Question

We first made a 1.0 Mcfarland inoculum with a 10ml volume from a few isolated colonies. Then we did the following dilutions:

10^6 dilution, add 0.1 mL of the Mcfarland inoculum to test tube #2 and add 10ml of water and mix.

10^4 dilution, add 0.1 mL of test tube #2 to test tube #3 and add 10ml of water and mix.

10^2 dilution, , add 0.1 mL of test tube #3 to test tube #4 and add 10ml of water.

Take 0.1 mL of the inoculum from test tube #4 and streak the plate.

Based on the dilutions made from the original inoculum, what is the expected colony count for the plate inoculated with the tube #4 specimen?

Answer 1

A 1.0 McFarland inoculum would have a cell density of 3.0 * 10⁸ CFU / mL.

now let us look at the dilutions from the original stock above and the corresponding cell densities as we make each dilution:

Test tube # 1 : Original suspension: 3.0 * 10⁸ CFU / mL.

Test tube #2 : Here, 0.1 mL from test tube #1 is taken and mixed in a total volume of 10 mL.

The dilution here would be : 0.1 / 10 = 1 / 100 = 10^-2, which means the colony count would be a 100 times less.

The CFU / mL here would be: 3.0 * 10⁸ * 10^-2 = 3.0 * 10⁶.

As we dilute the suspension further, theoretically, the number of cells is going to reduce that many times.  So, we just need to multiply the dilution with the CFU calculated for each dilution from the original value.

Test tube#3 : Here, 0.1 mL from test tube #2 is taken and mixed in a total volume of 10 mL.

The dilution here would be : 0.1 / 10 * 10^-2 = 1 / 100 * 10^-2 = 10^-2 * 10^-2 = 10^-4

The CFU / mL here would be: 3.0 * 10⁸ * 10^-4 = 3.0 * 10⁴ .

Test tube #4 : Here, 0.1 mL from the 10^-4 tube (test tube #3) is taken and mixed in a total volume of 10 mL.

The dilution here is : 0.1 / 10 * 10^-4 = 1/100 * 10^-4 = 10^-2 * 10^-4 = 10^-6

The CFU / mL here would be: 3.0 * 10⁸ * 10^-6 = 3.0 * 10² .

Given that 0.1 mL of the inoculum from test tube # 4 is streaked onto a plate. The expected number of colonies can be calculated using the formula for CFU / mL:

CFU / mL = no. of colonies * Dilution factor / volume plated.

No. of colonies = (CFU / mL) / Dilution factor * volume plated

The volume plated for test tube # 4 here is 0.1 mL; the dilution factor is the inverse of the dilution used for test tube # 4, which would be 10⁶ and the CFU / mL for test tube # 4 would be 3.0 * 10² .

Incorporating these values in the formula above, we get:

No. of colonies = 3.0 * 10² / 10⁶ * 0.1 = 3.0 * 10⁸ * 10^-6 * 0.1 = 3.0 * 10² * 0.1 = 3 * 10 = 30 colonies.

Therefore the number of colonies expected when 0.1 mL of 10^-6 dilution (Test tube #4) of Mcfarland is plated would be 30.