THE FEASIBILITY OF HAND SANITIZER IN PREVENTING THE GROWTH

OF MICROORGANISMS
OBJECTIVES:
1) Identify the number of colonies of microorganisms found on the
agar plate, and their morphologies (differences in color, shape,
and other properties).
2) Measure and compare the different zones of inhibition to
determine the effectiveness of the hand sanitizer
3) Know the principles behind the use of agar and a nutrient in
culturing microorganisms

GENERAL PROBLEM:
1) What is the antimicrobial property found in hand sanitizer that
inhibits the growth of microorganisms?

SPECIFIC PROBLEMS:
1. Are the sanitized paper disks masked by microorganisms?
2. What are the differences in the amount of bacteria in the four
zones of inhibitions?
3. What are the factors that caused a zero percent growth rate of
microorganisms in the different zones of inhibition on the
sanitized paper disks?

HYPOTHESIS:
a) There are no sanitized paper disks masked by microorganisms.
b) There are no differences in the amount of bacteria in the four
zones of inhibition.
c) There are no factors that caused a zero percent growth of
microorganisms in the different zones of inhibition on the
sanitized paper disks
MATERIALS:
In conducting the experiment, we used 200 mL of cold,
distilled water, beaker, paper towel, tweezers, hot plate, 1/8
nutrient, ruler, zip lock bag, teaspoon, cotton swabs, clear
tape to secure the agar plate, hand sanitizer as antibacterial
agent, small container for soaking paper disks in hand
sanitizer, 4 sanitized paper disks, bacteria collected from a
phone screen, and a glass petri dish for observing the cultured
microorganisms.
METHODOLOGY:
In preparing the nutrient agar mixture, we used a beaker
to blend a teaspoon of agar and one eighth of a nutrient with
200 mL of cold, distilled water. We brought this mixture to
simmer using a hot plate, then continuously boiled it for 3
minutes. At the same time we stirred it well with a teaspoon, to
completely dissolve the agar. We made sure that the mixture
was correctly mixed with no particles floating in it after boiling.
After, we allowed it to cool for 3 to 5 minutes.

To gather and grow our microorganisms, we took off the

lid of the Petri Dish and disinfected it with a hand sanitizer, and
a paper towel. Then, we carefully filled the bottom half of the
Petri Dish with warm agar mixture. We loosely covered the
bottom portion of the lid so that excess moisture can escape.
We also allowed the mixture to cool and set for a certain
amount of time to properly harden. In order to collect a good
sample of microorganisms, we used a cotton swab and swiped
it across the surface of our phone screen. Afterwards, we lifted
the lid of the petri dish and lightly drew a zigzag line in the
agar with the end of the cotton swab. We submerged four
paper disks in a small container of sanitizer and placed it on
the four quadrants of the petri dish using a tweezer. Next, we
tightly closed the lid, secured it with clear tape, and placed it in
a zipper lock bag. Finally, we positioned the petri dishes
upside down in a warm and dark place, to grow our cultured
microorganisms.
 To evaluate the effectiveness of the hand sanitizer, we
used a ruler to measure the distance of each zone of inhibition
from the nearest microorganism in terms of millimeters.

CONCEPTUAL FRAMEWORK
INDEPENDENT VARIABLE
DEPENDENT VARIABLE
Hand Sanitizer measurement of distance of colonies from the
zone of inhibition
Types of microorganisms
inoculated
Scope and Limitations
The Agar Plate, is a nutrient medium used for the cultivation of microbes, and is widely used because
it can grow a variety of types of microorganisms such as bacteria and fungi, utilizing nutrients for
bacterial growth. The intended purpose of our study is to determine and measure the
susceptibility of the microorganisms to the hand sanitizer under standard conditions.
However, due to limited scientific instruments/equipments, the specific nature and components of the
microorganisms cultivated on the agar plate will only be described in its outer appearance through
macroscopic and microscopic observations. The setting of the experiment is in the Physics Lab of
Baguio City High School and the time frame is within 2 - 3 weeks.

MACROSCOPIC OBSERVATIONS:

Day 1 Day 3

There were no colonies nor growth of microorganisms apparent.

Day 4 Day 5

The circumference of the petri dish starts to turn cloudy due to the growth and reproduction of
bacterial cells during incubation.

Day 6 Day 7

Evident patches of microorganisms start to grow.

Most bacterial colonies were white, cream, or yellow in color, and fairly circular in shape.

Some molds were whitish grey and other molds were variously greenish or blackish in color with
fuzzy edges.

MICROSCOPIC OBSERVATIONS:
CONCLUSION:

RRL:

http://www.biotopics.co.uk/microbes/tech.html

FINDINGS:

For this test, a culture medium, is uniformly and aseptically inoculated with the test organism and
then filter paper discs, which are impregnated with a specific concentration of a particular amount of
hand sanitizer, are placed on the medium. The organism will grow on the agar plate while the
antibiotic works to inhibit the growth. If the organism is susceptible to a specific antibiotic, there will
be no growth around the disc containing the antibiotic. Thus, a zone of inhibition can be observed
and measured to determine the susceptibility to an antibiotic for that particular organism. The
measurement is compared to the criteria set by the National Committee for Clinical Laboratory Studies
(NCCLS). Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I)
or Susceptible (S).

Principle of the method:

The media used in this test has to be the Mueller-Hinton agar because it is an agar that is thoroughly
tested for its composition and its pH level. Also, using this agar ensures that zones of inhibitions can
be reproduced from the same organism, and this agar does not inhibit sulfonamides. The agar itself
must also only be 4mm deep. This further ensures standardization and reproducibility.

The size of the inoculated organism must also be standardized (using the BBD Prompt system). The
reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than
what it is supposed to be (the antibiotics will have a distinct advantage) and if the inoculum is too
large, the zone of inhibition will be smaller.

The intended purpose of this assay is to avoid the introduction of microbial

contamination into in vitro cell cultures and in vivo animal studies utilizing the test-
nanomaterial, as microbial contamination will confound the results of these tests.
This assay is not intended to certify the tested material as sterile.