Question

You decide to dilute a broth culture by making a series of four 1:100 dilutions. You then pipet 1 ml of the final dilution onto one agar plate and 0.1 ml onto a second plate. After incubating overnight, you count 350 colonies on the first plate (with 1 ml) and 56 colonies on the second plate (with 0.1 ml). Which plate would you use for your calculations? Why? Calculate the concentration of the original culture and be sure to include the proper units in your answer. (Hint: Draw a diagram of the dilution series to help you get the answer.)

Answer 1

I will count the second plate (with 56 colonies) for near-accurate calculations, because,

(1) Counting 350 colonies on the agar plate is tedious, prone to mistakes and differentiate between distinct and fused colonies.

(2) Plating 1 ml of culture on a petri plate is not usually recommended, since spreading does not occur uniformly which might give some skewed/biased results.

Calculation:

There are four serial dilutions. Therefore, if we consider the stock solution to have a concentration of 1, then the last dilution of Tube number 5 should be 1: (100 x 100 x 100 x 100) = 1: 100000000 (see figure below).

Again, only 0.1 ml of culture from tube 5 was plated on Plate 2. Therefore, considering the unit of colonies to be CFU/ml, this plate was again diluted 10 times, that is 1: 1000000000 times.

Now, in this particular dilution, we observe 56 colonies. Therefore in the original stock solution, original concentration is 56 X 1000000000 = 56 x 10⁹ CFU/ml or 5.6 x 10¹⁰ CFU/ml.