TITLE: Calibration Protocol - Conversion of OD600 to Colony Forming Units (CFUs)
AUTHORS: Paul Rutten, Richard Tennant, Jacob Beal, Christopher Workman, Traci Haddock-Angelli, Natalie Farny, Vinoo Selvarajah

[DESCRIPTION]
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.

[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.

[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.

[STEPS]
SECTION: Sample Preparation
1. 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.
SECTION: Sample Preparation
2. 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
SECTION: Sample Preparation
3. Recommended plate setup is below. Each well should have 200 μL

 
SECTION: Sample Preparation
4. 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)
 

SECTION: Sample Preparation
5. Check the OD600 and make sure it is 0.1. 

Recommended plate setup is below. Each well should have 200 μL.

 
SECTION: Dilution Series
6. 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.

 
SECTION: Dilution Series
7. You will need 3 LB Agar + Cam plates (36 total)
SECTION: Dilution Series
8. Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3
SECTION: Dilution Series
9. Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5
SECTION: Dilution Series
10. Label each tube according to the figure above (Dilution 1, etc.) for each Starting Sample
SECTION: Dilution Series
11. Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs
SECTION: Dilution Series
12. Repeat Step 11 for each dilution through to Dilution 5 as shown above
SECTION: Dilution Series
13. Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5
SECTION: Dilution Series
14. Incubate at 37 °C overnight and count colonies after 18-20 hours of growth
SECTION: CFU/mL/OD Calculation
15. 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
SECTION: CFU/mL/OD Calculation
16. First, count the colonies on each plate with fewer than 300 colonies
SECTION: CFU/mL/OD Calculation
17. Next, multiply the colony count by the Final Dilution Factor on each plate, or use this Excel spreadsheet: 
 

 


SECTION: Congratulations!
18. You have now completed this calibration protocol