sterilisation

1. STERILISATION AND ITS
TECHNIQUES
GUIDED BY:
DR. PANKAJ KUKREJA PRESENTED BY:
(PROFRESSOR & HEAD) DR. NASIM
ORAL & MAXILLOFACIAL SURGERY PG I YEAR
ORAL & MAXILLOFACIAL SURGERY

2. CONTENTS
 DEFINITIONS
 TECHNIQUES
 PHYSICAL METHODS
 CHEMICAL METHODS
 RECENT ADVANCES IN STERILISATION TECHNIQUES
 CONCLUSION
 REFERENCES

3. 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.

4. 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

5. TECHNIQUES OF STERILISATION
PHYSICAL METHODS
 Sunlight
 Drying
 Dry heat
 Moist heat
 Filtration
 Radiation
 Ultrasonic and sonic vibrations
CHEMICAL METHODS
 Alcohols
 Aldehydes
 Dyes
 Halogens
 Phenols
 Surface active agents
 Metallic salts
 Gases

6. 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.

7. 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.

8. 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:
- Economical
- Does not rust metals
- Easily monitored
- Used for anhydrous oils & powder
 DISADVANTAGES:
- Hot air is bad conductor of heat
hence it has less penetrating power.

9. 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.

10. 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
• Saturated steam has penetrative power
• When steam comes in contact with a cooler surface it condenses to water and
gives up latent heat to that surface
 Three major factors for effective autoclave:
1. Pressure : 15 psi
2. Temperature : 121°C
3. Time : 15 minutes
Pressure(psi)
15
20
20
Temperature
(°C)
121
126
134
Time(mins)
15
10
3

11. 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

12. •Economical
•Good penetration
•Short cycle time
•Easily monitored
•No special chemical or exhausts required
ADVANTAGES
•Moisture retention
•Causes corrosion
•Carbon steel gets damaged
•Dulling of unprotected cutting edges
•Destruction of heat sensitive materials
DISADVANTAGES

13. 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.

15. FILTRATION
SINTERED GLASS FILTERS
MEMBRANE FILTERS
ASBESTOS FILTERS
CANDLE
FILTERS

16. 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

17. 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

18. CHEMICAL AGENTS
• Alcohols
• Aldehydes
• Phenols
• Halogens
• Heavy metals
• Surface active agents
• Dyes
LIQUIDS
• Formaldehyde
• Ethylene oxideGASES

19. 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

20. 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

21. 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

22. 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

23. 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.

24. 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)

25. 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

26. RECENT ADVANCES IN
STERILISATION
TECHNIQUES

27.  Plasmas
 Psoralens and UVA(PUVA)
 Pulsed light systems
 Ortho-phthalaldehyde
 Surfacine
 Superoxidized water
 Endoclens
 Attest Ethylene oxide(E0) Rapid readout

28. 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.

29. 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.

30. PLASMA STERILISER(STERRAD 50)
-To sterilise temperature-sensitive equipment
STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES
-Hydrogen peroxide gas plasma
steriliser
-Use of two hydrogen peroxide
diffusion plasma stage cycles is a
more effective process
-Reduced cycle time(45min)
-Various sized units available
-Leaves no toxic residues
-Cost??
-Endoscopes with lengths >40cm
or a diameter of <3mm cannot
be processed

31. 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.

32. 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

33. 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)
-Not a known irritant to eyes and
nasal passages
-No vapor ceiling limit
-Weak odor
-Stains protein gray
-Higher cost

34. 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
ammonium)
• Antiseptics(alcohol,iodophor,chlorhexid
ine gluconate)
-Antimicrobial
persistence(>13days)
-May be used on animate and
inanimate surfaces
-Broad antimicrobial spectrum
-Transfers active agent(silver)
to microbes on demand without
elution
-Resistant to forming biofilm
-No toxicity to mammalian cells
- Cost??

35. 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) inexpensive
-End product not damaging to
environment
-Production equipment expensive due
to monitoring
-Endoscope compatibility unknown
-Decreased efficacy in presence of
organic matter
- Limited-use life (must be freshly
generated)

36. ENDOCLENS
-New liquid chemical sterilisation system
-Liquid sterilant- performic acid(hydrogen peroxide + formic acid)
ADVANTAGES DISADVANTAGES
-Device automatically cleans
and sterilizes
-Rapid cycle time(<30min)
-Tests endoscope for channel
blockage and leaks
-Advantages of automated
process(eg, consistent exposure
to sterilant, filtered water
rinse,operator convenience)
-Cost??
-Used for immersible
instruments only
-Point of use systems, no long
term storage

37. ATTEST ETHYLENE OXIDE (EO) RAPID
READOUT
STANDARD TECHNOLOGY ADVANTAGES DISADVANTAGES
-48 hr spore readout biological
indicator
-Rapid(4 hr), reliable assessment
of sterilisation efficacy
-Prevents recalls of released
sterilisation loads
-Cost???
-Not tested with EO and CO2
mixture

38. 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.

39. 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