Difference between revisions of "Team:Lethbridge/InterLab"

 
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<h1>INTERLAB STUDY</h1>
 
<h1>INTERLAB STUDY</h1>
<br>
 
  
 
<div class="oneText-Wrapper">
 
<div class="oneText-Wrapper">
 
<div class="oneText-Text">
 
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<p class="f14">Synthetic biology interconnects the biological process and engineering techniques. The significance of reliable and repeatable measurement are one of the fundamentals of engineering, which synthetic biology must demonstrate. In 2018, GFP measurement is developed by the Measurement Committee to be experimented. GFP is the most common measurement marker in synthetic biology which is widely available in synthetic biology laboratories. Previously, Fluorescence, from GFP, data was collected in arbitrary settings which caused the technical difficulties in data comparison and interpretation. The purpose of the 2018 InterLab study is to standardizing the fluorescence measurement with a detailed protocol and data analysis form that produces absolute units for measurement in a plate reader.
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<p class="f12">Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results. <br><br>
 
+
The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).</p>
</p>
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<br><br>
 
<br><br>
  
<h1>QUESTION</h1>
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<br>
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<h1>Methods</h1>
  
 
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<p class="f14">Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD)?
 
<br></br>
 
<br>To answer this questions, two orthogonal approaches are proposed.</br>
 
  
<br>1. Converting between absorbance of cells to absorbance of a known concentration of beads </br>
+
<h3>Overview</h3>
    Absorbance measurement uses the light scattering by a sample of cells in liquid.The variable in this measurement is the concentration of the cell. With assumption that the silica beads at the size of typical E.coli cells would scatter the light in similar way, the absorbance measurement with known concentration of the beads can be standardized as the “equivalent concentration of beads” measurement.
+
<p class="f12"> Two approaches were used:<br><br>
<br></br>
+
<br>2. Counting colony-forming units (CFUs) from the sample.</br>
+
    Simply, growing cells on a plate and counting the number of colonies on the plate can determine the amount of cells in a sample of liquid. The concentration of the sample can be obtained with the number of singly grown cells in the plate, which indicates the amount of cells, and the volume of media that is plated out. The positive and negative control samples will be used to calculate a conversion factor from absorbance to CFU.
+
  
</p>
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<b>1. Converting between absorbance of cells to absorbance of a known concentration of beads.</b><br>
</div>
+
Absorbance is a measure of how light is scattered by cells in liquid, which can be used to estimate the concentration of cells in the sample. Assuming that silica beads approximately the same size and shape of <i>E. coli</i> cells would have similar optical properties, we can use the absorbance measurement of a known concentration of beads to produce a standardized “equivalent concentration of beads” measurement.
</div>
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<br><br>
<br></br>
+
<b>2. Counting colony-forming units (CFUs) from the sample.</b><br>
 +
We can estimate the number of individual cells in a volume of liquid media by plating a sample and quantifying colonies (which are each clones of a single cell). The number of colonies in positive and negative control samples can be used to calculate a conversion factor from absorbance to CFU.<br><br>
 +
For complete methods, refer to: <a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader"> https://2018.igem.org/Measurement/InterLab/Plate_Reader</a></p><br><br>
  
 +
<h3>Our Equipment</h3>
  
<h1>METHODS</h1>
+
<p class="f12">A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.</p><br><br>
<div class="oneText-Wrapper">
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<div class="oneText-Text">
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<br></br>
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<p class="f14"> The measurement must be done in a plate reader. Because the 96 well format of the plate reader is convenient for multiple measurement, the methods are written from the perspective of 96 well format.
+
<br></br>
+
All plate reading was done using a Molecular Devices Spectramax i3x. This device has variable temperature settings, pathlength correction and can measure both absorbance and fluorescence. All GFP measurements were taken at wavelengths of 532/25 for emission and 485/20 for excitation.<br></br>
+
  
Firstly, the standard curve for fluorescence using the sodium fluorescein reference material needs to be obtain. Since the reference material should have consistent result, the standard curve of fluorescein values can be used to calibrate the values in the plate reader. The instrument settings must be exactly the same as other labs to ensure the quality of the standard curve. Here is the list of settings that must be standardized; the amplitude of the signal collected; filters or monochromator settings; slit widths; gain settings; plates or cuvette type used; measurement from top or bottom (in plates); number of reads (integration time); orbital averaging. In order to further improve the design of the experiment, additional standard curve must be collected with different sensitivity settings. The additional standard curves will potentially improve the analysis of a cell based assays by matching the most suitable standard curve for the data.
 
<br></br>
 
For complete methods: <a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader"> https://2018.igem.org/Measurement/InterLab/Plate_Reader  </a>
 
<br></br>
 
</p>
 
  
<h2> Calibration </h2>
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<h3> Calibration </h3>
  
 +
<p class="f12">1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a
 +
conversion factor to transform absorbance data into a comparable OD measurement.<br><br>
  
<p class="f14">1. Measured a standard curve for fluorescein to allow this data to be standardized with the data from other iGEM labs. Fluorescein displays similar excitation and emission as GFP and could also be read by the plate reader. This allowed for measurements of GFP fluorescing cells to to be transformed into similar fluorescein readings. </p><br></br>
+
2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. This standard curve was later used to convert absorbance measurements to an estimated number of cells.<br><br>
<p class="f14">2. Followed a LUDOX protocol to serve as a reference point that functioned as a conversion factor. This conversion factor allowed for the absorbance values taken at 600 nm to be converted into OD600 measurements  </p><br></br>
+
<p class="f14">3. A plate was prepared with silica microspheres and read by the plate reader at 600 nm. These microspheres have similar functional characteristics to cells that allows for the absorbance measurements to accurately estimate cell counts. </p><br></br>
+
  
<h2> Cell Measurement Protocol </h2>
+
3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert cell-based readings to an equivalent fluorescein concentration.
 +
</p><br></br>
  
 +
<h3> Cell Measurement Protocol </h3>
  
<p class="f14">1. Measured a standard curve for fluorescein to allow this data to be standardized with the data from other iGEM labs. Fluorescein displays similar excitation and emission as GFP and could also be read by the plate reader. This allowed for measurements of GFP fluorescing cells to to be transformed into similar fluorescein readings. </p><br></br>
+
<p class="f12">Briefly, we measured the raw absorbance and fluorescence of diluted cells (transformed with the below testing devices) at 0 hours and after 6 hours of incubation at 37°C.</p><br></br>
<p class="f14">2. Followed a LUDOX protocol to serve as a reference point that functioned as a conversion factor. This conversion factor allowed for the absorbance values taken at 600 nm to be converted into OD600 measurements  </p><br></br>
+
<p class="f14">3. A plate was prepared with silica microspheres and read by the plate reader at 600 nm. These microspheres have similar functional characteristics to cells that allows for the absorbance measurements to accurately estimate cell counts. </p><br></br>
+
  
<h1> Testing Devices </h1>
 
  
<p class="f14"> The negative control did not contain fluorescent DNA parts and positive control is the fluorescent beads with known value. From the fluorescence values of two controls, the fluorescence value of each devices were calculated. Additionally, the kit plate and well locations were also pre-determined to minimize the detecting errors between trials. </p>
 
 
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   <tr class="f11">
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Device</th>
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     <th font-weight: bold;">Device</th>
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Pert number</th>
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     <th font-weight: bold;">Part number</th>
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Plate</th>  
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     <th font-weight: bold;">Plate</th>  
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Location</th>
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     <th font-weight: bold;">Location</th>
 
   </tr>
 
   </tr>
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<tr class="f10">
 
     <td>Negative Control</td>
 
     <td>Negative Control</td>
 
     <td>BBa_R0040</td>
 
     <td>BBa_R0040</td>
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     <td>Well 2D</td>
 
     <td>Well 2D</td>
 
   </tr>
 
   </tr>
<tr>
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<tr class="f10">
 
     <td>Positive Control</td>
 
     <td>Positive Control</td>
 
     <td>BBa_I20270</td>
 
     <td>BBa_I20270</td>
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     <td>Well 2B</td>
 
     <td>Well 2B</td>
 
   </tr>
 
   </tr>
  <tr>
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<tr class="f10">
 
     <td>Test Device 1 </td>
 
     <td>Test Device 1 </td>
 
     <td>BBa_J364000</td>
 
     <td>BBa_J364000</td>
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     <td>Well 2F</td>
 
     <td>Well 2F</td>
 
   </tr>
 
   </tr>
  <tr>
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<tr class="f10">
 
     <td>Test Device 2</td>
 
     <td>Test Device 2</td>
 
     <td>BBa_J364001</td>
 
     <td>BBa_J364001</td>
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     <td>Well 2H</td>
 
     <td>Well 2H</td>
 
   </tr>
 
   </tr>
<tr>
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<tr class="f10">
 
     <td>Test Device 3</td>
 
     <td>Test Device 3</td>
 
     <td>BBa_J364002</td>
 
     <td>BBa_J364002</td>
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     <td>Well 2J</td>
 
     <td>Well 2J</td>
 
   </tr>
 
   </tr>
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<tr class="f10">
 
     <td>Test Device 4</td>
 
     <td>Test Device 4</td>
 
     <td>BBa_J364007</td>
 
     <td>BBa_J364007</td>
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     <td>Well 2L</td>
 
     <td>Well 2L</td>
 
   </tr>
 
   </tr>
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<tr class="f10">
 
     <td>Test Device 5</td>
 
     <td>Test Device 5</td>
 
     <td>BBa_J364008</td>
 
     <td>BBa_J364008</td>
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     <td>Well 2N</td>
 
     <td>Well 2N</td>
 
   </tr>
 
   </tr>
<tr>
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<tr class="f10">
 
     <td>Test Device 6</td>
 
     <td>Test Device 6</td>
 
     <td>BBa_J364009</td>
 
     <td>BBa_J364009</td>
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</table>
 
</table>
 
</center>
 
</center>
 +
<br><br>
  
<h1> Result </h1>
 
  
<h2> Particle and Fluorescence Standard Curve protocol </h2>
+
<h1>Results</h1>
<p class="f14"> The data collected with Molecular Devices Spectramax i3x in 96 well format. The linear relationship between absorbance and particle counts and the linear relationship between fluorescence and fluorescein are desired by the iGEM InterLab committee. However, the log scale graph of the particle standard curve (Figure 1 B) and the standard curve (Figure 2 A) has exponential and square root relationship respectively. The possible source of error would be consistent pipetting error and oversaturation of the detector. </p>
+
  
<img src="https://static.igem.org/mediawiki/2018/c/c4/T--Lethbridge--Fluorescein_Standard_Curve_V2.png" width = 600 px; height=350px; padding: 30px;>
+
<div class="oneText-Wrapper">
 +
<div class="OneText-Text">
 +
<p class="f12">In our standard curves, we expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.</p><br><br>
 +
    </div>
 +
</div>
  
<img src="https://static.igem.org/mediawiki/2018/9/9c/T--Lethbridge--Fluorescein_Standard_Curve_log_scale_V2.png" width = 600 px; height=350px; padding: 30px;>
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<div style="clear:both"></div>
  
<p class="f11"> <b> Figure 1 Standard curve of Fluorescein </b> </p>
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<div class="twoImage-Wrapper">
<p class="f9">(A) Standard <br>
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<div class="twoImage-Image">
(B) Log scale </br></p>
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<img src="https://static.igem.org/mediawiki/2018/c/c4/T--Lethbridge--Fluorescein_Standard_Curve_V2.png" alt="">
 +
</div>
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/9/9c/T--Lethbridge--Fluorescein_Standard_Curve_log_scale_V2.png" alt="">
 +
</div>
 +
</div>
 +
<div style="clear: both"></div>
  
<img src="https://static.igem.org/mediawiki/2018/6/6a/T--Lethbridge--Particle_Standard_Curve_V2.png" width = 600 px; height=350px; padding: 30px;>
 
  
<img src="https://static.igem.org/mediawiki/2018/9/9d/T--Lethbridge--Particle_Standard_Curve_log_scale_V2.png"width = 600 px; height=350px; padding: 30px;>
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<div class="oneText-Wrapper">
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<div class="oneText-Text">
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<p class="f10"><b>Figure 1:</b> A) Fluorescein standard curve. B) Fluorescein standard curve (log-scale).</p>
 +
</div>
 +
</div>
 +
<div style="clear: both"></div><br><br>
  
<p class="f11"> <b> Figure 2 Standard curve of Particle </b> </p>
 
<p class="f9">(A) Standard <br>
 
(B) Log scale </br> </p>
 
<br> </br>
 
<p class="f14"> The conversion of average fluorescence to reference unit was designed to reduce the impact of  unorganized unit system.
 
<br>For fluorescence to OD conversion, fluorescence was converted to absorbance and, then, to OD.
 
<br>For fluorescence to particle conversion, Molecules of Equivalent of Fluorescence (MEFL) was converted to absorbance and, then, to particles. <br>The result indicated that device 1,2,4,5 contained higher fluorescence part than control in any colonies and Device 6 contained higher parts in Colony 1 but not in Colony 2, and Device 3 did not contained any Fluorescence part in any colonies.
 
<br>The trend showed that the device 2 and 5 had increased fluorescence part after 6 hours, but device 1,4, and 6 had decreased fluorescence part after 6 hours.
 
</br></br> </p>
 
<img src="https://static.igem.org/mediawiki/2018/9/99/T--Lethbridge--Average_Fluorescence_vs_OD.png" width = 600 px; height=350px; padding: 30px;>
 
  
<img src="https://static.igem.org/mediawiki/2018/2/2f/T--Lethbridge--Average_Fluorescence_vs_particle.png" width = 600 px; height=350px; padding: 30px;>
 
  
  
<p class="f11"> <b> Figure 3 Fluorescence conversion to reference units</b></p>
+
<div class="twoImage-Wrapper">
<p class="f9">
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<div class="twoImage-Image">
(A) Average Fluorescence per OD <br>
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<img src="https://static.igem.org/mediawiki/2018/6/6a/T--Lethbridge--Particle_Standard_Curve_V2.png" alt="">
(B) Average Fluorescence per particle </br></br></p>
+
</div>
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/9/9d/T--Lethbridge--Particle_Standard_Curve_log_scale_V2.png" alt="">
 +
</div>
 +
</div>
 +
<div style="clear: both"></div>
  
  
 +
<div class="oneText-Wrapper">
 +
<div class="oneText-Text">
 +
<p class="f10"><b>Figure 2:</b> A) Particle standard curve. B) Particle standard curve (log-scale).</p>
 +
</div>
 +
</div>
 +
<div style="clear: both"></div><br><br>
  
 +
<div class="twoImage-Wrapper">
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/9/99/T--Lethbridge--Average_Fluorescence_vs_OD.png" alt="">
 +
</div>
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/2/2f/T--Lethbridge--Average_Fluorescence_vs_particle.png" alt="">
 +
</div>
 
</div>
 
</div>
 +
<div style="clear: both"></div>
 +
 +
 +
<div class="oneText-Wrapper">
 +
<div class="oneText-Text">
 +
<p class="f10"><b>Figure 3:</b> A) Average fluorescence per optical density. B) Average fluorescence per particle.</p>
 +
</div>
 
</div>
 
</div>
<br></br>
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<div style="clear: both"></div><br><br>
  
  

Latest revision as of 23:33, 17 October 2018



Interlab Banner Image

INTERLAB STUDY

Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results.

The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).



Methods

Overview

Two approaches were used:

1. Converting between absorbance of cells to absorbance of a known concentration of beads.
Absorbance is a measure of how light is scattered by cells in liquid, which can be used to estimate the concentration of cells in the sample. Assuming that silica beads approximately the same size and shape of E. coli cells would have similar optical properties, we can use the absorbance measurement of a known concentration of beads to produce a standardized “equivalent concentration of beads” measurement.

2. Counting colony-forming units (CFUs) from the sample.
We can estimate the number of individual cells in a volume of liquid media by plating a sample and quantifying colonies (which are each clones of a single cell). The number of colonies in positive and negative control samples can be used to calculate a conversion factor from absorbance to CFU.

For complete methods, refer to: https://2018.igem.org/Measurement/InterLab/Plate_Reader



Our Equipment

A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.



Calibration

1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a conversion factor to transform absorbance data into a comparable OD measurement.

2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. This standard curve was later used to convert absorbance measurements to an estimated number of cells.

3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert cell-based readings to an equivalent fluorescein concentration.



Cell Measurement Protocol

Briefly, we measured the raw absorbance and fluorescence of diluted cells (transformed with the below testing devices) at 0 hours and after 6 hours of incubation at 37°C.



Device Part number Plate Location
Negative Control BBa_R0040 Kit Plate 7 Well 2D
Positive Control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P


Results

In our standard curves, we expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.



Figure 1: A) Fluorescein standard curve. B) Fluorescein standard curve (log-scale).



Figure 2: A) Particle standard curve. B) Particle standard curve (log-scale).



Figure 3: A) Average fluorescence per optical density. B) Average fluorescence per particle.