Question

Steps

a) add 20 grams of oysters to 20 mL of saline to give a 1:2 dilution slurry

b) Transfer 5 grams of the slurry into a conical tube.

c) Prepare a 1:10 serial dilution by adding 20mL of saline to the measured 5 g of slurry ( this step effects a 1:5 dilution but that sample was already diluted 1:2)

d) Prepare a 10^-1, 10^-2, and 10^-3 serial dilution in culture tubes with 9 mL of saline

e) plate 0.1 ml of each dilution onto an agar plate.

f) two plates from the 10^-3 dilution made colonies of 112 colonies and 58 colonies.

1. Show calculations for the serial dilutions with units

2. calculate the CFU/g for each plate with colonies with units

Answer 1

Note the following points before starting:

· Note that using this method, the answer in CFUs per one milliliter or per one gram is derived. Answers may need to be adjusted if the number of CFUs per sample (other than a milliliter or gram) is requested. Assume 1 gram = 1 ml. (1 ml of water does indeed weigh 1 gram. That is actually how the ml is defined.)

· Use only those plates with colony counts between 25 and 300. With duplicate or triplicate plating from the same dilution, take the average of the plate counts and then proceed.

· Note that all individual dilutions and the amount plated are multiplied together.

· The initial dilution is often different from the subsequent dilutions. This is generally due to the nature of the sample available for analysis.

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

The CFU/ml or CFU / g can be calculated using the formula:

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

Calculation of cell density in the original, undiluted stock culture

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

Dilution factor initially in Tube are

1st Dilution factor: 40 (total volume) / 20 = 2

2nd dilution factor: 25 (total volume) / 5 = 5

Then, two serial dilutions of 1/10 in 3 tubes (tube 1 to 3).

Dilution from 3rd to 5th: 10 x 10 x 10 = 1000 =1 x 10³

Total dilution factor of the original sample till tube 3rd: 2 x 5 x 10³ = 1 x 10⁴

Third plate:

1. CFU for 3^rd plate with 58 colonies:

No of colonies in 0.1 ml (100 µl) = 58

Then, the number of bacteria in 1 ml of the Tube 3 (plate having 58 colonies) can be calculated as follows:

Volume of the broth plated from Tube 3 is = 100 µl or 0.1 ml

CFU /ml or CFU / g = 58 / 0.1 ml = 5.8 x 10²

2. CFU for 3^rd plate with 112 colonies:

If number of colonies in 0.1 ml (100 µl) from the same dilution (third tube) = 112

Then, the number of bacteria in 1 ml of the Tube 3 (plate having 112 colonies) can be calculated as follows:

Volume of the broth plated from Tube 3 is = 100 µl or 0.1 ml

CFU /ml or CFU / g = 112 / 0.1 ml = 1.12 x 10³

3. Calculation of CFU / mlfor the original sample

If same dilution has given two different CFU value, then you have to take the average of both for the final result.

Average number of colonies: (58 + 112) / 2 = 170 / 2 = 85

Total dilution factor of the original sample till tube 3^rd: 2 x 5 x 10³ = 1 x 10⁴

Final CFU / g = (average of number of colonies x dilution factor) / volume of culture plate (ml)

                        = (85 x 10⁴) / 0.1 (ml)

                        = 8.5 x 10⁶ CFU / g