l. If you count 35 colonies on a plate after plating 150 L of a culture a. How m
ID: 143873 • Letter: L
Question
l. If you count 35 colonies on a plate after plating 150 L of a culture a. How many organisms were in l mL of that culture? b. How much culture would you need to plate to see only 5 colonies? 2. You purchase a sample of E.coli cells from a scientific supply company. The company ships a tube containing 50 uL of cells at a concentration of 5 x 108 CFU/mL. a. Are more or less than 5 x 10 CFU in the tube? b. How many cells are in the tube? c. Describe a step by step protocol that would allow you to generate a growth plate with roughly 50 colonies. (There is more than one way to perform this task, but all involve diluting the original sample somehow. Also, just a note: when diluting cells, media is the diluent, not water.) 3. I grew E.coli overnight in an attempt to generate a culture with a concentration of around 2.0 x 10 (or 20 million) CFU/mL. I performed a series of dilutions and plated 50 uL of each on a growth plate and counted the following number of colonies that appeared: 4 colonies on the 1:1e6 dilution plate 25 colonies on the 1:1e5 dilution plate " 215 colonies on the 1:1e4 dilution plate * TNTC colonies on the 1:1e3 dilution plate Calculate an average value for the concentration of the culture from this information. Did the culture reach my intended concentration, roughly? a. b.Explanation / Answer
1 a) In 150 muL there were 35 colonies. In 1 mL which is 1000 muL, there will be, 1000/150 * 35 = 233 colonies approx.
b) If you need to see 5 colonies you would need to plate 150/7 = 22muL. But this is not suggested normally. Rather you can dilute the 150 muL from the stock 7 times and then take 150 muL from the diluted solution and plate.
2 a) It says 5 * 10^8 CFU per mL which is 5 * 10^5 per muL. We have only 50 muL and thus the number of cells in the tube would be
50 * 5 * 10^5 = 250 * 10^5 cells. Thus it will be less.
b) Refer previous answer.
c) We will do what is called a serial dilution. Take 10 uL of the stock. This would contain 50 * 10^5 cells. Dilute it to 100 ul which is 10 times.
Now take 10 ul from this, (N=50*10^4) and dilute to 100 ul again.
From this take 10 ul and dilute to 100 ul, and repeat this for three more times and we would get 100 uL of sample containing 50 cells exactly. Dilution should be done with media.
3 a) In the first plate, we have 4 colonies in 50 uL, which means 4/50 * 10^3 colonies per mL. But this is 10^6 times diluted. So the concentration of stock is : 4/50 * 10^3 * 10^6 = 80,000,000 CFU/mL
In the same way, the second plate has 25/50*10^3*10^5 = 50,000,000 CFU/mL
The third, 215/50*10^3*10^4 = 43, 000, 000 CFU/mL
The average is 3.3 * 10^7 CFU/mL
b) We can say it is almost the concentration we want.
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