The purpose of the first calibration is to establish a conversion from “absorbance at 600nm” (Abs600) data in our 96 well plates into an “optical density at 600nm” (OD600) measurement, as would be obtained in a standard spectrophotometer. “Such conversion is necessary because plate reader measurements of absorbance are volume dependent; the depth of the fluid in the well defines the path length of the light passing through the sample, which can vary slightly from well to well.” To do so, we first measured the absorbance values of LUDOX-S30 versus deionized H2O. Reading the LUDOX-S30 absorbance value from our spectrophotometer, we subtract the dH2O absorbance to get a corrected absorbance value. Then, the LUDOX-S30 OD600 from iGEM’s reference spectrophotometer is divided by our corrected absorbance value to give a standardized conversion factor.

Our silica microspheres have similar size and optical characteristics as cells but can be mass produced and distributed globally so that all labs will have the same size and concentration of microspheres. Measuring the absorbance of these beads allowed our lab to construct a standard curve of particle concentration (seen below), “which can be used to convert Abs 600 measurements to an estimated number of cells.”

Per the protocol given by iGEM, we prepared out microsphere stock solution and serially diluted this solution over 11 wells each with a total of 100ul. Throughout this process we mixed by pipetvting up and down 3 times before each transfer of solution, as well as one final remixing before measurement by the plate reader.