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]
<div class = "text-blocks"><div class = "text-block"><span>This procedure can be used to calibrate OD</span><span style = "vertical-align:sub;">600</span><span> to colony forming unit (CFU) counts, which are directly relatable to the cell concentration of the culture, i.e. viable cell counts per mL. </span></div><div class = "text-block">This protocol assumes that 1 bacterial cell will give rise to 1 colony.
</div><div class = "text-block"><span>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 </span><a href="https://fankhauserblog.wordpress.com/1988/06/29/yeast-plate-count-protocol/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Yeast Plate Count Protocol</span></a><span style = ":;:UNDERLINE;">.</span></div></div>

[STEPS]
?. [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.
?. [Sample Preparation]
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
?. [Sample Preparation]
Recommended plate setup is below. Each well should have 200 μL
?. [Sample Preparation]
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 dilutionsC1 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)
Important: When calculating C1, subtract the blank from your reading and multiple by the dilution factoryou used.Example:    C1 = (1:8 OD600 - blank OD600) x 8 = (0.195 - 0.042) x 8 = 0.153 x 8 = 1.224Example:    (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
?. [Sample Preparation]
Check the OD600 and make sure it is 0.1. Recommended plate setup is below. Each well should have 200 μL.
?. [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.
?. [Dilution Series]
You will need 3 LB Agar + Cam plates (36 total)
?. [Dilution Series]
Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cam media for Dilutions 1, 2, and 3
?. [Dilution Series]
Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cam media for Dilutions 4 and 5
?. [Dilution Series]
Label each tube according to the figure above (Dilution 1, etc.) for each Starting Sample
?. [Dilution Series]
Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs
?. [Dilution Series]
Repeat Step 11 for each dilution through to Dilution 5 as shown above
?. [Dilution Series]
Aseptically spead plate 100 μL on LB + Cam plates for Dilutions 3, 4, and 5
?. [Dilution Series]
Incubate at 37 °C overnight and count colonies after 18-20 hours of growth
?. [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
?. [CFU/mL/OD Calculation]
First, count the colonies on each plate with fewer than 300 colonies
?. [CFU/mL/OD Calculation]
Next, multiply the colony count by the Final Dilution Factor on each plate, or use this Excel spreadsheet:
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!]
You have now completed this calibration protocol