Question 4 You are given three stock cultures – Escherichia at a concentration o

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Question 4 You are given three stock cultures – Escherichia at a concentration of 1.8 x 104 cfu/ml, Bacillus at a concentration of 3.6 x 104 cfu/ml, and Staphylococcus at a concentration of 4.8 x 104 cfu/ml. You have been asked to make 30 ml of a single mixed culture with a final concentration of 3 x102 cfu/ml of Escherichia, 9 x102 cfu/ml of Bacillus, and 80 cfu/ml of Staphylococcus. In the space below, show me how you would make this mixture.

(i) If you plate 0.4 ml of the mixture on a TSA plate, how many colonies each of Escherichia, Staphylococcus, and Bacillus would you expect to get? Don’t worry if this results in an uncountable plate.

(ii) If you plate 0.4 ml of the mixed culture above on a MacConkey plate, how many total colonies would you expect to get?                                               

Explanation / Answer

In order to make 30 ml mix culture we need to add all the orgainsm in following quantity.

1- E-coli- Given that the original cfu/ml of Ecoli is 1.8 x 104 cfu/ml and we need to dilute it to 3 x102 CFU/ml.

For 30 ml of mix culture we will need

1.8*104 * X = 30 * 3 * 102

X = 30 * 3 * 102/ 1.8*104

= 0.5 ml Ecoli culture

2- Bacillus - Bacillus at a concentration of 3.6 x 104 cfu/ml and we need to make  9 x102 cfu/ml

So we need to add

3.6*104 * X = 9* 102 * 30

X= 9* 102 * 30 / 3.6*104

0.75ml

3- Staphylococcus at a concentration of 4.8 x 104 cfu/ml and we need 80 CFU/ml

So we need to add

4.8*104 * X = 80 * 30

X = 80*30 / 4.8*104

=0.05 ml

We will add 0.5ml of Ecoli culture , 0.75 ml of Bacillus culture and 0.05ml of staphylococcus culture.

(i) in the 0.4 ml of culture we will have

300 * 0.4 = 120 Ecoli CFU

900 * 0.4 ml = 360 Bacillus CFU

80* 0.4 = 32 Staphylococcus

we will get 120+360+32 = 512 CFU on TSA plate

(ii) MacConkey agar different gram-negative with gram-positive bacterium as only gram-negative bacterium grows in MacConkey agar. Bacillus is gram positive so it won't grow.

So we will get 152 CFU on the plate

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