Apr 30, 2024
Calibration Protocol - Conversion of OD600 to Colony Forming Units (CFUs)
This protocol is a draft, published without a DOI.
- Paul Rutten1,
- Richard Tennant1,
- Jacob Beal1,
- Christopher Workman2,
- Traci Haddock-Angelli3,
- Natalie Farny4,
- Vinoo Selvarajah3
- 1iGEM Measurement Committee;
- 2Technical University of Denmark;
- 3iGEM;
- 4Worcester Polytechnic Institute
External link: https://2019.igem.org/Measurement
Protocol Citation: Paul Rutten, Richard Tennant, Jacob Beal, Christopher Workman, Traci Haddock-Angelli, Natalie Farny, Vinoo Selvarajah 2024. Calibration Protocol - Conversion of OD600 to Colony Forming Units (CFUs). protocols.io https://protocols.io/view/calibration-protocol-conversion-of-od600-to-colony-dcwh2xb6
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: In development
We are still developing and optimizing this protocol
Created: July 13, 2019
Last Modified: April 30, 2024
Protocol Integer ID: 98985
Abstract
This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL.
This protocol assumes that 1 bacterial cell will give rise to 1 colony.
For the CFU protocol, you will need to count colonies for your two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures. Protocol based on this Yeast Plate Count Protocol.
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.
Materials
MATERIALS
1.5 mL Eppendorf tubes
96 well plate
Chloramphenicol (25 mg/ml in EtOH)
LB Broth
2.0 mL Eppendorf tubes
Before start
Read through this entire protocol carefully before you start your experiment and prepare any materials you may need. See the "Results" section for an example of a completed data analysis spreadsheet. Please see disclaimer in guidelines section.
Sample Preparation
Sample Preparation
This protocol will result in CFU/mL for 0.1 OD600. Your overnight cultures will have a much higher OD600 and so this section of the protocol, called “Sample Preparation”, will give you the “Starting Sample” with a 0.1 OD600 measurement.
Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader.
e.g. Add 25 μL culture to 175 μL LB + Chloramphenicol (Cam) in a well in a black 96-well plate, with a clear, flat bottom
Recommended plate setup is below. Each well should have 200 μL
Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
Use (C1)(V1) = (C2)(V2) to calculate your dilutions
C1 is your starting OD600
C2 is your target OD600 (= 0.1)
V1 is the unknown volume in μL
V2 is the final volume (= 1000 μL)
Expected result
Important:
When calculating C1, subtract the blank from your reading and multiple by the dilution factor
you used.
Example: C1 = (1:8 OD600 - blank OD600) x 8 = (0.195 - 0.042) x 8 = 0.153 x 8 = 1.224
Example: (C1)(V1) = (C2)(V2)
(1.224)(x) = (0.1)(1000 μL)
x = 100/1.224 = 82 μL culture
Add 82 μL of culture to 918 μL media for a total volume of 1000 μL
Check the OD600 and make sure it is 0.1.
Recommended plate setup is below. Each well should have 200 μL.
Dilution Series
Dilution Series
Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 5. You should have 12 total Starting Samples - 6 for your Positive Controls and 6 for your Negative Controls.
You will need 3 LB Agar + Cam plates (36 total)
Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3
Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5
Label each tube according to the figure above (Dilution 1, etc.) for each Starting Sample
Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs
Repeat Step 11 for each dilution through to Dilution 5 as shown above
Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5
Incubate at 37 °C overnight and count colonies after 18-20 hours of growth
CFU/mL/OD Calculation
CFU/mL/OD Calculation
Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated
First, count the colonies on each plate with fewer than 300 colonies
Next, multiply the colony count by the Final Dilution Factor on each plate, or use this Excel spreadsheet:
iGEM Data Analysis Template - Plate Reader CFU Calibration - v1.xlsx Expected result
Example using Dilution 4 from above:
# colonies x Final Dilution Factor = CFU/mL
125 x (8 x 105) = 1 x 108 CFU ⁄ mL in Starting Sample (OD600 = 0.1)
Congratulations!
Congratulations!
You have now completed this calibration protocol