Dilute and measure

The overnight culture sample is ready for BactoBox® measurements.

An E. coli shake flask overnight culture typically has ~1×10¹⁰ cells/mL. A 1:10,000 dilution is suitable to hit the right concentration for BactoBox® measurements. Dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a 1:10 000 dilution.

How to make 1;10,000 dilutions

Dilution is done by two consecutive dilution steps. First a 1:100 dilution and then a sequential 1:10,000 dilution.

Tutorials 1 explained the importance of using the correct vortexing technique for 15 mL vials. If you need a recap, expand the section below. Skip ahead if you've already mastered the technique.

Proper vortex technique

When vortexing is done correctly, a strong vortex forms in the vial within a few seconds. Follow the recommendations in the green information box and avoid the common mistakes demonstrated in the below slow-motion video (✔ demonstrates correct. ✘ demonstrates incorrect).

Good vortexing technique

Choose a mixer platform that is compatible with your vial. Some platforms can accommodate multiple vial types and sizes.

Use a gentle grip on the vial cap while vortexing the tube. Start with a firm 3-finger hold on the cap and switch to a gentle press with one finger once the motion starts.

• The left panel demonstrates proper vortexing technique: The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.

• The center panel demonstrates improper technique: The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.

• The right panel demonstrates improper technique: A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.

Step by step

1

Label two dilution vials

Label first dilution vial 1:100.

Label second dilution vial 1:10,000.

2

Dilute 1:100

Transfer 101 µL of your sample to the 1:100 vial.

Suspend ×10 to rinse the pipette tip for residual bacteria.

Vortex 10 seconds at maximum speed.

3

Dilute 1:10,000

Transfer 101 µL of the 1:100 vial to the 1:10,000 vial.

Suspend ×10 to rinse the pipette tip for residual bacteria.

Vortex 10 seconds at maximum speed.

4

Measure the diluted sample on BactoBox®

The below video demonstrates how to do the bacterial measurement

• Transfer the tubing kit to the 1:10,000 vial.

• Press Measure to start a new measurement and wait for the results.

A measurement consists of three distinct stages

First stage is Starting up: Conductivity and flow is checked

Second stage is Measuring: Electric fields in the flow cell are turned on and cells are counted

Third stage is Emptying: Pump direction is reversed and the flow path is drained.

5

Create a table of the results

Note down your results in a table.

In this example, the concentration is 540 000 cells/mL out of 540 000 total/mL. The first number is the cell concentration; the latter is the total objects detected in the measurement. Cell concentration is reported as a subset of the total objects. The numbers are identical, i.e. all detected objects are cells.

Measurement ID	Dilution	Cells/mL	Total/mL
M77	10,000	540 000	540 000

6

Clean the device

Transfer the tubing kit to the disinfection vial and press Clean .

Always run a Clean after a measurement.

Summary

You have now measured the cell concentration in the E. coli overnight culture. Notice that the BactoBox® measurement was faster here than in the first tutorial Do a simple measurement. The fast results feature is triggered because the bacterial concentration in the diluted sample was higher.

In the next page, Summary, we will give an overview of the procedure and the obtained results.

Run your measurements in Access

In this tutorial we keep things simple and demonstrate BactoBox® as a stand-alone-device.

But a manual log like the above table belongs to last century. Manual logs may get lost and it is common to make typing mistakes when you transfer data to spreadsheets.

Access is a powerful tool to keep track of dilution factors, sample IDs and results. Data are stored on the device and can be exported easily as a csv file. See an example in Track growth curve.