Alternative Roots participated in the 2018 Interlab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD600. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:

1. 1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs₆₀₀ value measured by the plate reader to a comparable OD₆₀₀ value, considering path length and well volume (table 1). Abs₆₀₀ of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD₆₀₀ of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor.
Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD₆₀₀ readings for plate reader measurements.

2. A standard curve was prepared by measuring the OD₆₀₀ of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to E. coli cells. This was used to standardise OD readings across labs (Figure 1 A).
3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B).