WHY NEEDED? Microorganisms are ubiquitous. Since they cause contamination, infection and decay, it becomes necessary to remove or destroy them from materials or from areas. This is the object of sterilization.
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, and in many other situations. 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.
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)
Antisepsis – prevention of infection, usually by inhibiting the growth of bacteria in wounds or tissues. Antiseptics – chemical disinfectants which can be safely applied to skin or mucous membrane and are used to prevent infection by inhibiting growth of bacteria. Bactericidal – agent which can kill bacteria. Bacteriostatic – agent which only prevents multiplication of bacteria.
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.
IDEAL CHEMICAL DISNIFECTANT 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
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.
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. Spectrum: Effective against fungi, vegetative bacteria, Mycobacterium species and some lipid-containing viruses.
Limitation: Not effective against spores.Concentration: Most effective at 70% inwater.Caution: Do not use near flames due toflammability.May swell rubber or harden plastics.
ALDEHYDES FORMALDELYDE: 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.
Metal instruments, biosafetycabinets, clothing, bedding, furniture and books
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 Neutralise with ammonia following decontamination.
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 ascystoscopes, bronchoscopes, corrugated rubber anesthetictubes, face masks, endotracheal tubes, metalinstruments, polythene tubing.
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.
GlutaraldehydeAdvantages Numerous use studies published Relatively inexpensive Excellent materials compatibilityDisadvantages: Pungent and irritating odor Relatively slow mycobactericidal activity Coagulate blood and fix tissues to surfaces
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 range 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.
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.
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.
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.
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+cetrimid e) 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.
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.
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.
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) 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
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.
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.
ETO Machine 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.
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. 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.