STERILIZATIONS AND DISINFECTIONS:

METHODS OF STERILIZATION AND DISINFECTIONS;

Why we need Sterilization?

• Microorganisms capable of causing infection are constantly present in the external

environment and on the human body.
• Microorganisms are responsible for contamination and infection.
• The aim of sterilization is to remove or destroy them from materials or from surfaces.
How can microorganisms be killed?

 Denaturation of proteins (e.g. wet heat, ethylene oxide)

 Oxidation (e.g. dry heat, hydrogen peroxide)
 Filtration
 Interruption of DNA synthesis/repair (e.g. radiation)
 Interference with protein synthesis (e.g. bleach)
 Disruption of cell membranes (e.g. phenols)

Definitions:
Sterilization: It is a process by which an article, surface or medium is made free of all living
microorganisms either in vegetative or spore form.
Disinfection :Destruction of all pathogens or organisms capable of producing infections but not
necessarily spores.
All organisms may not be killed but the number is reduced to a level that is no longer harmful to
health.
Disinfectant: An agent that kills microorganisms by applying to non living objects.
Antisepsis: Prevention of infection, usually by inhibiting the growth of bacteria in wounds or
tissues
A sepsis : Technique by which the occurrence of infection in uninfected tissue is prevented.
Antiseptics : Chemical disinfectants which can safely be applied to skin or mucus membrane to
prevent infection by inhibiting the growth of microorganisms.
Bactericidal-agent which kill bacteria
Bacteriostatic-agent which only prevents multiplication of bacteria
Decontamination. Process that makes inanimate objects (non-living)safer to be handled by staff
before cleaning (i.e., inactivates HBV, HCV and HIV. The objects would include large surfaces,
surgical instruments and protective devices such as gowns gloves or goggles.

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Classification of Sterilization
There are two types of sterilization: physical and chemical.
1. Physical sterilization includes:
1. heat
2. radiation
3. filtration

2. Chemical sterilization includes:

1. Alcohols – ethyl, isopropyl, trichlorobutanol
2. Aldehydes – formaldehyde, glutaraldehyde
3. Dyes
4. Halogens
5. Phenols
6. Surface active agents
7. Metallic salts
8. Gases – ethylene oxide, formaldehyde, betapropiolactone.

Factors that influence efficacy of disinfection/sterilization

1 Contact time
2 Physico-chemical environment (e.g. pH)
3 Presence of organic material
4 Temperature
5 Type of microorganism
6 Number of microorganisms
7 Material composition

What to sterilize?
• It is mandatory to sterilize :
– all instruments that penetrate soft tissues and bone.
– Instruments that are not intended to penetrate the tissues, but that may come into
contact with oral tissues.
• If the sterilization procedure may damage the instruments, then, sterilization can be
replaced by Disinfection procedure

Physical methods of sterilization:

Physical methods:
1. Heat
a. Dry heat
b. Moist heat
2. Filtration

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3. Radiation
4. Ultasonic&Sonic vibrations
5. Sunlight
6. Drying
Dry Heat :
Principle:
– Dry heat kills the organism by
• denaturation of the bacterial proteins,
• oxidative damage
• Toxic effect of elevated levels of electrolytes.
1. Red heat:
Red heat: Materials are held in the flame of a Bunsen burner till they become red hot.
 Inoculating wires or loops
 Tips of forceps
 Needles
2. Flaming:
Flaming: Materials are passed through the flame of a Bunsen burner without allowing them to
become red hot.
 Glass slides
 scalpels
 Mouths of culture tubes
3. Hot air oven:
Most widely used method
Electrically heated and fitted with a fan to even distribution of air in the chamber.
Fitted with a thermostat that maintains the chamber air at a chosen temperature.
Temperature and time:
Dry heat:
 170°C (340°F) for 1 hour (total cycle time placing instruments in
Oven, heating to 170°C, timing for 1 hour, and then cooling is from
2–2.5 hours), or
 160°C (320°F) for 2 hours (total cycle time is from 3–3.5 hours)
Remember:
- Exposure time begins only after the sterilizer has reached the target
Temperature.
- Do not overload the sterilizer. (Leave at least 7.5 cm [3 inches] between the items and
walls of sterilizer.) Overloading alters heat convection and increases the time required to
sterilize.
Sterile instruments and other items should be used immediately unless they:
- were wrapped in a double layer of muslin, paper or other appropriate
Material prior to sterilization; or
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 - can be stored in a dry, sterile container with a tight-fitting lid.
The material used for wrapping instruments and other items must be porous enough to let
steam through but tightly woven enough to protect against dust
Particles and microorganisms. Wrapped sterile packs should remain sterile until some event
causes the package or container to become contaminated. An event can be a tear or worn
area in the wrapping, the package becoming wet or anything else that will allow
microorganisms to enter the package or container.
Uses of Hot Air Oven
Sterilization of:
1. Glassware like glass syringes, petri dishes, pipettes and test tubes.
2. Surgical instruments like scalpels, scissors, forceps etc.
3. Chemicals like liquid paraffin, fats etc.

4. Incineration:
• Materials are reduced to ashes by burning.
• Instrument used was incinerator.
• Soiled dressings
• Animal carcasses
• Bedding
• Pathological material

Moist heat:
Sterilizing below100°C
1. Temperature below 100°
1. Pasteurization of milk
2. Inspissation
3. Vaccine bath
1. Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used
to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.
u Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes.
u High Temperature Short Time Pasteurization (HTST): Used today. Milk is
exposed to 72oC for 15 minutes.
Pasteurization:
Used for milk, ice cream, yogurt, and fruit juices, Not sterilization, Heat-tolerant microbes
survive. Pasteurization of milk: Batch method, Flash pasteurization & Ultrahigh - temperature
pasteurization
2. Inspissation: Inspissations: Heating at 80 - 85°C for half an hour daily on three consecutive
days. Serum or egg media are sterilized

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3. Vaccine bath: Heating at 60°C for an hour daily in vaccine bath for several successive days.
Serum or body fluids can be sterilized by heating at 56°C for an hour daily for several successive
days.

1. Boiling at 100°C:
1 Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling.
2. Tyndallisation: Steam at 100C for 20 minutes on three successive days Used for egg , serum
and sugar containing media.

3. Steam sterilizer: Steam at 100°C for 90 minutes. Used for media which are decomposed at
high temperature?

A temperature above 100°C;

Autoclave:
Steam above 100°C: has a better killing power than dry heat. Bacteria are more susceptible to
moist heat.
Sterilization conditions:

– Temperature 121 °C
– Chamber pressure 15 lb per square inch.
– Holding time 15 minutes
– Others :
• 126°C for 10 minutes
• 133°C for 3 minutes
Uses:
1. Useful for materials which cannot withstand high temp.
2. To sterilize culture media, rubber material, gowns, dressings, gloves etc.

Instructions (Steam Sterilizer)

STEP 1: Decontaminate, clean and dry all instruments and other items to be sterilized.
STEP 2: All jointed instruments should be in the opened or unlocked Position, while instruments
composed of more than one part or sliding parts should be disassembled.
Note: To help prevent dulling of sharp points and cutting edges, wrap the sharp edges and needle
points in gauze before sterilizing. Repair (sharpen) or replace instruments as needed.
Note: Do not allow to boil dry. Steam should always be escaping from the pressure valve.
STEP 3: Instruments should not be held tightly together by rubber bands or any other means
that will prevent steam contact with all surfaces.
STEP 4: Arrange packs in the chamber to allow free circulation and
penetration of steam to all surfaces.
STEP 5: When using a steam sterilizer, it is best to wrap clean instruments or other clean items
in a double thickness of muslin or newsprint.

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 (Unwrapped instruments must be used immediately after removal from the sterilizer, unless kept
in a covered, sterile container.)
If using a pressure cooker or kerosene-powered (nonelectric) gravity displacement steam
sterilizer, bring the water to a boil and let steam escape from the pressure valve; then turn down
heat, but keep steam coming out of the pressure valve.
STEP 6: Sterilize at 121°C (250°F) for 30 minutes for wrapped items, 20 minutes for
unwrapped items; time with a clock.

2. Filtration:
Useful for substances which get damaged by heat.
To obtain bacteria free filtrates of clinical samples. To sterilize sera, sugars and antibiotic
solutions. Purification of water.
3. Radiations:
Ionizing radiations and Non - Ionizing radiations
Ionizing radiations: X rays, Gamma rays and Cosmic rays Gamma radiation are commercially
used for sterilization of disposable items. (Cold sterilization)

Non ionizing radiation: Wavelengths greater than 1 nm. Excites electrons, causing them to
make new covalent bonds Affects 3-D structure of proteins and nucleic acids. UV light causes
pyrimidine dimers in DNA; UV light does not penetrate well. Suitable for disinfecting air,
transparent fluids, and surfaces of objects. Used to disinfect operating rooms, nurseries,
cafeterias.
Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.
Infra red is used for rapid mass sterilization of syringes and catheters.

Chemical methods of sterilization:

MODES OF ACTION OF CHEMICAL DISINFECTANTS:

 Protein coagulation
 Disruption of cell membrane resulting in exposure, damage or loss of the contents.
 Removal of free sulphydryl groups essential for the functioning of the enzymes.
 Substrate competition – a compound resembling the essential substrate of the enzyme
diverts or misleads the enzymes necessary for the metabolism of the cell and causes cell
death.

1. ALCOHOLS;

Ethanol (80% v/v ethyl alcohol) or 2- propanol (60-70% v/v iso - propyl alcohol)
solutions are used to disinfect skin and decontaminate clean surfaces.

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 Spectrum: Effective against fungi, vegetative bacteria, Mycobacterium species and some
lipid-containing viruses.
Limitation: Not effective against spores.
Concentration: Most effective at 70% in water.
Caution: Do not use near flames due to flammability.
May swell rubber or harden plastics.

2. ALDEHYDES:

FORMALDEHYDE:
Precautions are required when handling formaldehyde
Formalin is 37% w/v formaldehyde gas in water.
Spectrum: Active against most microorganisms.
Bactericidal, sporicidal, virucidal.
13% v/v formalin is a good decontaminant (but has an irritating odour).
8% v/v formalin in 80% v/v alcohol is effective against vegetative bacteria, spores and
viruses.
Does not corrode stainless steel.
Uses:
 Formaldehyde is used to preserve anatomical specimens, and for destroying anthrax
spores in hair and wool.
 10% formalin containing 0.5% sodium tetraborate is used to sterilize clean metal
instruments.
 Formaldehyde gas is used to sterilize instruments, heat sensitive catheters and for
fumigation of wards, sick rooms and laboratories.
 It can also be used for clothing, bedding, furniture and books.
 To disinfect equipment such as centrifuges or biosafety cabinets.

GLUTARALDEHYDE:
 Concentration:Glutaraldehyde is commercially available as 2% w/v aqueous solution
which must be made alkaline to "activate" (e.g. by addition of 0.3% sodium bicarbonate).
 A 2% glutaraldehyde solution, for at least 10 hours, can be used to sterilize heat labile
items.
 Spectrum: Active against vegetative bacteria, spores, fungi and many viruses.
 Note: Also available in stable glycocomplexed form which does not require addition of
alkaline buffer.
Use: for instruments such as cystoscopes, bronchoscopes, corrugated rubber anesthetic tubes,
face masks, endotracheal tubes, metal instruments, polythene tubing.

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Advantages;
 Numerous use studies published
 Relatively inexpensive
 Excellent materials compatibility
Disadvantages:
 Pungent and irritating odor
 Relatively slow mycobactericidal activity
Coagulate blood and fix tissues to surface

3. CHLORINE COMPOUNDS;
Generally used in the form of sodium hypochlorite.
 Effective against a wide variety of microorganisms (vegetative bacteria and viruses).
Preferred disinfectant for HIV and hepatitis viruses.
 Concentration: Use at 0.1% as a general disinfectant.
 Effective between a pH ranges of 6-8.
Limitations:
Less suitable in the presence of organic matter (such as blood). Concentration must be increased
to retain action (0.5%).
Strength decreases on standing (make fresh solutions daily).
High concentrations corrode metal surfaces, and bleach and damage fabrics
.
HYPOCHLORITE:
These are inexpensive, broad spectrum chlorine releasing disinfectants of choice against viruses,
including hepatitis B virus.
 Aqueous solution of sodium hypochlorite (5.25%) is called household bleach.
 It is used in a concentration of 0.2-1% depending upon the circumstances.
 Caution: hypochlorites are inactivated by organic matter. They corrode metals, hence
contact of the chemical with the metallic instruments and equipment must be avoided.

4. IODINE COMPOUNDS;

 Iodine is used in aqueous or alcoholic solution.

 Rapidly effective against most microorganisms.
 Concentration: Usually diluted to 1% w/v free iodine, optimum pH neutral to acid.
 Caution: Not suitable in the presence of organic matter.
 Stains skin and may cause irritation.
 Decomposes when heated above 40ºC.
 Do not use on aluminium or copper.
 Method of use: Dilute in alcohol for washing hands, or use as a sporicide.
 Prepare dilutions daily.
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  Use: Most commonly used for skin disinfection and decontaminating clean surfaces.

Iodophores:

They have largely replaced the aqueous and tincture forms of iodine since the side effects like
staining and irritation are far less in iodophores than in aqueous or tincture iodine.

Iodophores are chemical complexes with iodine bound to a carrier such as polyvinylpyrolidone
(povidone, PVP) or ethoxylated nonionic detergents.
Free microbicidal iodine is gradually released from these compounds.
Uses:iodophors are widely used for antisepsis of skin, mucosa and wounds. A 2.5% ophthalmic
solution is a useful prophylactic against neonatal conjunctivitis.

5. CHLORHEXIDINE;
(hibitane) Chlorhexidine as chlorhexidinegluconate is dissolved in 70% alcohol.
 Effective against Gram-positive organisms and HIV.
 Active in ph range 5.5 - 8.0.
 Limitation: Not recommended as a general disinfectant.
 Not active against sporulating bacteria or non-lipid-containing viruses.
 Incompatible with soap and anionic detergents.
Use: savlon (chlorhexidine + cetrimide) is widely used in burns, wounds, as bladder irrigant, for
surgical instruments and pre - operative disinfection of skin.
Use as antiseptic. Apply alcoholic chlorhexidine to the skin in the event ofaccidental
contamination.

6. HYDROGEN PEROXIDE;
A concentration of 3% w/v generally used for disinfection.
 Active against a range of microorganisms.
 Fungi, spores and enteric viruses require higher concentration.
 No toxic end-products of decomposition.
Use: H2O2 is applied to disinfect plastic implants, contact lenses, and surgical prostheses.

7.PHENOLICS;

Synthetic phenolics (clear soluble fluids) can be used as general disinfectants in the laboratory.

 Spectrum: Active against bacteria and lipid-containing viruses.

 Not active against spores and non-lipid-containing viruses.

 Active in presence of organic matter.

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  Use: for disinfecting floors, walls, benches and other furniture.

 Cresols: (methyl phenols, lysol)

 Limitation: toxic to skin and tissues.

Use: mainly used for preliminary sterilization of infected glasswares in laboratory,

disinfection of excreta, cleaning floors of wards and operation room in hospital.

Chloroxylenol (dimethyl phenol) is active ingredient of dettol.

Hexachlorophane:

it is bacteriostatic at very high dilutions and used in soap and powder form. It is more effective
against gram positive than gram negative bacteria and is applied on skin as prophylaxis against
staphylococcal infection.

Chlorhexidine: mentioned previously

8. QUATERNARY AMMONIUM COMPOUNDS;

Quaternary ammonium compounds are positively charged (cationic) surface-active disinfectants.
 Effective against Gram-positive bacteria and lipid-containing viruses.
 Not recommended as general disinfectants (they have a narrow antibacterial spectrum).
 Inactivated by proteins, soap and anionic detergents.
 Eg. Benzalkonium chloride, alkyldimethylbenzyl ammonium chloride, and
cetylpyridinium chloride.
 Used for cleaning of floors of hospitals.

9. ETHYLENE OXIDE;
It is an alkylating agent.
 Exerts lethal effect on proteins of bacteria.
 It is gas at ordinary room temperature and active against all types of bacteria and spores.
 It has got a good degree of penetration power, even through plastics.
Uses: Plastic goods, polythene tube, artery and bone grafts, cystoscopes, vaccines and
culture media can be sterilized by ethylene oxide.

 These objects are kept in a cabinet from which air is removed by a vacuum pump and
then a mixture of ethylene oxide and carbon dioxide is introduced in the cabinet.

10. BETAPROPIOLACTONE (BPL);

It is a condensation product of ketone and formaldehyde having a boiling point of 163’C.
 It is capable of killing all microorganisms including viruses.

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  Uses: Although BPL has a low penetrating power as a gas, but it is believed to be more
efficient for the purpose of fumigation.
 Its biocidal action is very rapid and only 0.2% BPL is used for the sterilization of
biological products.
 Limitation: unfortunately BPL has carcinogenic effect.

Select the antiseptic solution from the following recommended products:

 Alcohol-based solutions (tinctures) of iodine or chlorhexidine
 Alcohols (60–90% ethyl,or “methylated spirit”)
 Chlorhexidinegluconate (2–4%) (e.g, Hibitane, Hibiscrub, Hibiclens
 Chlorhexidinegluconate and cetrimide, various concentrations at least 2% (e.g, Savlon)
 Iodine (3%); aqueous iodine and alcohol-containing (tincture of iodine products
 Iodophors (7.5–10%), various other concentrations (e.g, Betadine)
 Chloroxylenol (Para-chloro-metaxylenol or PCMX) (0.5–3.75%), various other
concentrations (e.g., Dettol)

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