Check growth medium

Certain growth media have unexpectedly high particle concentrations. This can lead to confusing results, particularly during the lag phase when bacterial concentration is still low.

Here, you will measure a 1:100 dilution of the growth medium prior to inoculation. If the cells/mL concentration is below 30,000 cells/mL the growth medium contribution is negligible and you can skip ahead to the next section. If the cells/mL exceeds 30,000 cells/mL a suitable workaround must be chosen. See the info box at the bottom of this page for more information.

Prepare the growth medium for BactoBox® measurement

1. Use a serological pipette to collect a small sample from the shake flask.

2. Return the shake flask to the incubator shaker.

3. Vortex 30 seconds at max speed to ensure an even distribution of particles

4. Dilute 1:100 in diluent by adding 101 µL of sample to 10 mL of diluent. Vortex 10 sec., max speed.

5. Transfer the tubing kit to the diluted sample.

Measure the growth medium

1. Click the Measure button in Access.

2. Add metadata:

   1. Dilution: 100.

   2. Label: medium_ctrl.

Do you get the error conductivity too high?

Some cultivation media have very high conductivity and a 1:100 dilution in standard diluent is not sufficient to bring the diluted sample within the accepted conductivity limits.

You can typically solve this issue by using a 1:201 dilution: Simply add 50 µL of sample to 10 mL of diluent. For more information, please see the section Hit the right concentration.

Clean the device

1. Transfer the tubing kit to the disinfection vial.

2. Click Clean.

Summary

Now you know the particle contribution from the growth medium. Proceed to the next step to calculate the volume of starter culture needed to inoculate the growth curve flask.

Substantial background in growth medium

Does the cells/mL exceed 30,000 cells/mL at 1:100 dilution?

Several strategies can be employed if the particle concentration in the medium is substantial.

A. Remove particles by filtering the medium through 0.2 µm membrane prior to autoclaving

B. Remove particles by filtering the medium through 0.1 µm membrane instead of autoclaving

C. Subtract the cells/mL background from the growth curve results.

D. Investigate stricter gating approaches. Note that this is a power-user feature. Options A and B apply only if the particulates do not play a critical role in bacterial growth. For further assistance, contact SBT.