I am performing experiments with the yeast Pichia pastoris where it will be beneficial to calculate product yield per cell. I plan to use a plate reader to measure OD600 of my cultures and then somehow relate this to cell number.
The Pichia pastoris expression manual from ThermoFisher says that 1 OD600 is equivalent to 5x10^7 cells/ml, and Weis et al (2004) have a similar number of 1.5x10^8 cells per 1 OD.
However, there are now calibration protocols (Beal et al 2020) and packages (Fedorec et al 2020) for using absorbent beads to convert OD measurements to cell density in E.coli. These rely on the beads scattering the light in a way that is equivalent to cells, so that one can compare measurements of OD with a standard curve of known amounts of beads to estimate cell number. Pichia is about 5µm in size size, compared to E. coli which is about 1-2µm, therefore I do not believe that using the same protocol will work for me.
Is it necessary for me to develop a new protocol, or can I just convert by multiplying OD by the numbers given above? If it is necessary, how can I develop such a protocol? Can I just use the E. coli protocol that I linked with different size beads?
- Weis, Roland, Ruud Luiten, Wolfgang Skranc, Helmut Schwab, Marcel Wubbolts, and Anton Glieder. ‘Reliable High-Throughput Screening with Pichia Pastoris by Limiting Yeast Cell Death Phenomena’. FEMS Yeast Research 5, no. 2 (1 November 2004): 179–89. https://doi.org/10.1016/j.femsyr.2004.06.016.
- Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten 2020. Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles. protocols.io https://dx.doi.org/10.17504/protocols.io.bgy6jxze
- Fedorec, Alex J. H., Clare M. Robinson, Ke Yan Wen, and Chris P. Barnes. ‘FlopR: An Open Source Software Package for Calibration and Normalization of Plate Reader and Flow Cytometry Data’. ACS Synthetic Biology, 27 August 2020. https://doi.org/10.1021/acssynbio.0c00296.