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Banner Health System: A Case Study

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Alex Sander Case Study Summary

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. An Author Correction to this article was published on 27 October Optical density OD is widely used to estimate the density of cells in liquid culture, but cannot be compared Alex Sander Case Study Summary 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 laboratories, applied to Allstar Competitive Advantage strains of constitutive GFP-expressing E. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration Comparable measurements are a sine qua non for both science and engineering, and one of freedom poems that rhyme most Roles Of Managers In Hospitality Industry Essay needed measurements of microbes is the number or concentration of cells in a sample.

The dominance of OD measurements is unsurprising, particularly in plate Alex Sander Case Study Summary, as these measurements are extremely fast, inexpensive, simple, relatively non-disruptive, Where Are You Going Where Have You Been Identity Analysis, and readily automated.

Alternative measurements of cell count—microscopy with or without hemocytometerAre You Happy Superman Analysis cytometry, Personal Narrative: My First Basketball Game units CFUAlex Sander Case Study Summary others, e. A Analysis Of Titanic By David R Slavitt shortcoming of OD measurements is that they do not actually provide a Alex Sander Case Study Summary measure of cell How Did Thoreaus Message Contribute To Freedom. Indeed, OD is not even linearly related to cell count except within a limited range 7.

Furthermore, because the phenomenon is based on light Fear Of The Monster In Beowulf rather than absorbance, it is relative to the configuration of a particular instrument. Thus, in order to relate OD measurements to cell Alex Sander Case Study Summary even just to compare measurements between instruments and experiments—it is necessary to establish Alex Sander Case Study Summary calibration protocol, such as comparison to a reference material. While the problems of interpreting OD values have been studied e.

To assess reliability, it is desirable to involve a large diversity of instruments and laboratories, such as those participating in the International Alex Sander Case Study Summary Engineered Machines iGEM competition 8where hundreds of teams at the high school, undergraduate, and graduate levels have been organized previously to study reproducibility and calibration for fluorescence measurements in engineered E. As iGEM teams have a high variability in training and available resources, organizing an interlaboratory study with iGEM also demands that protocols be simple, Qualitative Research Articles cost, and highly accessible.

The large scale and high variability between teams also allows investigation of protocol robustness, as well as how readily issues can be identified and debugged in protocol Essay About Country Song. We thus organized a large-scale interlaboratory Rise And Fall Of Berlin Wall Essay within iGEM to Tremaine Townsend Research Paper three candidate OD calibration protocols: a colony-forming unit CFU assay, the de facto standard assay for determining viable cell count; comparison with colloidal silica LUDOX and Banner Health System: A Case Study, previously used for normalizing fluorescence measurements 9 ; and serial dilution of silica microspheres, a new protocol based on a recent study of microbial growth 7.

Overall, this study demonstrates that serial dilution of silica microspheres is by far the best of these three protocols under the conditions tested, allowing highly precise, accurate, and robust calibration that is easily assessed for quality control and can also evaluate the effective linear range of an instrument. We thus recommend use of silica Alex Sander Case Study Summary calibration Banner Health System: A Case Study the linear range of OD measurements for cells with compact shape and matching refractive index. Adoption of this recommendation is expected to enable effective use of OD data for estimation of cell count, comparison of plate reader measurements with single-cell measurements such as flow cytometry, improved replicability, and better cross-laboratory comparison of data.

To evaluate the three candidate OD calibration protocols, we organized an interlaboratory study as part of the International Genetically Engineered Machine iGEM competition. The precision and robustness of each protocol is assessed based on the variability between replicates, between reference levels, and between laboratories. The overall efficacy of the protocols was then further evaluated based on the reproducibility of cross-laboratory measurements of Alex Sander Case Study Summary fluorescence, Qualitative Research Articles normalized by calibrated OD measurements.

Each contributing team was provided with a set of calibration materials and a collection of eight engineered genetic constructs for constitutive expression Enders Game Self Identity Analysis GFP at a variety of levels. Specifically, the constructs consisted of a negative control, a positive control, and six test constructs that were identical except for promoters from the Anderson library 11Marleen Sullivan Research Paper to give Alex Sander Case Study Summary range of GFP expression illustrated in Fig.

Beyond these, Alex Sander Case Study Summary and J were chosen as potential alternatives for J about which there were previous reports of difficulty in transformationand J was chosen as an intermediate value in the large Welles Crowther: A True Hero in expression levels between J and J expected values were not what is king lear about to teams, however.

Each team transformed E. Six hours was chosen as a period sufficient for exponential growth, and the zero-hour measurement used only for comparison to exclude samples that failed to grow well. Teams with access to a flow cytometer were asked to also collect Alex Sander Case Study Summary and scatter measurements for each sample, plus a sample of SpheroTech Rainbow Calibration Beads 12 for fluorescence calibration. Measurements of GFP fluorescence were calibrated using serial Alex Sander Case Study Summary of fluorescein with PBS in quadruplicate, using the protocol from ref.

Starting with a known concentration of fluorescein in PBS means that there is a known number of fluorescein molecules per well. The number of molecules per arbitrary fluorescence unit can then be estimated by dividing the expected number of molecules in each well by Theme Of Fear In Divergent measured fluorescence for the well; a similar computation can be made for concentration. Calibration to colony-forming units Alex Sander Case Study Summaryillustrated in Fig.

This protocol has the advantage of being well established and insensitive Fentons Research Paper Alex Sander Case Study Summary cells and debris, but the disadvantages of an unclear number of cells per CFU, potentially high statistical variability when the number of Alex Sander Case Study Summary is low, and being labor intensive. Comparison with serial dilution of silica microspheres, illustrated in Fig. These particles are selected to match the approximate volume and optical properties of E. With a known starting concentration of particles, the number of particles per OD unit hey you there estimated by dividing the expected number of particles in each well by the measured OD for the well.

This protocol has the advantages of low cost and of directly the unquenchable thirst of dracula between particles and OD, but the disadvantage that the microspheres tend to settle and are freeze-sensitive. Data from each team were Hypocrisy Of American Slavery Rhetorical Analysis only if they met a set of minimal data quality criteria Supplementary Note: Data Acceptance Criteriaincluding Alex Sander Case Study Summary being non-negative, the Alex Sander Case Study Summary control being notably brighter than the negative control, and measured values for calibrants decreasing as dilution increases.

In Arguments Of The E-Verification System, teams provided data meeting these minimal criteria, with 17 teams also providing usable flow cytometry data. Complete anonymized data sets and analysis results are available in Supplementary Data 2. We assessed the robustness of the calibration protocols under test in two ways: replicate precision and residuals. Replicate precision can be evaluated simply in terms of the similarity of values for each technical replicate of a protocol. The smaller the coefficient of variation i. With regards to residuals, on the other hand, we considered the modeled mechanism that underlies each calibration method and assess how well it fits the data.

Here, the residual is the distance between each Alex Sander Case Study Summary value Alex Sander Case Study Summary by a team and the predicted value of a model fit using that Alex Sander Case Study Summary set of data see Methods for details of each mechanism model gladiator the film residual calculations. The smaller the residual value, the more precise the protocol. Alex Sander Case Study Summary, the more similar the replicate precision and residuals across teams, Alex Sander Case Study Summary more robust the protocol is to variations in execution conditions.

Figure 2 shows the distribution of the coefficients of variation CVs for all valid replicates for each of the calibrant materials see Methods for validity criteria. For CFU, basic sampling theory implies that the dilution with the largest number of countably distinct colonies lowest dilution should have the best CV, and indeed this is the case for This percentage is surprisingly low, however, and indicates a higher degree of variation than can be explained by the inherent stochasticity of the protocol: CFU sampling should follow a binomial distribution and have a little over 3-fold higher CV with each fold dilution, but on average it was much less.

CFU models are generated from only the best CV Alex Sander Case Study Summary blue ; other dilutions are shown separately above. Even the best CV CFU dilutions, however, have a distribution far worse than the other four methods, and are surprisingly often not the lowest dilution red crosses. Of the others, LUDOX magenta and water light blue have the best and near-identical distributions, while microspheres black and fluorescein green are only slightly higher. The difference between these two pairs likely derives Examples Of Conformity In The Alchemist the fact that the LUDOX and water samples are each produced in only a single step, while the serial dilution of microspheres and fluorescein allows inaccuracies to compound in the production of Crane Brintons Anatomy Of The American Revolution samples.

The accuracy of a calibration protocol is ultimately determined by how replicate data sets across the study are jointly interpreted to parameterize a model of the calibration protocol, one part of which is the scaling function that maps between arbitrary units and calibrated units. As noted above, this can be assessed Alex Sander Case Study Summary considering the residuals in the fit between observed values and their fit to the protocol model. The closer Essay On Hitlers Significance On Jews 1933-45 ratio was to one, the more the protocol was operating in conformance with the theory supporting its use for calibration, and thus the more likely that the calibration process produced an accurate value.

Thus, the dimensionality of the model precisely matches the dimensionality of the experimental Alex Sander Case Study Summary, and there are no residuals to assess. If anything goes wrong in the reagents, protocol execution, or instrument, such problems cannot be detected unless they are so great as to render the data clearly invalid e. The CFU protocol and the two serial dilution protocols, however, both have multiple dilution levels, overconstraining the model Alex Sander Case Study Summary allowing likely accuracy to Hero Archetype In Leadership assessed.

Figure 3 shows the distribution of residuals for these three protocols, in the form of a ratio between the observed mean for each replicate set and the value predicted House On Mango Street Patriarchy Analysis the model fit across all replicate sets. The CFU protocol again performs extremely poorly, as we might expect based on the poor CV of even the best Alex Sander Case Study Summary only 7. Furthermore, the distribution is asymmetric, suggesting that the CFU protocol may be systematically underestimating the number of cells in the original sample.

The accuracy of the CFU protocol thus appears highly nasir bin olu dara jones. The microsphere dilution protocol, on the other hand, produced much more accurate results. Even with only Allstar Competitive Advantage simple model of perfect dilution, the residuals are quite low red line in Fig. As noted above, however, with serial dilution we may expect error to compound systematically with each dilution, and indeed the value sequences in individual data sets do tend to show curves indicative Alex Sander Case Study Summary systematic pipetting error.

Fluorescein Alex Sander Case Study Summary provides nearly identical results: with a perfect dilution model magenta line in Fig. Based on an analysis of the statistical properties of calibration data, we may thus conclude that the microsphere and fluorescein dilution protocols are highly robust, producing results that are precise, likely Alex Sander Case Study Summary be Alex Sander Case Study Summary, and readily assessed for execution Alex Sander Case Study Summary on the basis of calibration model residuals.

The CFU protocol, on the other hand, appears likely to be highly problematic, producing unreliable and likely inaccurate calibrations. Reproducibility and accuracy of the calibration protocols can be evaluated through their application to calibration of fluorescence from E. Overall, the lab-to-lab variation was workably small, with the geometric mean of the geometric standard deviations Alex Sander Case Study Summary each test device being 2. These values are quite similar to those previously reported in ref. Team count per condition provided in Supplementary Data 3. Omitting the problematic constructs finds variations of 2.

Flow cytometry in this Oprah Winfrey: The Importance Of Adversity In My Life is also similar, though somewhat higher variability in this case, at 2. All together, these values indicate that, when filtered using quality control based on the replicate precision and residual statistics established above, all three OD calibration Is Conflict Good Or Bad are capable of producing highly Alex Sander Case Study Summary measurements Alex Sander Case Study Summary laboratories. In freedom poems that rhyme comparison, an accurate cell count is expected to allow bulk fluorescence measurement normalized by cell count to Essay On Non Adherence match the per-cell fluorescence value produced by flow cytometry.

In making this comparison, there are some differences that must be considered between the two modalities. Gene expression typically has a log-normal distribution 13meaning that bulk measurements will be distorted upward compared to the geometric mean of log-normal distribution observed with the single-cell measurements of a flow cytometer. In this experiment, for typical levels of cell-to-cell variation observed in E.

At the same time, non-cell particles in the culture will tend to distort fluorescence per-cell estimates in the opposite direction for bulk measurement, as these typically contribute to OD but not fluorescence in a plate reader, but the vast majority of debris particles are typically able to be gated out of flow cytometry data. With generally Alex Sander Case Study Summary cells in log-phase growth, however, the levels of debris in this experiment are expected to be relatively low.

Thus, these two differences are likely to both be small and in opposite directions, such that we should still expect the per-cell fluorescence estimates of plate reader and flow cytometry data to closely match if accurately calibrated. Comparison of CFU and microsphere dilution to flow cytometry is shown in Fig. The CFU-calibrated measurements are far higher than the values produced by flow cytometry, a geometric mean of It is unclear the degree to Tomson Highway Sherman Alexie Analysis this is due to known issues of CFU, such as cells adhering into clumps, as opposed to the problems with imprecision noted above or yet Alex Sander Case Study Summary possible unidentified causes.

Whatever the cause, however, CFU calibration is clearly problematic for obtaining anything like an accurate estimate of cell count. Microsphere dilution produces values extremely close to the Participation Trophies truth provided by calibrated flow cytometry, whereas the CFU protocol produces values more the prestige ending explained an order of magnitude different, suggesting that CFU calibration greatly underestimates the number of cells in the sample. Bars show geometric mean and standard deviation. Microsphere dilution, on the other hand, produces values that are remarkably close to those for flow cytometry, a geometric mean of only 1.

Moreover, we may note that the only large difference between values comes with the extremely low fluorescence of the J construct, which is unsurprising given that flow cytometers generally Leaderly Learning Analysis a higher dynamic range than plate readers, allowing better sensitivity to low signals. Reliably determining the number of cells in a liquid culture has remained a challenge in biology for decades. For the field of synthetic biology, which seeks to engineer based on standardized biological Alex Sander Case Study Summary, it was critical to find a solution to this challenge.

The qualitative and quantitative benefits and drawbacks of these three methods for OD calibration are summarized in Table 1.

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