1:100 dilutions

The lag phase, acceleration phase, and early exponential growth phase are typically monitored using 1:100 dilutions prepared in 15 mL centrifuge tubes. The procedure for preparing these dilutions is described below.

Special emphasis is placed on proper vortexing of the 15 mL tubes, as inadequate mixing is one of the most common pitfalls in sample preparation for BactoBox® analyses.

How to make 1:100 dilutions

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

Tutorials 1 explained the importance of using the correct vortexing technique for 15 mL vials. If you need a refresher, 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

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 like the one below.

Measurement ID	Sampled at	Dilution	Cells/mL	Total/mL
Q17	11:30	100	89 000	130 000

4

Clean the device

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

5

Repeat 1:100 dilutions for subsequent samples

When the 30-minute timer expires, repeat the same sampling, disaggregation, 1:100 dilution, measurement, and cleaning procedure for the subsequent samples.

Proceed to the 1:1,000 dilutions when the measured cell concentration of the last sample is ≥ 1,500,000 cells/mL.

Summary

You measured the first time point after ~5 hours of incubation. Proceed to 1:1,000 dilutions to determine cell concentration with BactoBox®.

Workarounds to analyze dilute bacterial cultures

To keep this tutorial as simple as possible, only 1:100, 1:1,000, and 1:10,000 dilutions are used. However, custom diluents and dilution factors can also be applied.

Lower dilution factors provide higher sensitivity when measuring dilute bacterial cultures. See Dilute 1:10 for more information.