1:1,000 dilutions

As the bacteria multiply, the cell concentration increases, and a 1:100 dilution may eventually exceed the maximum concentration of 5,000,000 total cells/mL permitted for a BactoBox® measurement.

At this point, switch from the initial 1:100 dilution to a 1:1,000 dilution.

Rule of thumb for when to switch to 1:1,000 dilutions

To avoid the “too many particles” error during the exponential growth phase, follow this simple rule of thumb: if the previous 1:100 dilution measurement, taken approximately 30 minutes earlier, exceeded 1,500,000 cells/mL, switch to a 1:1,000 dilution for the next measurement.

How to make 1:1,000 dilutions

A 1:1,000 dilution is prepared by performing two sequential dilution steps. The first step is the standard 1:100 dilution, which is prepared by transferring 101 µL of sample to 10 mL of diluent.

The second step is a 1:10 dilution of the first vial. To prepare this dilution, transfer 1 mL of the 1:100 dilution to a second dilution vial. Because the dilution vials contain a standard volume of 10 mL, you must first discard 1 mL of diluent from the second vial.

Step by step

Remember to disaggregate: Vortex each culture sample for 1 min at max speed.

Set a 30 min timer after each sampling point.

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.

Add your results to the measurement table.

4

Clean the device

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

5

Repeat 1:1,000 dilutions for subsequent samples

Repeat the sampling, disaggregation, 1:1,000 dilution, measurement, and cleaning procedure for the next samples.

Proceed to the 1:10,000 dilutions when the measured cell concentration of the last sample is ≥ 1,500,000 cells/mL.

Summary

Once the cell concentration increased it was necessary to switch from 1:100 to 1:1,000 dilutions. As the cell concentration continues to rise you will need to use 1:10,000 dilutions.