STERILISATION AND ITS

TECHNIQUES

GUIDED BY:
DR. PANKAJ KUKREJA PRESENTED BY:
(PROFRESSOR & HEAD) DR. NASIM
ORAL & MAXILLOFACIAL SURGERY PG I YEAR
ORAL & MAXILLOFACIAL
SURGERY
CONTENTS

• DEFINITIONS
• TECHNIQUES
• PHYSICAL METHODS
• CHEMICAL METHODS
• RECENT ADVANCES IN STERILISATION TECHNIQUES
• CONCLUSION
• REFERENCES
STERILISATION
• It is derived from latin word ‘STERILIS’ which means unable to
produce offspring.
• The process by which an article surface or medium is freed of all living
microorganisms either in vegetative or spore state.
DEFINITIONS
DISINFECTION:
Destruction of all pathogenic organisms capable of giving rise to infection.
ANTISEPSIS:
Prevention of infection usually by inhibiting the growth of bacteria in wounds
or tissues.
BACTERICIDAL AGENTS:
Those which are able to kill bacteria.
BACTERIOSTATIC AGENTS:
Only prevents the multliplication of bacteria which may however remain
alive
TECHNIQUES OF STERILISATION

PHYSICAL METHODS CHEMICAL METHODS

• Sunlight • Alcohols
• Drying • Aldehydes
• Dyes
• Dry heat
• Halogens
• Moist heat
• Phenols
• Filtration • Surface active agents
• Radiation • Metallic salts
• Ultrasonic and sonic vibrations • Gases
PHYSICAL METHODS
SUNLIGHT
Active germicidal effect due to the combined effect of UV rays and heat rays.
Eg:-river, tanks and lakes
DRYING
4/5th weight of bacterial cell consists of water
Hence drying has deleterious effect on many bacteria
INCINERATION
Rapidly destroying material by the use of incinerator
Eg:- soiled dressings, bedding, animal carcasses, pathological materials etc.
FLAMING
Inoculating loops or wires, tip of forceps & needles and spatulas are held in Bunsen flame
till they become red hot in order to be sterilised.
DRY HEAT
• PRINCIPLE
- Protein denaturation
- Oxidative damage
- Toxic effects of elevated levels of electrolytes

HOT AIR OVEN

- Most widely used
- Temp: 160 degree Celsius for 45 min
- 170 degree Celsius for 18 min
- 180 degree Celsius for 7.5 min
- USES: Glasswares like syringes, petridishes, flasks, pipettes & test tubes.
- surgical instruments like scalpels, scissors, forceps etc.
- chemical such as liquid paraffin, fats, greases,sulphonamides etc.
 PRECAUTIONS

- Not to be overloaded.
- Fitted with fans for even distribution of air.
- Materials to be sterilised perfectly dry.
- Rubber materials will not withstand the temperature.
- Allowed to cool for 2 hrs before opening the doors.

• ADVANTAGES: • DISADVANTAGES:
- Economical - Hot air is bad conductor
- Does not rust metals of heat hence it has less
penetrating power.
- Easily monitored
- Used for anhydrous oils &
powder
MOIST HEAT
TEMPERATURE BELOW 100°C: PASTEURIZATION
-HOLDER METHOD: 63°C for 30 min
- FLASH PROCESS: 72°C for 2o sec….rapid cooling to 13°C

TEMPERATURE AT 100°C: BOILING

- 90-100°C for 10 min
- Sporing bacteria required prolonged periods of boiling-24 hrs
- Sterilisation may be promoted by 2% Na bicarbonate

TYNDALLISATION or INTERMITTENT STERILISATION

- Used for media containing sugars or gelatin.
- Exposure for 100 degrees for 20 minutes on three successive days.
- First exposure kills all vegetative bacteria.
- Subsequent exposure will kill the spores present.
AUTOCLAVE
The autoclave was invented by Charles Chamberland in 1884
• PRINCIPLE
• Boiling water alone is insufficient to kill spores and viruses
• Water boils when its vapour pressure equals to that of
surrounding atmosphere
• According to BOYLE’ law, when volume of steam is is kept
constant the temperature is directly proportional to
pressure
• Hence when pressure increases inside closed vessel
• Temperature at which water boils increases Temperature
Pressure(psi) Time(mins)
(°C)
• Saturated steam has penetrative power 15 121
15
10
• When steam comes in contact with a cooler20 surface
126 it
3
20 134
condenses to water and gives up latent heat to that
surface
 CONSIDERATIONS DURING AUTOCLAVING

• Ensure complete air removal for temperature to reach

121 degree Celsius.
• Ensure loose packing in the chamber.
• Tighly sealed materials may become dangerously
pressurised causing injury during removal.
USES:
• Disposable syringes, nondisposable syringes, glassware
• Metal instruments
• Surgical dressing
• Surgical instruments
• Laboratory equipment
• Culture media
• Pharmaceutical products
 • Economical
• Good penetration

ADVANTAGES •
•
Short cycle time
Easily monitored
• No special chemical or exhausts required

• Moisture retention
• Causes corrosion

DISADVANTAGES •
•
Carbon steel gets damaged
Dulling of unprotected cutting edges
• Destruction of heat sensitive materials
FILTERATION
-Sterilize solutions that may be damaged or denatured by high temperatures or
chemical agents.

- Used for the sterilization of heat labile materials such as sera, sugar solutions, and
antibiotics.

AIR FILTERS
• Air can also be sterilized by filtration
• Large volumes of air may be rapidly freed from infection
by passage through high efficiency particulate air
(HEPA) filters.
• They are used in laminar air flow system in microbiology
laboratories.
• HEPA filters can remove particles of 0.3 µm or larger.
FILTRATION
SINTERED GLASS FILTERS

MEMBRANE FILTERS

ASBESTOS FILTERS

CANDLE
FILTERS
RADIATION
• NON-IONISING RADIATION
• Electromagnetic rays with
wavelengths longer than those of
visible light are used.
• Infrared radiation- rapid mass
sterilization of prepacked items eg.
Syringes,catheters.
• UV radiation- disinfecting closed
areas like operation theatres,
laboratories.
• IONISING RADIATION
• Short wavelength
• Lethal action – breakdown of single
stranded or sometimes double-
stranded DNA and effect on other
vital cell components.
• Cold sterilisation.
• X-rays, gamma rays and beta rays.
• Sterilizing plastics, swabs, metal
foils etc
 BIOLOGICAL CONTROLS FOR DIFFERENT
STERILIZATION METHODS

METHODS OF STERILISATION BIOLOGICAL CONTROL

Hot air oven Bacillus subtilis subsp.,
Clostridium tetani
Autoclave Bacillus stearothermophilus
Thermocoples
Browne tube
Autoclave tapes
Filteration Serratia marcescens
Pseudomonas diminuta
Ionising radiation Bacillus pumilis
CHEMICAL AGENTS

• Alcohols
• Aldehydes
LIQUIDS • Phenols
• Halogens
• Heavy metals
• Surface active agents
• Dyes

GASES • Formaldehyde
• Ethylene oxide
MODE OF ACTION OF CHEMICAL AGENTS
• Protein coagulation
• Disruption of cell membrane resulting in exposure, damage or loss of
the contents.
• Removal of free sulphydryl groups essential for functioning of
enzymes.
• Substrate competition
ALCOHOLS

• Denaturation of proteins
• Isopropyl alcohol & 70% ethyl alcohol used as skin disinfectant
• Methyl alcohol is active against the fungal spores and used to treat
cabinets and incubator.
• Suitable for skin preparation before venipuncture
ALDEHYDES

• FORMALDEHYDE(FORMALIN) –acts as a bactericidal and sporicidal.

• Active against Gram –ve bacteria, spores, viruses(HB , HIV) & fungi
• AQUEOUS SOLUTION:- FORMALIN(37% SOLUTION) to clean metal
instrument
• GASEOUS FORM:- fumigation of wards/corridors/ICU
GLUTARALDEHYDE/CIDEX(2% Alkaline NaHCO3)

• High level disinfectant

• Active against tubercle bacilli, fungi and viruses.
• Less toxic
• To treat corrugated rubber, anesthetic tubes,face masks, metal
instruments
• Exposure time:-less than 10 hrs
PHENOLS

• Cell membrane damage

• Eg : cresol(LYSOL), chlorhexidine(SAVLON), chloroxylenol(DETTOL)
and hexachlorphene.
• Decontamination of the hospital environment including lab surfaces
and noncritical medical items.
HALOGENS

• CHLORINE COMPOUNDS:- bleaching powder or hypochlorite solution

for HIV infected material.
• IODOPHORS & IODINE:- active against bacteria, spores & some
viruses.
• Suitable for skin preparations, mouthwash and as a surgical scrub
• (7.5% POVIDONE+ IODINE = BETADINE)
 ETHYLENE OXIDE(ETO)
• Colourless liquid with a boiling point of 10.7 degree Celsius
• Highly lethal to all kinds of microbes including spores.
• Action is due to its alkylating the amino, carboxyl, hydroxyl and
sulphydryl groups in protein molecules.
• in addition it reacts with DNA and RNA
• Highly inflammable and in concentrations (>3%) highly explosive.
• By mixing with inert gases such as CO2, its explosive tendency can be
eliminated.
• Used for sterilising plastic and rubber articles, respirators, heart-lung
machines, sutures, dental equipments and clothing, prepackaged
materials.
DISADVANTAGES:
• EO sterilisers combined with a chlorofluorocarbon stabilizing agent
that are linked to destruction of earth ozone’s layer.
• Explosion risks of the ETO
 RECENT ADVANCES IN
STERILISATION TECHNIQUES
• Plasmas
• Psoralens and UVA(PUVA)
• Pulsed light systems
• Ortho-phthalaldehyde
• Surfacine
• Superoxidized water
• Endoclens
• Attest Ethylene oxide(E0) Rapid readout
PLASMAS
• Fourth state of matter, and as such is distinguished from solids, liquids,
and gases.
• Produced at very high temperatures, or at low temperatures in strong
electromagnetic fields.
• Plasma usually consists of a reactive cloud of ions, electrons, free radicals,
and other neutral species.
• Produce a sterilizing effect using lower concentrations of sterilant with a
higher reactivity.
• Sterrad Process is a plasma system that uses hydrogen peroxide as the
source of the active species.
• Overcome the inhibitory effect of packaging materials by using a gas-
diffusion phase to allow gas to penetrate to all parts of the load before the
plasma is created.
 STERRAD
USES
• Non-hollow loads, such as
electrocautery instruments,
dopplers, laser probes,
defibrilator paddles,
thermometers, Ophthalmic
lenses, and harmonic cables
• Hollow loads, such as
Laryngoscopes and their
blades, shaver handpieces,
fiber optic light cables, and
surgical power drills
• Endoscopes, such as rigid and
flexible endoscopes.
ADVANTAGES
• No chemical residues
• Safety of handling
• Safety for the environment
• Short aeration time.
DISADVANTAGES
• Inability to sterilize: liquids,
powders, and strong
absorbers
• Requires specific synthetic
packaging of the load
• Sterilization chamber is
relatively smaller than that of
an EtO sterilizer.
 PLASMA STERILISER(STERRAD 50)
-To sterilise temperature-sensitive equipment

STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES

-Hydrogen peroxide gas plasma -Use of two hydrogen peroxide -Cost??

steriliser diffusion plasma stage cycles is a
more effective process
-Reduced cycle time(45min)
-Various sized units available
-Leaves no toxic residues
-Endoscopes with lengths >40cm or
a diameter of <3mm cannot be
processed
PSORALENS & UVA(PUVA)
• Psoralens are naturally occurring substances found in a wide range of
plants, in which their role is to fight infection from pathogenic fungi.
• Use of ultraviolet light in combination with psoralens to purge blood
plasma and platelets of pathogenic organisms.
• The use of UV is also noted for its ability to inactivate viruses while
preserving their antigenic properties for the preparation of vaccines.
• The psoralens form a labile bond with DNA and RNA which, upon
exposure to UV light, becomes a firm bond and hence synthetic
psoralens and UV irradiation can be used to destroy infectious agents
such as HIV, hepatitis viruses, and toxemia-inducing bacteria.
PULSED LIGHT SYSTEMS(PureBright system )
• High-power electrical energy to produce intense pulses of light that are claimed to
provide unique bactericidal effects.
• High-voltage, high-current pulse applied to the lamp
• Emit an intense pulse of light, which typically lasts for a few hundred microseconds.
• The light produced by the lamp includes a broad spectrum of wavelengths, from
ultraviolet to infrared, with an intensity some 20,000 times greater than sunlight.
• Highly successful in killing microorganisms, viruses, and spores, as well as in deactivating
enzymes.
• CLARANOR
USES:
• Surface sterilization of packaging materials
• Terminal sterilization of parenterals packed in transparent plastic bags or bottle
 ORTHO-PHTHALALDEHYDE: A NEW CHEMICAL
STERILANT
-Clear, pale-blue liquid(Ph-7.5)
-Mycobactericidal activity
-

STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES

-GLUTARALDEHYDE -Shorter process time (12 vs. 45 min) -Stains protein gray
-Not a known irritant to eyes and -Higher cost
nasal passages
-No vapor ceiling limit
-Weak odor
 SURFACINE: A NEW ANTIMICROBIAL
AGENT
-Effective against vancomycin resistant Enterococcus spp.(VRE), methicillin-resistant
STAPHYLOCOCCUS aureus(MRSA), Clostridium difficile

STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES

• Disinfectants(phenolics quaternary -Antimicrobial - Cost??

ammonium) persistence(>13days)
• Antiseptics(alcohol,iodophor,chlorhexidine -May be used on animate and
gluconate) inanimate surfaces
-Broad antimicrobial spectrum
-Transfers active agent(silver) to
microbes on demand without
elution
-Resistant to forming biofilm
-No toxicity to mammalian cells
 SUPER-OXIDISED WATER (STERILOX)
-Use of electrolyzing saline as a disinfectant
-Mode of action: formation of oxidising species (hypochlorous acid and free chlorine
radicals)
-Effective against bacteria, viruses,fungi and spores

STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES

-High or low level disinfectants
-Antiseptics
-Basic materials(saline and electricity) -Production equipment expensive due to
inexpensive monitoring
-End product not damaging to -Endoscope compatibility unknown
environment -Decreased efficacy in presence of
organic matter
- Limited-use life (must be freshly
generated)
ENDOCLENS
-New liquid chemical sterilisation system
-Liquid sterilant- performic acid(hydrogen peroxide + formic acid)

ADVANTAGES DISADVANTAGES

-Device automatically cleans and -Cost??

sterilizes -Used for immersible instruments
-Rapid cycle time(<30min) only
-Tests endoscope for channel -Point of use systems, no long term
blockage and leaks storage
-Advantages of automated
process(eg, consistent exposure to
sterilant, filtered water
rinse,operator convenience)
 ATTEST ETHYLENE OXIDE (EO) RAPID
READOUT
STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES

-48 hr spore readout biological -Rapid(4 hr), reliable assessment of -Cost???

indicator sterilisation efficacy -Not tested with EO and CO2 mixture
-Prevents recalls of released
sterilisation loads
CONCLUSION
• “Prevention is better than cure” a proverb well suited to sterilization.
• Sterilization has major share in success of surgical management.
• Thorough understanding of the application of sterilization will help
ensure safety from the invisible but deadly world of microbial
pathogens.
• Hence utilization of proper sterilization, disinfectants and aseptic
procedures help us achieve the safety of our professional demands.
REFERENCES
• Textbook of Microbiology, 7th edition –Ananthanarayan and Paniker
• Textbook of Microbiology – C.P. Baveja
• Recent Developments in Sterilization Technology-David J. Hurrell
• New Disinfection and Sterilization Methods- William A. Rutala and
David J. Weber