1:10,000 dilutions

When the culture reaches the deceleration and stationary growth phases you should switch to a 1:10,000 dilution.

The rule of thumb still applies when deciding whether to increase the dilution factor: switch to the next dilution scheme if the measurement obtained approximately 30 minutes earlier was ≥ 1,500,000 total cells/mL.

How to make 1:10,000 dilutions

A 1:10,000 dilution is obtained by two sequential dilutions. First dilution is the standard 1;100 dilution done by transferring 101 µL to 10 mL of diluent. Second dilution is an additional 1:100 dilution from the first 1;100 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

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.

Add your results to the measurement table.

5

Clean the device

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

6

Repeat 1:10,000 dilutions for subsequent samples

Repeat the sampling, disaggregation, 1:10,000 dilution, measurement, and cleaning procedure for the next samples.

Conclude the sampling when two subsequent data points are within ±25%.

Summary

In deceleration and stationary phase 1;10,000 dilutions are necessary to hit the right concentration. By now you have gathered sufficient data points to plot a nice growth curve and calculate the growth rate. Proceed to Data analysis.