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Precise, High-throughput Analysis of Bacterial Growth
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Robust estimation of bacterial cell count from optical density.

Jacob Beal1, Natalie G Farny2, Traci Haddock-Angelli3

  • 1Raytheon BBN Technologies, Cambridge, MA, USA. jakebeal@ieee.org.

Communications Biology
|September 18, 2020
PubMed
Summary
This summary is machine-generated.

Standardizing optical density (OD) measurements is crucial for cell density estimation. Calibrating OD to cell count using silica microspheres offers a precise, accessible method for interlaboratory data comparison.

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Area of Science:

  • Biotechnology
  • Microbiology
  • Cell Biology

Background:

  • Optical density (OD) is a common but unstandardized method for estimating cell density in liquid cultures.
  • Lack of standardized calibration protocols hinders inter-instrument comparison and accurate cell count determination.
  • Relating OD measurements to actual cell numbers remains a significant challenge in biological research.

Purpose of the Study:

  • To compare the effectiveness of three simple, low-cost OD calibration protocols across multiple laboratories.
  • To establish a standardized method for calibrating optical density measurements to actual cell counts.
  • To enable direct comparison and data fusion of cell density measurements across different instruments and laboratories.

Main Methods:

  • An interlaboratory study involving 244 laboratories was conducted.
  • Three distinct, accessible OD calibration protocols were evaluated.
  • Eight strains of constitutive GFP-expressing Escherichia coli (E. coli) were used in the study.
  • Silica microspheres were employed for serial dilution calibration to estimate cell count.

Main Results:

  • The silica microsphere calibration protocol demonstrated high precision, with 95.5% of residuals less than 1.2-fold.
  • This method allows for quality control assessment and evaluation of the instrument's effective linear range.
  • Combining OD calibration with fluorescence calibration enabled reporting in Molecules of Equivalent Fluorescein (MEFL) per cell.
  • Fluorescence per cell measurements showed a minimal mean difference (1.07-fold) between plate reader and flow cytometry data.

Conclusions:

  • Calibrating optical density to estimated cell count using serial dilution of silica microspheres is recommended for standardization.
  • This approach ensures precise, reproducible cell density measurements and facilitates data fusion with other methods like flow cytometry.
  • The proposed calibration method enhances comparability of biological data across different research settings.