A Simple Experiment on Unicellular Growth Kinetics Exemplifying Simple Life History

Objective: To determine the growth kinetics of non-pathogenic strain Escherichia coli

Materials:

Pure culture of non-pathogenic strain Escherichia coli

Wire loop

Bunsen burner

Sterile Plate count agar, PCA (slant/plate)

Incubator (35 ⁰C)

Alcohol as sanitizer

Methods:

1.      Obtain pure culture non-pathogenic strain Escherichia coli

2.      Transfer aseptically a loopful of non-pathogenic strain Escherichia coli to a sterile plate count agar slant.

3.      Incubate at 35 ⁰C for 20 minutes.

4.      Pour plate the culture. Note: This  will be the initial population of the culture. Dilute serially using replicate at 10¹ up 10⁶

5.      Incubate the plates.

6.      Incubate further the slant for 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 and 78 hours.

7.      Pour plate the culture as stated in Step 4 for every time interval as mentioned in Step 6. Incubate the plates for each time interval.

8.      Log the results using the Table below. Consider the colonies with 25 to 250 cfu/ml. How many viable bacteria are there in your sample?

Time	Dilution Factor	Plate Counts (CFU)	Average Plate Count (CFU)	Nunber of living cells in the culture
20 minutes
1 hour
6 hours
12 hours
18 hours
24 hours
...
78 hours

                                                                                                        

9.   Graph the results of the experiment, put time (in minutes) on the x-axis and numbers of living bacteria on the y-axis (log).

10.  Calculate a growth rate and generation time using this formula:

k= [log (xt) - log (x0)] / 0.301 * t

k= the number of population doublings in one hour (called the growth rate constant)

x0= the number of cells/milliliter at the beginning of the log phase

xt= the number of cells/ml at some later time

t= the number of hours between the beginning and the later time

1/k= the Generation Time (the amount of time it takes for a population to double in number)        

Upon plotting, the theoretical results will appear as:

Source of illustration: http://en.wikipedia.org/wiki/Bacterial_growth

i.        During lag phase, the E.coli cells adapt and adjust themselves to growth conditions. It is the period where the microbial cells are maturing and not yet able to divide. This is the period in bacterial growth cycle wherein DNA, RNA, enzymes and other molecules occurs.

ii.      Log phase is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and waste accumulation occurs.

iii.    The growth rate slows during stationary phase as a result of nutrient depletion and accumulation of toxic products. This phase is reached as the bacteria begin to exhaust the resources that are available to them. It is said that rate of bacterial growth is equal to the rate of bacterial death during stationary phase

iv.    At death phase, bacteria run out of nutrients and die.