Introduction:

Colony-forming units (CFUs) are used to estimate the number of viable microorganisms, such as bacteria and yeast, in a sample. This measurement is widely used in microbiology to analyze cell concentration and growth. In this article, we will walk you through the process of calculating CFUs using the dilution plating method.

Step 1: Create serial dilutions

Serial dilutions are performed to obtain a countable range of colonies on a plate. Begin by preparing a set of tubes containing diluent (sterile broth or saline). Typically, 9 mL of diluent is used for each tube.

1. Take 1 mL of your original undiluted sample and add it to the first tube containing 9 mL of diluent. This creates a 10-fold (10^-1) dilution.

2. Mix well by vortexing or pipetting up and down.

3. Take 1 mL from the 10^-1 diluted tube and transfer it to the second tube containing 9 mL of diluent. This creates a 100-fold (10^-2) dilution.

4. Repeat this process for additional dilutions as needed.

Step 2: Plating dilutions

Using sterile technique, spread 0.1 mL or 100 µL aliquots of each dilution onto labeled agar plates:

1. Dispense the appropriate volume of diluted sample onto the center of each plate.

2. Gently spread the sample over the entire surface using a sterile bent glass rod or disposable spreader, creating a thin layer.

3. Incubate the plates at an appropriate temperature for your organism until colonies are visible.

Step 3: Counting colonies

After incubation, count the number of colonies on each plate:

1. Select plates with countable colony numbers, ideally between 30 and 300 colonies.

2. Record the colony count and corresponding dilution factor.

Step 4: Calculating CFUs

Use the following formula to calculate CFUs:

CFU/mL = (Number of Colonies x Dilution Factor) / Volume plated (mL)

For example, if you count 125 colonies from a plate made using a 10^-5 dilution and you plated 100 µL, the calculation would be:

CFU/mL = (125 x 10^5) / 0.1 = 1.25 x 10^7

Conclusion:

Calculating colony-forming units is an essential skill in microbiology, as it provides an estimate of viable cell concentration in a sample. Through creating serial dilutions, plating them on agar, counting colonies, and applying the CFU/mL formula, you can effectively determine the concentration of viable microorganisms in your sample.