Question

1. You have an E. coli culture of which you are trying to determine the cell concentration. You need to perform a serial dilution. You take 100 µL of the undiluted stock culture, add it to 9.9 mL water in tube A, and mix. Then you take 100 µL of the material in tube A, add it to 9.9 mL water in tube B, and mix. Then you take 100 µL of the material in tube B, add it to 9.9 mL water in tube C, and mix. You then take 100 µL from tube C, plate it on a petri dish, incubate overnight, and the next day you count 47 colonies. Show all work, including units.
   • What was the concentration of cells in tube C?
   • What was the concentration of the original, undiluted stock culture?

Answer 1

For calculating the colony forming unit bacteria, please see below explanations:

Colony-forming unit (CFU or cfu) is a measure of viable bacterial cells. For convenience the results are given as CFU/mL (colony-forming units per milliliter) for liquids.

Calculate the number of bacteria (CFU) per milliliter by dividing the number of colonies by the dilution factor.

The CFU/ml can be calculated using the formula:

cfu/ml = (no. of colonies x dilution factor) / volume of culture plate

you can also use the following for calculating the CFU

Answer: 1 (concentration of cells in tube C)

Number of colonies in 100 µl (0.1 ml) of broth of Tube C = 47 colonies

Then, the number of bacteria in 1 ml of the Tube C can be calculated as follows:

Volume of the broth plated from Tube C is = 100 µl or 0.1 ml

cfu /ml (in Tube C) = 47 / 0.1 ml = 4.70 x 10² colonies/ml

Answer: 2 (concentration of the original, undiluted stock culture)

First thing we need to know is the Dilution Factor, or how much the original sample is diluted till tube C:

Initially in tube A 100 µl in 9900 µl (9.9 ml) = 10000 (total volume)/100 = 100

Then, two serial dilutions of 1/100 in tube B and C.

Total Dilution Factor = 100 x 100 x 100 = 10⁶

CFU/mL = (no. of colonies x dilution factor) / volume of culture plate (ml)

= (47 x 10⁶) / 0.1 (100 µl)

= 4.7 x 10⁸

So Total colony forming units in the original sample will be = 4.7 x 10⁸ CFU/mL

You can convert CFU/mL to Log value using log table.