Stephanie Yin and Karen Lin

Science Fair
General Procedure
Question/Research Problem: What is the effect of non-alcoholic wines in reducing the amount of E.
coli?
Hypothesis: If we use substances containing proancyanidins, such as non-alcoholic wines, then the
concentration of E. coli in LB agar plates will decrease.
Materials:
 Filter paper
 (3) Non-alcoholic wines: Chardonnay, Brut, White Zinfadel, (Merlot)
 E. coli bacteria
 (~40) Clear petri dishes
 (2) 500mL flasks
 Microwave
 Tweezers
 Incubator/freezer/refrigerator (for bacteria)
 Bleach
 Alcohol
 LB Agar (growth medium for bacteria)
One liter of the agar contains:
-20g of premix LB Agar powder
-1000 mL of deionized H2O
 Balance
 Thermometer
 Sterile bacterial spreaders
 Pipettes
 Clear glass stirring rod
General Procedure: In preparation for experimentation, lab benches will be wiped down with alcohol
before and after following the procedure. Safety goggles, aprons, and gloves will be worn and aseptic
techniques will be employed.
1. Prepare 1000mL of LB agar from LB broth and agar mix.
2. Let the plates cure at room temperature for 48 hours, then store plates upside down in refrigerator to
avoid condensation.
3. Grow the colonies for 24 hours and streak each agar plate evenly with sterile bacteria spreaders for a
total of about 10 times to generate single colonies from a concentrated suspension of bacteria.
4. Apply one type of non-alcoholic wine, the antimicrobial agent, to the center of the agar plate (in a
fashion such that the antimicrobial doesn't spread out from the center) on filter paper, and incubate the
plates.
5. Observe growth of bacteria around a circular hole cut out of filter paper in the center of the plate. If
substantial antimicrobial activity is present, then a zone of inhibition will appear around the test product.
The zone of inhibition is simply the area on the agar plate that remains free from microbial growth. The
size of the zone of inhibition is usually related to the level of antimicrobial activity present in the sample
or product-a larger zone of inhibition usually means that the antimicrobial is more potent.
6. Repeat steps 3-4 for with 2 additional types of non-alcoholic wine. Test first on a control group,
containing plates streaked only with E. coli.
7. Record the results and compare the qualitative change of E. coli present before and after being dipped in
non-alcoholic wine. (If the treatment killed the bacteria, no colonies will grow on the agar plate.)
8. Repeat steps 5-6 for (10) trials per wine flavor. Record observations/size of zone of inhibition. In
addition, follow procedure by testing various amounts of Chardonnay and Brut Cuvee wine, as well as
by testing on various other non-alcoholic wines (e.g. Chardonnay, White Zinfandel, Merlot) to see how
reliable and repeatable the experiment is. Additional trials will help determine how non-alcoholic wines
affect the growth of E. coli as a result of different variables seen by comparing each trial with the control
group.
9. To measure the zone of inhibition, measure the diameter around the filter paper circles in millimeters,
with the plate cover still on. If the diameter is not uniform in all directions, then the circles were not set
right or the bacteria was not evenly spread. (use another plate in this case)
10. Either wrap each Petri dish/agar plate in Parafilm after labeling, or place them in a resealable plastic bag
to prevent drying out and to control odors. Turn the plates upside down and put them in a warm place
(ideal temperature for incubation is 32°C or 90°F). Bacterial plates should be stored in a refrigerator for
short-term storage. They will be good for up to one month under these conditions. For long–term storage
of bacterial strains, the bacterial stock can be placed in the freezer in a regular -20°C environment
without further treatment.
11. After experimentation, all glassware will be treated with 10% bleach solution. The petri dishes will be
flooded with bleach solution and disposed of in biohazard bags.

Non-alcoholic wines–[NAW], [NAW1], [NAW2]

LB Agar-[A]
Bacteria-[B]

Control experiment:
[A] + [B] [A] + [B] + [NAW]

Varying amounts of NAW:

[A] + [B] [A] + [B] + 0.5 [NAW]
[A] + [B] [A] + [B] + 2 [NAW]

Varying types of NAW:

[A] + [B] [A] + [B] + [NAW2]
[A] + [B] [A] + [B] + [NAW3]