Colony Forming Units Method and Calculation

CFU/mL (Colony-Forming Units per milliliter) is a measure used to quantify the number of viable bacterial cells in a liquid sample. It is commonly determined by plating serial dilutions of a bacterial culture on agar plates and counting the resulting colonies after incubation.

 

Here's how you can calculate CFU/mL of bacteria:

 

Materials Needed:

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Bacterial culture
Sterile saline solution or sterile growth medium
Agar plates (appropriate agar medium for the bacteria)
Incubator
Pipettes and tips
Bunsen burner or alcohol burner
Colony counter or magnifying glass
Calculator

Procedure:

 

Serial Dilution:
a. Prepare a series of dilutions of your bacterial culture using sterile saline solution or sterile growth medium. For example, you can prepare 10-fold serial dilutions (1:10, 1:100, 1:1000, etc.).
b. Label each dilution tube with the dilution factor.

 

Inoculation of Agar Plates:
a. Sterilize the pipettes and spreader or glass rod by passing them through a flame (Bunsen burner or alcohol burner).
b. Pipette a known volume (e.g., 0.1 mL or 1 mL) of each diluted bacterial sample onto separate agar plates.
c. Spread the liquid evenly over the agar surface using a sterile spreader or glass rod.

 

Incubation:
a. Incubate the agar plates at the appropriate temperature and conditions for the bacteria you are working with (e.g., 37°C for most common bacteria).
b. Allow the plates to incubate for a suitable time (typically 24-48 hours) to allow visible colonies to form.

 

Counting Colonies:
a. After incubation, examine each plate and count the visible colonies. Consider using a colony counter, magnifying glass, or other suitable means to ensure accuracy.
b. Keep track of the colony counts for each dilution.

 

Calculating CFU/mL:
a. Calculate the CFU/mL for each dilution by dividing the number of counted colonies by the volume of the sample plated.
b. For example, if you plated 0.1 mL of a 1:100 dilution and counted 50 colonies, the calculation would be:

CFU/mL = (Number of Colonies) / (Volume Plated in mL)
CFU/mL = 50 colonies / 0.1 mL = 500 CFU/mL

 

Dilution Factor Adjustment (if applicable):
a. Since you performed serial dilutions, you'll need to adjust the calculated CFU/mL values to account for the original dilution factor.
b. For example, if you performed a 1:100 dilution, you would multiply the calculated CFU/mL by 100 to account for the original undiluted sample.

 

Final Calculation:
a. Calculate the average CFU/mL from all your plates if you plated the same dilution multiple times.

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Remember that CFU/mL represents viable, culturable bacterial cells. Some bacterial cells might not form colonies under certain conditions, so this method provides an estimate of viable cells under the specific culture conditions used. Always follow proper aseptic techniques and safety guidelines when working with bacterial cultures and agar plates.