Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

This procedure was 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, counting colonies is performed for the two Positive Control (BBa_I20270) cultures and the two Negative Control (BBa_R0040) cultures.

Step 1: Starting 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 “Starting Sample Preparation”, will give you the “Starting Sample” with a 0.1 OD600 measurement.

1.Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader, e.g. to 0.05 – 0.5 OD600 range. Include blank media (LB + Cm) as well. For an overnight culture (16-18 hours of growth), we recommend diluting your culture 1:8 (8-fold dilution) in LB + Cm before measuring the OD600.

Preparation

LB + Cm before measuring the OD600. Preparation:Add 25 μL culture to 175 μL LB + Cm in a well in a black 96-well plate, with a clear, at bottom.

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

2.Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm 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 of 0.1

V1 is the unknown volume in μL

V2 is the final volume of 1000 μL

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)(1000μL)

x = 100/1.224 = 82 μL culture

Add 82 μL of culture to 918 μL media for a total volume of 1000 μL

3.Check the OD600 and make sure it is 0.1 (minus the blank measurement). Recommended plate setup is below. Each well should have 200 μL .

Step 2: Dilution Series Instructions

Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 1. You should have 12 total Starting Samples - 6 for your Positive Controls and 6 for your Negative Controls.

For each Starting Sample (total for all 12 showed in italics in paraenthesis):

1. You will need 3 LB Agar + Cm plates (36 total).

2. Prepare three 2.0 mL tubes (36 total) with 1900 μL of LB + Cm media for Dilutions 1, 2, and 3 (see figure below).

3. Prepare two 1.5 mL tubes (24 total) with 900 μL of LB + Cm media for Dilutions 4 and 5 (see figure below).

4. Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.

5. Pipet 100 μL of Starting Culture into Dilution 1.Discard tip.Do NOT pipette up and down. Vortex tube for 5-10 secs.

6. Repeat Step5 for each dilution through to Dilution 5 as shown below.

7. Aseptically spead plate 100 μLon LB +Cm plates for Dilutions 3, 4, and 5.

8. Incubate at 37°C overnight and count colonies after 18-20 hours of growth.

Step 3: CFU/mL/OD Calculation Instructions

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 as follows:

1. Count the colonies on each plate with fewer than 300 colonies.

2. Multiple the colony count by the Final Dilution Factor on each plate.

Example using Dilution 4 from above

 # colonies x Final Dilution Factor = CFU/mL

 125 x (8 x 105) = 1 x 100000000 CFU ⁄ mL in Starting Sample (OD600 = 0.1)

Result

Colony Forming Units per o.1 OD600 E.coli cultures