Hit the right concentration

Mini course: How to dilute samples prior to BactoBox® measurements.

BactoBox® has a working range from 30,000 to 5,000,000 objects/mL. Cultures may be as concentrated as 1×10¹¹ cells/mL, that is 100,000,000,000 cells/mL. Therefore, cultures must be diluted prior to BactoBox® measurements.

Dilutions of bacterial cultures

To get conductivity right, the minimal dilution factor when using BactoBox® diluent is typically 1:100. This dilution is done by a single pipetting step and works well for the early growth stages.

Dilute at least 1:100 to hit the right conductivity.

Most growth media must be diluted at least 1:100 to reach the conductivity range required for BactoBox® measurements (1,500–2,200 µS/cm).

As the cell concentration of a culture changes so does the required dilution. The table below summarizes the dilution factors used for various growth stages.

Dilution	Growth stage
1:100	Lag, acceleration and early-exponential
1:1,000	Mid- and late-exponential
1:10,000	Deceleration, stationary, and decline

Dilution schemes

Below dilution schemes provide an overview of how you obtain the different dilutions. The expandable () in each section reveals step by step videos.

1:100 dilutions

1:100 dilution is done by a single transfer of 101 µL culture of 10 mL of diluent.

Hands-on demonstration of 1:100 dilutions (click to show more)

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.

1:1,000 dilutions

1:1,000 dilution is done by two consecutive dilution steps. First a 1:100 and then a sequential 1:1,000 dilution. Note that 1 mL is removed from the second dilution vial before transferring the sample from the 1:100 vial.

Hands-on demonstration of 1:1,000 dilutions (click to show more)

1

Dilute 1:100

Label a dilution vial 1:100 .

Vortex sample vial briefly (it has already been thoroughly vortexed when sampling).

Transfer 101 µL of your sample to the 1:100 vial. Pipette up and down ×10 in the 1:100 vial to rinse the pipette tip for residual bacteria.

Cap tightly and vortex 10 seconds at maximum speed.

2

Dilute 1:1,000

Label a second dilution vial 1:1,000.

Aspirate and discard 1 mL diluent from the vial labeled 1:1,000

Transfer 1 mL of the 1:100 vial to the 1:1,000 vial. Pipette up and down ×10 in the 1:1,000 vial to rinse the pipette tip for residual bacteria. Cap tightly and vortex 10 seconds at maximum speed.

3

Measure the diluted sample on BactoBox®

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

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

1:10,000 dilutions

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

Hands-on demonstration of 1:10,000 dilutions (click to show more)

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.

Cap tightly and vortex 10 seconds at maximum speed.

4

Measure the diluted sample on BactoBox®

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

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

Learn the ropes

This mini course provided you with the essentials on how to dilute bacterial cultures prior to BactoBox® measurements. Try tutorial 3 for a hands-on example on how to adjust the dilution factors when tracking a growth curve.

Track growth curve