Bacterial cell calculation involves determining the number of viable bacteria present in a sample, typically expressed as colony forming units (CFU). Here's a breakdown of how it's done, incorporating information from the provided reference:
Understanding the Process
Calculating bacterial cells often involves serial dilution and plating techniques.
- Serial Dilution: A series of dilutions is made from the original sample to ensure that the number of bacteria plated is within a countable range (usually 30-300 colonies).
- Plating: A known volume of the diluted sample is spread on agar plates and incubated. Each viable bacterial cell will grow into a visible colony.
- Counting Colonies: After incubation, the colonies on each plate are counted.
The Calculation
The primary method for determining bacterial count involves using the following formula, based on the reference:
CFU/ml or CFU/gram = (Number of colonies) / (Dilution factor x Volume of specimen plated)
Step-by-step Breakdown:
- Count the Colonies: Use a Quebec colony counter or similar method to accurately count the number of colonies on a plate that falls within the countable range (ideally 30-300 colonies).
- Determine the Dilution Factor: The dilution factor reflects how much the original sample was diluted before plating. For example, a 1:100 dilution has a dilution factor of 100. For multiple serial dilutions, you must multiply all dilution factors, e.g., a 1:10 dilution followed by a 1:100 dilution yields a dilution factor of 10 x 100 = 1000.
- Determine the Plating Volume: This is the volume of the diluted sample (in milliliters, or grams if using a solid sample) that was plated onto the agar.
- Apply the Formula: Divide the number of colonies counted by the product of the dilution factor and the plating volume.
Example
Let's say:
- You counted 150 colonies on a plate.
- The plate was from a 1:1000 dilution.
- You plated 0.1 ml of diluted sample.
Calculation:
CFU/ml = 150 colonies / (1000 x 0.1 ml)
CFU/ml = 150 / 100
CFU/ml = 1500 CFU/ml
Therefore, your original sample had an estimated 1500 colony forming units per milliliter.
Key Considerations:
- Colony Forming Units (CFU): The result represents viable bacterial cells, meaning those that were able to grow and form a colony.
- Accurate Counting: Using a colony counter and counting plates within the 30-300 range is critical for accuracy.
- Appropriate Dilutions: Making appropriate serial dilutions is essential to achieve a countable number of colonies. Too many colonies on a plate make counting impossible, and too few colonies may lead to inaccurate results.
- Homogeneous Sampling: Ensure the sample is thoroughly mixed before plating to achieve uniform bacterial distribution.
By carefully following these steps, one can effectively calculate the concentration of viable bacteria in a sample.
