> 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/optical-drawbacks/od-calibration-factors-drift.md). # OD calibration factors drift OD600 is often used as an inexpensive, real-time proxy for cell concentrations. A conversion factor is needed to convert the arbitrary OD600 units to cell concentrations. The factor is determined once by making a [standard curve](https://www.eppendorf.com/dk-en/lab-academy/life-science/microbiology/how-to-quantify-bacterial-cultures/). That only works if the factor stays put. It does not. OD600 is turbidity, so the signal per cell scales with cell size, and cells change size as a culture grows. The conversion factor moves with them. This study tracked one *E. coli* culture in LB medium with BactoBox®, plate counts (CFU), and OD600 at the same time points It asks one question. Which ratio between two methods holds still? ### Take-home messages * Cells-per-OD moved 11.4x across the run, from 1.6×108 to 1.8×109 cells/mL per OD unit. That is a 11× change during the growth curve * The BactoBox®-to-CFU ratio held at a geometric mean of 1.01 with a geometric CV of 21%. It varied 2.2x in total, five times less than cells-per-OD. * The drift in cells-per-OD and cells-per-CFU is not noise. It rose steadily with time as cell size decreased and scaled with CIZE⁻²·⁵ . * CFU-per-OD drifted the same way, 6.6x. The problem is OD600, not the counting method it is paired with. ### Study design * A single *E. coli* ATCC 8739 LB medium shake-flask culture inoculated at 1×106 cells/mL and sampled 28 times over 11.6 hours. Cultivated at 37 °C, 200 RPM. * Every sample was measured with BactoBox® (cells/mL and CIZE), by compact dry TC plate counts (CFU/mL), and by OD600 using an Inplen Diluphotometer and standard cuvettes. * Samples were diluted into the linear range of each instrument before measurement. A single measurement was done for each method. * We report ratios as geometric means, because a ratio is a multiplicative quantity. Span is max divided by min. #### The first nine OD readings sit in the blank The photometer was read at every time point, including the first nine. Those nine readings came back at 0.00 ± 0.01 AU, which is below the lower limit of detection. The detection limit, taken as the blank mean plus three standard deviations, is 0.03 AU. The first reading above that limit arrived at 2.2 hours, at 0.07 AU. By then the culture had passed 1×107 cells/mL. This means that the first decade of growth carries no usable OD600 signal at all.

An E. coli shake flask culture measured by BactoBox® (lavender), plate counts (yellow) and OD600 (red). CIZE (green) from BactoBox® measurements is also shown (secondary axis). The y-axis is symlog, i.e. linear below the limit of detection (grey box) and logarithmic beyond 0.027 AU.

## Stability during different growth stages Ratios between the three different measurands are presented below. Values before 2.2 hours are not available for the OD method as OD below the detection limit would be division by noise.

Ratio between methods as a function of incubation time. The BactoBox®-to-CFU ratio stays flat across four log decades. The per-OD ratios climb with the inverse square of CIZE.

### OD600 ratios are unstable The geometric mean of OD-to-CFU and OD-to-cells is 8.6×108 CFU/AU and 8.9×109 cells/AU, respectively. A curve centered at 1× would represent a stable calibration factor. This is clearly not the case. For OD-to-CFU comparisons the factor rose 6.6x between early exponential phase and stationary phase. For OD-to-cells the factor rose 11.4×. The drift is one-directional. It is not scatter around a stable mean that more replicates would average away. Pick your calibration point early and every later number is too low. Pick it late and every earlier number is too high. | Phase | Cells-per-OD (cells/mL per OD) | CIZE (µm) | | ----------------------------- | :----------------------------: | :-------: | | Early exponential, 2.2–3.35 h | 2.5×108 | 1.74–2.08 | | Late exponential, 4.42–5.53 h | 9.0×108 | 1.05–1.34 | | Stationary, 6.12–11.63 h | 1.7×109 | 0.85–0.94 | ### BactoBox to CFU ratios are stable The BactoBox®-to-CFU ratio is stable, and it is stable at 1.01. On average, one BactoBox® cell equals one colony-forming unit here, with no fitted factor in between. The span is 2.2× which is expectable when single measurements are compared, especially for a method that is as imprecise as CFU determination. In this study, frequent sampling points were preferred over precision. In any case the 2.2× span is much lower than the 6× and 11× spans observed in the OD-to-CFU and OD-to-CFU results. ## Cell size explains the difference CIZE peaked at 2.13 µm after 1.6 hours and fell to 0.85 µm by the end of the run. Turbidity is scattering, and scattering follows the cross-sectional area of the cell. Halve the diameter and each cell contributes roughly a quarter of the signal. The number of cells behind one OD unit therefore rises as cells shrink. The data match that. Over this curve, cells-per-OD scaled as CIZE⁻²·⁵ (log-log r = -0.98) and CFU-per-OD as CIZE⁻² (r = -0.98). Both exponents sit near -2, which is the inverse-square behaviour you expect from an area term. BactoBox® counts cells one at a time. Its ratio to plate counts compares one count to another count, so no size term enters. That is why it holds still while the per-OD ratios do not. ## Implications A cells-per-OD factor is only valid for one growth phase, in one medium, with one strain. Carry it outside those bounds and it brings an error you cannot see in the OD600 reading itself. The error is largest exactly where people want the factor most: comparing a culture early against the same culture late, or comparing two conditions that grow at different rates. Cell size differs in both cases. This means that growth rates are often not reliable when derived from OD600 data. OD600 also confounds growth medium screening, since one condition may have few, but large cells, while another condition has small but copious cells. When you need a number that means cells, count cells. BactoBox® reports cells/mL directly, matched CFU/mL one to one across this entire curve, and needs no calibration factor to do it. --- # 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/mpd/cell-growth/optical-drawbacks/od-calibration-factors-drift.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.