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
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 and LLOQ, respectively. X-axis represents concentration calculated from the known dilution factor relative to the 12 hour culture.
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.
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.
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.
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