Shake flask or fermenter

Measure bacteria from a shake flask or fermenter

Overall approach

The overall approach for shake flasks or liquid samples from fermenters is the same:

1. Disaggregate to ensure that your sample consists of single cell bacteria.

2. Dilute your sample until it is within the limits of detection. Target 0.5–4 million cells/mL.

We do not know the cells/mL concentration beforehand, and we don't want to clog your BactoBox® setup. Therefore, it is best to start with the most diluted sample and then work your way up to the 0.5–4 million cells/mL target range.

Note that this is similar to the approach for plate harvest.

Don't know the concentration of your sample?

Make a dilution series and measure the most diluted sample first. This avoids clogging your BactoBox® setup. It also avoids carry-over from your previous sample vial.

Once you are above the lower limit of detection (30 000 cells/mL), target 0.5–4 million cells/mL.

Detailed protocol

Dilution series

1

Prepare your sample

Make sure that your sample is agitated sufficiently prior to sampling.

Grab 10 mL of sample and transfer it to an empty vial.

If your sample is very viscous, don't trust the pipette volume. Instead, weigh your sample.

2

Disaggregate your sample

Disaggregate clumps and chains until you have a single-cell suspension:

1. Add 3 mm glass beads to your sample vial until the liquid level rises 1 mL.

2. Vortex your sample vial (1 min, max RPM)

3

Dilute your sample

Prepare three dilution vials: Take three empty vials and add 10 mL of diluent to each.

1st dilution (10^-2)

Dilute 1:100: Transfer 101 μL from your disaggregated sample into the first dilution vial. Vortex (15 sec., Max RPM).

2nd dilution (total 10^-4)

Dilute 1:100: Transfer 101 μL from your first dilution vial to your second dilution vial. Vortex.

3rd dilution (total 10^-6)

Dilute 1:100: Transfer 101 μL from your second dilution vial to your third dilution vial. Vortex.

4

Measure your sample

Measure with BactoBox®. Start with the least concentrated sample and move towards higher concentrations.

First, clean your BactoBox® setup with a fresh disinfection vial.

Measure on the 10^-6-diluted sample

If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.

Measure on the 10^-4-diluted sample

If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.

Measure on the 10^-2-diluted sample

Is even the most-concentrated sample below the limit of detection? See Concentrate your sample for next steps.

Multiply the cells/mL concentration with the dilution factor to calculate the cells/mL concentration in your sample stock.

If you need technical replicates, perform these at step 1 to include variation from the Mix and sample step.