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- Beal, Jacob, et al.
(author)
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Robust estimation of bacterial cell count from optical density
- 2020
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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2. |
- Bowman, Jennifer, et al.
(author)
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Evaluation of the reliability of indicators of consciousness during CO2 stunning of rainbow trout and the effects of temperature
- 2020
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In: Aquaculture Research. - : Hindawi Limited. - 1355-557X .- 1365-2109. ; 51, s. 5194-5202
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Journal article (peer-reviewed)abstract
- A two-part experiment was conducted to determine whether visual indicators of consciousness such as equilibrium, eye-roll reflex and ventilation are reliable for evaluating whether CO2 stunning of rainbow trout (Oncorhynchus mykiss) is humane. In part 1, the time taken until the loss of visual indicators in rainbow trout during CO2 stunning was monitored under field conditions at 14, 8 and 2 degrees C. Here, we clearly demonstrate that it takes longer for visual indicators to disappear as temperature decreases, with significant differences in the time taken until the loss of equilibrium between 2 and 14 degrees C, and significant differences between all temperatures in the time taken until the loss of eye-roll reflex and ventilation. In part 2, rainbow trout were equipped with external non-invasive electrodes for recording EEG prior to, and following, CO2 stunning to assess the presence or absence of visually evoked responses (VERs), which are indicative of brain function and sensibility. The resulting EEG recordings during CO2 stunning at 10 degrees C demonstrated a poor relationship between visual indicators of consciousness and loss of sensibility, as VERs were present up to 3.5 min after ventilation was lost and up to 6.5 min after the fish lost equilibrium. Collectively, these results show that cold-water temperatures prolong the time taken until loss of consciousness and that visual indicators are insufficient for determining when sensibility is lost in rainbow trout during CO2 stunning.
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