> 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/drifts-in-od-to-cfu-ratios.md). # Drifts in OD to CFU ratios OD600 is often used as a real-time stand-in for plate counts. You prepare different dilutions of the same cultures, measure with OD and CFU pairs, draw a standard curve, and read cell concentration off that curve from then on. The curve rests on one assumption: that a given OD always means the same number of cells. It does not. The conversion factor drifts as the culture grows. Across one growth curve the OD-to-CFU factor rose **6.6×**, from 2.6×108 to 1.7×109 CFU/mL per OD unit. Read the highlights below. The full story is available in [OD calibration factors drift](/mpd/cell-growth/optical-drawbacks/od-calibration-factors-drift.md). ## The same OD, three different CFU results An *E. coli* culture was followed from \~1×106 to 1×1010 cells/mL, measuring OD600 and plate counts side by side. The left chart shows that BactoBox® cells (lavender) is practically superimposed on the CFU data (green). OD axis is adjusted to balance the data points below and over the CFU curve. Initially, at 2.2 hours, cells are big and effective at scattering light. This leads to relatively few cells per OD; 1 OD600 unit corresponds to 2.57×108 CFU/mL. At the end of incubation cells as cells starve, they get smaller and scatter less light. At 11.6 hours 1 OD600 unit corresponds to 1.69×109 CFU/mL. That's a factor **6.6×** difference. Same reading, same culture, same instrument. The only difference is when the calibration was made.

Three OD-to-CFU standard curves from one E. coli culture. Each line uses the conversion factor measured at that point in the run. The dots mark the measured OD and plate-count pairs. Reading one OD off all three lines gives answers that span 6.6×.

## No single factor fixes it Averaging does not rescue the method. The geometric mean of the CFU-to-OD ratio across the whole run is 8.59×108 CFU/mL per OD unit. Apply it everywhere and it over-reads by 3.3× at 2.2 hours and under-reads by 2.0× at 11.6 hours. Every constant you could pick lands somewhere in that same trade-off. ## Implications When OD measurements are used to infer bacterial concentration, this assumes a constant calibration factor. It is not. Therefore OD is not a suitable proxy for standardizing actively growing bacterial cultures. Use BactoBox® instead. BactoBox® measures bacteria directly and does not need calibration factors. BactoBox® demonstrates close to 1:1 agreement with plate counts during the entire growth curve. --- # 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/drifts-in-od-to-cfu-ratios.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.