General calibration of microbial growth in microplate readers
Open accessPublished version of a preprint
Abstract
Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from studies of antibiotic efficacy to investigations of growth under different nutritional or stress environments, to characterization of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions, and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.
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OD alone is an unreliable metric and is only proportionately accurate to cell titers up to about an OD of 0.1.
“However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.1.”
From Estimating microbial population data from optical density (2022) · cites this paper as [7]
The paper says OD is only approximately proportional to cell number under limited, low-density conditions and that calibration matters. But its Abstract places the approximate proportional range at OD600 ≲ 0.2, not only up to about 0.1; it also makes that relationship conditional on factors such as instrument geometry and cell properties. No page number is available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measuredAbove OD 0.1, some of the light scattered away from the detector by one cell is subsequently scattered back to the detector by another cell, so OD does not increase as fast as cell titer.
“Above that range, some of the light that is scattered away from the detector by one cell is subsequently scattered back to the detector by another cell [7]. As a result, the OD does not increase as fast as does the cell titer and therefore, one cannot rely on OD alone to accurately measure bacterial population densities.”
From Estimating microbial population data from optical density (2022) · cites this paper as [7]
The passage labeled Abstract (page unavailable) says that multiple scattering becomes more likely as cell number increases and that OD then ceases to be proportional to cell number. It does not specify that light scattered away by one cell is scattered back to the detector by another, or establish OD 0.1 as the threshold; it describes approximate proportionality through OD600 ≲ 0.2. (LLM, conf=0.84)
Experimental — accuracy not yet measuredThe same considerations of calibration apply to microtiter plate readers as to spectrophotometers.
“However, the same considerations of calibration apply to microtiter plate readers as to spectrophotometers [7].”
From Estimating microbial population data from optical density (2022) · cites this paper as [7]
This relationship is consistent with Koch's 1961 and Stevenson et al.'s 2016 finding.
“This is consistent with Koch’s 1961 and Stevenson et al.’s 2016 finding [2, 7].”
From Estimating microbial population data from optical density (2022) · cites this paper as [7]
The quoted sentence does not identify what “This” refers to, so the relationship being attributed to Stevenson et al. cannot be determined. The Results section discusses relationships among optical density, cell concentration, and cell size, but without the preceding statement there is no way to tell whether any of them is the one claimed to be consistent with this paper. (LLM, conf=0.72)
Experimental — accuracy not yet measured
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References 29 · Cited by 407
Research works printed but not linked: 1
- Ishimaru A. Approximate solutions for tenuous media in Wave Propagation and Scattering in Random Media. (Wiley-IEEE Press, 1997).
- Authors
- Stevenson, K., McVey, A. F., Clark, I. B. N., Swain, P. S., & Pilizota, T.
- Affiliations
- University of Edinburgh (GB)
- Venue
- Scientific Reports
- Published
- 2016-12-13
- Volume
- 6
- Issue
- 1
- Pages
- 38828-38828
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC5153849
- OpenAlex
- W2952668961
- Cited by
- 407
- References
- 29
- Citations by year
- 2026: 44 · 2025: 51 · 2024: 56 · 2023: 52 · 2022: 52 · 2021: 50 · 2020: 55 · 2019: 29 · 2018: 13 · 2017: 3 · 2016: 1
- FWCI
- 12.39
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Edinburgh Research Explorer (University of Edinburgh) (publishedVersion, cc-by)
- Funders
- Medical Research Council
- Engineering and Physical Sciences Research Council
- Biotechnology and Biological Sciences Research Council (1602789, BB/M018040/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Physical Sciences › Chemistry › Physical and Theoretical Chemistry › thermodynamics and calorimetric analyses
- Keywords
- microbial growth, optical density, microplate reader, calibration, mutant strain
- MeSH terms
- Calibration, Escherichia coli, Nephelometry and Turbidimetry, Colony Count, Microbial, Reproducibility of Results
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