Sterlization techniques.

Presented by:-
arun patel
m.sc abt 4nd sem.
md university rohtak

DEFINITIONS
 Sterilization – process by which an article, surface or
medium is freed of all living microorganisms either in the
vegetative or spore state.
 Disinfection – destruction or removal of all pathogenic
organisms. (sanitization w.r.t. Food processing and
catering)

The process of sterilization is used
 in microbiology for preventing contamination by
extraneous organisms,
 in surgery for maintaining asepsis,
 in food and drug manufacture for ensuring safety from
contaminating organisms.
The methods of sterilization employed depend on
 the purpose for which it is carried out,
 the material which has to be sterilized and
 the nature of the microorganisms that are to be removed or
destroyed.

PHYSICAL AGENTS
 Sunlight
 Drying
 Dry heat – flaming, incineration, hot air.
 Moist heat – pasteurization, boiling, steam
under normal pressure, steam under pressure.
 Filtration – candles, asbestos pads,
membranes.
 Radiation
 Ultrasonic and sonic vibrations.

CHEMICAL AGENTS
 Alcohols – ethyl, isopropyl, trichlorobutanol
 Aldehydes – formaldehyde, glutaraldehyde
 Dyes
 Halogens
 Phenols
 Surface active agents
 Metallic salts
 Gases – ethylene oxide, formaldehyde, betapropiolactone.

Sunlight:
 Action primarily due to UV rays.
Eg: In tropical country, the germicidal effect is better than
seasoned countries.
Bacteria in water are readily destroyed by sunlight.
Drying:
 Moisture is essential for growth of bacteria.
 Drying in air has deleterious effect on many bacteria.
 However, spores are unaffected.
Therefore, it is not really unreliable.
Physical methods of Sterilization
6

Heat:
 Most reliable method of sterilization and should be the method of
choice.
The factors influencing sterilization by heat:
 Nature of heat-dry or moist
 Temperature and time
 Number of microorganisms present
 Characteristics of organisms –species, strain, sporing capacity
 Type of material from which organism have to be eliminated.
 Killing effect is due to protein denaturation, oxidative damage and
toxic effect of elevated level of electrolytes.
 Killing effect of moist heat due to denaturation and coagulation of
proteins.
Dry Heat & Moist Heat.

Dry heat
 Flaming:-
 Heating over fire, till they become red hot.
 Instruments like: Point of Forceps, Spatulas,
Inoculating loops and Wires.
 (Inoculating loop is better dipped in disinfectant
first before flaming to prevent spattering)

Incineration:
 It is a process that involves the combustion
of organic substances contained in waste materials.
 Items: contaminated cloth, animal carcasses and
pathological material.
(However, polystyrene will emit black smoke. Hence should be
autoclaved in appropriate container.)

Hot air oven
 Hot air ovens are electrical devices used in sterilization.
 The oven uses dry heat to sterilize articles.
 Generally, they can be operated from 50 to 300 °C (122 to 572 °F) .
 There is a thermostat controlling the temperature.
 This is the most widely used method of sterilization by dry heat.
 Items: glassware, forceps, scissors, scalpels, all-glass syringes,
swabs, liquid paraffin, dusting powder, fats, grease.
(Materials should be properly arranged to allow free
circulation of air )
10

Temperature
(c)
Holding time(in
minutes
160 45
170 18
180 7.5
190 1.5
Precautions:
Glass wares should be dry.
Oven should not be over loaded.
Articles are to be arranged in a manner to allow free circular of air.
Door of the Oven should be opened after it cools down (2Hours).

Advantages & Disadvantages:
They do not require water and there is not much pressure
build up within the oven, unlike an autoclave, making
them safer to work with.
Suitable to be use in a laboratory environment.
They are much smaller than autoclaves but can still be as
effective.
 As they use dry heat instead of moist heat, some
organisms like prions, may not be killed by them every
time.

Moist heat:
Moist heat can be categorized into 3 groups:
 Temperature below 100°C
 Temperature at 100°C
 Temperature above 100°C
Pasteurisation of milk
 Holding period: 63°C, 30 minutes (holder method) ; or 72°C, 15-
20 minutes followed by cooling quickly to 130c or lower.
 Target: all nonsporing pathogens

Temperature at 100°C:
Boiling
Not recommended for sterilising but used for disinfection.
sterilization may be promoted by addition of 2% sodium
bicarbonate to the water.
Holding period: 10-30 minutes.
Steam at atmospheric pressure (100°C)
Used to sterilize culture media.
This is an Inexpensive method
Holding period: 100°C, 20 minutes on three succesive days
Principle: first exposure kills vegetative bacteria and then the
next exposure will kill vegetative bacteria that matures from
the spore.

Steam under pressure
 Autoclave/steam sterilizer--: autoclave is a device that
uses steam to sterilize equipment and other objects.
 This means that all bacteria, viruses, fungi, and spores are
inactivated.
 However, prions may not be destroyed by autoclaving at the typical
134 °C for 3 minutes or 121 °C for 15 minutes.
Principle:
Water boils when its vapour pressure equals the surrounding
atmosphere. Thus, when pressure inside closed vessels increases, the
temperature at which water boils increases too.
Holding period: varies.
Temperature: between 108°C and 147°C.
Items: dressings, instruments, laboratory ware, media and
pharmaceutical products.

filtration
 Filtration helps to remove bacteria from large volumes of fluid,
especially fluid containing heat-labile components such as sera,
solution of sugars and antibiotics.
 A pore size of 0.2 m is effective because filters act not only
mechanically but by electrostatic adsorption of particles to their
surface.
 The filters are of two types (deep and membrane).

FILTERS
DEEP FILTERS
Made up of Examples Comments and Uses
Candle filters 1.Diatomaceous earth
2.Unglazed porcelain
Berkefeld filters
Chamberlain filters
They are available in different porosity.
Mostly used for the purification of
drinking water and industrial uses.
Asbestos filters Asbestos (magnesium
silicate)
Seitz and Sterimat
filters
They have high adsorbing capacity and
tend to alkalinize the filtered fluid.
These are disposable , single use discs
available in different grades.
Asbestos is carcinogenic hence its use
is discouraged.
Sintered glass
filters
Fusing finely powdered
glass particles
They are expensive and brittle.
They have low absorptive property.
MEMBRANE
FILTERS
Cellulose esters
Other polymers
They are routinely used for water
purification and analysis, sterilization,
sterility testing and solutions of
parenteral use.
Wide range of average pore
diameters(APD) are available but
0.2mm size is widely used.

RADIATION
Non Ionisizing radiation:
Types Produced by Mode of action Uses Comments
Low energy
(Infrared and
ultraviolet rays)
UV lamps DNA damage Infrared rays:
Sterilization of prepacked
items such as syringes
and catheters.
Ultraviolet rays:
Used for disinfecting
operation threatres and
laboratories.
Use of UV light
is limited by
penetration and
hazardous.
Ionisizing radiation:
High energy
ionizing type
(Gamma rays and
high energy
electrons such as
X-rays and
cosmic rays)
Cobalt-60
based
instruments
DNA damage For the sterilization of
antibiotics, hormones
and other prepacked
disposable items such
as catheters, gloves,
syringes, infusion sets,
oils, animal feeds, etc.
They are
expensive.
They are very
effective due to
high penetrative
power.

Ultrasonic and sonic vibration:
 High frequency sound waves beyond the sensitivity of human ears
are called as ultrasonic waves.
 They have the property to disrupt the cells but the results have
been variable.
 Gram negative rods are more sensitive to ultrasonic vibration
whereas Gram positive cocci, spores of fungi and bacteria are
resistant to the vibration.
 Ultrasonic devices are used in dental.
 However, most sonic machines are not reliable for routine use.

Chemical Agents:
Properties of Ideal antiseptics or disinfectants should
• Effective against all microorganisms and have a wide spectrum of activity.
•Have speedy action.
•Be effective in presence of organic matter.
•Be effective in varying pH(acidic and alkaline medium).
•Be active in presence of organic matter.
•Be stable.
•Also compatible with other antiseptics and disinfectants.
•Have high penetrating power.
•Not corrode metals.
•Not cause local irritation or sensitization.
•Not interfere with healing.
•Not be toxic if absorbed into circulation.
•Be cheap and safe.

Factors influencing antiseptics or disinfectants:
Time of action.
Temperature.
Concentration of the substance.
pH of the medium.
Nature of the organisms.
Presence of foreign material.
Mode of action
Protein coagulation.
Disruption of cell membrane.
Removal of free sulphydryl groups which is essential for the functioning of the
enzymes.
Substrate competition.

alcohols
 Ethyl alcohol, isopropyl alcohol, and n-propanol exhibit
rapid, broad-spectrum antimicrobial activity against
vegetative bacteria, viruses, and fungi but are not
sporicidal.
 Activity is optimal when they are diluted to a
concentration of 60–90% with water.
 Protein slows its action whereas 1% mineral acid or alkali
enhances the action.

aldyhyde
• Formaldehyde is bactericidal, sporicidal, and virucidal.
• It is active against the amino group in the protein
molecule.
• Formaldehyde gas is used for sterilizing instruments and
heat sensitive catheters and for fumigating wards, sick
rooms and laboratories.
• Glutaraldehyde is used for low-temperature disinfection
and sterilization of endoscopes and surgical equipment.
• It is normally used as a 2% solution to achieve sporicidal
activity.

dyes
 Two groups of dyes are there
 1.Aniline dyes
 eg: brilliant green, malachite green, crystal violet
2.Acridine dyes
 eg: proflavine, acriflavine, euflavine and aminacrine.
 They are used as skin and wound antiseptics.
 They are more active against Gram positive organisms
than Gram negative organisms

halogens
 Iodine is an effective disinfectant that acts by iodinating
or oxidizing essential components of the microbial cell.
 Iodine is rapidly bactericidal, fungicidal, tuberculocidal,
virucidal, and sporicidal.
 2% iodine in 50% alcohol (tincture), which kills more
rapidly and effectively than alcohol alone.
 The most important types of chlorine-releasing agents
are sodium hypochlorite, chlorine dioxide, and sodium
dichloroisocyanurate, which are oxidizing agents that
destroy the cellular activity of proteins

Phenolic compounds:
 The use of phenolic compounds are introduced by the Lister.
 The mode of action is due to their capacity to disrupt the cell membrane.
 Phenol(carbolic acid) is a potent protein denaturant and bactericidal agent.
 It is used as disinfectants for various hospital purposes.
 Cresol and Lysol are active against a wide range of organisms but they are toxic
and corrosive.
 They are used to disinfect surgical instruments, contaminated materials and
floors.
 Halogenated diphenyl compounds such as hexachlorophene and chlorohexidine.
 Hexachlorophene is toxic and should be handled with care.
 Chlorohexidene is nontoxic, hence used as skin antiseptic which is more
effective against Gram positive organisms than Gram negative organisms.

gases
 Ethylene oxide is an inflammable and potentially explosive gas.
 By mixing with inert gases like CO2 and N2 to concentration of
10% its explosive tendency is eliminated.
 It is an alkylating agent that inactivates microorganisms by
replacing labile hydrogen atoms on hydroxyl, carboxy, or
sulfurhydryl groups, particularly of guanine and adenine in DNA.
 Under controlled condition it is used to sterilize materials such as
glass, artificial heart valves, respirators, lung machines, equipments
and clothing.
 Formaldehyde gas and Betapropiolactone(BPL) are used for
fumigation of operation theatres and other rooms.
 BPL is more efficient than formaldehyde gas.

Surface active agents
 These compounds have two regions in their molecular structures, one a water-
repelling (hydrophobic) group and the other a water-attracting (hydrophilic)
group.
 They are classified into four groups; anionic, cationic, nonionic and amphoteric.
 Cationic detergents, particularly the quaternary ammonium compounds
(“QACs") such as acetyl trimethyl ammonium bromide are highly bactericidal.
 They are effective against Gram +ve organisms than Gram –ve organisms.
 They are more active in alkaline pH.
 They are inactive against spores and viruses, tubercle bacilli.
 Anionnic detergents Eg; common soap.
 Amphoteric or ampholytic compounds or tego compounds are active against
Gram+ve organisms than Gram –ve organisms.

Mettalic salts
 Salts of mercury, copper and silver are used as
disinfectant.
 Mercuric chloride is highly toxic. Hence organic
compounds such as mercurochrome, phenyl mercury
nitrate and thiomersal are used as antiseptics(less
toxic).
 Copper salts are used as fungicides.
 Silver salts in aqueous solution have a limited use.

Sterlization techniques.

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