Summary

This tutorial used an E. coli Vitroids™ disc to inoculate a shake flask containing LB medium. The initial cell concentration is below the limit of quantification, and approximately 5 hours of incubation are required before the culture can be reliably measured using a 1:100 dilution.

Although it may be tempting to use a lower dilution factor, this will typically result in conductivity errors when using the standard diluent because most growth media have relatively high conductivity.

As the cell concentration increases, the initial 1:100 dilution scheme is replaced by 1:1,000 and later 1:10,000 dilutions.

Screening for suitable dilution factors

Selecting an appropriate dilution is often the most challenging aspect of BactoBox® measurements. We recommend using the following screening approach.

Prepare a dilution series up to a 1:10,000 dilution and begin by measuring the most diluted sample.

• If the 1:10,000 dilution yields a valid cell concentration, no further measurements are required.
• If the measured concentration is below 30,000 cells/mL, repeat the measurement using the 1:100 dilution instead.

The advantage of measuring the most diluted sample first is that it minimizes the risk of clogging the external filter.

Measure the lowest dilution first when screening for suitable dilution factors

For cultures with unknown bacterial concentration you often screen, try a 1:10,000 dilution first. If the cell concentration is < 30,000 try a 1:100 dilution.

BactoBox® skills

After successfully completing this tutorial you have acquired the following skills:

Disaggregrate bacterial clumps by vortexing

Hit the right conductivity

Hit the right concentration

Plot a growth curve

Calculate growth rate and generation time

Next steps

Tutorials 1, 2, and 3 provide the basic skills needed to use BactoBox® as a stand-alone device.

After successfully completing these tutorials, you are ready to move on to more advanced studies involving your own cultures and growth media. See Workflows for inspiration on potential use cases.

We recommend you see see the Track growth curve mini course for a brief introduction to tracking growth curves via Access. This provides a powerful way to see your growth curves in real time and skip the manual logs.