CHEM525 Experiment 1

Bacterial growth and supplement dependence.

Bacteria display a characteristic four-phase pattern of growth in liquid culture. The initial Lag phase is a
period of slow growth during which the bacteria are adapting to the conditions in the medium. This is
followed by a Logarithmic or Log Phase, during which growth is exponential, with cells doubling every
replication cycle. Stationary Phase occurs when the nutrients become limiting or the concentration of
metabolic waste in the medium becomes high, and the rate of replication is equal to the rate of cell
death. Death Phase occurs when cells die faster than they are replaced.

We will study the patterns of growth of E. coli BL21(DE3) strain in a variety of different conditions.

Sample Growth Medium1,2 Temperature Light Antibiotic3

1 LB 37 °C Yes -
2 M9 37 °C Yes -
3 LB 25 °C Yes -
4 LB 37 °C Yes Amp
5 LB 37 °C Yes Kan
6 LB 37 °C No -
1
LB (Lauria Bertani) is a mixture of peptides, yeast extract and NaCl
2
M9 media is a chemically defined media containing all essential components (phosphate,
glucose, NH4Cl, NaCl, MgSO4 and CaCl2
3
Antibiotics should be added to a final concentration of 50 μg/mL.

The bacterial population in the culture will be estimated by measuring its turbidity with a
spectrophotometer. Traditionally, turbidity is defined as the absorbance of the culture at a wavelength of
600 nanometers, commonly referred to as the OD600 (or optical density at 600 nm). This value can then
be converted to a useful concentration value using the standard conversion factor where 1 OD600 = 1 x
109 cells/mL i.

You should record the collected data and make two graphs of it in your notebook: one on a linear scale,
and one on a log scale. Label the different phases on your graph and determine the doubling times of the
cells for each medium. Presentation of experimental data is a critical skill to master; see the instructor
for specific tips and strategies.

Safety Precautions:
• Always wear glove, safety coat and goggles when in the lab.
• Although the strain of E. coli used in this experiment is not inherently dangerous because it lacks
the ability to colonize outside of a laboratory cultures, it is always good practice to use care when
handling living organisms.
• Antibiotics should never be handled without gloves.
• If a significant amount of any chemical is spilled, immediately seek the instructor for clean-up
protocols.

i th
Fred Ausubel et al., Short protocols in Molecular Biology, 5 ed. Vol. 1 pp. 1-6
Equipment/Reagents Needed:
250 mL Erlenmeyer flasks containing 100 mL of growth medium (1 for each type experiment)
100 mg/mL antibiotic stocks
foil
37° C Gyrotory shaker
25° C Gyrotory shaker
Timer
Sterile pipettes (5 & 1 mL)
Spectrophotometer
Plastic cuvettes
Overnight Escherichia coli BL21 (DE3) cultures (20 mL per culture)
Blank samples of uninoculated media

Procedure:
1) Label each of your Erlenmeyer flasks with the type of medium it contains, your group name and the
date.
2) Add any necessary antibiotic or wrap foil around your flask for the dark culture.
3) Obtain 10 mL of the overnight cultures of E. coli BL21(DE3) and transfer each culture to an
Erlenmeyer flask and start the timer. Swirl the flasks and transfer 1 mL of each to a plastic cuvette.
Blank the spectrophotometer with one of the two media blanks and then read the absorbance of the
cultures made of the same type of medium. Repeat for the other medium type.
4) Repeat the measurement procedure every 30 minutes for 3 hours.
5) Prepare a plot of Absorbance at 600 nm versus time using a standard linear graph. Prepare the same
plot using a logarithmic scale on the appropriate axis.
6) Determine the doubling time of the culture using each plot. Be certain to note any differences
between the 2 different plot types.
7) Label the different phases of the linear plot. If your plot does not show all of the different phases,
label the ones you do have.

Suggested discussion topics:

• Both growth media contain all essential components for bacterial growth. Why does the growth
of this organism vary with the growth medium?
• Which temperature provides the optimal growth condition? Justify this by thinking about where
E. coli traditionally colonizes. Make sure to cite any external resource you use.
• The BL21(DE3) strain does not have any natural antibiotic resistances. Based on what you have
learned in you background reading, propose a reason for any observed resistances.
• Does this strain of E. coli need light to grow efficiently? Justify your answer.
• What other variables do you think would influence bacterial growth?
• Next week we will use IPTG (Isopropyl β-D-1-thiogalactopyranoside) to induce gene expression
and protein production. Will this influence how your bacteria grow?