Jul 13, 2019

Public workspaceCalibration Protocol - OD600 Inter-equipment Conversion with LUDOX V.4

DOI
dx.doi.org/10.17504/protocols.io.5gig3ue
  • 1iGEM Measurement Committee;
  • 2iGEM;
  • 3Worcester Polytechnic Institute;
  • 4Imperial College London
Paul Rutten
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DOI: dx.doi.org/10.17504/protocols.io.5gig3ue
External link: https://2019.igem.org/Measurement
Protocol Citation: Paul Rutten, Richard Tennant, Jacob Beal, Traci Haddock-Angelli, Natalie Farny, Geoffrey Baldwin, Marko Storch, Ari Dwijayanti 2019. Calibration Protocol - OD600 Inter-equipment Conversion with LUDOX. protocols.io https://dx.doi.org/10.17504/protocols.io.5gig3ue
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 13, 2019
Last Modified: July 13, 2019
Protocol Integer ID: 25834
Abstract
With this protocol you will use LUDOX CL-X (a 45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform absorbance (OD600) data from your plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. This conversion is necessary because plate reader measurements of absorbance are volume dependent; the depth of the fluid in the well defines the path length of the light passing through the sample, which can vary slightly from well to well. In a standard spectrophotometer, the path length is fixed and is defined by the width of the cuvette, which is constant. Therefore this conversion calculation can transform OD600 measurements from a plate reader (i.e. absorbance at 600 nm, the basic output of most instruments) into comparable OD600 measurements. The LUDOX solution is only weakly scattering and so will give a low absorbance value.
Guidelines
Disclaimer: The 2018 InterLab study found that this protocol gave very variable results. We therefore advise teams treat this protocol with some caution, and encourage them to find ways to improve it.

  • Many plate readers have an automatic path length correction feature. This adjustment compromises the accuracy of measurement in highly light scattering solutions, such as dense cultures of cells. You must therefore turn off pathlength correction if it can be disabled on your instrument.

  • This calibration should be completed before your team takes OD600 cell measurements.

  • Optical density (OD) measures the amount of light that is absorbed, scattered and reflected as it passes from source to detector. The OD600 (i.e. OD at 600 nm) of a bacterial culture that has been blanked against growth media serves as an approximation for the number of bacteria in the culture. Spectrometers should be calibrated if numbers of bacteria are important to the experiment. A detailed description of optical density can be found here.

  • General information about calibrating microbial growth in microplate readers can be found here.
Materials
MATERIALS
ReagentAgar
Reagent96 well plate
ReagentddH20
Reagent1ml LUDOX CL-X
ReagentiGEM Measurement Kit 2019
STEP MATERIALS
ReagentddH20
Reagent1ml LUDOX CL-X
LUDOX-CL is no longer provided in the iGEM Measurement Kit. The 96-well plate should preferably be black with a clear flat bottom.
Protocol materials
Reagent1ml LUDOX CL-X
In Materials, Materials, Step 1
ReagentiGEM Measurement Kit 2019
Materials
ReagentddH20
In Materials, Materials, Step 2
ReagentAgar
Materials
Reagent96 well plate
Materials
Before start
Read through this entire protocol carefully before you start your experiment and
prepare any materials you may need. Please see disclaimer in guidelines section. See the "Results" section for an example of a completed data analysis spreadsheet.
Data collection: OD 600 Reference point - LUDOX Protocol
Data collection: OD 600 Reference point - LUDOX Protocol
Add 100 μl LUDOX CL-X into wells A1, B1, C1, D1

Reagent1ml LUDOX CL-X


Add 100 μl of ddH20 into wells A2, B2, C2, D2

ReagentddH20

Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for
cell measurements

Equipment
Plate reader
NAME
Generic
BRAND
.
SKU

Record the data in your notebook or in the table below:

LUDOX CL-XddH2O
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Table for recording OD600 values from your plate.

Once you've collected your results, open this spreadsheet to process them. Download iGEM 2019 Plate Reader LUDOX Calibration.xlsxiGEM 2019 Plate Reader LUDOX Calibration.xlsx

Data processing and analysis
Data processing and analysis
Input your raw values from Step 5 into the blue cells in the table.Go togo to step #4

Yellow cells will automatically populate with values calculated from your raw data.
Expected result
Here is an example of a completed table:
Example of a completed data analysis table for this experiment. Blue cells contain raw data, yellow cells are the result of automatic calculations based on that data. The corrected Abs600 is calculated by subtracting the H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer (as provided to you in the Excel sheet). The correction factor to convert measured Abs600 to OD600 is thus the Reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by (in this example) 1.585.


Congratulations!
Congratulations!
You have now completed this calibration protocol