CLINICAL BACTERIOLOGY

Topic: Sterilization and Disinfection

Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

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STERILIZATION AND DISINFECTION

Sterilization is a process that kills all forms of microbial life, including bacterial endospores.
Disinfection is a process that destroys pathogenic organisms, but not necessarily all
microorganisms, endospores, or prions. Many factors limit the success or degree of
sterilization, disinfection, or decontamination in a health care setting, such as organic load
(organisms and other contaminating materials such as blood or body fluids), the type of
organisms present, the concentration and exposure time to the germicide, the physical and
chemical nature of the surface (hinges, cracks, rough or smooth surfaces), temperature, pH,
humidity, and presence of a biofilm. These processes may be accomplished by a variety of
physical or chemical methods.

I. OBJECTIVE:
● To learn principles and methods of sterilization.
● To know the different types of sterilizers.
● To stress importance of reliable sterilization procedures.
● To emphasize importance of aseptic techniques.
● To demonstrate readily available test methods.

II. MATERIALS:
1. Autoclave 7. Bar of soap
2. Nutrient agar/broth 8. Syringe
3. Normal Saline Solution 9. Inoculating loop
4. Sterile cotton swabs 10. Towelette/ tissue paper
5. 70% alcohol 11. Bleach
6. Detergent powder

III. PROCEDURE:

1. Testing sterility of commercially packaged sterile isotonic saline

solution.
i. Disinfect the surface of the rubber stopper placed in the top portion
of the vial.
ii. Air dry until no obvious excess alcohol can be seen.
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

iii. Using a brand-new syringe, penetrate the vial by puncturing the

rubber stopper.
iv. Aspirate a small amount of the solution, and inoculate it in the agar
broth or plate.
v. Incubate for 18-24 hours at 37 degrees Celsius.
vi. Observe any sign of growth.

2. Comparison of sterility between a newly washed hands using a bar soap

hands and hands with no hand washing.

i. Hands with proper handwashing

1. Wash hands using bar soap. Follow the basic steps in
handwashing.
2. After handwashing, do not touch any object or even some
parts of your body or your groupmates.
3. Using sterile cotton swab, swab the surface of your hands.
4. Inoculate it in the agar broth or plate.
5. Incubate for 18-24 hours at 37 degrees Celsius.
6. Observe any sign of growth.

ii. Hands with no handwashing

1. Touch any object or even some parts of your body or your
groupmates.
2. Using sterile cotton swab, swab the surface of your hands.
3. Inoculate it in the agar broth or plate.
4. Incubate for 18-24 hours at 37 degrees Celsius.
5. Observe any sign of growth

3. Testing sterility of hands disinfected with 70% alcohol

1. Using a 70-75% alcohol, rub your hands and let it dry for 1
minute.
2. Using sterile cotton swab, swab the surface of your hands.
3. Inoculate it in the agar broth or plate.
4. Incubate for 18-24 hours at 37 degrees Celsius.
5. Observe any sign of growth.

4. Testing the sterility of laboratory table tops before and after application
of bleach solution.

i. Before application of bleach solution

1. Using a sterile cotton swab, swab the surface of the
laboratory table tops.
2. Inoculate the cotton swan in a culture media plate or broth.
3. Incubate of 18-24 hours at 37 degrees Celsius
4. Observe any sign of growth.
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

ii. After application of bleach solution

1. Making of bleach solution
a. Follow the directions on the bleach bottle for
preparing a diluted bleach solution.
b. If your bottle does not have directions, you can
make a bleach solution by mixing:
i. 5 tablespoons (1/3 cup) of bleach per gallon
of room temperature water or
ii. 4 teaspoons of bleach per quart of room
temperature water
2. Using the bleach solution, wiped the laboratory table tops.
3. Wait for 5-10 minutes to dry up the surface.
4. Using a sterile cotton swab, swab the surface of the
laboratory table tops.
5. Inoculate the cotton swan in a culture media plate or broth.
6. Incubate of 18-24 hours at 37 degrees Celsius
7. Observe any sign of growth.

IV. DRAW AND LABEL:

A. Laboratory sterilizers used in the experiment, its use and different functions of its
parts.
1. Autoclave
Use: to decontaminate certain biological waste and sterilize media,
instruments and lab ware.
Different Function of its Part:
● Vessel
- The vessel, the main body of an autoclave, consists of an inner
chamber and outer jacket. Laboratory and hospital autoclaves
have "jacketed" chambers filled with steam, reducing
sterilization time and condensation. Full jackets improve
temperature uniformity, reduce wet packs, and minimize steam.
● Control System
- Modern autoclaves have a sophisticated controller interface
similar to microwaves or ovens, following a preprogrammed
software formula for sterilization. These systems can be simple
with a microprocessor or complex with a programmable logic
controller.
● Thermostatic Trap
- Autoclaves use thermostatic traps or steam traps to remove air
and water from the chamber, preventing dry steam passage.
These temperature-sensitive valves close when heated past a set
point, making them crucial for well-designed autoclaves.
● Safety Valve
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

-Autoclaves, operating under high pressure, require robust

construction and safety features, including a safety valve as a
final fail-safe device. It must be inspected, tested, and verified
according to manufacturer recommendations, local inspection
and insurance agencies, and sterilizer recommendations.
● Waste-Water Cooling Mechanism
- Autoclaves often have systems to cool effluent before it enters
drains piping, preventing damage to drain piping. To cool
steam, mix it with cold tap water. Some autoclaves have
systems designed to reduce or eliminate water consumption,
ensuring efficient water usage.
● Vacuum System
- The vacuum system must replace all air in the autoclave
chamber with steam for proper sterilization. Porous materials or
small containers with air pockets can harbor microorganisms,
preventing sterility.
● Steam Generator
- An autoclave typically uses a central house boiler as its steam
source, but if house steam is insufficient, an electric steam
generator is used.

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2. Incubator
Use: is a useful tool for culturing microorganisms and cells. It can also
regulate the temperature and humidity levels in an environment. This allows
for optimal growth conditions for the cultures being incubated.
Different Function of its Part:
● Cabinet
- The incubator's main body is a double-walled cuboidal
enclosure with a capacity of 20 to 800L, made of stainless steel
sheets and aluminum. Glass wool insulation prevents heat loss
and reduces electric consumption. The inner wall supports
shelves.
● Door
- Incubators feature an insulated door with a glass for incubation
visualization, and a handle for easy door maneuvering.
● Control Panel
-The outer wall of the incubator features a control panel with
switches and indicators for controlling incubator parameters
and a witch for controlling the device's thermostat.
● Thermostat
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

- A thermostat is utilized to set the desired incubator

temperature, which is automatically maintained until the
temperature is adjusted again.
● Perforated shelves
- Perforated shelves in incubators allow hot air movement and
can be removable for proper cleaning.
● Asbestos door gasket
- The asbestos door gasket creates an airtight seal between the
door and cabinet, preventing outside air from entering, thereby
creating an isolated hot environment.
● L-shaped thermometer
-A thermometer with gradations is placed on the outer wall of
the incubator, with one end outside and the other slightly
protruding into the chamber.
● HEPA filters
-Advanced incubators with HEPA filters reduce airflow
contamination, creating a closed-loop system with an air-pump
to minimize airborne contaminants.
● Humidity and gas control
- CO2 incubators feature a reservoir containing water for
humidity control and gas chambers for adjusting CO2
concentration within the chamber.
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B. Final results obtained on each of the culture media agar and broth.
a. Over all culture media observation
- After 2 days of incubation, the physical appearance show color
changes in the agar, some are orange, yellow and white thus,
nutrient agar grow bacilli bacteria colony as seen in the
microscopic examination.
b. Color of the media before and after inoculation
- Before inoculation nutrient agar shows clear yellow color. After
inoculation and incubation there are some bacaterial growth occur
and changes the structure of the media.
c. Turbidity of the culture media before and after
- Before inoculation nutrient media shows clear transparency
structure. After the inoculation, due to moisture and environment
the turbidity slightly alter into hazy transparency.
V. QUESTIONS:
1. Explain the principle of autoclave and give different use of this equipment
according to:
a. Laboratory use
b. Industrial use
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

Principle of Autoclave:
According to Rodriguez of Review Handbook in Diagnostic
Bacteriology, the Principle of Autoclaving is STEAM UNDER
PRESSURE.
Steam under pressure according to the Centers for Disease
Control and Prevention is a principle that uses pressure to
obtain high temperature to kill microorganisms or to expose
each item to direct steam contact at the required temperature
and pressure for the specified time.
In simple terms, Pressure serves as a means to obtain the high
temperatures necessary to quickly kill microorganisms.
Specific temperatures must be obtained to ensure the
microbicidal activity.

Use of Equipment according to

● Laboratory use
- In laboratory, it is used to sterilize biohazardous trash
and heat-stable objects.
- It is used as a method for sterilization which all
organisms (except for prions), including those that
contain spores are killed within 15 minutes (Rodriguez,
Diagnostic Bacteriology)
● Industrial use
- In medical settings, autoclave is used to sterilize solids,
liquids and instruments like surgical equipment.
- In food preparation or industry, it is used in canned
goods, drinks and beverages and also in dairy products
to preserve food.

2. Differentiate moist heat sterilization, dry heat sterilization, and cold sterilization.

According to Review Handbook in Diagnostic Bacteriology of Rodriguez

.l.
● Moist heat sterilization
- Moist heat sterilization is a process where it can destroy or kill
different microorganisms through the process of coagulation of
enzymes and structural proteins and through the degradation of nucleic
acids.

● Dry heat sterilization

- From the word itself, dry heat. It requires no water in sterilization
process. However, it can kill microorganisms by the process of protein
denaturation. Dry heat sterilization process is the recommended
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

process for the sterilization of glasswares materials, oil products and

powder.

● Cold Sterilization
- In medical instruments, cold sterilization can be used. It can occur for
15 to 30 minutes and the materials are submerged in a sterilant
solution like Glutaraldehyde

3. Give one scenario in the Bacteriology laboratory on how to apply Thermal Death
Time.
- Thermal death time is a concept used to determine how long it takes to kill a
specific bacteria at a specific temperature. It was originally developed for food
canning and has found applications in cosmetics, and in producing salmonella-free
feeds for animals (e.g. poultry, and pharmaceuticals).

In the Bacteriology laboratory, this concept is used with bacteria that causes food
poisoning, such as: Staphylococcus aureus, Salmonella, Clostridium perfringens,
Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus
cereus, and Entero-pathogenic Escherichia coli. Various heat treatments can be
used in determining the TDT.

4. Give 5 common bacteria that can be isolated in the following:

a. Hands
Staphylococcus aureus: A common bacterium found on human skin.
Reference: "Staphylococcus aureus in the Community: Colonization
Versus Infection" - NCBI

Escherichia coli (E. coli): Typically found in the gastrointestinal tract but
can also be present on hands. Reference: "Escherichia coli in Humans" -
Microbiology Society

Enterococcus species: These bacteria are commonly found in the intestines

and can be transferred to hands. Reference: "Enterococci in Human Health
and Disease" - Microbiology and Molecular Biology Reviews

Pseudomonas aeruginosa: Often found in moist environments and can be

present on hands. Reference: "Pseudomonas aeruginosa: The
Strain-Induced Switch to the Stringer Cell Phenotype and Dispersal" -
NCBI

Enterobacter species: These bacteria are part of the Enterobacteriaceae

family and can be isolated from various sources, including hands.
Reference: "Enterobacteriaceae: Friend or Foe?" - Clinical Microbiology
Reviews
CLINICAL BACTERIOLOGY
Topic: Sterilization and Disinfection
Activity No.: 3.0
Laboratory Proctor: Melford L. Teodoro, RMT

b. Laboratory table tops

Staphylococcus aureus: Staphylococcus aureus is a common bacterium
found on human skin and can be transferred to surfaces through contact. It
is a well-known pathogen and is often isolated in environmental
monitoring.

Escherichia coli (E. coli): E. coli is commonly found in the gastrointestinal

tracts of humans and animals. Contamination with E. coli can occur when
handling samples or materials.

Bacillus spp.: Various species of Bacillus can be found in soil and dust,
which may settle on laboratory surfaces. Bacillus species are often used as
indicators for cleanliness and sterility testing.

Pseudomonas aeruginosa: Pseudomonas aeruginosa is a ubiquitous

bacterium that can thrive in moist environments. Laboratory sinks and
drainage systems can be sources of this bacterium.

Micrococcus spp.: Micrococcus species are often found on human skin and
mucous membranes. They can be transferred to tabletops through contact
or aerosolization.

c. Sterile Normal saline solution

Escherichia coli (E. coli): E. coli is a common bacterium found in the
human intestine and is often used in laboratory settings. It can survive in
Sterile Normal Saline for short periods.

Staphylococcus aureus: This bacterium can be isolated in Sterile Normal

Saline and is frequently used in microbiological studies. It's a common
cause of skin and respiratory infections.

Salmonella spp.: Some strains of Salmonella can be isolated in Sterile

Normal Saline. These bacteria are associated with foodborne illnesses.

Pseudomonas aeruginosa: Pseudomonas is a hardy bacterium that can be

stored in Sterile Normal Saline. It's often studied due to its role in
healthcare-associated infections.

Enterococcus faecalis: Enterococcus species can be isolated and preserved

in Sterile Normal Saline. These bacteria are found in the intestines and are
associated with various infections.