Measure a dense culture

Measure a dense overnight culture, summarized to key steps.

This is the Measure a dense culture tutorial, summarized to its key steps. First growing, and later using a dense E. coli culture grown overnight, we disaggregate, dilute 1:10,000 in two steps, and read a concentration off BactoBox®.

Where you start: a Vitroids™ disc dropped into LB medium for an overnight culture.

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1

Start an overnight culture

In the afternoon, drop a Vitroids™ disc (or 50 µL of an in-house E. coli glycerol stock) into 50 mL autoclaved LB in a baffled shake flask. Incubate 16 hours at 37 °C, 200 RPM.

2

Sample and disaggregate

Swirl the flask, aspirate 2 mL into a centrifuge vial, and vortex 1 minute at maximum speed.

Take a 2 mL sample of the overnight culture.

3

Dilute 1:100

Transfer 101 µL of the sample into the 1:100 vial, suspend ×10 to rinse the tip, and vortex 10 seconds.

First 1:100 dilution.

Vortex properly between dilution steps

Form a visible cyclone in the vial.

Allow enough time for the liquid to homogenize.

Hold the vial cap gently while vortexing.

4

Dilute 1:10,000

Transfer 101 µL of the 1:100 vial into the 1:10,000 vial, suspend ×10, and vortex 10 seconds. Two 1:100 steps give a 1:10,000 dilution.

Second 1:100 step lands you at 1:10,000.

5

Measure

Move the tubing kit to the 1:10,000 vial and press Measure. The dense sample triggers fast results. Write down the cells/mL and total/mL.

Press Measure and wait for the result.

6

Clean

Move the tubing kit to the disinfection vial and press Clean. Always clean after a measurement.

Finish with a Clean.

Multiply the reading by the dilution factor to get the concentration of your undiluted culture.

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Do it yourself

That is the whole loop. The full tutorial covers the overnight culture, the two-step dilution, and the calculation, step by step.

Measure a dense culture