Optical methods for Κ

Turbidimetric methods like optical density (OD) and backlight scattering are often used as proxies for cell concentration. The methods conveniently integrates with 96-well plate readers or as online sensors for frequent automated measurements. This is often used for relative assessment of carrying capacity, but note that the liquid movement and oxygen transfer in small 96 well plate does not rival shake flask or bioreactor experiments.

At higher cell concentrations detector saturation may occur. To get the measurements within the more reliable linear range of the instruments culture samples are diluted and measured off-line in a cuvette or similar.

Optical methods depend on object size

While optical methods are easy and fast, the output is an arbitrary unit and calibration is needed to convert OD to cell concentrations. Calibration is tricky because optical methods are dependent on other factors than cell concentration. The cell size changes throughout a growth curve. In early-exponential growth phase plenty of nutrients are available and the fat cells lead to a high degree of light scattering and less light reaching the detector (high OD per cell ratio). Contrarily, in stationary growth phase the nutrient are scarce and cells adapt by shrinking in size. This leads to less light scattering (low OD per cell ratio). For more information, see Understanding OD600.

Optical methods are strongly dependent on the size of objects. The left figure depicts a cuvette containing large, exponential stage bacteria. The right figure illustrates the same bacteria in stationary stage, where they are considerably smaller and scatter less light per cell.

In theory, different calibration factors are needed for each stage of the growth curve, but it is often difficult to determine the specific growth phase of bacteria. The industry standard is to apply a single calibration factor for each species, despite potential variations across growth phases and media.

Real-life example of misleading OD data

Sometimes the influence of size and concentration leads to misleading conclusions when optical density is taken as a proxy for cell concentration. The first figure in the E. coli dataset below (A) shows the relationship between the added carbon source (glycerol) and max OD during the growth curve. R² ≈1 suggests a strong linear relationship. If you are tasked with optimization of a growth medium for max cell concentrations, you would get a clear indication that higher glycerol is better.

But - surprisingly - the R² is extremely poor (0.0003) when the dependency between glycerol concentration and direct cell counts is investigated by BactoBox® analyses (B). Data presented in the peer-reviewed performance qualification shows a strong correlation between BactoBox® and total cell concentrations for E. coli so OD must be the odd one out. The data shows that more glycerol is not the solution for higher cell concentrations; the cultures are likely constrained by other factors than the carbon source.

The CIZE metric (D) offers an (at least partial) explanation to why OD is deceiving. The CD medium with 4 mL/L glycerol has a very low CIZE at max OD. Even though the concentration is high the small CIZE may result in relatively low light scattering. The 7 mL/L has lower cell concentration, but substantially higher CIZE, which likely explains the higher max OD. Finally, the 10 mL/L CD culture has similar concentration as the 4 mL/L but the CIZE is substantially bigger.

Also note that the different growth curves reach max OD at different incubation times. For BactoBox® the cultures hit max value at same incubation time and the cell concentration is relatively constant after reaching the max value.

Effect of glycerol-concentration investigated with OD and direct cell counts. E. coli cultivated in shake flasks with Bacto™ CD Supreme fermentation production medium. Glycerol was added as carbon source at a concentration of 4, 7, or 10 mL/L. Dashed lines in figure D correspond to CIZE (secondary axis)

Practical recommendations

Be cautious when using OD to determine which growth medium has the highest carrying capacity. Optical methods serve well as quick and easy screening tools to exclude poor-performing media recipes. But once the promising growth media recipes are shortlisted, you should use direct cell counts from microscopic or flow cytometric methods for the further evaluation.

Finally, it is worth noting that optical methods cannot distinguish between bacterial and non-bacterial objects. Optical methods are likely not fit for purpose if your culture has a high background of non-bacterial objects .