> For the complete documentation index, see [llms.txt](https://help.sbtinstruments.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.sbtinstruments.com/mpd/cell-growth/carrying-capacity-k/overview-of-methods-for-k/optical-methods-for-k.md). # 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](https://sbtinstruments.com/knowledge/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](https://www.sciencedirect.com/science/article/pii/S0167701225002003) 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](/software/access/pages/measurement-group/growth-curve-group-type.md#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 . --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://help.sbtinstruments.com/mpd/cell-growth/carrying-capacity-k/overview-of-methods-for-k/optical-methods-for-k.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.