Question

#4. You have been given a tube of E. coli. You are asked to make 1 mL total volume of 10^-1 dilution of the bacterial culture. Explain how you would do this. Show all necessary calculations.

#5 You have bacteria at a concentration of 5 x 10⁸ CFU/mL. You spread 1 mL of this sample on an agar plate to obtain isolated colonies. How many colonies do you expect to find the next day after incubation at 37⁰C? Can you count these colonies? Can you use this plate for determining concentration?

#6. You have bacteria at a concentration of 1 x 10³ CFU/mL (in real life – you don’t know this, but we are just working on math skills here). You transfer 1 mL of this sample into 9 mL of water and then spread 1 mL on a plate of agar. How many colonies do you expect to find the next day after incubation at 37⁰C? Can you count these colonies? Can you use this plate for determining concentration?

#7 You take 0.05 mL of a culture of bacteria at a concentration of 4 x 10⁷ CFU/mL, and add 4.95 mL of water to it. What is the dilution that you have performed? What is the concentration of bacteria (CFU/mL) in the diluted culture?

#8. You have diluted a sample by 1000 fold (1/1000) and plated 1 mL on an agar plate. You observe 55 colonies. What was the concentration of the original sample in CFU/mL?

#9. A bacterial sample has a concentration of 3 x10⁷CFU/mL. You make serial dilutions of 10^-3 followed by 10^-2 and 10^-1 dilutions. You finally plate 1 mL of the last dilution on an agar plate and incubate it at 37⁰C.   What is the total dilution? How many colonies do you expect to see on the plate?

Total dilution:

# of colonies expected on plate:

#10. This time, you see 10 times fewer colonies than you had expected to see.What could have gone wrong? How will you fix this problem?

Answer 1

#4 Answer: 0.9ml of medium added to 0.1 ml of original E.coli bacterial culture. Explanation: 10 raised to the minus 1 dilution is 1 part in 10. As the total volume of the solution is 1 mL, 1ml/10 = 0.1 ml, the solution will be made adding 0.9 ml of medium to 0.1 ml of the original E.coli bacterial culture.

#5. Answers: How many colonies do you expect to find the next day after incubation at 37^°C? 2.36x10³⁰ CFU, Can you count these colonies? No, they are too many to count, Can you use this plate for determining concentration? No

Explanation: E. coli bacteria division time (doubling time) is 20 minutes. To calculate the final amount of bacteria you can use the formula N = N₀2ⁿ, where N is the final number of CFU, N₀ is the initial number of CFU, and n is the number of generations. Knowing we spread 5x10⁸ CFU (1 mL contains 5x10⁸ CFU) on an agar plate, and let it grow for 24 hours:

24 hours x 60 = 1440 minutes/20 = 72 generations = n

N = N₀2ⁿ ---> N = 5x10⁸ CFU (2⁷²) = 5x10⁸ CFU (4.72x10²¹) = 2.36x10³⁰ CFU

6# Answers: How many colonies do you expect to find the next day after incubation at 37^°C? 4.72x10²³ CFU, Can you count these colonies? No, they are too many to count, Can you use this plate for determining concentration? No

1 x 10³ CFU/mL = (1,000 CFU/mL)/10 = 100 CFU (2⁷²) = 4.72x10²³ CFU

#7 Answers: What is the dilution that you have performed? 100, What is the concentration of bacteria (CFU/mL) in the diluted culture? 4 x 10⁵ CFU/ml.

Explanation: To get the dilution you need to divide the total final volume (4.95ml +0.05ml = 5ml) by the initial volume to dilute (0.05ml), 5ml/0.05ml = 100. To calculate the final concentration after the dilution, taking into account that you have 4 x 10⁷ CFU in 1 mL, by the rule of three, you will have 2x10⁶ CFU in 0.05ml, and after adding 4.95 mL of water, you will have 2x10⁶ CFU/5ml = 4 x 10⁵ CFU/ml.

#8 What was the concentration of the original sample in CFU/mL? Answer: 55 colonies x 1000 = 55,000 colonies/mL = 5.5x10⁴ CFU/ml

#9 Answers: What is the total dilution? 10^-6 (1/1000000 or 1:1000000) How many colonies do you expect to see on the plate? 30  

Explanation: To calculate the total dilution you need to multiply the three serial dilutions: 10^-3 x 10^-2 x 10^-1 = 10^-6. To calculate the final number of colonies, taking into account that the original concentration is 3 x10⁷CFU/mL, after the first dilution of 10^-3 (1part of the initial concentration culture/1000parts of total volume), you will have (3 x10⁷CFU/mL)/1000 = 3 x10⁴CFU/mL, then from that dilution after a 10^-2 (1/100) dilution, you will have (3 x10⁴CFU/mL)/100 = 3 x10²CFU/mL, from that dilution after a 10^-1 (1/10)dilution, you will have (3 x10²CFU/mL)/10 = 30 CFU/mL, which is the samw you will get by using the total dilution of 10^-6 as (3 x10⁷CFU/mL)/1000000 = 30 CFU/mL

#10 Answers: What could have gone wrong? Doing an extra 10^-1 dilution, or increasing by 10 the dilution mistakenly in one of the serial dilutions How will you fix this problem? So seeing 10 times fewer colonies in the last problem would be 3 colonies/ml, we just need to do a new 10^-1 dilution of the 10^-2 dilution​.