Sterilization and Disinfection

• Sterilization: It is the process of destruction of

microorganisms either in the vegetative form or spore
form.
• It is a process of complete destruction of all
microorganisms present in a system.
• The products free from living organisms those are
called “Sterile products”
• Sterilization refers to any process that eliminates,
removes, kills, or deactivates all forms of life and other
biological agent such as bacteria, fungi, viruses, spore
forms, prions present in a specified region such as , a
volume of fluid, medication or in a compound such as
biological culture media.
• Sterilization is the process by which an article, surface
or medium is made free of all microorganisms either in
vegetative or spore form.
• Disinfection: a process that removes the infection
potential by destroying the microorganism but not
generally bacterial spore.
• Disinfectant: usually a chemical agent (but
sometimes a physical agent) that destroys disease-
causing pathogens or other harmful microorganisms
but might not kill bacterial spores. It refers to
substances applied to inanimate objects.
• Antiseptics: These are the chemical disinfectants
that either kill or inhibit the multiplication of
organisms. It can be safely applied to living tissues.
• Fungicide: Agent that destroys fungi (including
yeasts) and/or fungal spores pathogenic to humans
or other animals in the inanimate environment.
• Bactericide : a substance that kills the bacteria.
• Bacteriostatic : The substance that arrest or retards
the growth of bacteria.
• Germicide : A substance that kills pathogenic
bacteria but not bacterial spores.
• Virucide: An agent that kills viruses to make them
non infective.
• Sterility : The absence of viable microorganism is
called sterility.
• Sporicidal: any agent with the ability to kill spores.
• Sanitizer: Agent that reduces the number of
bacterial contaminants to safe levels as judged by
public health requirements. According to the
protocol for the official sanitizer test, a sanitizer is a
chemical that kills 99.99% of the specific test
bacteria in 30 seconds under the conditions of the
test.
• Sterilizer: Apparatus used to sterilize medical de-
vices, equipment, or supplies by direct exposure to
the sterilizing agent.
• Decontamination : to make safe by removing or
reducing contamination by infectious organisms or
other harmful substances.
• Antisepsis :The term antisepsis is used to indicate the
prevention of infection, usually by inhibiting
the growth of bacteria. Chemical disinfectants,
which can be safely applied to skin or mucous
membrane to prevent infection are called
antiseptics.
• Asepsis: Technique employed in preventing the
infections from gaining access into uninfected
tissues.
• Thermal death point: It is the lowest temperature at which
an organism is completely killed in aqueous
suspension within 10 minute. For most non- sporing
mesophilic species thermal death point lies between 50° to
56°C. For most sporing species thermal death point lies
between 100° to 120°C.
• Thermal death time:
It is the shortest time, at which an organism is
completly killed at a given temperature.
Methods of sterilization
Sunlight Drying
 Physical Methods

1. Sunlight:
• It has an active germicidal effect due to its content
of ultraviolet rays.
• This is one of the natural methods of sterilization in
case of water in tanks,laundered clothes,sweeming
pools,lakes etc.
• Sunlight is not sporicidal, hence it does not sterilize.
• Medically it has no importance.
 2. Drying
• Moisture is essential for growth of bacteria.
• 80% of a bacterial cell contains water.
• So drying in air has deleterious effect on many
bacteria.
• Spores are unaffected by drying.
• So it is unreliable method.
 3.Heat
• Heat is the most reliable and commonly
employed method of sterilization.
• Two types of heat are used
– Dry heat
– Moist heat
 Dry heat method
• Dry heat kills the microorganisms by
denaturation of bacterial protein, oxidative
damage and by the toxic effect of elevated levels
of electrolyte.
• Method of dry heat:
• A) Red heat
• B) Flaming
• C) Incineration
• D) Hot Air Oven
Red heat:
• Heating till the article become red hot is called red
heat sterilization method.
• Inoculating wires or loops, tips of forceps needle can
be sterilized by passing them into the flame of a
Bunsen burner till they become red hot.
• Flaming:
• Glass slides, scalpels and mouths of culture tubes
are passed through the flame of a Bunsen burner
without allowing them to become red hot .It is
not reliable method.
• Incineration:
• This is a method of destroying contaminated
material by burning them in incinerator.
Temperature ranges from 800-1600 degree
centigrade.
• This method is mainly used for the destruction
hospital waste disposal such as soiled dressings;
animal carcasses, pathological material and
bedding and laboratory waste material are
destroyed by this method.
• By this method, infective material is reduced to
ashes by burning.
• The temperature of incineration depends on the
type of waste and the incineration process and
type of incinerator used.
• Municipal waste: 850-1100 oC
• Medical waste :1000-1200oC
• Hazardous waste: 1200-1600oC
• Cremation(Human/Animal Remains):760-980oC
Hot Air Oven
• Introduced by Louis Pasteur
• most widely used method of sterilization by
dry heat.
• The oven is electrically heated and is fitted
with a fan to ensure adequate and even
distribution of hot air in the chamber.
• It is also fitted with a thermostat that
maintains the chamber air at a chosen
temperature.
• Principle:
• It is based on the concept of dry heat.
• Dry heat kills the microorganisms by
denaturation of bacterial protein, oxidative
damage of essential cell constituents.
• The heat in oven is usually supplied by
electricity whose chosen temperature is
thermostatically controlled. It has a fan to assist
the circulation of air and so ensure rapid,
uniform heating of the load.
• Sterilization temperature and time
– 160 degrees Celsius for 120minutes,
– 170 degrees Celsius for 60 minutes, and
– 180 degrees Celsius for 30 minutes.
 Method of using hot air oven
• Place the article to be sterilized on the shelves in the
cold oven, without packing too tight, so that the air
may circulate to the articles.
• Close the door firmly and set the temperature at 160
degree celcius.
• Switch on the oven and put on the indicator light
• Take the time for sterilizing from the time oven reaches
the required temperature ie. 160° degree celcius for 2
hour.
• Turn off the heat and allow the oven to cool and
remove the material from the oven when the
temperature falls below 40° celcius.
 Uses:
• Metallic instruments (like forceps, scalpels,
scissors)
• Glassware's (such as petridishes, pipettes,
flasks, all-glass syringes)
• Swabs, oils, grease, petroleum jelly and some
pharmaceutical products
• Unsuitable for rubber and plastics
 MOIST HEAT:
• Moist heat acts by coagulation and denaturation of proteins.
At temperature below 100oC:
• ƒPasteurization:
• 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 63oC for 30 minutes) and flash method (heated at
72oC for 15 seconds) followed by quickly cooling to 4oC.
• Other pasteurization methods include Ultra-High
Temperature (UHT), 140oC for 15 sec and 149 oC 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.
• V
ƒ accine bath: The contaminating bacteria in a
vaccine preparation can be inactivated by heating in
a water bath at 60oC for one hour. Only vegetative
bacteria are killed and spores survive.

• Sƒ erum bath: The contaminating bacteria in a serum

preparation can be inactivated by heating in a water
bath at 56oC for one hour on several successive
days. Proteins in the serum will coagulate at higher
temperature. Only vegetative bacteria are killed and
spores survive.
• Inspissation: This is a technique to solidify as well
as disinfect egg and serum containing media.
• The medium containing serum or egg are placed in
the slopes of an inspissator and heated at 80-85oC
for 30 minutes on three successive days.
• On the first day, the vegetative bacteria would die
and those spores that germinate by next day are
then killed the following day.
• The process depends on germination of spores in
between inspissation.
• If the spores fail to germinate then this technique
cannot be considered sterilization.
• At temperature 100oC:
• Boiling:
• Boiling water (100oC) 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 100oC:
• Instead of keeping the articles in boiling water, they are subjected to
free steam at 100oC.
• 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 100oC :
• Autoclave:
• Sterilization can be effectively achieved at a temperature above 100oC using
an autoclave.
• Water boils at 100oC 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 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
121oC .
• 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;
• 126oC (20lb/in2) for 10 minutes or 134oC (30lb/in2) for 3 minutes
recommended.
• Advantages of steam:
• It has more penetrative power than dry air, it moistens the spores (moisture
is essential for coagulation of proteins), condensation of steam on cooler
surface releases latent heat, condensation of steam draws in fresh steam.
 Methods of using autoclave
• Pour water to a point just the level of the bottom of the basket
and check the water level every time the autoclave is used.
• Place the article to be sterilized on the perforated tray, cotton
wool plugs to be wrapped with craft paper and bottles.
• Close the lid and tight the screw.
• Turn on source of heat and allow stem and air mixture to escape
through the air cock till the autoclave becomes air free.
• Close the air cock.
• Allow the steam pressure to rise until it reaches the desired level
i.e. 121oC at 15 lbs pressure for 15 minutes is over, the heat source
is turned off and autoclaved is allowed to cool till the pressure
guage indicates that the inside is at atmospheric pressure.
• The discharge tap is opened slowly and air is allowed to enter the
autoclave .
• When pressure gauge is at zero ,open the ventcock slowly.
 Advantages of moist heat sterilization
• The penetration power of steam is much more
than that of dry heat. So microorganism are killed
more efficiently in lesser time at lower
temperature than dry heat.
• In large size autoclaves large quantities of
materials can be sterilized in one batch.
• Solutions packed in sealed containers as ampoles
are readily sterilized by this method.
• Bulk solutions, glasswares ,surgical dressing
,rubber gloves and surgical instruments are
effectively sterilized by this method.
 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
Non-ionizing rays :
– low energy rays, poor penetrative power
– Rays of wavelength longer than visible light
– Microbiocidal
– wavelength of UV rays: 200-280 nm, 260 nm most effective
– UV rays induce formation of thymine-thymine dimers,
ultimately inhibits DNA replication
– readily induces mutations in cells
– Don’t kill spores
Uses of UV radiation:
– Disinfection of closed areas in microbiology laboratory,
inoculation hoods, laminar flow, and operating theaters
– Harmful to skin and eyes
– Doesn't penetrate glass, paper or plastic
– Of use in surface disinfection
– Generated using a high-pressure mercury vapor lamp
• Ionizing rays
• High-energy rays, good penetrative power
• Radiation does not generate heat- "cold
sterilization“
• e.g. (a) X-rays, (b) gamma rays, and (c) cosmic rays
• Gamma radiation from cobalt-60 source
sterilization of antibiotics, hormones, vitamins,
sutures, catheters, animal feeds, metal foils, and
plastic disposables, such as syringes, petri dishes
• kills all bacteria, fungi, viruses and spores
• Also used for meat and other food items
• Damage the nucleic acid of the microorganism
 Mechinical method(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.
• Filtration is aided by using either positive or negative pressure
using vacuum pumps.
• The older filters made of earthenware or asbestos are called
depth filters.
 Chemical Methods of Sterilization
• Disinfectants are those chemicals that destroy
pathogenic bacteria from inanimate surfaces.
• Some chemical have very narrow spectrum of
activity and some have very wide.
• Those chemicals that can sterilize are called
chemisterilants.
• Those chemicals that can be safely applied over
skin and mucus membranes are called antiseptics.
• An ideal antiseptic or disinfectant should have
following properties:
Properties of Ideal Disinfectant
• Should have wide spectrum of activity
• Should be able to destroy microbes within practical period of time
• Should be active in the presence of organic matter
• Should make effective contact and be wettable
• Should be active in any pH
• Should be stable
• Should have long shelf life
• Should be speedy
• Should have high penetrating power
• Should be non-toxic, non-allergenic, non-irritative or non-
corrosive
• Should not have bad odour
• Should not leave non-volatile residue or stain
• Efficacy should not be lost on reasonable dilution
• Should not be expensive and must be available easily
• Action of Disinfectants :
• Damage to the cell wall and alter permeability of the cell
membrane, resulting in
• exposure, damage, or loss of the cellular contents
• Alter proteins and form protein salts or cause coagulation of
proteins
• Inhibit enzyme action, nucleic acid synthesis or alter nucleic acid
molecules
• Cause oxidation or hydrolysis
Factors Influencing Activity of Disinfectants
• Temperature: Increase in temp. increases the efficiency of
disinfectants
• Type of microorganism: Vegetative cells more susceptible than
spores. Spores may be resistant
• Physiological state of cell: Young and metabolically active cells
more sensitive
• Environment: Physical or chemical properties of medium or
substance
• e.g., pH of the medium and presence of extraneous materials
 Types of Disinfectants
(a) alcohols
(b) Aldehydes
(c) phenolic compounds
(d) halogens
(e) gases
(f) surface active agents
(g) oxidizing agents
(h) dyes
(i) heavy metals
(j) acids and alkalis
 1. ALCOHOLS:
• Mode of action:
• Alcohols dehydrate cells, disrupt membranes and cause
coagulation of protein.
• Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
• Application:
• 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, damages nucleic acids.
• It kills all microorganisms, including spores
• Bactericidal, sporicidal, and also effective against viruses
• Can also be used as chemical sterilants
• Examples: Formaldehyde, Glutaraldehyde

Application:
40% Formaldehyde (formalin):
• for surface disinfection and fumigation of room chambers, operation theatres, biological
safety cabinets, wards, sick rooms etc.
• Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin
with potassium permanganate. It also sterilizes bedding, furniture and books.
• 10% formalin with 0.5% tetraborate sterilizes clean metal instruments
2% glutaraldehyde:
• It is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic
equipments etc.
• An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde.
• Especially effective against tubercle bacilli, fungi, and viruses
• 2% formaldehyde at 40o C for 20 minutes is used to disinfect wool and 0.25% at 60o C for
six hours to disinfect animal hair and bristles.
 PHENOL:
Mode of action:
• Act by disruption of membranes, precipitation of
proteins and inactivation of enzymes
• Examples: 5% phenol, 1-5% Cresol, 5% Lysol,
hexachlorophene, chlorhexidine, chloroxylenol
Applications:
As disinfectants at high concentration and as
antiseptics at low concentrations.
Bactericidal, fungicidal, but are inactive against spores
and most viruses.
Effective in the presence of organic material and
remain active on surfaces long after application
 HALOGENS:
Mode of action:
• Oxidizing agents and cause damage by oxidation of essential sulfydryl groups of
enzymes
• Chlorine reacts with water to form hypochlorous acid, microbicidal
• Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine
compounds (tincture iodine, iodophores)
Application:
• Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic.
• Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone
to prepare iodophores such as povidone-iodine.
• Iodophores permit slow release and reduce the irritation of the antiseptic.
• For hand washing iodophores are diluted in 50% alcohol.
• 10% Povidone Iodine is used undiluted in pre and postoperative skin disinfection.
• Chlorine gas is used to bleach water.
• Household bleach can be used to disinfect floors.
• Household bleach used in a stock dilution of 1:10.
• In higher concentrations chlorine is used to disinfect swimming pools.
• 0.5% sodium hypochlorite is used in serology and virology.
• Used at a dilution of 1:10 in decontamination of spillage of infectious material.
• Mercuric chloride is used as a disinfectant.
 HEAVY METALS:
Mode of action:
• Act by precipitation of proteins and oxidation of sulfydryl groups
• They are bacteriostatic
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic
mercury, salts (e.g., mercurochrome, merthiolate)

Applications:
• 1% silver nitrate solution can be applied on eyes as treatment for
opthalmia neonatorum.
• Silver sulphadiazine is used topically to help to prevent
colonization and infection of burn tissues.
• Mercurials are active against viruses at dilution of 1:500 to 1:1000.
• Merthiolate at a concentration of 1:10000 is used in preservation
of serum.
• Copper salts are used as a fungicide.
 SURFACE ACTIVE AGENTS:
Mode of actions:
• Disrupt membrane resulting in leakage of cell
constituents
• Examples: Soaps or detergents, anionic or cationic,
Anionic detergents- soaps and bile salts , Cationic
detergents are known as quaternary ammonium
compounds (or quat), Cetrimide and benzalkonium
chloride act as cationic detergents
Application:
• active against vegetative cells, Mycobacteria and
enveloped viruses
• as disinfectants at dilution of 1-2% for domestic use
and in hospitals
 DYES:
Mode of action:
• Acridine dyes are bactericidal because of their interaction with
bacterial nucleic acids.
• Examples:
• Aniline dyes such as crystal violet, malachite green and brilliant
green.
• Acridine dyes such as acriflavin and aminacrine. Acriflavine is a
mixture of proflavine and euflavine.
• Only euflavine has effective antimicrobial properties.
• A related dye, ethidium bromide, is also germicidal. It intercalates
between base pairs in DNA.
• They are more effective against gram positive bacteria than gram
negative bacteria and are more bacteriostatic in action.
Applications:
• Topically as antiseptics, on skin treat bacterial skin infections
• The dyes are used as selective agents in certain selective media.
 HYDROGEN PEROXIDE:
Mode of action:
• It acts on the microorganisms through its release of nascent oxygen.
Hydrogen peroxide produces hydroxyl-free radical that damages proteins
and DNA.
• Application:
• 6% concentration to decontaminate the instruments, equipments such as
ventilators
• 3% for skin disinfection and deodorising wounds and ulcers
• Strong solutions are sporicidal
BETA-PROPIOLACTONE (BPL):
Mode of action:
• Acts through alkylation of carboxyl- and hydroxyl- groups
Application:
• Effective sporicidal agent, broad-spectrum activity, 0.2% to sterilize
biological products.
• It is more efficient in fumigation that formaldehyde.
• It is used to sterilize vaccines, tissue grafts, surgical instruments and
enzymes
 ETHYLENE OXIDE (EO):
Mode of action:
• by alkylating sulfhydryl-, amino-, carboxyl- and
hydroxyl- groups
Application:
• Effective chemisterilant, capable of killing spores
rapidly •
• Highly flammable, used as 10% CO2+ 90% EO) or
dichlorodifluoromethane
• Used to sterilize heat labile articles such as bedding,
textiles, rubber, plastics, syringes, disposable petri
dishes, respiratory and dental equipment's
• Highly toxic, irritating to eyes, skin, highly flammable,
mutagenic and carcinogenic
 Class work
• 1) Difference between sterilization and
pasteurization.
• 2) Difference between moist heat sterilization
and dry heat sterilization.
• 3) Difference between Antiseptic and
disinfectant.