Dilute and measure

By now the Vitroids™ disc is solubilized and a single-cell suspension is ready for BactoBox® measurements. The bacterial concentration is low so we use as little dilution factor as possible to hit the right conductivity. This is typically 1:100.

How to make 1:100 dilutions

1:100 dilutions are done by transferring 101 µL sample to 10 mL of diluent.

Proper vortexing of the 15 mL vial is important for reliable results

Proper vortexing of the 15 mL vials is critical. It isn't trivial. It requires a suitable vortex mixer attachment and a proper hold of the vial.

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

Dilute 1:100 in BactoBox® diluent

The below video demonstrates how to do a 1:100 dilution.

• Transfer 101 µL of your sample to 10 mL of diluent.

• Suspend ×10 to rinse the pipet tip for residual bacteria

• Vortex 10 seconds at maximum speed.

2

Measure the diluted sample on BactoBox®

The below video demonstrates how to do the bacterial measurement

• Transfer the tubing kit to the 1:100 vial.

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

3

Create a table of the results

Note down your results in a table.

In this example, the concentration is 89 000 cells/mL out of 130 000 total/mL. The first number is the bacterial concentration; the latter is the total objects detected in the measurement. Bacterial concentration is reported as a subset of the total objects and therefore 41 000 objects were something other than bacterial cells. The concentration may differ between different Vitroids™ discs.

Measurement ID	Dilution	Cells/mL	Total/mL
Q17	100	89 000	130 000

Full measurement cycle is used for samples with low bacterial concentration

Because the bacterial concentration is in the low end of the BactoBox® working range, the full measurement time is used.

4

Clean the device

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

Always run a Clean after running a measurement.

Summary

You have now measured the cell concentration in the liquid derived from the solubilized Vitroid™ disc. 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.