Question

You receive a yeast culture of an unknown concentration, so you decide to determine the concentration by performing a direct count using a counting chamber. You count the number of yeast in seven 1/400 mm² boxes and get the following results: 5, 8, 19, 8, 4, 3 and 6.

a. How many cells per ml are in your unknown sample? (6 points)

b.If you diluted your unknown sample 1:100, then 1:20, then 1:2 and then plated 0.2 ml onto a YEPD plate, about how many colonies would you expect?

Answer 1

a)

Number of cells in 7 1/400 mm2 boxes = 5 + 8 + 19 + 8 + 4 + 3 + 6 = 53 cells.

Volume of 7 1/400 mm2 boxes =Number of chambers * Area of chamber * Height of Chamber
= 7 * 1/10 * 1/400 mm3 = 7/4000 mm3
= 1.75 * 10-3 * (10-1 cm)3 = 1.75 * 10-6 cm3 = 1.75 * 10-6 mL

Therefore, cell concentration = 53 cells/(1.75 * 10-6 mL) = 3.0286 * 107 cells/mL

b)

The dilutions performed are 1:100, 1:20, 1:2. Therefore, the total dilution is 1:4000. 0.2 mL of this dilution is plated onto YEPD plates and therefore the volume correction factor is 1/5.

Thus,

Number of colonies expected on plate = Original CFU count * Dilution Factor * Volume Correction Factor
= 3.0286 * 107 * 1/4000 * 1/5 = ‭0.00015143‬ * 107 cells = 1514.3 cells

Thus, for the given dilution, 1514 colonies would be expected.