Sterilization and Disinfection PDF

By Dr/ Shimaa M. Ghanem Types and uses of radiation for sterilization Types Uses Comments Ionizing radiation For sterilization of Though expensive administered using pharmaceuticals and fraught with Cobalt-60-based...

By Dr/ Shimaa M. Ghanem Types and uses of radiation for sterilization Types Uses Comments Ionizing radiation For sterilization of Though expensive administered using pharmaceuticals and fraught with Cobalt-60-based like antibiotics, safety risks, it is very instruments hormones, sutures; effective due to better and prepacked penetration power disposable items, such as syringes, infusion sets, catheters, etc. Nonionizing radiation Only for disinfection Hazardous and not administered through of clear surfaces in as effective as ionizing UV lamps OTs, laminar flow radiation hoods, etc.  Air also can be sterilized by filtration.  Two common examples are surgical masks and cotton plugs on culture vessels that let air in but keep microorganisms out.  Laminar flow biological safety cabinets are most widely used air filtration systems in hospitals and industries.  In this method, air is passed through high-efficiency particulate air (HEPA) filters that remove nearly 99.97% of 0.3 µm particles from the filtered air.  Radiations  The ionizing and nonionizing radiations are the two types of radiation used for sterilization  Ionizing radiations:  Ionizing radiation is an excellent sterilizing agent with very high penetrating power.  These radiations penetrate deep into objects and destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic. These are, however, not that effective against viruses.  Ionizing radiations include:  (a) X-rays  (b) gamma rays  (c) cosmic rays.  Gamma radiation from a cobalt-60 source  It is used for sterilization of antibiotics, hormones, sutures, catheters, animal feeds, metal foils, and plastic disposables, such as syringes.  This has also been used to sterilize and ‘pasteurize’ meat and other food items.  Irradiation usually kills Escherichia coli O157:H7, Staphylococcus aureus, Campylobacter jejuni, and other pathogens.  Since there is no detectable increase of temperature in this method, this method is commonly referred to as cold sterilization.”  Both the Food and Drug Administration and the World Health Organization have approved food irradiation and declared it safe  Nonionizing radiations:  Nonionizing radiations include infrared and ultraviolet radiations.  Infrared radiations are used for rapid and mass sterilization of disposable syringes and catheters.  Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite lethal and has a marked bactericidal activity. o It acts by denaturation of bacterial protein and also interferes with replication of bacterial DNA. o UV radiation is used primarily for disinfection of closed areas in microbiology laboratory, inoculation hoods, laminar flow, and operating theaters. o It kills most vegetative bacteria but not spores, which are highly resistant to these radiations. o However, it does not penetrate glass, dirt films, water, and other substances very effectively. o Since UV radiations on prolonged exposure tend to burn the skin and cause damage to the eyes, UV lamps should be switched off while people are working in such areas.   Sound (sonic) waves:  High-frequency sound (sonic) waves beyond the sensitivity of the human ear are known to disrupt cells.  Sonication transmits vibrations through a water-filled chamber (sonicator) to induce pressure changes and create intense points of turbulence that can stress and burst cells in the vicinity.  Sonication also forcefully dislodges foreign matter from objects.  Heat generated by the sonic waves (up to 80°C) also appears to contribute to the antimicrobial action.  Gram-negative rods are most sensitive to ultrasonic vibrations, while Gram- positive cocci, fungal spores, and bacterial spores are resistant to them.  Ultrasonic devices are used in dental and some medical offices to clear debris and saliva from instruments before sterilization and to clean dental restorations.  However, most sonic machines are not reliable for regular use in disinfection or sterilization.  STERILITY TESTING  A sterility test may be defined as - 'a test that critically assesses whether a sterilised pharmaceutical product is free from contaminating microorganisms'.  The purpose of the test for sterility is to independently provide a means of verifying that any substance, preparation or article is sterile, in accordance with the requirements of the Pharmacopoeia.  Two methods to perform sterility tests depending on the nature of the product:  1- Direct inoculation (immersion).  2- Membrane Filtration. 1. Direct inoculation (immersion)  A small volume of sample is removed aseptically from the sample unit and inoculated directly into a suitable volume of growth medium prior to incubation.  Using two separate media. These are usually fluid thioglycollate medium {FTM}, to culture anaerobic bacteria and soybean casein digest medium (SCDM) to culture fungi and aerobic bacteria.  The cultures are incubated at 30-35˚C and 20-25˚C and then examined.  Any turbidity in the culture may indicate growth and must be investigated.  This method has some significant disadvantages:  1-Only small volumes of product can be inoculated into the culture medium, limiting the sensitivity of the test.  2- If the sample appears milky or turbid, it can be very difficult to detect turbidity caused by microbial growth at the end of the incubation period.  Advantages of the direct inoculation method are: — 1. sterility testing for materials that cannot be easily filtered. — 2. smaller volumes of test article can be used.  Membrane Filtration  Bulk articles or effluent from vials of final products are passed through a 0.45 µm membrane filter and about 50 mm in diameter designed to retain microbial contaminants.  Cellulose nitrate filters, are used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for strongly alcoholic solutions.  Specially adapted filters may be needed for certain products, e.g. for antibiotics.  The filters are rinsed to remove inhibitors and then incubated in two types of media and assayed exactly as in the direct inoculation method.  Aqueous solutions; if appropriate, transfer a small quantity of a suitable, sterile diluent such as neutral solution of meat or casein peptone pH 6.9 to 7.3 onto the membrane in the apparatus and filter.  The diluent may contain suitable neutralizing substances and/or appropriate inactivating substances for example in the case of antibiotics.  If the product has antimicrobial properties, wash the membrane not less than three times.  Transfer the whole membrane to the culture medium or cut it aseptically into two equal parts and transfer one half to each of two suitable media. Incubate the media for not less than 14 days.  The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test.  Advantages of the membrane filtration method include:  1-accommodation of large volume samples (up to 500 ml).  2-Removal of inhibitory substances such as antibiotics or preservatives that inhibit the growth of microorganisms by rinsing the filter membrane with a suitable agent.  3-Filtration also provides an opportunity to rinse away components in the sample that may cause turbidity.  Sampling:  Samples must be representative (size, site and no.).  Sample size is determined according to pharmacopoeia  No. of items &quantity taken are prescribed in the tables.  Use for each medium not less than the quantity of the product prescribed in the table.  If the batch size is not known, use the maximum number of items prescribed.  Sterility tests for different pharmaceutical products  1-Aquous solutions:  by immersion or filteration.  2-Soluble solids  The product dissolved in a suitable solvent such as the solvent provided with the preparation, water for injections , sodium chloride (9 g/l) or peptone (1 g/l) and proceed with the test as described above for aqueous solutions.  3-Insoluble solids:  Prepare asuspension then make direct inoculation in both FTM and SCDM and make subculture during incubation to decrease the effect of turbidity on the result.  4-Oils and oily solutions  Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate ( a clear, colorless, oily liquid).  Allow the oil to penetrate the membrane by its own weight then filter, applying the pressure or suction gradually.  Wash the membrane at least three times by filtering through it each time about 100 ml of a suitable sterile solution such as peptone (1 g/l) containing a suitable emulsifying agent at a concentration shown to be appropriate , for example polysorbate 80.  Transfer the membrane or membranes to the culture medium or media as described above for aqueous solutions, and incubate at the same temperatures and for the same times.  5-Ointments and creams  Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent in isopropyl myristate as described above, by heating, if necessary, to 40 °C - 44 °C. Filter as rapidly as possible and proceed as described above for oils and oily solutions.  OR;  Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as peptone (1 g/l).  Transfer the diluted product to the culture media of the test medium.  Incubate the inoculated media for not less than 14 days.  Observe the cultures several times during the incubation period.  Shake cultures containing oily products gently each day.  However when fluid thioglycollate medium is used for the detection of anaerobic microorganisms keep shaking or mixing to a minimum in order to maintain anaerobic conditions.  Chemical Methods of Sterilization  Several chemical agents are used as antiseptics as well as disinfectants.  All these chemical agents (e.g., alcohols, aldehydes ,etc.) are described later in detail under disinfection.  The effects of cold and desiccation: The main benefit of cold treatment is to slow the growth of cultures and microbes in food during processing and storage.  It is essential to know that cold merely retards the activities of most microbes. Although it is true that cold temperatures kill some microbes, gradual cooling, long-term refrigeration, or deep-freezing does not adversely affect most of the microorganisms.  In fact, freezing temperatures, ranging from -70 to -135°C, provide an environment that can preserve cultures of bacteria, viruses, and fungi for longer periods.  Some psychrophiles grow very slowly even at freezing temperatures and can continue to secrete toxic products.  S. aureus, Clostridium species (spore formers), Streptococcus species, and several types of yeasts, molds, and viruses are the pathogens that can survive for several months in the refrigerated food items.  Lyophilization is a process of freezing and drying. It is the most common method of preserving microorganisms and other cells in a viable state for many years.  Pure cultures are frozen instantaneously and exposed to a vacuum that rapidly removes the water (it goes right from the frozen state into the vapor state).  This method avoids the formation of ice crystals that would damage the cells. Although not all cells survive this process, lot many of them survive after reconstitution of lyophilized culture.  As a general rule, chilling, freezing, and desiccation are not considered as methods of disinfection or sterilization because their antimicrobial effects are erratic and uncertain, and one cannot be sure that pathogens subjected to these procedures have been killed.  Disinfection  Disinfection is the process of inactivating microorganisms by direct exposure to chemical or physical agents.  Disinfectants are products or biocides that destroy or inhibit the growth of microorganisms on inanimate objects or surfaces. Disinfectants can be sporistatic but are not necessarily sporicidal.  Antiseptics are biocides or products that destroy or inhibit the growth of microorganisms in or on living tissue.  Antiseptics and disinfectants are used extensively in hospitals for a variety of topical and hard surface applications. They are an essential part of infection control practices and aid in the prevention of nosocomial infections. Differences between sterilization and disinfection sterilization disinfection Definition Freeing an article, surface, or Process that reduces the medium from all living organisms number of contaminating including viruses, bacteria and microorganisms, liable to cause their spores, and fungi and their infection to a level which is spores. deemed no longer harmful to health. Spores are not killed. Uses Objects or instruments coming in Objects or instruments coming direct contact with a break in skin in direct contact with mucous or mucous membrane or entering membrane but tissue is intact or a sterile body area. via intact skin. Examples Surgical instruments, needles, Endotracheal tubes, aspirators, syringes, parenteral fluid, gastroscopes, bed pans, urinals, arthroscopes, media, reagents and etc. equipments in laboratory use.  Properties of Ideal Disinfectant  An ideal disinfectant or antiseptic has the following characteristics:  1. Ideally, the disinfectant should have a wide spectrum of antimicrobial activity. It must be effective against a wide variety of infectious agents (Gram-positive and Gram- negative bacteria, acid-fast bacteria, bacterial endospores, fungi, and viruses) at high dilutions.  2. It should act in the presence of organic matter.  3. It should not be toxic to human or corrosive. In practice, this balance between effectiveness and low toxicity for animals is hard to achieve. Some chemicals are used despite their low effectiveness, because they are relatively nontoxic.  4. It should be stable upon storage and should not undergo any chemical change.  5. It should be odorless or with a pleasant odor.  6. It should be soluble in water and lipids for penetration into microorganisms.  7. It should be effective in acidic as well as in alkaline media.  8. It should have speedy action.  9. If possible, it should be relatively inexpensive. Action of Disinfectants  Disinfectants act in many ways as discussed below.  1. They produce damage to the cell wall and alter permeability of the cell membrane, resulting in exposure, damage, or loss of the cellular contents.  2. They alter proteins and form protein salts or cause coagulation of proteins.  3. They inhibit enzyme action and inhibit nucleic acid synthesis or alter nucleic acid molecules.  4. They cause oxidation or hydrolysis.  Factors Influencing Activity of Disinfectants  Various conditions influencing the efficiency of disinfectant are as follows: Temperature: Increase in temperature increases the efficiency of disinfectants. Type of microorganism: Vegetative cells are more susceptible than spores. Spores may be resistant to the action of disinfectants. Physiological state of the cell: Young and metabolically active cells are more sensitive than old dormant cells. Non-growing cells may not be affected. Environment: The physical or chemical properties of the medium or substance influence rate as well as efficiency of disinfectants, e.g., pH of the medium and presence of extraneous materials.  Types of Disinfectants  Disinfectants include the following:  (a) phenolic compounds, (b) halogens,  (c) alcohols, (d) aldehydes,  (e) gases, (f) surface active agents,  (g) oxidizing agents, (h) dyes,  (i) heavy metals, (j) acids and alkalis. Phenolic compounds  In 1867, Joseph Lister employed phenolic compounds to reduce the risk of infection during operations.  Phenolic compounds are the most widely used antiseptics and disinfectants in laboratories and hospitals worldwide.  They are bactericidal or bacteriostatic and some are fungicidal also.  They act by denaturing proteins and disrupting cell membranes.  They are effective in the presence of organic material and remain active on surfaces long after application.  Different phenolic compounds are as follows: Phenol:  It is effective against vegetative forms of bacteria, Mycobacterium tuberculosis, and certain fungi.  It is an excellent disinfectant for feces, blood, pus, sputum, etc.  It has a low degree of activity as compared to other derivatives.  It is not suitable for application to skin or mucous membrane. Cresol:  Cresols are more germicidal and less poisonous than phenol but corrosive to living tissues.  They are used for cleaning floors (1% solution), for disinfection of surgical instruments, and for disinfection of contaminated objects.  Lysol is a solution of cresols in soap.  Halogenated diphenyl compounds:  These compounds include hexachlorophene and chlorhexidine.  They are highly effective against both Gram-positive and Gram-negative bacteria.  They are used as skin antiseptics and for the cleaning of wound surfaces.  Hexachlorophene has been one of the most popular antiseptics because once applied it persists on the skin and reduces growth of skin bacteria for longer periods.  However, it can cause brain damage and is now used in hospital nurseries only after a staphylococcal outbreak.  Halogens  Halogens are fluorine, bromine, chlorine, and iodine—a group of nonmetallic elements that commonly occur in minerals, sea water, and salts.  Although they can occur either in the ionic (halide) or nonionic state, most halogens exert their antimicrobial activity primarily in their nonionic state, but not in the halide state (e.g., chloride, iodide).  These agents are highly effective disinfectants and antiseptics, because they are microbicidal and not just microbistatic.  They are also sporicidal with longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of all antimicrobial chemicals currently marketed.  Chlorine and iodine are the only two routinely used halogens because fluorine and bromine are dangerous to handle.   Chlorine and its compounds:  Chlorine has been used for disinfection and antisepsis for approximately 200 years.  The major forms used in microbial control are  (a) liquid and gaseous chlorine  (b) hypochlorites.  In solution, these compounds combine with water and release hypochlorous acid (HOCl), which oxidizes the sulfhydryl (S–H) group on the amino acid cysteine and interferes with disulfide (S– S) bridges on numerous enzymes.  The resulting denaturation of the enzymes is permanent.  Gaseous and liquid chlorine are used almost exclusively for large-scale disinfection of drinking water, sewage, and wastewater from sources, such as agriculture and industry.  Chlorine kills not only bacterial cells and endospores but also fungi and viruses.  Treatment of water with chlorine destroys many pathogenic vegetative microorganisms without unduly affecting its taste.  Chlorination at a concentration of 0.6–1.0 part of chlorine per million parts of water makes water potable and safe to use.  Hypochlorites are perhaps the most extensively used of all chlorine compounds.  They are used for:  Sanitization and disinfection of food equipment in dairies, restaurants, and canneries,  Treatment of swimming pools, spas, drinking water, and even fresh foods,  Treatment of wounds; and  Disinfection of equipments, beddings and instruments.  Common household bleach is a weak solution (5%) of sodium hypochlorite that is used as an all-around disinfectant, deodorizer ,and stain remover. It is frequently used as an alternative to pure chlorine in treating water supplies.  However, the major limitations of chlorine compounds are that they are:  a) Ineffective if used at an alkaline pH,  b) Less effective in the presence of excess organic matter,  c) Relatively unstable, especially if exposed to light.  Iodine and its compounds:  Iodine is a pungent black chemical that forms brown-colored solutions when dissolved in water or alcohol.  Iodine rapidly penetrates the cells of microorganisms ,where it apparently disturbs a variety of metabolic functions.  It acts by interfering with the hydrogen and disulfide bonds of proteins (similar to chlorine).  It kills all types of microorganisms if optimum concentrations and exposure times are used.  Iodine activity, unlike chlorine, is not as adversely affected by organic matter and pH.  The two primary iodine preparations are free iodine in solution and iodophors.  Free iodine in solution: Aqueous iodine contains 2% free iodine in solution and 2.4% sodium iodide. It is used as a topical antiseptic before surgery and also occasionally as a treatment for burnt and infected skin.  A stronger iodine solution (5% iodine and 10% potassium iodide) is used primarily as a disinfectant for plastic items, rubber instruments, cutting blades, and thermometers.  Iodine tincture is a 2% solution of iodine and sodium iodide in 70% alcohol that can be used in skin antisepsis. Because iodine can be extremely irritating to the skin and toxic when absorbed, strong aqueous solutions and tinctures (5–7%) are no longer considered safe for routine antisepsis.  Iodine tablets are available for disinfecting water during emergencies or for destroying pathogens in impure water supplies.  Iodophors: Iodophors are complexes of iodine and a neutral polymer, such as polyvinyl alcohol. This formulation permits the slow release of free iodine and increases its degree of penetration.  These compounds have largely replaced free iodine solutions in medical antisepsis because they are less prone to staining or irritating tissues.  Betadine, povidone, and isodine are the common iodophor compounds that contain 2–10% of available iodine.  They are used to prepare skin and mucous membranes for surgery and in surgical hand scrubs.  They are also used to treat burns and to disinfect equipments.  A recent study has shown that betadine solution is an effective means of preventing eye infections in newborn infants, and it may replace antibiotics and silver nitrate as the method of choice.