Chemical method sterilization - In this slide liquid and gas, both are chemicals included that are used in sterilization.

1. CHEMICAL
METHODS
STERILIZATION

2. • Chemical Sterilization is the process of removal of microorganisms by the use of chemical bactericidal agents.
• Even if physical methods of sterilization are more appropriate for effective sterilization, it is not always
appropriate to use for heat-sensitive materials like plastics, fiber optics, and biological specimens.
• Under such conditions, chemical either in liquid or gaseous state can be used for sterilization. However, it is
crucial to ensure that the materials undergoing sterilization are compatible with the chemical being used.
• Besides, it is important to adopt safety rules in the workplace safety during the use of chemical agents.
• The chemical method of sterilization can be categorized as liquid and gaseous sterilization.
Ideal Chemical Disinfectant :
An ideal chemical antiseptic or disinfectant should have the following properties:
Wide spectrum of activity
Active in the presence of organic matter
Effective in acid as well as alkaline media
Speedy action
High penetrating power
Stable
Compatible with other antiseptics and disinfectants
Not corrode metals
Not cause local irritation or sensitization
Not interfere with healing
Not be toxic if absorbed into circulation
Cheap and easily available
Safe and easy to use

3. 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.
Liquid Sterilization
• Liquid sterilization is the process of sterilization which involves the submerging of equipment in the liquid sterilant
to kill all viable microorganisms and their spores.
• Although liquid sterilization is not as effective as gaseous sterilization, it is appropriate in conditions where a low
level of contamination is present.
• Different liquid chemicals used for liquid sterilization includes the following:

4. 1. Alcohols:
• Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein. Examples: Ethyl alcohol, isopropyl
alcohol and methyl alcohol.
• Ethyl alcohol and isopropyl alcohol are most frequently used. They act by denaturing bacterial proteins. But they are significantly
active only when there concentration is more that 60 to 70 % in water.
• 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.
Spectrum:
• Effective against fungi, vegetative bacteria, Mycobacterium species and some lipid-containing viruses.
• They have no virucidal or sporicidal activity
Application:
• 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.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable.
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.

5. 2. Aldehydes:
• Probably damages nucleic acids and kills all microorganisms, including spores.
• Different concentration of different Aldehydes are used against different kind of microbes.
• Examples: Formaldehyde, Glutaraldehyde.
• 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 is used to sterilize thermometers, Centrifuges, anasethetic equipment's etc.
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.

6. 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.
• Metal instruments, biosafety cabinets, clothing, bedding, furniture and books
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation
of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc.
Fumigation technique
After sealing the windows and other outlets, formaldehyde gas is generated by adding 250g of KMnO4
to 500 ml formalin for every 1000 cu.ft of room volume.
Caution: The reaction produces considerable heat, and so heat resistant vessels should be used.
After starting generation of formaldehyde vapour, the doors should be sealed and left unopened for 48
hours.
Caution: Formaldehyde can react with free chlorine to produce toxic gas. Remove hypochlorite
solutions and hydrochloric acid from spaces to be decontaminated.
Irritant vapours are released hence Neutralize with ammonia following decontamination.

7. 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).
• 2% glutaraldehyde solution, for at least 10 hours, can be used to sterilize heat labile items.
• Glutaraldehyde is an accepted liquid sterilizing agent which requires comparatively long immersion time. For the
removal of all spores, it requires as long as 22 hours of immersion time.
• The presence of solid particles further increases the immersion time.
• The penetration power is also meager as it takes hours to penetrate a block of tissues.
• The use of glutaraldehyde is thus limited to certain surfaces with less contamination.
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 ascystoscopes, bronchoscopes, corrugated rubber anesthetic tubes, face masks, endotracheal
tubes, metal instruments, polythene tubing.

8. Caution:
• Glutaraldehyde is known to cause dermatitis and asthma.
• Less irritating than formaldehyde.
• Glutaraldehyde should not be used in an area with little or no ventilation.
• Eye protection, a plastic apron, and gloves must be worn.
• Should be stored away from heat sources and in containers with close-fitting lids.
• The length of time that glutaraldehyde solutions can be used varies but they are usually good for up to 14
days.
• Solutions should be replaced any time they become cloudy.
Advantages:
• Relatively inexpensive
• Excellent materials compatibility
Disadvantages:
• Pungent and irritating odor
• Relatively slow mycobactericidal activity
• Coagulate blood and fix tissues to surfaces

9. 3. Phenol:
Mode of action:
• Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.
• The lethal effect of phenol is due to cell membrane damage, thus releasing cell contents and causing
lysis.
Examples: hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications:
• They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses.
• Chlorhexidine can be used in an isopropanol solution for skin disinfection.
• It is often used as an antiseptic hand wash.
• Chlorhexidine gluconate is also mixed with quaternary ammonium compounds such as cetrimide to
get stronger and broader antimicrobial effects (eg. Savlon)
Disadvantages:
• It is toxic, harsh and skin irritant.
• Chlorhexidine is inactivated by anionic soaps.
• Chloroxylenol is inactivated by hard water

10. 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.
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)
• It is active ingredient of Dettol.

11. Halogens: Chlorine and iodine are two commonly used.
4. Chlorine
 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.
 Effective between a pH range of 6-8.
Concentration: Use at 0.1% as a general disinfectant.
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.
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.

12. 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.
• Hypochlorite solution, which is also called liquid bleach, is another liquid chemical that can be used as a disinfectant,
even though sterilization is difficult to obtain with this chemical.
• Submerging devices for a short period in liquid bleach might kill some pathogenic organisms but to reach
sterilization submersion for 20-24 hours is required.
• It is an oxidizing agent and thus acts by oxidizing organic compounds which results in the modification of proteins in
microbes which might ultimately lead to death.
• Appropriate concentrations of hypochlorite can be used for the disinfection of workstations and even surfaces to
clean blood spills and other liquids.
Caution:
Hypochlorites are inactivated by organic matter.
They corrode metals, hence contact of the chemical with the metallic instruments and equipment must be avoided.
In case of heavy soilage eg blood spillage, a concentration of 10000 ppm (1:5 dilution of household bleach) of available chlorine
is recommended.
Uses:
It has great widespread use as a laboratory disinfectant on surfaces of bench and in discard spots.

13. 5. 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.
Use:
• Most commonly used for skin disinfection and decontaminating clean surfaces.

14. 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.
• Commercial PVP in a dilution of 1:2 to 1:100 kills most bacteria including S.aureus.
Limitation:
• May become contaminated by organisms like pseudomonas.
Uses:
• Iodophors are widely used for antisepsis of skin, mucosa and wounds.
• A 2.5% ophthalmic solution is an useful prophylactic against neonatal conjunctivitis.

15. 6 . Biguanide - CHLORHEXIDINE (hibitane)
• Chlorhexidine as chlorhexidine gluconate 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 of accidental contamination.

16. 7. Oxidizing agents- 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.
• Hydrogen peroxide, per acetic acid, etc. are used by releasing free radicals which are the active ingredients in these methods.
• Hydrogen peroxide is a liquid chemical sterilizing agent which is a strong oxidant and can destroy a wide range of microorganisms.
• It is useful in the sterilization of heat or temperature-sensitive equipment like endoscopes. In medical applications, a higher concentration (35-
90%) is used.
• H2O2 has a short sterilization cycle time as these cycles are as short as 28 minutes where ethylene oxide has cycles that as long as 10-12 hours.
• However, hydrogen peroxide has drawbacks like low material compatibility, lower capacity of penetration, and associated health risks.
• Vaporized hydrogen peroxide (VHP) is used to sterilize largely enclosed and sealed areas, such as entire rooms and aircraft interiors.
Caution:
Do not use on aluminium, copper, zinc or brass.
Mechanism:
H2O2 on decomposition liberates free hydroxyl radical which is the active ingredient in the process.
Use:
H2O2 is applied to disinfect plastic implants, contact lenses, and surgical prostheses.

17. 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.
Betapropiolactone (bpl)
• It is a condensation product of ketone and formaldehyde having a boiling point of 163’C.
• t is capable of killing all microorganisms including viruses.
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.
Points to ponder
• Prepare fresh dilutions daily
• Use clean, dry containers
• Use clean water for dilutions
• Use higher concentration or for longer duration if spillage is heavy.
• Avoid using corrosive disinfectants on metal surfaces.

18. Gaseous Sterilization:
• Gaseous sterilization involves the process of exposing equipment or devices to different gases in a closed heated or
pressurized chamber.
• Gaseous sterilization is a more effective technique as gases can pass through a tiny orifice and provide more effective
results.
• Besides, gases are commonly used along with heat treatment which also facilitates the functioning of the gases.
• However, there is an issue of release of some toxic gases during the process which needs to be removed regularly from
the system.
• The mechanism of action is different for different types of gases.
 The Chemically reactive gases ethylene oxide and formaldehyde possess broad spectrum biocidal activity.
 Have found application in the sterilization of reusable surgical instruments.
 Certain medical diagnostic and electrical equipments and the surface sterilization of powder.
 Sterilization processes using ethylene oxide sterilization are far more commonly used on an international bases then
those employing formaldehyde.
• Some of the common gases used for gaseous sterilization are explained below:

19. 1. Ethylene oxide:
• Ethylene oxide (EO) gas is a common gas used for chemical treatment applied to sterilize, pasteurize, or disinfect
different types of equipment and surfaces because of its wide range of compatibility with different materials.
• EO treatment often replaces other sterilization techniques like heat, radiation, and even chemicals in cases where the
objects are sensitive to these techniques.
• This method is a widespread method used for almost 70% of all sterilizations and around 50% for disposable medical
devices.
• The mechanism of antimicrobial action of this gas is assumed to be through the alkylation of sulphydryl, amino,
hydroxyl, and carboxyl groups on proteins and imino groups of nucleic acids.
• EO treatment is usually conducted at the temperature range of 30-60°C for several hours which aids in the activity of
the gas.
• The efficacy of the gas depends on the concentration of gas available for each article which is greatly assisted by the
good penetrating nature of the gas, which diffuses readily into many packaging materials including rubber, plastics,
fabric, and paper.
• Ethylene oxide kills all known microorganisms, such as bacteria (including spores), viruses, and fungi (including
yeasts and molds), and is compatible with almost all materials even when repeatedly applied.
• This process, however, is not without drawbacks as the level of gas in the sterilizer goes on decreasing due to
absorption, and the treated articles need to undergo a process of desorption to remove the toxic residual wastes.
• Organisms are more resistant to ethylene oxide treatment in a dried state, as are those protected from the gas by
inclusion in crystalline or dried organic deposit.

20. • Recognized sterilization method in BP and BPC( British pharmaceutical codex).
• This gas is highly explosive in a mixture of less than 3.6 % v/v in air
• In order to reduce the explosion hazard it is usually supplied for sterilization purposes as a 10% mixed with C02 or
8.6% mixture with HFC(Hydro floro carbon) which has replaced fluorinated hydrocarbon.
• Alternatively pure ethylene oxide gas can be used below atmospheric pressure in sterilizer chamber from which all
air has been removed.
• Used almost exclusively to sterilize medical products that cannot be steam sterilized or sensitive to radiation.
• It is a colorless liquid with boiling point of 10.7 ˚C
• It is effective against all types of microorganisms including viruses and spores.
• It has a potential toxicity towards humans including mutagenicity, carinogenicity.
• It is highly flammable.
• It is specially used for sterilizing plastic and rubber articles, respirators, heart lung machine, sutures, Dental
equipments, cloths,etc.
Principle
• The sterilization action of EtO is based on an alkylation reaction.
• Sterilizer design and operation: An ethylene oxide sterilizer consist of a leak proof and explosion proof steal chamber
normally of 100-300 liter capacity

21. Mechanism of action:
• It destroys micro-organisms by alkylation and cause denaturation of nucleic acids of micro-organisms.
• At 30 °C - 60°C with relative humidity above 30 % and gas conc. between 200 and 800 mg/l for at least 3 hours.
• Ethylene oxide is a colorless liquid with a boiling point of 10.7 °C.
• Highly penetrating gas with sweet ethereal smell.
• Highly inflammable & in conc. greater than 3%, highly explosive.
• By mixing with inert gases such as CFC or CO2, explosive tendency is eliminated.
• Plastics, rubber & photographic equipments can be sterilized by this method.
• Also used for mass sterilization of disposable items, plastic syringes, needles, catheters, blades etc.
Sterilizer design and operation(contd):
• Forced gas circulation is often employed to minimize variations in conditions through out the sterilizer chamber.
• Absorption of ethylene oxide by the load is enhanced by the introduction of excess gas at the beginning or by the
addition of more gas as the pressure drops during the sterilization process.
• After treatment, the gases are evacuated either directly to the outside atmosphere or through the special exhaust
system.
• Filtered sterile air is then admitted either for a repeat of the vacuum or for air purging until the chamber is opened.
• In this way safe removal of the ethylene oxide is achieved reducing the toxic hazards to the operator.

22. Disadvantages
• Low penetrating power
• Pungent
• Teratogenic
• Inflammable
• Toxic
• carcinogenic
• Mutagenic
• Acute toxicity including irritation of skin, conjunctiva and nasal mucosa.
• Lengthy cycle time
• Cost
• Potential hazards to patients & staff
Advantage:
• Can sterilize heat or moisture sensitive medical equipments

23. Clinical Notes :-
• ETOX gas is the best way to sterilize rotary handpieces.
• Automatic devices sterilize items in several hours and operate at elevated temperature below 100˚C.
• Less expensive devices operate overnight to produce sterilization at room temperature.
• Porous and plastic materials absorb gas and require aeration for 24 hours or more before it is safe for them to
contact skin or tissues.
• Units with large chambers sizes hold more instruments or packs per cycle; however they are expensive.
2. Formaldehyde
• Formaldehyde gas for use in sterilization is produced by heating formalin to a temperature of 70-75 C with steam
leading to the process known as LTSF.
• Formaldehyde has a similar toxicity to ethylene oxide.
• Although absorption to material appear to be low.

24. • Formaldehyde is another important highly reactive gas which is used for sterilization.
• This gas is obtained by heating formalin (37%w/v) to a temperature of 70-80°C.
• It possesses broad-spectrum biocidal activity and has found application in the sterilization of reusable surgical
instruments, specific medical, diagnostic and electrical equipment, and the surface sterilization of powders.
• Formaldehyde doesn’t have the same penetrating power of ethylene oxide but works on the same principle of
modification of protein and nucleic acid.
• As a result of the low penetrating power, its use is often limited to paper and cotton fabrics.
• Formaldehyde can generally be detected by smell at concentrations lower than those permitted in the atmosphere
and thus can be detected during leakage or other such accidents.
Sterilizer design and operation:
• A LTSF sterilizer is designed to operate with sub atmospheric pressure steam.
• Air is removed by evacuation and steam is admitted to the chamber to allow heating of the load and to assist in air
removal.
• The sterilization period starts with the release of formaldehyde by vaporization from formalin.
• The chamber temperature is maintained by a thermostatically controlled water jacket and steam and condensate is
removed via drain channel and an evacuated condenser.
• At the end of the treatment period formaldehyde vapor is expelled by steam flushing and the load is dried by
alternating stages of evacuation and admission of sterile filtered air.

25. ROOM TEMPERATURE
ETHYLENE OXIDE STERILIZER

26. 3. Nitrogen dioxide (NO2)
• Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of common bacteria, fungi, and even spores.
• NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard temperature.
• This property of NO2 enables the use of the gas at standard temperature and pressure.
• The biocidal action of this gas involves the degradation of DNA by the nitration of phosphate backbone, which results in lethal effects on the
exposed organism as it absorbs NO2.
• An advantage of this gas is that no condensation of the gas occurs on the surface of the devices because of the low level of gas used and the
high vapor pressure. This avoids the need for direct aeration after the process of sterilization.
4. Ozone
• Ozone is a highly reactive industrial gas that is commonly used to sterilize air and water and as a disinfectant for surfaces.
• Ozone is a potent oxidizing property that is capable of destroying a wide range of organisms including prions, without the use of
hazardous chemicals as ozone is usually generated from medical-grade oxygen.
• Similarly, the high reactivity of ozone allows the removal of waste ozone by converting the ozone into oxygen by passing it through
a simple catalyst.
• However, because ozone is an unstable and reactive gas, it has to be produced on-site, which limits the use of ozone in different
settings.
• It is also very hazardous and thus only be used at a concentration of 5ppm, which is 160 times less than that of ethylene oxide.
• It is a technique of low temperature sterilization ,i.e., 25˚C to 35˚C
• Ozone sterilizer uses oxygen, water and electricity to produce ozone within the sterilizer and provide sterilization without
producing toxic chemicals.

27. HAND WASHING
Three types of hand washing-
• Social hand washing
• Clinical hand washing
• Surgical hand washing
Social hand washing
• Recommended following social-type contact with clients, after going to the toilet and after covering a cough or
sneeze.
• A plain liquid soap is often used.
Clinical hand washing
• A clinical hand wash is used before clinical procedures on clients, when a client is being managed in isolation, or in
outbreak situations.
• An anti-microbial soap, containing an antiseptic agent, is used.
Surgical hand washing
• A surgical hand wash is required before any invasive or surgical procedure requiring the use of sterile gloves.
• An antimicrobial skin cleanser, usually containing chlorhexidine or detergent-based povidone-iodine, is used.

28. Doubts