How to Improve Your Bacterial Expression System With BactoBox

BactoBox is an essential analytical tool to use when working with bacterial expression systems - particularly when the expression system has to be scaled. Use BactoBox to optimize your yield, and save valuable time and costs through better data in your engineering and production runs.

The Challenge: Relying on OD600 Makes Your Job Difficult

Optical Density at 600 nm (OD600) is a widely used spectroscopic method for estimating the concentration of bacterial cells in bacterial expression systems. OD600 readings guide critical decisions, from inoculum transfer to induction and later process conclusion. While OD600 is known to lack viability information, it is less commonly understood that it poorly represents cell concentrations, a crucial parameter in bacterial expression systems.

In microbiology, it's often believed that the "1.0" OD600 unit represents a specific cell concentration. However, scientists frequently encounter inconsistencies with the same bacterial strain, even under seemingly identical conditions. The problem is that the identical OD600 values can show up to a threefold difference in actual cell concentration. This variability can result in errors in overall experimental design and interpretation. 

ICC vs CFU v3

In the above experiment, an E. coli strain was grown in three different media and sampled at two different time points. All six collected samples were standardized by dilution to an OD600 value of 1 on the same spectrophotometer. The six OD600 = 1 samples were subsequently analyzed for concentrations. The figure above shows the immense variability an OD600 reading can have. The actual cell concentrations in the six different standardized samples were found to vary between 5e8 cells/mL and 1.7e9 cells/mL, a threefold difference! The cell concentrations were measured using both BactoBox (intact cells/mL) and CFUs. The scientific explanation for why there is a difference is the size of the

The Solution: Exact Cell Counts and Viability Information Save Costs

While OD600 is a fundamental tool for estimating bacterial cell concentrations, its limitations make advanced alternatives necessary for precision-critical applications. Despite being fast and cost-effective, OD600 cannot differentiate viable cells, is prone to interference from particles and metabolites, and relies on indirect measurements. These issues compromise data accuracy, especially in complex expression systems and biotechnological applications where precise monitoring is essential.

BactoBox accurately reports the exact cell concentration in a sample, irrespective of the morphology of the cells, presence of metabolites, and any of the other pitfalls that are attributed to OD600. Furthermore, BactoBox can be used to provide viability information on the cells, as demonstrating in this peer-reviewed article that compares BactoBox to fluorescence flow cytometry. And BactoBox can do all this within 2 minutes.

Conclusively, when you work with tricky bacterial expression systems, BactoBox is a must-have tool in your analytical toolpark to obtain better data, improve consistency, and reduce the number of required engineering runs for your process. Use BactoBox to:

  • Make critical decisions based on exact cell concentrations.
  • Monitor culture viability.
  • Obtain detailed insights into the state of your culture by monitoring any change in cell sizes.

The figure above illustrates that the cell concentration in an E. coli cultivation process plateaus after 7-8 hours, while the OD600 value continues to increase beyond 24 hours. In complex processes, OD600 rarely tracks cell concentration perfectly. However, it has been the only feasible tool for monitoring growth curves in real-time until now.

Reach Out to Learn More or Get a Trial

We would love to connect with you to discuss how BactoBox can be used to speed up your development work and improve the consistency of your bacterial expression systems. Please fill out the form below, and a representative from SBT will reach out to you soon.