STERILIZATION AND DISINFECTION

Historical background
• The scientific use of disinfection and
sterilization methods originated more than
100 years ago

• •Ignatz Semmelweis(1816-1865) and Joseph

Lister(1827-1912)
• –important pioneers for the promotion of
infection control
More than 100 years ago, Semmelweis demostrated that routine hand
washingcan prevent the spread of disease
•He worked in a hospital in Vienna when maternity patients were dying an
alarming rate
•He recognized that medical students worked on cadavers during an anatomy
class and afterwords they went to the maternity ward.
•Students did not wash their hands between touching the dead and the
living!!!
•After administrating the handwashing before examining the maternity
patients the mortality rate decreased
Lister, for the first time,used carbolic acid in operating theatres that
significantly reduced mortality rates.
•Later when it was accepted that microorganisms were the causative agents
of infections
in 1867, Lister introduced British surgery to hand washing and the use of
phenol as antimicrobial agent for surgical wound dressings
•His priciples were gradually and adopted in Britain and later in US
•This was the beginning of infection control
 Critical Items
• Critical items confer a high risk for infection if they are contaminated with
any microorganism. Thus, objects that enter sterile tissue or the vascular
system must be sterile because any microbial contamination could
transmit disease. This category includes surgical instruments, cardiac and
urinary catheters, implants, and ultrasound probes used in sterile body
cavities
 Semicritical Items
• Semicritical items contact mucous membranes or nonintact skin. This
category includes respiratory therapy and anesthesia equipment, some
endoscopes, laryngoscope blades 24, esophageal manometry probes,
cystoscopes 25, anorectal manometry catheters, and diaphragm fitting
rings. These medical devices should be free from all microorganisms;
however, small numbers of bacterial spores are permissible. Intact mucous
membranes, such as those of the lungs and the gastrointestinal tract,
generally are resistant to infection by common bacterial spores but
susceptible to other organisms, such as bacteria, mycobacteria, and
viruses. Semicritical items minimally require high-level disinfection using
chemical disinfectants. Glutaraldehyde, hydrogen peroxide,
 Noncritical Items
• Noncritical items are those that come in contact with intact skin but not
mucous membranes. Intact skin acts as an effective barrier to most
microorganisms; therefore, the sterility of items coming in contact with
intact skin is "not critical.
• Examples of noncritical patient-care items are bedpans, blood pressure
cuffs, crutches and computers.
 Sterilization

• Sterilization (or sterilisation) is a term referring to any process that

eliminates (removes) or kills (deactivates) all forms of life and other
biological agents (such as viruses which some do not consider to be alive
but are biological pathogens nonetheless), present in a specified region,
such as a surface, a volume of fluid, medication, or in a compound such as
biological culture media. Sterilization can be achieved with one or more of
the following: heat, chemicals,irradiation, and filtration.
 Applications

Foods:
• One of the first steps toward sterilization was made by Nicolas Appert who
discovered that thorough application of heat over a suitable period slowed the
decay of foods and various liquids, preserving them for safe consumption for a
longer time than was typical. Canning of foods is an extension of the same
principle, and has helped to reduce food borne illness ("food poisoning").
Medicine and surgery:
• In general, surgical instruments and medications that enter an already aseptic
part of the body (such as the bloodstream, or penetrating the skin) must be
sterile. Examples of such instruments include scalpels, hypodermic needles
and artificial pacemakers. This is also essential in the manufacture of
parenteral pharmaceuticals.
• Preparation of injectable medications and intravenous solutions for
fluid replacement therapy requires sterility.
 Quantification

• The aim of sterilization is the reduction of initially present microorganisms

or other potential pathogens. The degree of sterilization is commonly
expressed by multiples of the decimal reduction time, or D-value,
denoting the time needed to reduce the initial number to one tenth of its
original value. Then the number of microorganisms after sterilization
time is given by:
– The D-value is a function of sterilization conditions and varies with the type of
microorganism, temperature, water activity, pH etc..
• Theoretically, the likelihood of survival of an individual microorganism is
never zero. To compensate for this, the overkill method is often used.
Using the overkill method, sterilization is performed by sterilizing for
longer than is required to kill the bioburden present on or in the item
being sterilized.
 Sterilization by Heat
1. Steam
2. Dry Heat
3. Flaming
4. Incineration
5. Tyndallization
 Steam
• A widely used method for heat sterilization is the autoclave, sometimes
called a converter or steam sterilizer. The invention of the autoclave sterilizer
is atributed to Charles Chamberland, in 1879. An autoclave is used to
sterilize surgical equipment, laboratory instruments, pharmaceutical items,
and other materials. It can sterilize solids, liquids, and instruments.
• Autoclaves vary in size, shape and functionality. A very basic autoclave is
similar to a pressure cooker; both use the power of steam to kill bacteria,
spores and germs resistant to boiling water and powerful detergents.
• Autoclaves use steam heated to 121-134 °C under pressure. To achieve
sterility, the article is heated in a chamber by injected steam until the article
reaches a time and temperature setpoint. The article is then held at that
setpoint for a period of time which varies depending on the bioburden
present on the article being sterilized and its resistance (D-value) to steam
sterilization. A general cycle would be anywhere between 3 to 15 minutes,
(depending on the generated heat) at 121 °C at 100 kPa.
• Following sterilization, liquids in a pressurized autoclave must be cooled
slowly to avoid boiling over when the pressure is released. This may be
achieved by gradually depressurizing the sterilization chamber.
• Proper autoclave treatment will inactivate all resistant bacterial spores in
addition to fungi, bacteria, and viruses, but is not expected to eliminate
all prions, which vary in their resistance. For prion elimination, various
recommendations state 121-132 °C for 60 minutes or 134 °C for at least
18 minutes
• Most autoclaves have meters and charts that record or display
information, particularly temperature and pressure as a function of time.
The information is checked to ensure that the conditions required for
sterilization have been met. Indicator tape is often placed on packages of
products prior to autoclaving, and some packaging incorporates indicators.
The indicator changes color when exposed to steam, providing a visual
confirmation.
• Steam sterilization process is not appropriate for many materials due to
the high temperatures involved. Sterilized packages need to be allowed to
dry before being removed from the autoclave to prevent contamination.
Once removed, they must be allowed to cool to ambient temperatures,
which may take several hours.
• Biological indicators can also be used to independently confirm autoclave
performance. Simple bioindicator devices are commercially available
based on microbial spores Biological indicators may take the form of glass
vials of spores and liquid media, or as spores on strips of paper inside
glassine envelopes. These indicators are placed in locations where it is
difficult for steam to reach to verify that steam is penetrating there.
• For autoclaving, cleaning is critical. Extraneous biological mater or grime
may shield organisms from steam penetration. Proper cleaning can be
achieved through physical scrubbing
• Moist heat causes destruction of micro-organisms by denaturation of
macromolecules, primarily proteins. This method is a faster process than
dry heat sterilization.
 Dry Heat

• Dry heat was the first method of sterilization, and is a longer process than
moist heat sterilization. The destruction of microorganisms through the
use of dry heat is a gradual phenomenon. With longer exposure to lethal
temperatures, the number of killed microorganisms increases. At higher
temperatures, shorter exposure times are required to kill organisms. This
can reduce heat-induced damage to food products.
• The standard setting for a hot air oven is at least two hours at 160 °C. A
rapid method heats air to 190 °C for 6 minutes for unwrapped objects and
12 minutes for wrapped objects. Dry heat has the advantage that it can be
used on powders and other heat-stable items that are adversely affected
by steam (e.g. it does not cause rusting of steel objects).
 Flaming

• Flaming is done to loops and straight-wires in microbiology labs. Leaving

the loop in the flame of a Bunsen burner or alcohol lamp until it glows red
ensures that any infectious agent gets inactivated. This is commonly used
for small metal or glass objects, but not for large objects.
 Incineration

• Incineration is a waste treatment process that involves the combustion of

organic substances contained in waste materials. This method also burns
any organism to ash. It is used to sterilize medical and other biohazardous
waste before it is discarded with non-hazardous waste. Bacteria
incinerators are mini furnaces used to incinerate and kill off any micro
organisms that may be on an inoculating loop or wire
 Tyndallization

• Named after John Tyndall, Tyndallization is an obsolete and lengthy

process designed to reduce the level of activity of sporulating bacteria
that are left by a simple boiling water method. The process involves
boiling for a period (typically 20 minutes) at atmospheric pressure,
cooling, incubating for a day, then repeating the process a total of three to
four times. The incubation periods are to allow heat-resistant spores
surviving the previous boiling period to germinate to form the
heat-sensitive vegetative (growing) stage, which can be killed by the next
boiling step. This is effective because many spores are stimulated to grow
by the heat shock. The procedure only works for media that can support
bacterial growth, and will not sterilize non-nutritive substrates like water.
 Chemical sterilization
1. Ethylene oxide
2. Nitrogen dioxide
3. Ozone
4. Glutaraldehyde and formaldehyde
5. Hydrogen peroxide
6. Peracetic acid
7. Potential for chemical sterilization of prions
 Chemical sterilization

• Chemicals are also used for sterilization. Heating provides a reliable way to
rid objects of all transmissible agents, but it is not always appropriate if it
will damage heat-sensitive materials such as biological materials,
fiber optics, electronics, and many plastics. In these situations chemicals,
either as gases or in liquid form, can be used as sterilants. While the use of
gas and liquid chemical sterilants avoids the problem of heat damage.
• Users must ensure that article to be sterilized is chemically compatible
with the sterilant being used.
• In addition, the use of chemical sterilants poses new challenges for
workplace safety, as the properties that make chemicals effective
sterilants usually make them harmful to humans.
 Ethylene oxide

• Ethylene oxide (EO or EtO) gas is commonly used to sterilize objects that are sensitive
to temperatures greater than 60 °C and / or radiation such as plastics, optics and
electrics. Besides moist heat and irradiation, ethylene oxide is the most common
sterilization method, used for over 70% of total sterilizations.
• Ethylene oxide penetrates well, moving through paper, cloth, and some plastic films
and is highly effective. Ethylene oxide can kill all known viruses, bacteria (including
spores) and fungi, and is compatible with most materials even when repeatedly
applied.
• However, it is highly flammable, toxic and carcinogenic with a potential to cause
adverse reproductive effects.
• Ethylene oxide is still widely used by medical device manufacturers for larger scale
sterilization, but ethylene oxide is becoming less popular in hospitals. Since ethylene
oxide is explosive at concentrations from 3% to 100%.
• Other reasons for the decrease in use of ethylene oxide are the well-known health
effects. In addition to being a primary irritant, ethylene oxide is now classified by the
IARC as a known human carcinogen.
 Nitrogen dioxide

• Nitrogen dioxide (NO2) gas is a rapid and effective sterilant for use against
a wide range of microorganisms, including common bacteria, viruses, and
spores. The unique physical properties of NO2 gas allow for sterilant
dispersion in an enclosed environment at room temperature and ambient
pressure.
• The mechanism for lethality is the degradation of DNA in the spore core
through nitration of the phosphate backbone, which kills the exposed
organism as it absorbs NO2. This degradation occurs at even very low
concentrations of the gas
 Ozone

• Ozone is used in industrial settings to sterilize water and air, as well as a

disinfectant for surfaces. Ozone offers many advantages as a sterilant gas;
ozone is a very efficient sterilant because of its strong oxidizing properties
capable of destroying a wide range of pathogens, including prions
 Glutaraldehyde and formaldehyde
• Glutaraldehyde and formaldehyde solutions (also used as fixatives) are
accepted liquid sterilizing agents, provided that the immersion time is
sufficiently long.
• To kill all spores in a clear liquid can take up to 22 hours with
glutaraldehyde and even longer with formaldehyde. The presence of solid
particles may lengthen the required period or render the treatment
ineffective.
• Sterilization of blocks of tissue can take much longer, due to the time
required for the fixative to penetrate.
• Glutaraldehyde and formaldehyde are volatile, and toxic by both skin
contact and inhalation.
• Glutaraldehyde has a short shelf life (<2 weeks), and is expensive.
• Formaldehyde is less expensive and has a much longer shelf life if some
methanol is added to inhibit polymerization to paraformaldehyde, but is
much more volatile.
• Many vaccines, such as the original Salk polio vaccine, are sterilized with
formaldehyde.
 Hydrogen peroxide

• Hydrogen peroxide, in both liquid and as vaporized hydrogen peroxide

(VHP), is another chemical sterilizing agent.
• Hydrogen peroxide is used to sterilize heat or temperature sensitive
articles such as rigid endoscopes.
• In medical sterilization hydrogen peroxide is used at higher
concentrations, ranging from around 35% up to 90%.
• The biggest advantage of hydrogen peroxide as a sterilant is the short
cycle time. Whereas the cycle time for ethylene oxide may be 10 to 15
hours, some modern hydrogen peroxide sterilizers have a cycle time as
short as 28 minutes.
• The penetrating ability of hydrogen peroxide is not as good as ethylene
oxide.
 Peracetic acid

• Peracetic acid (0.2%) is a recognized sterilant by the FDA for use in

sterilizing medical devices such as endoscopes.
 Radiation sterilization
Sterilization can be achieved using , X-rays, gamma rays
 Non-ionizing radiation sterilization

• Ultraviolet light irradiation (UV, from a germicidal lamp) is useful for

sterilization of surfaces and some transparent objects.
• UV irradiation is routinely used to sterilize the interiors of
biological safety cabinets between uses.
• It also damages some plastics, such as polystyrene foam if exposed for
prolonged periods of time.
 Ionizing radiation sterilization

• Gamma radiation is very penetrating, and is commonly used for

sterilization of disposable medical equipment, such as syringes, needles,
cannulas and IV sets, and food.
 Sterile filtration
• Fluids that would be damaged by heat, irradiation or chemical sterilization,
such as drug products, can be sterilized by microfiltration using
membrane filters.
• This method is commonly used for heat labile pharmaceuticals and protein
solutions in medicinal drug processing. A microfilter with pore size 0.2 µm
will usually effectively remove microorganisms.
• In the processing of biologics, viruses must be removed or inactivated,
requiring the use of nanofilters with a smaller pore size (20 -50 nm) are used.
Disinfectant
 Definition
• Disinfectants are antimicrobial agents that are applied to non-living objects to
destroy microorganisms that are living on the objects.
• Disinfection does not necessarily kill all microorganisms, especially resistant
bacterial spores; it is less effective than sterilization, which is an extreme physical
and/or chemical process that kills all types of life.
• Disinfectants are different from other antimicrobial agents such as antibiotics,
which destroy microorganisms within the body, and antiseptics, which destroy
microorganisms on living tissue. Disinfectants are also different from biocides —
the later are intended to destroy all forms of life, not just microorganisms.
• Disinfectants work by destroying the cell wall of microbes or interfering with the
metabolism.
• Disinfectants are frequently used in hospitals, dental surgeries, kitchens, and
bathrooms to kill infectious organisms.
• Bacterial endospores are most resistant to disinfectants, but some viruses and
bacteria also possess some tolerance.
 Properties

• A perfect disinfectant would also offer complete and full microbiological

sterilisation, without harming humans and useful form of life, be
inexpensive, and noncorrosive. However, most disinfectants are also, by
nature, potentially harmful (even toxic) to humans or animals.
• The choice of disinfectant to be used depends on the particular situation.
Some disinfectants have a wide spectrum (kill many different types of
microorganisms), while others kill a smaller range of disease-causing
organisms but are preferred for other properties (they may be
non-corrosive, non-toxic, or inexpensive)
• There are arguments for creating or maintaining conditions that are not
conducive to bacterial survival and multiplication, rather than atempting
to kill them with chemicals. Bacteria can increase in number very quickly,
which enables them to evolve rapidly.
• Some bacteria survive a chemical atack, they give rise to new generations
composed completely of bacteria that have resistance to the particular
chemical used. Under a sustained chemical atack, the surviving bacteria
in successive generations are increasingly resistant to the chemical used,
and ultimately the chemical is rendered ineffective.
Types of Disinfectants
 Air disinfectants

• Air disinfectants are typically chemical substances capable of disinfecting

microorganisms suspended in the air. Disinfectants are generally assumed
to be limited to use on surfaces, but that is not the case.
• An air disinfectant must be dispersed either as an aerosol or vapour at a
sufficient concentration in the air to cause the number of viable infectious
microorganisms to be significantly reduced.
• In the 1940s and early 1950s, further studies showed inactivation of
diverse bacteria, influenza virus, and mold fungus using various glycols,
principally propylene glycol and triethylene glycol.
• In principle, these chemical substances are ideal air disinfectants because
they have both high lethality to microorganisms and low mammalian
toxicity.
 Alcohols

• Alcohols, usually ethanol or isopropanol, are sometimes used as a

disinfectant, but more often as an antiseptic (the distinction being that
alcohol tends to be used on living tissue rather than nonliving surfaces).
They are non-corrosive, but can be a fire hazard. They also have limited
residual activity due to evaporation. Alcohols are most effective when
combined with distilled water to facilitate diffusion through the cell
membrane; 100% alcohol typically denatures only external membrane
proteins.
• A mixture of 70% ethanol or isopropanol diluted in water is effective
against a wide spectrum of bacteria, though higher concentrations are
often needed to disinfect wet surfaces. Additionally, high-concentration
mixtures (such as 80% ethanol + 5% isopropanol) are required to
effectively inactivate lipid-enveloped viruses (such as HIV, hepatitis B, and
hepatitis C)
 Aldehydes

• Aldehydes, such as formaldehyde and glutaraldehyde, have a wide

microbiocidal activity and are sporicidal and fungicidal.
• Some bacteria have developed resistance to glutaraldehyde, and it has
been found that glutaraldehyde can cause asthma and other health
hazards.
 Oxidizing agents

• Oxidizing agents act by oxidizing the cell membrane of microorganisms,

which results in a loss of structure and leads to cell lysis and death. A
large number of disinfectants operate in this way. Chlorine and oxygen
are strong oxidizers.
• Sodium hypochlorite is very commonly used. Common household bleach
is a sodium hypochlorite solution and is used in the home to disinfect
drains, toilets, and other surfaces. In more dilute form, it is used in
swimming pools, and in still more dilute form, it is used in drinking water.
When pools and drinking water are said to be chlorinated, it is actually
sodium hypochlorite or a related compound—not pure chlorine—that is
being used.
• Other hypochlorites such as calcium hypochlorite are also used, especially
as a swimming pool additive.
• Chloramine is often used in drinking water treatment.
• Hydrogen peroxide is used in hospitals to disinfect surfaces and it is used
in solution alone or in combination with other chemicals as a high level
disinfectant.
• Iodine is usually dissolved in an organic solvent or as Lugol's iodine
solution. It is used in the poultry industry. It is added to the birds'
drinking water. In human and veterinary medicine, iodine products are
widely used to prepare incision sites prior to surgery. Although it increases
both scar tissue formation and healing time, tincture of iodine is used as
an antiseptic for skin cuts and scrapes, and remains among the most
effective antiseptics known
 Phenolics

• Phenolics are active ingredients in some household disinfectants. They are

also found in some mouthwashes and in disinfectant soap and
handwashes.
• Phenol is probably the oldest known disinfectant as it was first used by
Lister, when it was called carbolic acid. It is rather corrosive to the skin.
• Chloroxylenol is the principal ingredient in Detol, a household
disinfectant and antiseptic.
• Amylmetacresol is found in Strepsils, a throat disinfectant.
 Quaternary ammonium compounds

• Quaternary ammonium compounds ("quats"), such as benzalkonium

chloride, are a large group of related compounds. Some concentrated
formulations have been shown to be effective low-level disinfectants.
Typically, quats do not exhibit efficacy against difficult to kill
non-enveloped viruses such as norovirus, rotavirus, or polio virus.
 Silver

• Silver has antimicrobial properties, but compounds suitable for

disinfection are usually unstable and have a limited shelf-life. Silver
dihydrogen citrate (SDC) is a chelated form of silver that maintains its
stability. SDC kills microorganisms by two modes of action: 1) the silver ion
deactivates structural and metabolic membrane proteins, leading to
microbial death; 2) the microbes view SDC as a food source, allowing the
silver ion to enter the microbe. Once inside the organism, the silver ion
denatures the DNA, which halts the microbe's ability to replicate, leading
to its death. This dual action makes SDC highly and quickly effective
against a broad spectrum of microbes. SDC is non-toxic, non-caustic,
colorless, odorless, and tasteless, and does not produce toxic fumes.