You begin a culture of Escherichia coil by inoculating 1 ml from a mid-log-phase
ID: 33185 • Letter: Y
Question
You begin a culture of Escherichia coil by inoculating 1 ml from a mid-log-phase culture that is 1.28 x10^6 cells per ml into 99 ml of the same fresh medium. This culture is shaken at 37 degree C and allowed to grow. The new culture population density increases exponentially over the next 12 hours. At 12 hours, you perform a spread plate count, plating dilutions of 10^-21^-3010^-5 and 10^-5 to triplicate plates; after counting the colonies and averaging the three numbers at each dilution you obtain the following results: 10^-2--confluent lawn 10^-3-- 100 colonies 10^-5--10 colonies 10^-5-- 2 colonies information you may need: and so on.. What is the population density (viable cells per ml of culture) at time of inoculation? What is the population density (viable cells per ml of culture) at 12 hours? . How many generations occurred during the 12 hours? What is the generation time? Was your culture heavily turbid (essentially opaque) at any time during the growth? Is Escherichia coli growing at a rate approximating its maximum under optimal growth conditions.?Explanation / Answer
E.coli has a generation rate or doubling time of 20 minutes. i.e. it takes each E.coli cell 20minutes to divide by binary fission into two cells.
if there are 1.28 x 10^6 cells/ml den after initial 20 minutes the cell density will double to 2.56 x10^6. thus, after 12 hrs it will be 46.08 x 10^6 cells/ml.
However, after 12 hrs, when inoculum is collected from the incubated culture the cell density will be same as above. i.e. 46.08 x 10^6 cell/ml of culture.
after the dilutions are plated and the plates are incubated under standard experimental condition and the triplicates are counted, from the given reading it can be calculated that the number of cells at the end of the experiment are 1.5 x 10^5cfu/mL. i.e. 1.5 colony forming units/mL which are the viable cells/mL of culture.
during the 12hours of incubation time, 36 generations of E.coli occured.
Formula for generation time (G)= t log 2/[log b ? log B]
G is generation time, t time interval, b is population density at end of time interval and B is density at initial time.
t=12hrs=720 minutes
b=46 x 10^6 cells/mL
B=1.28 x 10^6cells/mL
log 2=0.3010
log B=log (1.28 x 10^6)= 6.107
log b= log (46 x 10^6) =7.663
thus, G= 720 (log2) 7.663-6.107
G= 216.72 1.556
G= 139.280 minutes
Thus, generation time for the given experiment is 139.280minutes.
The culture is 1mL in 100mL thus, after 12 hrs time period it may not appear heavily turbid but slight opacity will be seen.
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