> 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/inoc/explainers/od-is-blind-at-low-cell-concentrations.md). # OD is blind at low cell concentrations Optical methods such as OD₆₀₀ offer a convenient approach to frequent tracking of bacterial cultures. Despite these advantages, the sensitivity is relatively low and OD₆₀₀ is often insufficient for tracking changes during lag, deceleration and early-exponential growth stages. This study aimed to investigate the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for OD₆₀₀ and compare these with the BactoBox® method. For the full details, see [OD misses low cell concentrations](/mpd/cell-growth/optical-drawbacks/od-misses-low-cell-concentrations.md) We diluted a stationary stage culture of *E. coli* (ATCC 25922) to different concentrations.

## BactoBox® quantifies 50–500× lower concentrations than OD₆₀₀ Limit of detection and quantification is assessed by determining the "noise level" of a sample that contains no analytes (a blank sample). The grey bands on the plot represents the "blank band", i.e. the arithmetic mean value of the blank measurements ± standard deviation (σ). The dashed yellow and red lines represent the LLOD[^1] and LLOQ[^2], respectively. X-axis represents concentration calculated from the known dilution factor relative to the 12 hour culture.

Lower limit of detection (LLOD) and lower limit of quantification (LLOQ) investigated for OD₆₀₀ and BactoBox®. Each data point represents arithmetic mean ± standard deviation. The dashed red lines represent LLOQ whereas the dashed yellow lines represents LLOD. The lavender line represents BactoBox® lower limit defined by the technical specifications. The concentrations on the x-axis are calculated from the dilution factors relative to the original culture. Prior to BactoBox® measurements, the bacterial stocks were further diluted to hit the right conductivity; the labels on the BactoBox® plot show which dilution was used for the given stock concentrations.

### Sensitivity of the OD₆₀₀ method The OD₆₀₀ measurement for the culture diluted to 1×10⁸ cells/mL is above the LLOD, but it is still not safely above the LLOQ. The OD measurement for the culture diluted to 5×10⁷ cells/mL is in principle beyond the LLOD, but the error bands are hugging the yellow border. The table summarizes the key metrics. In OD units the LLOQ is 0.123 AU. Converting to a cell concentration requires interpolation. We used the three highest data points from the OD measurements for a linear interpolation in the linear space (not log transformed, results not shown). By this approach LLOQ is \~2×10⁸.

Method	Upper blank band (mean + σ)	LLOD (mean + 3σ)	LLOQ (mean + 10σ)
OD₆₀₀	0.003 + 0.012 = 0.015	~0.04 AU ~5×10⁷ cells/mL	~0.123 AU ~2×10⁸ cells/mL

### Sensitivity of the BactoBox® method The technical specification for BactoBox® uses a lower limit of detection for the measured sample of 30,000 cells/mL, i.e. 3×10⁴ cells/mL. This is represented by the dashed lavender line in the above figure. The lavender line sits safely beyond the upper blank band, LLOD and LLOQ. This demonstrates that the specified BactoBox® lower limit of detection is reliable. In practice, to hit the right conductivity, most cultures must be diluted prior to BactoBox® measurements. This affects the *practical* sensitivity when performing BactoBox® measurements. * A dilution factor of 1:100 is the typical lowest dilution factor when using standard diluent. In this case the practical lower limit for the BactoBox® method is 3×10⁶ cells/mL. This is **\~50× better** than the LLOQ of the OD₆₀₀ method. * A dilution factor of 1:10 is often possible if sterile-filtered low conductivity water is used instead of BactoBox® diluent. This improves practical lower limit for BactoBox® to 3×10⁵ cells/mL. This is **\~500× better** than the LLOQ of the OD₆₀₀ method. We compare OD₆₀₀'s statistical LLOQ against BactoBox®'s specified lower limit, because that is the number you work to in practice. The results are summarized in the table below.

Method	Upper blank band (mean + σ)	LLOD (mean + 3σ)	LLOQ (mean + 10σ)
BactoBox® 1:100	6.2×10⁵ cells/mL	9.3×10⁵ cells/mL	2.0×10⁶ cells/mL
BactoBox® 1:10	6.2×10⁴ cells/mL	9.3×10⁴ cells/mL	2.0×10⁵ cells/mL

## Implications *E. coli* has a relatively normal bacterial cell size. Yet for a stationary stage culture \~5×10⁷ cells/mL cell are needed before readings are distinguishable from a blank measurement. Cultures with smaller cells like *Mycoplasma* spp may require even higher concentrations to be detectable by OD. In conclusion, OD is bad at assessing cultures in the early, dilute, growth phases. [^1]: Lower limit of detection: Mean_blank + 3σ. [^2]: Lower limit of quantification: LLOQ: Mean_blank + 10σ --- # 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, and the optional `goal` query parameter: ``` GET https://help.sbtinstruments.com/inoc/explainers/od-is-blind-at-low-cell-concentrations.md?ask=&goal= ``` `ask` is the immediate question: it should be specific, self-contained, and written in natural language. `goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal. 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.