ACTIVITY

5:
CONTROL OF
MICROBIAL GROWTH
ACTIVITY 5.2
EVALUATION OF ANTISEPTIC

Jens Martensson 2
 SPECIFIC LEARNING OBJECTIVES:
At the end of the experiment, the students are expected to:

❖Define the terms antiseptic, disinfectant, sterilant,

sporocide, sanitizer, bacteriostatic, and bacteriocidal;
❖Demonstrate the relative effectiveness of common
antiseptics and disinfectants such as bleach, hydrogen
peroxide, and mouthwash using the paper disk method.

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Everyday we use several different chemical agents to
control or kill microorganisms.
Use in:
1. Hospital set-ups – disinfect contaminated areas (strong
oxidizing agents such as sodium hydroxide) 1 2

- alcohol applied before surgery

1. Common households – bleach
2. Cities and municipalities’ water supplies – adding
chlorine to water supplies to prevent the spread of
potential pathogens such as salmonella typhi in the
drinking water 3 4

3. Food productions – to retard spoilage of food by

microbes, to increase shelf life of food products in the
supermarket, and to prevent growth of pathogenic
bacteria such as Clostridium botulinum, which
causes a deadly from of food poisoning.
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 Substances that inhibit microbial growth or
kill microorganisms and applied to living Alcohol and
Antiseptic tissue. However, they do not destroy betadine
endospores

• Applied to inanimate objects

• More harsh than antiseptic Ethylene
Disinfectant • Also known as sterilant or sporocide oxide

• Agents that reduce microbial Chlorine

Sanitizers numbers to a safe level.

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 • Inhibit growth of bacterial
Bacteriostatic cells
Clindamycin

Bacteriocidal • Kill bacterial cells Penicillin

Bacteriocides,
fungicides, and • Specific for certain group Chlorine
viricides

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KIRBY BAUER TECHNIQUE Determine in an antiseptic or
disinfectant inhibits the growth of a
test organism

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METHODS
Chemical reagents used : 3% Hydrogen peroxide, 5% Lysol,
5% Iodine, 1:10 bleach, mouthwash, betadine

Bacteria used: P. aeruginosa, and S. aureus

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 3. Sterilize the forceps by dipping it in
2. Inoculate the loop in alcohol and flaming. Using the flamed
one of the organism forceps, pick up a sterile filter paper disk
1. Liquefy a nutrient and streak it in the and dip it halfway into a disinfectant.
agar pour in sterile prepared dish.
petri dish. Refrigerate
until solidify

4.Repeat the
same procedure
for Pseudomonas
aeruginosa

5.Incubate the plate for 48hrs at

370°C
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RESULTS:
Disinfectant/ Antiseptic Millimeters of Inhibition
Staphylococcus aureus Pseudomonas aeruginosa
3% Hydrogen Peroxide 1.6 cm 1.4 cm
5% Lysol 4 mm 3 mm
5% Iodine 2 mm none
1:10 Bleach 4.0 cm 3.0 cm
Mouthwash None None
Betadine 3 mm None

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1. Which chemical was the most effective for inhibiting
growth of S aureus? Of P. aeruginosa?
1. Bleach inhibits the growth of both microorganism
2. Which chemical was the least effective for inhibiting the
growth of S. aureus? Of P. aeruginosa?
1. The least effective is the mouthwash
3. What do your results indicate about the relative
chemical resistances of these two species?
1. The two species resist some of the chemical reagents used in
activity such as in mouthwash and betadine, yet the rest of the
reagents inhibit the growth of these species. Hence, resistance
of samples differ in the concentration of the reagents.

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CONCLUSION
Antiseptic is a substances that inhibit microbial growth or kill
microorganisms and applied to living tissue. However, they do not destroy
endospores. While, disinfectant is applied to inanimate objects, and is also
known as sterilant or sporocide since it destroy microorganisms including
those with endospores.
In the reagents used in the experiment, bleach has the largest zone of
inhibition both in P. auruginosa and S. aureus and the mouthwash has the
least zone of inhibition. With the results obtained, we can compare the
effectivity of disinfectant and antiseptic. This prove that disinfectant is
more harsh than antiseptic.

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1. DIFFERENTIATE BETWEEN ANTISEPTIC AND
DISINFECTANT. INCLUDE EXAMPLES OF EACH IN YOUR
ANSWER. INDICATE WHETHER ANY CHEMICALS CAN BE
USED AS BOTH
 Antiseptics are substances such as alcohol or iodine that inhibit microbial
growth or kill microorganisms and are gentle enough to be applied on
living tissues. Disinfectant are chemical agents such as bleach that are
applied to inanimate objects such as floors, walls, and tabletops to kill
microorganisms. Disinfectant are usually more harmful than antiseptics and
cannot be used as an antiseptics because they can damage living tissue.

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2. WHAT FACTORS INFLUENCE THE SIZE OF THE ZONE
OF INHIBITION PRODUCED BY A CHEMICAL?
- some technical factors:
❖Timing of disc application - If the plates, after being seeded with the test strain, are left at
room temperature for periods longer than the standard time, multiplication of the inoculum
may take place before the discs are applied
❖Temperature of incubation - Susceptibility tests are normally incubated at 35 °C for optimal
growth. If the temperature is lowered, the time required for effective growth is extended and
larger zones result
❖Incubation time – Most techniques adopt an incubation period of between 16 and 18 hours.
❖Size of plate, depth of agar medium, and spacing of the disc – eexcessively large
inhibition zones may be formed on very thin media; the converse is true for thick media.
Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or
deformation near the edge of the plates.

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3. HOW MIGHT THE PHYSICAL DIFFERENCES BETWEEN
GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA
CONTRIBUTE TO DIFFERENCES IN CHEMICAL
RESISTANCE?
- Gram-negative bacteria have an outer membrane that keeps chemicals from
reaching the inside of the cell

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DOCUMENTATION

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THANK YOU