Question

Derrick has a flask with 50 ml of LB medium and 1 x 10^9 bacteria (or Colony Forming Units - cfus). He is trying to plate between 10 and 100 colonies per plate. He must construct a dilution series. So he adds 50 µL of his bacteria to a new tube with 9.5x10^-1 ml of fresh LB medium. Derrick now transfers 10^-4 litres from this tube to a plate.

Jenny laughs at Derrick for having done this. She tells him that he “should add a few tubes to his dilution series before transferring 0.1 ml to a plate”, if he wants to get about 30 cfu on the plate. At his current rate of dilution, how many more tubes will he need to get about 30 cfus on the plate?

Answer 1

Answer

Given data - flask contains 1 x 109 cfu in 50 ml LB broth.

plating requires 10-100 cfu of bacteria.

Derrick adds 50 µl of bacteria culture to 9.5 x 10-1 ml (950 µl) LB broth

                    (i.e. 1:20 dilution)

He now has 1 x 106 cfu in 1 ml culture.

Which means 1 x 105 cfu in 0.1 ml culture.

We require about 30 cfu

So, we need to do 3 more serial dilution of 1 ml culture prepared by Derrick.

Dilution 1– 50 µl culture + 950 µl broth = 5 x 104 cfu in 1 ml

Dilution 2 – 50 µl of diln 1 + 950 µl broth = 2500cfu in 1 ml

Dilution 3 – 50 µl of diln 2 + 950 µl broth = 125 cfu in 1 ml

Therefore, 100 µl of culture will have 12.5 cfu.

So Derrick have to plate 240 µl to get 30 cfu in one plate.