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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?

Refs:

  • 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.
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  • $\begingroup$ Is there a reason you can't plate serial dilutions of your culture, and then create a CFU/mL vs. OD curve for your given growth conditions? $\endgroup$ – acvill Mar 9 at 17:14
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    $\begingroup$ @acvill CFU/mL is very noisy, per citation #2 $\endgroup$ – jakebeal Mar 9 at 23:36
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From a technical perspective, for the FlopR package you mention (and I believe also for Jacob Beal's protocol) you can simply change the bead size and perform a new calibration. The only information that you need is the bead count at each point of your calibrant curve.

However, using calibration beads of a given size assumes that your cells will remain approximately the size of the beads for the duration of your experiment. We know that cell size and shape vary based on growth phase, antibiotics etc. [1] This can cause substantial misreporting of cell counts but is currently brushed under the carpet. I would suggest that if your Pichia change significantly in size or shape during their growth, it would be worth using a range of beads sizes and attempting to calibrate different growth phases of your samples against beads that correspond to cell size at during that phase.

[1] K. Stevenson, A. F. McVey, I. B. N. Clark, P. S. Swain, and T. Pilizota, “General calibration of microbial growth in microplate readers,” Sci. Rep., vol. 6, no. 1, p. 38828, Dec. 2016.

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    $\begingroup$ It's worth noting that the calibration of different bead sizes hasn't been formally verified yet, but there's not reason to expect it won't scale. $\endgroup$ – jakebeal Mar 11 at 17:57

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