SELALE UNIVERSITY

COLLEGE OF HEALTH SCIENCE

DEPARTMENT OF
MEDICAL LABORATORY
SCIENCE
BACTERIOLOGY GROUP ASSIGNMENT
TITLE: STERILIZATION AND DISINFECTION
GROUP MEMBERS ID NUMBERS
1. AMLAKU GEDIFEW 0797/13
2. ASAYE GEDEFAW 0508/13
3. AWASSA ALEMAYEHU 2210/13
4. AYUB ABDULBAR 0152/13
5. BASAZIN ZEREFU 1225/13
6. DANIEL MEBRATU 1483/13

Submitted to: Abdi Negash (Msc)

Submission Date: June 14 2023
Table content Page
1. Introduction 3
2. Definition of Terms 4
3. Sterilization 5
3.1 Methods of sterilization 5
4. Disinfection 11
4.1 Classification of disinfectant 12
5. Summary 14
6.Reference 15

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 INTRODUCTION
Disinfection and sterilization are essential for ensuring that medical and surgical instruments do
not transmit infectious pathogens to patients. Because sterilization of all patient-care items is not
necessary, health-care policies must identify, primarily on the basis of the items’ intended use,
whether cleaning, disinfection, or sterilization is indicated.

Sterilization is defined as the process where all the living microorganisms, including bacterial
spores are killed. Sterilization can be achieved by physical, chemical and physiochemical means.
Chemicals used as sterilizing agents are called chemisterilants.

Disinfection is the process of elimination of most pathogenic microorganisms (excluding

bacterial spores) on inanimate objects. Disinfection can be achieved by physical or chemical
methods. Chemicals used in disinfection are called disinfectants. Different disinfectants have
different target ranges, not all disinfectants can kill all microorganisms. Some methods of
disinfection such as filtration do not kill bacteria, they separate them out. Sterilization is an
absolute condition while disinfection is not. The two are not synonymous.

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 DEFINITION OF TERMS
Sterilization: is the total destruction of all microbes, including the more resilient forms such as
bacterial spores, mycobacteria, nonenveloped (nonlipid) viruses, and fungi. This can be
accomplished using physical, gas vapor, or chemical sterilant.

Disinfection: is the process of elimination of most pathogenic microorganisms (excluding

bacterial spores) on inanimate objects. Disinfection can be achieved by physical or chemical
methods. Chemicals used in disinfection are called disinfectants. Different disinfectants have
different target ranges, not all disinfectants can kill all microorganisms. Some methods of
disinfection such as filtration do not kill bacteria, they separate them out. Sterilization is an
absolute condition while disinfection is not. The two are not synonymous.

Decontamination: is the process of removal of contaminating pathogenic microorganisms from

the articles by a process of sterilization or disinfection. It is the use of physical or chemical
means to remove, inactivate, or destroy living organisms on a surface so that the organisms are
no longer infectious.

Sanitization: is the process of chemical or mechanical cleansing, applicable in public health

systems. Usually used by the food industry. It reduces microbes on eating utensils to safe,
acceptable levels for public health.

Asepsis: is the employment of techniques (such as usage of gloves, air filters, uv rays etc) to
achieve microbe-free environment.

Antisepsis: is the use of chemicals (antiseptics) to make skin or mucus membranes devoid of
pathogenic microorganisms.

Bacteriostasis: is a condition where the multiplication of the bacteria is inhibited without killing
them.

Bactericidal: is that chemical that can kill or inactivate bacteria. Such chemicals may be called
variously depending on the spectrum of activity, such as bactericidal, virucidal, fungicidal,
microbicidal, sporicidal, tuberculocidal or germicidal.

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Antibiotics: are substances produced by one microbe that inhibits or kills another microbe.
Often the term is used more generally to include synthetic and semi-synthetic antimicrobial
agents.

STERILIZATION
Sterilization: Sterilization describes a process that destroys or eliminates all forms of microbial
life and is carried out in health-care facilities by physical or chemical methods.
Sterilization should be used for instruments, surgical gloves and other items that come in direct
contact with the blood stream or normally sterile tissues (Spaulding 1939). It can be achieved by
high-pressure steam (autoclave), dry heat (oven), chemical sterilant (glutaraldehyde’s or
formaldehyde solutions) or physical agents (radiation). Because sterilization is a process, not a
single event, all components must be carried out correctly for sterilization to occur.

Methods of sterilization
The various methods of sterilization are:
1. Physical Method
(a) Thermal (Heat) methods
(b) Radiation method
(c) Filtration method
2. Chemical Method

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PHYSICAL METHODS OF STERILIZATION
Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is
responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more
effective in killing germs due to combination of ultraviolet rays and heat. By killing bacteria suspended in
water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes. Sunlight
is not sporicidal; hence it does not sterilize.

Heat: Heat is considered to be most reliable method of sterilization of articles that can withstand heat.
Heat acts by oxidative effects as well as denaturation and coagulation of proteins. Those articles that
cannot withstand high temperatures can still be sterilized at lower temperature by prolonging the duration
of exposure.

Factors affecting sterilization by heat are:

o Nature of heat: Moist heat is more effective than dry heat
o Temperature and time: temperature and time are inversely proportional. As temperature
increases the time taken decreases.
o Number of microorganisms: More the number of microorganisms, higher the
temperature or longer the duration required.
o Nature of microorganism: Depends on species and strain of microorganism, sensitivity
to heat may vary.
Spores are highly resistant to heat.
o Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure.
Certain heat sensitive articles must be sterilized at lower temperature.
o Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.
DRY HEAT:
Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame.
Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure. This method too is limited to those
articles that can be exposed to flame. Cracking of the glassware may occur.
Incineration: This is a method of destroying contaminated material by burning them in
incinerator. Articles such as soiled dressings; animal carcasses, pathological material and
bedding etc should be subjected to incineration. This technique results in the loss of the article,

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hence is suitable only for those articles that have to be disposed. Burning of polystyrene
materials emits dense smoke, and hence they should not be incinerated.
Infra red rays: Infrared rays bring about sterilization by generation of heat. Articles to be
sterilized are placed in a moving conveyer belt and passed through a tunnel that is heated by
infrared radiators to a temperature of 180o C. The articles are exposed to that temperature for a
period of 7.5 minutes. Articles sterilized included metallic instruments and glassware. It is
mainly used in central sterile supply department. It requires special equipments, hence is not
applicable in diagnostic laboratory.
MOIST HEAT:
Moist heat acts by coagulation and denaturation of proteins.
At temperature below 100 ℃:
Pasteurization: This process was originally employed by Louis Pasteur. Currently this
procedure is employed in food and dairy industry. There are two methods of pasteurization, the
holder method (heated at 63℃ for 30 minutes) and flash method (heated at 72℃ for 15 seconds)
followed by quickly cooling to 13℃. Other pasteurization methods include Ultra-High
Temperature (UHT), 140℃ for 15 sec and 149℃ for 0.5 sec. This method is suitable to destroy
most milk borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and
Brucella, however Coxiella may survive pasteurization. Efficacy is tested by phosphatase test
and methylene blue test.
At temperature 100 ℃:
Boiling: Boiling water (100 ℃) kills most vegetative bacteria and viruses immediately. Certain
bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores
are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing
activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not
required, certain metal articles and glasswares can be disinfected by placing them in boiling
water for 10-20 minutes. The lid of the boiler must not be opened during the period.
Steam at 100℃: Instead of keeping the articles in boiling water, they are subjected to free
steam at 100℃. Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with
discharge tap open) can also serve the same purpose. A steamer is a metal cabinet with
perforated trays to hold the articles and a conical lid. The bottom of steamer is filled with water
and heated. The steam that is generated sterilizes the articles when exposed for a period of 90
minutes. Media such as TCBS, DCA and selenite broth are sterilized by steaming. Sugar and
gelatin in medium may get decomposed on autoclaving, hence they are exposed to free steaming
for 20 minutes for three successive days. This process is known as tyndallisation (after John
Tyndall) or fractional sterilization or intermittent sterilization. The vegetative bacteria are killed
in the first exposure and the spores that germinate by next day are killed in subsequent days. The
success of process depends on the germination of spores.
At temperature above 100℃:
Autoclave: Sterilization can be effectively achieved at a temperature above 100 ℃ using an
autoclave. Water boils at 100℃ at atmospheric pressure, but if pressure is raised, the
temperature at which the water boils also increases. In an autoclave the water is boiled in a

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closed chamber. As the pressure rises, the boiling point of water also raises. At a pressure of 15
lbs inside the autoclave, the temperature is said to be 121℃. Exposure of articles to this
temperature for 15 minutes sterilizes them.
To destroy the infective agents associated with spongiform encephalopathies (prions), higher
temperatures or longer times are used; 135℃ or 121℃ for at least one hour are recommended.
RADIATION:
Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy
rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative
power. Since radiation does not generate heat, it is termed "cold sterilization". In some parts of
Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.
Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most
effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this
wavelength that the absorption by the microorganisms is at its maximum, which results in the
germicidal effect. UV rays induce formation of thymine-thymine dimers, which ultimately
inhibits DNA replication.
Ionizing rays: Ionizing rays are of two types, particulate and electromagnetic rays: these are
Electronic beams and Electromagnetic rays
 Electron beams are employed to sterilize articles like syringes, gloves, dressing packs,
foods and pharmaceuticals. Sterilization is accomplished in few seconds. Unlike
electromagnetic rays, the instruments can be switched off. Disadvantage includes poor
penetrative power and requirement of sophisticated equipment.
 Electromagnetic rays such as gamma rays emanate from nuclear disintegration of
certain radioactive isotopes (Co60, Cs137). They have more penetrative power than
electron beam but require longer time of exposure. These high-energy radiations damage
the nucleic acid of the microorganism. A dosage of 2.5 megarads kills all bacteria, fungi,
viruses and spores. It is used commercially to sterilize disposable petri dishes, plastic
syringes, antibiotics, vitamins, hormones, glass wares and fabrics. Disadvantages include;
unlike electron beams, they can’t be switched off, glass wares tend to become brownish,
loss of tensile strength in fabric.

Filtration
Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes
between 0.2-0.45 µm are commonly used to remove particles from solutions that can't be
autoclaved. It is used to remove microbes from heat labile liquids such as serum, antibiotic
solutions, sugar solutions, urea solution. Various applications of filtration include removing
bacteria from ingredients of culture media, preparing suspensions of viruses and phages free of
bacteria, measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria,
clarifying fluids and purifying hydatid fluid.

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 CHEMICAL STERILIZATION
An alternative to physical sterilization is chemical sterilization (often called “cold
sterilization”). If objects need to be sterilized, but using high-pressure steam or dry-heat
sterilization would damage them or equipment is not available (or operational), they can be
chemically sterilized.
Some high-level disinfectants will kill endospores after prolonged (10–24 hour) exposure.
Common disinfectants that can be used for chemical
sterilization include glutaraldehyde’s and formaldehyde. Sterilization takes place by soaking for
at least 10 hours in 2–4% glutaraldehyde solution or at least 24 hours in 8% formaldehyde.
Glutaraldehyde’s, such as Cidex, are often in short supply and very expensive, but they are the
only practical sterilant for some instruments, such as laparoscopes, which cannot be heated.
Both glutaraldehyde’s and formaldehyde require special handling and leave a residue on treated
instruments; therefore, rinsing with sterile water is essential if the item must be kept sterile. Also,
if not rinsed off, this residue can interfere (cause sticking) with the sliding parts of the
laparoscope and cloud the lens.
Although formaldehyde is less expensive than glutaraldehyde’s, it is also more irritating to the
skin, eyes and respiratory tract and is classified as a potential carcinogen.
. When using either glutaraldehyde’s or formaldehyde, wear gloves to avoid skin contact, wear
eyewear to protect from splashes, limit exposure time and use both chemicals only in well-
ventilated areas.
Advantages:
Glutaraldehyde’s and formaldehyde solutions are not readily inactivated by organic materials.
Both can be used for items that will not tolerate heat sterilization such as laparoscopes.
Formaldehyde solutions can be used for up to 14 days (replace sooner if cloudy); some
glutaraldehyde’s can be used for up to 28 days.
Disadvantages
 Glutaraldehyde’s and formaldehyde are chemicals that cause skin irritation; therefore, all
equipment soaked in either solution must be thoroughly rinsed with sterile water after
soaking.
 Because glutaraldehyde’s work best at room temperature, chemical sterilization cannot
be assured in cold environments (temperatures less than 20(C/68(F), even with
prolonged soaking.
 Glutaraldehydes are expensive.
 Vapors from formaldehyde (classified as a potential carcinogen), and to a lesser degree
glutaraldehydes, are irritating to the skin, eyes and respiratory tract. Wear gloves and
eyewear, limit exposure time and use both chemicals only in well-ventilated areas.
Formaldehyde cannot be mixed with chlorine or chlorinated water because a dangerous
gas (bis-chloromethyl-ether) is produced

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ETHYLENE OXIDE (EO): Mode of action: It is an alkylating agent. It acts by alkylating
sulfhydryl-, amino-, carboxyl- and hydroxyl- groups. Properties: It is a cyclic molecule, which is
a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is
flammable. Application: It is a highly effective chemisterilant, capable of killing spores rapidly.
Since it is highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or
dichlorodifluoromethane. It requires presence of humidity.
It has good penetration and is well absorbed by porous material. It is used to sterilize heat labile
articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, complex
apparatus like heart-lung machine, respiratory and dental equipment’s. Efficiency testing is done
using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.
Peracetic acid (peroxyacetic acid). The acid is rapidly effective against all microorganisms,
organic matter does not diminish its activity and it decomposes into safe products. When diluted,
it is very unstable and must be used with a specially designed automatic sterilizer (APIC 2002).
It is usually used for sterilizing different types of endoscopes and other heat-sensitive
instruments.
Paraformaldehyde. This solid polymer of formaldehyde may be vaporized by dry heat in an
enclosed area to sterilize objects (Taylor, Barbeito and Gremillion 1969). This technique, called
“self-sterilization” (Tulis 1973), may be well suited for sterilizing endoscopes and other heat-
sensitive instruments.
MONITORING STERILIZATION PROCEDURES
Sterilization procedures can be monitored routinely using a combination of biological, chemical
and mechanical indicators as parameters.
Biological Indicators-Monitoring the sterilization process with reliable biological indicators at
regular intervals is strongly recommended. Measurements should be performed with a biological
indicator that employs spores of established resistance in a known population. The biological
indicator types and minimum recommended intervals should be:-

 Steam sterilizers: Bacillus stearothermophilus, weekly and as needed.

 Dry-heat sterilizers: Bacillus subtilis, weekly and as needed.
Chemical Indicators:- Chemical indicators include indicator tape or labels, which monitor
time, temperature and pressure for steam sterilization, and time and temperature for dry-heat
sterilization. These indicators should be used on the inside and outside of each package or
container.
External indicators are used to verify that items have been exposed to the correct conditions of
the sterilization process and that the specific pack has been sterilized.
. Internal indicators are placed inside a pack or container in the area most difficult for the
sterilization agent to reach (i.e., the middle of a linen pack). This is the indicator that tells if the
item has been sterilized.

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Chemical indicators, such as heat sensitive tape or glass vials containing pellets that melt at
certain temperatures for a given time, do not guarantee that sterilization has been achieved.
Mechanical Indicators :- Mechanical indicators for sterilizers provide a visible record of the
time, temperature and pressure for that sterilization cycle. This is usually a printout or graph
from the sterilizer, or it can be a log of time, temperature and pressure kept by the person
responsible for the sterilization process that day.

DISINFECTION
Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. It’s
the use of physical procedures or chemical agents to destroy most microbial forms; bacterial
spores and other relatively resistant organisms (e.g., mycobacteria, viruses, fungi) may remain
viable; disinfectants are subdivided into high-, intermediate-, and low-level.
Even the classification of disinfectants by their level of activity is misleading. The effectiveness
of these procedures is influenced by the nature of the item to be disinfected, number and
resilience of the contaminating organisms, amount of organic material present (which can
inactivate the disinfectant), type and concentration of disinfectant, and duration and temperature
of exposure.
High-level disinfectants are used for items involved with invasive procedures that cannot
withstand sterilization procedures (e.g., certain types of endoscopes and surgical instruments
with plastic or other components that cannot be autoclaved). Disinfection of these and other
items is most effective if cleaning the surface to remove organic matter precedes treatment.
Examples of high-level disinfectants include treatment with moist heat and use of liquids such as
glutaraldehyde, hydrogen peroxide, peracetic acid, and chlorine compounds.
Intermediate-level disinfectants (i.e., alcohols, iodophor compounds, phenolic compounds) are
used to clean surfaces or instruments on which contamination with bacterial spores and other
highly resilient organisms is unlikely. These have been referred to as semicritical instruments
and devices and include flexible fiberoptic endoscopes, laryngoscopes, vaginal specula,
anesthesia breathing circuits, and other items.
Low-level disinfectants (i.e., quaternary ammonium compounds) are used to treat noncritical
instruments and devices, such as blood pressure cuffs, electrocardiogram electrodes, and
stethoscopes. Although these items come into contact with patients, they do not penetrate
through mucosal surfaces or into sterile tissues. The level of disinfectants used for environmental
surfaces is determined by the relative risk these surfaces pose as a reservoir for pathogenic
organisms. For example, a higher level of disinfectant should be used to clean the surface of
instruments contaminated with blood than that used to clean surfaces that are “dirty,” such as
floors, sinks, and countertops. The exception to this rule is if a particular surface has been
implicated in a nosocomial infection, such as a bathroom contaminated with Clostridium difficile
(spore-forming anaerobic bacterium) or a sink contaminated with Pseudomonas aeruginosa. In
these cases, a disinfectant with appropriate activity against the implicated pathogen should be
selected.

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Classification of disinfectants:
1. Based on consistency
(a) Liquid (E.g., Alcohols, Phenols)
(b) Gaseous (Formaldehyde vapour)
2. Based on spectrum of activity
(a) High level
(b) Intermediate level
(c) Low level.
3. Based on mechanism of action
(a) Action on membrane (E.g., Alcohol, detergent)
(b) Denaturation of cellular proteins (E.g., Alcohol, Phenol)
(c) Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)
(d) Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Formaldehyde)
(e) Damage to nucleic acids (Formaldehyde)

Alcohols
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause
coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than
absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin.
Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces.
It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores,
hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

Aldehydes
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group,
and probably damages nucleicacids. It kills all microorganisms, including
spores.
Examples: Formaldehyde, Gluteraldehyde

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Application: 40% Formaldehyde (formalin) is used for surface disinfection and
fumigation of rooms, chambers, operation theatres, biological safety cabinets,
wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating
paraformaldehyde or treating formalin with potassium permanganate.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration,
leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde
requires alkaline pH and only those articles that are wettable can be sterilized.

Phenol
Mode of action: Act by disruption of membranes, precipitation of proteins and
inactivation of enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene,
chlorhexidine, chloroxylenol (Dettol)

Halogens
Mode of action: They are oxidizing agents and cause damage by oxidation of
essential sulfydryl groups of enzymes. Chlorine reacts with water to form
hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture
iodine, iodophores)

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 Summary
Sterilization is a physical or chemical process that destroys all life forms of microbial life,
including spores. Disinfection Aims at the destruction of microorganisms by chemical agents
(disinfectants), in order to reduce the number of vegetative forms to minimum levels.
Disinfectant It is the chemical substance that inhibits or destroys microorganisms when applied
to inert material without significantly altering it. To be effective, sterilization requires time,
contact, temperature and, with steam sterilization, high pressure.
Disinfection processes have been categorized as high level, intermediate level, and low level.
High-level disinfection can generally approach sterilization in effectiveness, whereas spore
forms can survive intermediate-level disinfection, and many microbes can remain viable when
exposed to low-level disinfection

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 References
1. Patrick R. Murray, PhD, F(AAM), F(IDSA) 2021 Medical Microbiology 9th edition page(12-16)
2. Bermand D.Davis, Renanto Dulbecco, Herman N.Eisen and Harolds S.Ginsberg(1990). Microbiology 4 th
edition. Lipincott Company.

3. From the internet especially (microrao.com)

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