours. This is optional, but very convenient.

Summary

You have now determined the cell concentration in deceleration phase. Next step is to Track hourly.