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Calibration 1: OD600 Reference point

• Purpose of this calibration: To transform absorbance data to a OD₆₀₀ measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD₆₀₀ from Abs₆₀₀ calculated for Ludox CL-X on a mass spectrophotometer.
• Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance:

• This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.
• Results: Cell density readings can thus be converted to OD₆₀₀ by multiplying correction factor value, 4.138.

Table1.

Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs₆₀₀ is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD₆₀₀ is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD₆₀₀/Abs₆₀₀ is the correction factor to convert Abs₆₀₀ to OD₆₀₀, calculated by dividing Reference OD₆₀₀ by Abs₆₀₀.

Calibration 2: Particle Standard Curve

Construct a standard curve of Abs₆₀₀ for microsphere particle concentration Purpose of calibration: iGEM distributed microspheres that mimic the size, shape and volume of cells which have a known amount of microspheres per volume. This calibration was required to generate a Particle Standard Curve which helped us determine the number of cells (as modelled by microspheres) in a sample.

• Use standard curve to convert Abs₆₀₀ measurements to an estimate of number of cells.
• Monodisperse silica microspheres are used in the calibration because they have similar size and optical characteristics as cells.

Figure 1. Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs₆₀₀ for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).