EXPERIMENT 4

BASIC PRINCIPLES OF FERMENTATION SHAKE FLASK TECHNIQUE

TOPIC OUTCOMES:

Student should be able to

1. To determine the growth rate of bacteria under different temperature
OR pH conditions
2. To establish growth curves for an unknown bacterial species and
observe the different phases of growth.

MATERIAL

- 10 ml culture of YEAST OR ECOLI culture

- 2 x 50 ml LB broth or NB broth in 125-ml flasks
- Spectrophotometer
- Cuvettes
- Sampling bottles or universal bottle
- Sterile Distilled Water 100 mL for each group
- Bunsen burner

METHODOLOGY

1. Your group will be assigned one of the following growth conditions:

a. 37C incubator with shaking

b. 37C incubator without shaking
c. 37C with pH medium of 7
d. 37C with pH medium of 5
2. Obtain a flask of LB or NB broth and transfer 5 ml of broth to a
small test tube. This is your blank.
3. Adjust the wavelength to 600 nm on the spectrophotometer and
calibrate the machine to 0.0 O.D. or 100% transmittance with your
blank. Take the blank out but do not discard it.
4. Inoculate your LB or NB flask with 10 ml of the culture provided and
swirl the flask well.
5. Work quickly to withdraw 5 ml of the sample into a clean small test
tube, using a pipette.
6. Place the culture flask immediately at the assigned incubating
temperature condition and record the time.
7. Measure the O.D. of your sample next. Record the 0 time
absorbance. Discard the contents of your sample test tube. This
test tube can be re-used for your next sample without washing.
8. At 15-minute intervals for up to 90 minutes, shake your flask
vigorously and transfer 5 ml of the culture to your sample test tube.
9. Record the optical density at each time period. Use your blank each
time to make sure your readings are correct and other students
have not altered the machines calibration.
10. Plot the optical density (y-axis) versus time (x-axis) for each growth
condition on the same regular graph paper.
11. To determine the generation time, plot the optical density (y-axis)
versus time (xaxis) on semi-log paper. Draw a straight line of best fit
through the points making up the log phase. Next draw two
horizontal lines cutting the y-axis where the optical density has
doubled and then draw two vertical lines where the horizontal lines
intersect the curve and note the time period between the two points
on the x-axis. This is the generation time.

QUESTIONS

1. Create a proper table to show your results

2. Draw a graph from the results that shows the different phases
occurs.
3. Discuss in terms of the obtained results and whether the objectives
of the experiment is achieved or any disturbance that may influence
the whole fermentation.

Note: Include the answers of these questions in your discussion

section.