William and Mary was honored to contribute to the fifth International Interlaboratory Measurement Study in synthetic biology. This year the interlab study focused on the discrepancies in comparing fluorescence data. In providing a detailed protocol and data analysis form, the Interlab protocol intends to answer the question “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?”
Following the Plate Reader and CFU protocol, we computed the cell count in our sample by both converting the absorbance of cells to absorbance of a known concentration of beads. Using our Synergy H1 microplate reader (Biotek) we took calibration measurements and proceeded to the cell measurement protocol. Excitation filters of 485 nm and Emission filter of 525 were used. Additionally, the team also completed the flow cytometry portion of the study using the FL1 channel of our S3e cells sorter (Bio-Rad) and submitting calibration measurements of RCP-30-5A SpheroTech Rainbow calibration beads.
The interlab study was a success for the team and we had a lot of fun completing it. However, our team did have one minor comment regarding the “Colony Forming Units per 0.1 OD 600 E.coli cultures” section. During the CFU calibration, the protocol instructs us to dilute the cells to an OD of 0.1 and check the plate to verify they are at an OD of 0.1. However, after our dilutions, our plate reader measurements were ~0.1 +/- ~0.02. Since no acceptable margin of error was given in the protocol, we assumed (and confirmed with the interlab measurement committee) that this was acceptable. We think in the future it may be useful to give an acceptable margin of error, especially given that different groups work with equipment that might be differently sensitive.