Question

You have prepared a culture that you expect to have a concentration of 96 x 10⁵ CFU/ml. Show how you would plate the culture out to confirm this. Use 9 ml blanks. Use the least number of blanks possible.

Answer 1

To confirm the culture concentration of 96 x 10⁵ CFU/ml, perform serial dilutions to achieve a 1:10,000 dilution, plate 1 ml, and count the colonies to extrapolate the original concentration.

Step-by-step explanation:

To confirm the expected concentration of 96 x 10⁵ CFU/ml by plating the culture, we would employ serial dilution and the plate count method. Given that microbiologists consider 30-300 colonies optimal for counting, the least amount of dilution necessary to bring the concentration within this range would be ideal.

First, 1 ml of the original culture is added to a 9 ml blank, making a 1:10 dilution (10⁶ CFU/ml). To further dilute, take 1 ml from this and add it to another 9 ml blank, creating a 1:100 dilution (10⁵ CFU/ml). Since the expected concentration is already at 96 x 10⁵ CFU/ml, this should be diluted again in the same manner to achieve a 1:1000 dilution (10⁴ CFU/ml). Finally, one more dilution to a 1:10,000 (10³ CFU/ml) would be advisable to ensure the count is within the desired range. From this dilution, 1 ml is plated on a solid medium, usually using the spread plate method.

If 50 colonies are averaged from the 1:10,000 dilution, to calculate back to the original concentration, we multiply by the total dilution factor (10 x 10,000) and the volume plated (0.1 ml), resulting in 5,000,000 CFU/ml, confirming our expected concentration.