Sterilization and

Disinfection
Dalia Sukmawati
Outline

• Principles Of Sterilization And Disinfection

• Methods Of Sterilization : Chemical & Physical Control
 PRINCIPLES OF STERILIZATION
AND DISINFECTION
• Sterilization is the killing or removal of all microorganisms in a
material or on an object. There are no degrees of sterility—sterility
means that there are no living organisms in or on a material.
• Disinfection: reducing the number of pathogenic organisms on objects
or in materials so that they pose no threat of disease.
• Agents called disinfectants are typically applied to inanimate objects,
and agents called antiseptics are applied to living tissue.
Methods of Sterilization :
Chemical & Physical Control
Chemical Control Physical Control
• Reaction that affect 1. Heat – moist and dry
protein 2. Cold temperatures
• Reaction that affect 3. Desiccation
membranes
4. Radiation
• Specific chemical
antimicrobial agents 5. Filtration

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Chemical Control
 Antimicrobial Agents’ Modes of Action

Cellular targets of physical and chemical agents:

1. The cell wall – cell wall becomes fragile and cell lyses; some
antimicrobial drugs, detergents, and alcohol
2. The cell membrane - loses integrity; detergent surfactants
3. Cellular synthetic processes (DNA, RNA) – prevention of
replication, transcription; some antimicrobial drugs, radiation,
formaldehyde, ethylene oxide
4. Proteins – interfere at ribosomes to prevent translation,
disrupt or denature proteins; alcohols, phenols, acids, heat

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CHEMICAL ANTIMICROBIAL
AGENTS
• The potency, or
effectiveness, of a
chemical anti microbial
agent is affected by
time, temperature, pH,
and concentration.
• High concentrations
may be bactericidal
(killing), whereas
lower concentrations
may be
bacteriostatic
(growth inhibiting).
Mechanisms of Action
of Chemical Agents

• Reaction that affect protein

• Reaction that affect membranes
• Specific chemical antimicrobial agents
Mechanisms of Action
of Chemical Agents
• Chemical antimicrobial agents kill microorganisms by
participating in one or more chemical reactions that
damage cell components. Whether they affect
proteins, membranes, or other cell components.

Much of a cell is
made of protein,
and all its
enzymes
are proteins.
Alteration of
protein structure
is called
denatura tion
Reaction that affect membranes

Membranes contain proteins and lipid. Surfactants are

soluble compounds that reduce surface tension.
Surfactant as soaps alcohol, and detergents break
up grease particles in dishwater and which dissolve
lipids(Figure 12.3).. Phenols, which are alcohols,
dissolve lipids and also denature proteins. Detergent
solutions, also called wetting agents, are often used
with other chemical agents to help the agent penetrate
fatty substances.
 Specific chemical antimicrobial agents

• Soaps and detergents remove microbes, oily

substances, and dirt.
• Soaps contain alkali and sodium and will kill
many species of Streptococcus, Micrococcus, and
Neisseria and will destroy influenza viruses.
• ALCOHOLS. When mixed with water, alcohols
denature protein. They are also lipid solvents and
dissolve membranes. Ethyl and isopropyl
alcohols can be used as skin antiseptics.
Isopropyl alcohol is more often used because of
legal regulation of ethyl alcohol.
Evaluating a chemical agent
• The filter paper method of
evaluating a chemical agent is uses
small filter paper disks, each soaked
with a different chemical agent. The
disks are placed on the surface of an
agar plate that has been inoculated
with a test organism.
• The dilution test. A broth culture of
one of these bacteria is coated onto
small stainless steel cylinders and
allowed to dry. Each cylinder is then
dipped into one of several dilutions of
the chemical agent for 10 minutes,
removed, rinsed with water, and placed
into a tube of broth.
Sterile Technique
➢When culturing bacteria or other microorganisms, it is
important to keep your work area as clean as possible.
➢This prevents the introduction of other microorganisms
from the environment into your culture.
➢The techniques used to prevent contamination are
referred to as sterile techniques.
 Physical Control

1. Heat – moist and dry

2. Cold temperatures
3. Desiccation
4. Radiation
5. Filtration
 Heat
Mode of Action and Relative
Effectiveness of Heat

• Moist heat – lower temperatures and shorter exposure

time; coagulation and denaturation of proteins
• Dry heat – moderate to high temperatures; dehydration,
alters protein structure; incineration
 Moist Heat Methods

• Boiling water destroys vegetative cells of most

bacteria and fungi and inactivates some viruses,
but it is not effective in killing all kinds of spores.

• Autoclave. Steam under pressure –

sterilization Autoclave 15 psi/121oC/10-40min
Steam must reach surface of item being sterilized
Item must not be heat or moisture sensitive

• Mode of action – denaturation of proteins,

destruction of membranes and DNA
PASTEURIZATION
• Pasteurization, a process invented by Pasteur to destroy
organisms that caused wine to sour, does not achieve sterility.
• It does kill pathogens, especially Salmonella and
Mycobacterium, that might be present in milk, other dairy
products, and beer. Even “organic” milk is pasteurized now.
• Milk is pasteurized by heating it to 71.6°C for at least 15 seconds
in the flash method
• by heating it to 62.9° C for 30 minutes in the holding method.
• Ultrahigh temperature (UHT) processing raises the
temperature from 74° to 140°C and then drops it back to 74°C
in less than 5 seconds. A com plex cooling process that keeps
the milk from ever touching a surface hotter than itself prevents
develop ment of a “cooked” flavor.
DRY HEAT
• Dry heat using higher
temperatures than moist
heat (above 1600C) ex:
metal instrument
• Incineration – flame or
electric heating coil and
reduces microbes and other
substances

• Dry ovens – 150-180oC-

coagulate proteins
 Physical Control

1. Heat – moist and dry

2. Cold temperatures
3. Desiccation
4. Radiation
5. Filtration
Cold temperatures: Refrigeration;
Freezing, Freeze-drying

• REFRIGERATION is used to prevent food spoilage.

• at 5°C (ordinary refrigerator temperature). However, storage
should be limited to a few days because some bacteria and
molds continue to grow at this temperature. Clostridium
botulinum (anaerobic microbe)

• FREEZING. at -20°C is used to preserve foods in homes and

in the food industry. Although freezing does not sterilize
foods, it does significantly slow the rate of chemical reactions
so that microorganisms do not cause food to spoil.
• Frozen foods should not be thawed and refrozen. The
texture of foods is thus altered, and they become less
palatable. It also allows bacteria to multiply . Using glycerol
or protein for protection. Freezing can -80 0C, -180 0C,
Cold temperatures: Refrigeration; Freezing,
Freeze-drying
• Freeze-drying . Freeze-drying, or lyophilization, is the drying of a
material from the frozen state
 Physical Control

1. Heat – moist and dry

2. Cold temperatures
3. Desiccation
4. Radiation
5. Filtration
Desiccation

• Desiccation is the state of extreme dryness, or the

process of extreme drying. A desiccant is
a hygroscopic (attracts and holds water) substance that
induces or sustains such a state in its local vicinity in a
moderately sealed container.
 Physical Control

1. Heat – moist and dry

2. Cold temperatures
3. Desiccation
4. Radiation
5. Filtration
4. Radiation
• Ionizing radiation – deep penetrating power that has sufficient
energy to cause electrons to leave their orbit, breaks DNA,
• gamma rays, X-rays, cathode rays
• used to sterilize medical supplies and food products

FIGURE 12.15 Ultraviolet radiation. The effects of UV

radiation can be seen in this Petri plate of Serratia marcescens.
The V-shaped area was exposed to UV, resulting in death of the
cells. The remainder of the plate was shielded from exposure to
the UV radiation, and cells remain alive. (© Gary E Kaiser,
Community College of Baltimore County)

Ultraviolet light is of limited use because it does not

penetrate glass, cloth, paper, or most other materials, and
it does not go around corners or under lab benches.
 Filtration

Filtration has been used since Pasteur’s time to separate bacteria

from media and to sterilize materials that would be destroyed by
heat. Using Membrane filters 25 µm to less than 0.025µm .