Chapter .. 7 STERILIZATION « LEARNING OBJECTIVES ¢ After completing thls chapter, reader should be able to understand: © ‘The different methods of Sterilization with Principle, Advantages, Disadvantages and Applications © Different Equipments Employed in Lange Scale Sterifization © The technique used for Evaluation of Tfficiency of Sterilization Methods ee 7Z.AINTRODUCTION Sterilization is an essential stage in the processing of any product used for parenteral administration, broken skin, mucosal surfaces or internal organs. Sterilization of microbiological materials, surgical dressings and equipments and other contaminated items is necessary to minimize the health hazard associated with these articles. The main reasons for controlling microorganisms are: 1. To prevent contamination in sterile products. 2. To prevent transmission of pathogenic microorganisms which are responsible for causing diseases in plants, animals and human beings. 3, To prevent decomposition and spoilage of food and food products. 4, To prevent the contamination of unwanted microbes in pure cultures and other microbiological experiments performed for research studies. 5, To prevent unwanted microbial contamination in antibiotic, enzyme, vitamin fermentation and other industrial processes. 6. To prevent contamination in aseptic areas which are used for the preparation of sterile dosage forms and sterility testing. Microorganisms can be killed, eliminated or inhibited by various physical and chemical agents. Several terms used to describe the physical processes and chemical agents employed in controlling microorganisms are: Sterilization: Sterilization is a process by which an article, surface or medium is freed of all microorganisms either in the vegetative or spore state. @ay Scanned with CamScannerPharmaceutical Microbiology (8.Pharm. Sem. 111) 72 Sterilization Disinfection: Disinfection means the destruction of all pathogenic organisms or organisms capable of giving rise to infections. In this process, vegetative cells are killed but not heat-resistant spores. Disinfection is usually accomplished by chemical agents called disinfectants. A disinfectant is normally applied to in- animate objects such as floors, buildings, equipment, laundry etc. Sanitization: Sanitization is the process of disinfection including cleansing action. Sanitizers are commonly applied to inanimate objects. Antiseptic: The term antiseptic is used to designate any substance which would prevent sepsis, either by killing microorganisms or by inhibiting their growth. An antiseptic can be applied to body tissues without causing injury to the tissue. Germicide: A germicide is an agent that kills vegetative cells but not necessarily the resistant spore forms of germs. The terms bactericide, fungicide, virucide and sporicide refer to agents that kill bacteria, fungi, viruses and spores respectively. Microbiostasis: Microbiostasis is the process of preventing the growth, reproduction and multiplication of microorganisms but not of killing them. Preservative: A preservative is a substance that prevents the growth of microorganisms. These substances are mainly added in food and pharmaceuticals to prevent microbial growth. Preservatives are not harmful to living tissues. The various methods used in sterilization can be classified as follows: L_ Physical methods: (a) Dry heat sterilization: eg. Incineration, direct flame, red heat, hot air etc. (b) Moist heat sterilization/steam sterilization e.g. Pasteurization, tyndallization, autoclave etc. (©) Radiation/cold sterilization: @ —_Use of ultra-violet rays: UV light. (ii) Ionising radiations: X-rays, gamma rays, beta rays. (d) Filtration / mechanical methods: (i) Asbestos filter (seitz filter). (ii) Sintered glass filter (morton filter). (ii) Filter candles (ceramic/Berkefeld filter) (iv) Membrane filter (millipore/ultra filter).Pharmaceutical Microb! [B.Pharm. Sem. 73 Sterilization IL Chemical methods: (a) Gaseous sterilization: e.g. Formaldehyde, ethylene oxide etc. (b) By using disinfectants: eg. Cresol, phenol etc. L_ Physical Methods These methods involve processes by the use of physical means. These may involve the utilisation of heat in the presence or in the absence of moisture or the applications of radiations or mechanical filtration. (a) Dry heat sterilization: Heat is the most reliable and rapid method of sterilization. The killing effect of dry heat is due to protein denaturation, oxidative damage and the toxic effect of elevated levels of electrolytes. The factors influencing sterilisation by heat are nature of heat, number of microorganisms present, temperature and time, characteristics of the microorganisms etc. The time required for sterilization is inversely proportional to the temperature of exposure. This can be expressed as thermal death time, which is the minimum time required to kill a suspension of microorganisms at a prescribed temperature and under specific conditions. Sterilization time is directly related to the number of microorganisms in the suspension, presence and nature of spores, the strain and characteristics of the microorganisms. Microorganisms are more resistant to dry heat as compared to moist heat and therefore, this process requires higher temperatures and longer exposure times. 1. Sunlight and drying: Sunlight possesses ultraviolet rays which along with heat rays are responsible for appreciable germicidal activity. These rays cannot penetrate through glass. This is a natural method for sterilisation of water in tanks, rivers and lakes. Drying in air has deleterious effect on many bacteria. Spores are unaffected by drying. Hence it is a very unreliable method. 2. Red heat: It is used to sterilize metallic objects by holding them on a flame till they are red hot e.g. inoculating wires, needles, forceps etc. 3. Flaming: The article is passed over flame without allowing it to become red hot eg. mouth of culture tubes, glass slides, scalpels, needles, coverslips etc. These items may be sometimes immersed in spirit in a tray and the spirit is then burnt off. However, it does not produce sufficient heat and destroys only vegetative microorganisms. 4. Incineration: This is an excellent method for rapidly destroying materials e.g. pathological material, bedding, animals carcasses, soiled dressing etc. Polystyrene type of materials emit clouds of dense black smoke and hence should be autoclaved in appropriate containers.Pharmaceutical Microb! Pharm. Sem. 7A Sterilization 5. Hot air oven: This is the most widely used method of sterilization by dry heat. The modern hot air ovens consist of a double walled chamber of aluminium or stainless steel separated from the outer case by a thick layer of insulation made of fibreglass (Fig. 7-1). Insulation is also filled in the hollow flanged door, which carries an asbestos jacket that provides a tight seal. Heating is affected by electrical heating elements and thermostats automatically contro! temperature. The material should be arranged in a manner which allows free circulation of air between the objects and it should not be overloaded. Glassware should be perfectly dry and wrapped in Kraft paper before being placed in the oven. The oven must be allowed to cool slowly for about two hours before the door is opened, since the glassware may get cracked by sudden cooling. Fig. 7.1: Hot alr oven Substances that are not heat-labile and can tolerate temperature upto 250°C may be sterilised by hot air oven. Normally the spores as well as the vegetative forms of all microorganisms are killed in two hours at a temperature of 160°C. The relation between the temperature and relative time required for sterilization in hot air oven is given in Table 7.1. Table 7.1: Temperature and time relationship for hot alr oven Temperature (°C) Time (minutes) 170 - 60 / 160 120 150 150 140 180Pharmaceutical Microbiol [B.Pharm. Sem. 7.5 Sterilization Hot air oven is used to sterilize glassware forceps, scalpels, scissors, spatula, swabs, some pharmaceutical substances such as glycerin, fixed oil, liquid paraffin, propylene glycol, sulphonamides and dusting powders such as kaolin, talc, zinc oxide, starch etc. It is not suitable for surgical dressings, rubbers, plastics, volatile and heat labile substances. Hot air is a bad conductor of heat and its penetration power is low as compared to moist heat. Hot air ovens are commonly available in pharmaceutical laboratories for drying and sterilization. (b) Moist heat sterilization: Sterilization by moist heat means killing of microorganisms with hot water or steam. The lethal effect of moist heat is due to the denaturation and coagulation of proteins. Moist heat sterilization is divided into three forms. () Temperature below 100°C. (i) Temperature at 100°C. (il) Temperature above 100°C. (1) Temperature below 100°C: Heat-labile fluids may be disinfected by heating at temperatures below 100°C. The temperature employed is either 63°C for 30 minutes (holder method) or 72°C for 20 seconds (flash method) followed by rapid cooling to 13°C or lower. This methods is known as pasteurization and it is widely applied in dairy products like milk and butter. By this method non-sporing microorganisms such as mycobacteria, brucella and salmonellae are destroyed. Heat-labile fluids such as serum may be disinfected by heating at 56°C for one hour. Vaccines prepared from non-sporing bacteria may be inactivated in a water bath at 60°C for one hour. Among the most heat resistant cells are the spores of Clostridium botulinum which require 120°C for four minutes. (li) Temperature at 100°C: Boiling at 100°C for 10 to 30 minutes kills all vegetative bacteria and some bacterial spores. Therefore, it is not recommended for sterilization of instruments for surgical procedures. Addition of small quantity of acid, alkali or washing soda markedly increases the sterilizing power of boiling water. An atmosphere of free steam is used to sterilize culture media which may decompose if subjected to higher temperatures. A Koch or Amold steam sterilizer is usually used. This steam sterilizer consists of a vertical metal cylinder with a removable conical lid having a small opening for the escaping steam. Water is added on the bottom and a perforated shelf above the water level is present. Single exposure to steam for 90 minutes ensures complete sterilization but media containing sugar and gelatin, which may get decompose on long heating. Hence, such materials may be exposed at 100°C for 20 minutes on three successive days. This is known as ‘tyndallization’ or ‘intermittent’ or ‘fractional sterilization’. First exposure to steam kills all vegetative bacteria and at second exposure all spores germinate in a favourable medium and are killed on subsequent occasions. Therefore, non-nutrient media cannot be sterilized by this method. (iil) Temperature above 100°C: Heat in the form of saturated steam under pressure is the most practical and dependable agent for sterilization. The laboratory apparatus designed to use steam under regulated pressure is called an autoclave. Saturated steam is aPharmaceutical Microb! Pharm. Sem. 76 Sterilization more efficient sterilizing agent than hot air because (i) it provides greater lethal action of moist heat (ii) it is quicker in heating up the exposed articles and (iii) it can penetrate easily porous material such as cotton wool stoppers, paper and cloth wrappers. The laboratory autoclave or pressure cooker type autoclave consists of a vertical (Fig. 7.2) or horizontal (Fig. 7.3) cylinder of gun metal or stainless steel in a supporting frame or case. The lid is fastened by screw clamps and rendered airtight by a asbestos gasket. The autoclave has on its lid or upper side a discharge tap for air and steam, a pressure gauge and a safety valve that can be set to blow off at any desired pressure. Steam valve Adjustable safety valve <3 Pressure gauge. Perforated metallic basket Stainless steel hollow vessel Fig. 7.2: Vertical autoclave Safety and air valves Steam jacket and chamber gauges ‘Operating valve ‘Steam to chamber Fig. 7.3: Horizontal autoclavePharmaceutical Microbiology (8.Pharm. Sem. I) 27 Sterilization Water is added on the bottom of the autoclave and articles to be sterilized are placed in a perforated shelf. The lid is closed, discharge tap is opened and safety valve is adjusted to the required pressure. When the air bubbles stop emitting from the discharge tap it indicates all the air from inside the autoclave has been removed. At this stage, the discharge tap is closed. Steam pressure rises inside and when it reaches the desired set level (15 p.s.i) the safety valve opens and excess steam escapes. From this point the holding time (15 minutes) is counted. When the holding time is over, the heating is stopped and autoclave is allowed to cool till pressure gauze indicates that the inside pressure has reached to the atmospheric pressure. The discharge tap is opened slowly and air is allowed to remove from the autoclave. The lid is opened and the sterilized articles are removed. The holding time needed for sterilization are indicated below (Table 7.2) in terms of the temperatures and pressures. However, the choice of the combination used depends mostly ‘on the ability of the material to withstand the imposed conditions. Table 7.2: Autoclaving conditions (temperature/time/pressure relationship) Temperature (C) Steam pressure (Ib/sq. Holding time (minutes) _ inch) | 115-118 10 30 121-124 1s 15 126-129 20 | 10 135-138 30 3 Autoclave is the most desired apparatus used for sterilizing bacteriological media, heat stable liquids, saline solutions, heat resistant equipments and instruments, glasswares, filters, ampoules, syringes, rubber products, surgical dressings and instruments etc. Autoclaving is one of the method of sterilization, recommended in Indian Pharmacopoeia for many official injections. Autoclaving is unsuitable for anhydrous materials such as powders, oils, fats, ointments and also for materials, which cannot withstand heating at 115°C and above. (c) Radiation: Energy transmitted through space in a variety of forms is generally called radiation. This method is also called cold sterilization because ionizing radiations produce relatively little heat in the material being irradiated. Thus, it is possible to sterilize heat-sensitive substances and such techniques are being developed in the food and pharmaceutical industries. Based on their wavelength and penetration power, radiation can be divided into two categories as non-ionizing radiations and ionizing radiations. Non-ionizing radiations have less energy and do not disturb the atomic configuration of the target molecule. Ionizing radiations have high energy and ionize the target molecules.Pharmaceutical Microbiology (B.Pharm. Sem. I) 78 Sterilization {l) Non-ionizing radiations (ultra violet radiations): Ultraviolet (UV) radiations in the region of 2537 A° have been shown to possess the greatest activity in destroying microorganisms. The part of the electromagnetic spectrum between the wavelength of 150 - 3900 A” constitutes UV radiation. It is commonly employed in the reduction of air-borne contamination in the maintenance of aseptic areas and rooms within the pracessing environment in the pharmaceutical industry. The penetrating power being negligible, its effectiveness is limited to exposed surfaces only. The most common source of artificial UV radiation is UV lamp. These UV lamps are called sterilizing lamps or germicidal lamps. Most vegetative bacteria are susceptible to UV radiations and spores are highly resistant. UV light is absorbed by the nucleic acids of the cell where it does the greatest damage. These rays induce the production of abnormal nucleotides such as thymine dimers. These interface in the process of DNA replication. Ultraviolet rays are used extensively in hospital operating rooms, in aseptic filling rooms, in the pharmaceutical industry (sterile product preparation), food and dairy industries for treatment of contaminated surfaces. UV rays have also been employed in sterilizing biological fluids such as blood plasma and vaccines. {li} Ionizing radiations (cold sterilization): X-rays, gamma rays and cathode rays are highly lethal to DNA and other vital cell constituents. They have very high penetration power and considerable energy. The factors that effect the lethal activity of ionizing radiations are oxygen, protective compounds, sensitizing agents, pH of culture, freezing, moisture and recovery conditions. X-rays: X-rays have considerable energy and penetration ability that is used to produce lethal effect on microorganisms. They are impractical for purposes of controlling microbial populations because they are very expensive and difficult to utilize efficiently. X-rays have been widely employed experimentally to produce microbial mutants. Gamma rays: Gamma rays are similar to X-rays but have higher energy and shorter wavelength. Gamma rays are commonly obtained using radioactive isotopes of Co. Two gamma rays are emitted in a succession as a result of disintegration of almost all of the unstable atoms of this isotope. The radiant energy particle makes a “direct hit” ‘on some essential substances such as DNA within the bacterial cell, causing ionization which results in the death of cell. Because of their high penetrating ability and microbicidal effect, gamma rays are ideal for sterilization of bulk materials such as packaged food, medical instruments etc. (iii) Cathode rays (electron-beam radiation): When a high-voltage potential is established between a cathode and an anode in an evacuated tube, the cathode emits beams of electrons, called cathode rays or electron beams. Special instruments arePharmaceutical Microblology (B.Pharm. Sem. HI) 7.9 Sterilization designed to produce electron beams of high intensity and velocity. One such instrument is the electron accelerator, which is extensively used for sterilization of drugs, surgicals and other materials. Ionizing radiation is a satisfactory method of sterilization of antibiotics like benzyl pencillin, streptomycin sulphate, polymyxin sulphate etc., vitamins such as ascorbic acid, sulphonamides, lactose, talc etc. Irradiations have also been applied to inactivate suspensions of influenza, vaccinia, rabies and poliomyelitis viruses for use as vaccines. (d) Filtration (mechanical) methods: This is a non-thermal method of sterilization used widely in the pharmaceutical industry where heat-labile solutions are to be sterilized. This is useful for large volume solutions, eye drops, antibiotic solutions, sera and carbohydrate solutions. This method is also useful for separation of bacteriophages and bacterial toxins from bacteria and for the isolation of microorganisms which are scanty in fluids. The process of sterilization by filtration consists of the following main stages for solutions. 1. Passage of the solution through a previously sterilized bacteria-proof filter unit. 2. Aseptic transfer of the filtrate to sterile containers which are then sealed aseptically. 3. Testing of sample for sterility. Filter efficiencies are affected by their pore size, wall thickness, filtration rate, positive or negative pressures and nature of the liquid to be filtered. When a pharmaceutical product is sterilized by filtration, a sterile technique must be maintained throughout the operation and the filters together with all of the assembly must be sterilized before use. The following types of filters have been used for sterilization of different pharmaceuticals. (I) Asbestos filter (Seitz filter): They are disposable, single-use discs made up of asbestos (magnesium trisilicate). It is supported on a perforated metal disc within a metal funnel (Fig. 7.4). It is then fitted on to a sterile flask through a silicone rubber bung. The fluid to be sterilized is put into the funnel and flask is connected to the exhaust pump. After completion of filtration, the filter is discarded and the entire unit sterilized. The pore size of filters range from 0.01 to 5 microns. (ii) Sintered glass filters (fritted glass filter/morton filters): Borosilicate glass is finely powdered in a ball mill and packed into disc moulds and heated until suitable adhesion takes place between the granules. The sintered discs are finally fused into funnels of a suitable size and shape (Fig. 7.5). Sintered glass filters are available in several different porosities but for filtration sterilization a number or grade 5 or 5 on 3 must be used. They have a low adsorptive property and can be cleaned easily. They are brittle and expensive and have a small area of filtration.Pharmaceutical Microb! Sem. 7.10 Sterilization | Sintered discs Funnel Fig. 7.4: Seitz filter Fig. 7.5: Sintered glass filter (iii) Filter candles (ceramic/Berkefield filter): These are manufactured in different grades of porosity and have been used widely for purification of water for industrial and drinking purposes. They are made of either porous porcelain or kieselguhr. They are usually encountered as cylindrical candles with comparatively thick walls (Fig. 7.6). These are depth filters with cellular walls and are available in various sizes. Fig. 7.6: Filter candies The filter is fixed to the filter assembly and placed in a mantle. The liquid to be filtered is poured into the mantle where vacuum forces it through the filter. After filteration, filter candle is removed from the assembly and filterate is transferred to a sterile container. These filters are inexpensive and available in different sizes. They are easily clogged and blocked and require high pressure for filtration.Pharmaceutical Microblology (B.Pharm. mm) 711 Sterilization (iv) Membrane filter (millipore/ultra filter): These are made up of various types of cellulose and cellulose esters. They are 150 ym thick and contain millions of microscopic pores ranging from 0.01 to 10 ym in diameter. The pore sizes most often used for sterilization are 0.45 ym + 0.02 ym (Millipore grade, HA) or 0.22 pm + 0.02 pm (Millipore grade, GS), particularly for very small bacterial contaminants. They are sterilized by autoclaving, in the holder or packed between thick filter pads to prevent curling. They are also available at ready sterilised form (by ethylene oxide or ionizing radiation). Membrane filters are supported on a rigid base of perforated metal, plastic or coarse sintered glass (Fig. 7.7). The HA grade filters are approximately 65 mi/min,/sq.cm. (GS - 22 ml/min/sq.cm) with a differential pressure of 70 cm mercury across the membrane. ee ‘~ S+—Fitter Fitter holder ‘Cotton ~ To suction pump Flask Filtrate (Sterile) (a) Components of filter (b) Membrane filtration assembly Fig. 7.7: Membrane filter Advantages of membrane filters are as follows: 1. All microorganisms are separated by process of sieving. 2. Membranes have a high and uniform porosity permitting a rapid rate of filtration. 3. Membranes are disposable. Hence, there is no cross contamination between filtered products. 4. Adsorption is very less. Disadvantages of membrane filters are as follows: 1. Prefilter is used before the membrane filter to avoid clogging and breaking. 2. They have less chemical resistance to certain organic solvents such as chloroform, ketones and esters. Membrane filters are routinely used in water purification and analysis, sterilization, sterility testing and for the preparation of solutions for parenteral use. They are also been used for the identification and enumeration of microorganisms from water samples and other materials.Pharmaceutical Microb! 7.12 Sterilization I. Chemical Methods (a) Gaseous sterilization: Gaseous sterilization may be defined as the destruction of all living microorganisms with a chemical in a gaseous or vapour state. Material substances, which are adversely affected by dry and moist heat are than sterilised by this method. All these gases are toxic to human beings above certain concentrations and may exhibit other unpleasant or undesirable side effects. Although ethylene oxide is the most widely used gaseous sterilization agent in pharmaceutical and medical fields, other chemicals used are formaldehyde and B- propiolactone. In addition to these, various glycols, methyl bromide and alcohol have been used for room sterilization. () Formaldehyde (HCHO): This gas is generated by heating a concentrated solution of formaldehyde. Formaldehyde in aqueous solution is known as formalin and contains 37 to 40% formaldehyde. Vapourisation of formaldehyde, either from formalin or paraformaldehyde, is used to sterilize an enclosed area. Formaldehyde gas is generated by adding 150 gm of KMnO, to 280 ml formalin for every 1,000 cu. ft. of room volume. After the start of generation of formaldehyde vapour, the doors should be sealed and left unopened for 48 hours. About 70% relative humidity and 22°C temperature gives best sterilization results. Formaldehyde is an extremely reactive chemical. It combines readily with vital organic nitrogen compounds such as proteins and nucleic acids. It is a bactericidal agent with poor penetrating power. It kills both vegetative cells and spores. Formaldehyde in gaseous form can be used for disinfection and sterilisation of enclosed areas such as operation theatres, hospital rooms, aseptic area and microbiology laboratories. (i) B-Propiolactone (BPL): This heterocyclic ring compound is a colourless liquid at room temperature with a high boiling point (155°C). H,C - CH, | | Oo - C=O It is capable of killing all microorganisms and is very active against viruses. BPL vapour is approximately 25 times more active as a disinfectant than formaldehyde gas, about 4000 times more active than ethylene oxide and about 50,000 times more active than methyl bromide. It is highly bactericidal and used in concentrations of 2 to 5 mg/litre. It has a low power of penetration and shows irritation and carcinogenic properties. Hence, it is not recommended for use in pharmaceutical applications.Pharmaceutical Microbiology (B.Pharm. Sem.) 7.13 _Sterilzation. (ili) Ethylene oxide: It is a colourless liquid with a boiling point of 10.8°C. It is highly inflammable and may be explosive when mixed with air in concentrations greater than 3%. “?* oO Its mixtures with carbon dioxide or fluorinated hydrocarbons (freons) in certain proportions makes ethylene oxide non-inflammable. The carbon dioxide and the freons act as Inert diluents which prevent flammability. Effect of ethylene oxide as a sterilizing agent depends on concentration of gas, temperature, moisture, time, conditions and accessibility of the micoorganisms. Concentration and time relationship commonly used for sterilization is given in Table 7.3. Table 7.3: Concentration and time relationship of ethylene oxide as a sterilizing agent : Concentration (mg/lit.) | Exposure time (hours) 44 24 88 10 442 4 884 | 2 Action of ethylene oxide is due to its power of alkylating the amino, carboxyl, hydroxyl and sulphydryl groups in the enzymes and protein molecule. It reacts with DNA and RNA. In this reaction the ring in the ethylene oxide molecule splits and attaches itself where the hydrogen is present CH, —CH, + enzyme — SH -» enzyme — S — CH, CH, OH \Z oO active inactive Ethylene oxide is a powerful sterilizing agent for heat and moisture sensitive materials. It can be used for sterilization of medical and biological preparations, catgut, plastic equipments, antibiotics, plaster bandages, culture media, hospital bedding, food stuff, heavy equipment, books, clothing and soil. Different methods used to control the microbial growth are summarized in Table 7.4.Pharmaceutical Microbiology (B.Pharm. Sem. 11) __7.14 Sterilization Table 7.4: Methods of sterilization used to control microbial growth | Sterilization — Mechanism Instrument/chemical | Temperature/ | Applications method ___ of action | Dose + | Dry heat sterilization — s —> | (@) Red heat Burning Bunsen burner Till red hot Inoculating aoe bumer Suttabie temp. to | Contaminated Incineration Burning to Bunsen or any jut temp. (b) I adil other burn Gressings, animal carcasses, | bags. (c) Hot alr Oxidation Hot alr oven 160°C for 2 hrs. Instruments, sterilization | needles, glassware, sealed materials like olls, dry | powder etc. | Moist heat sterilization . , , (a) Below 100°C Protein Water bath 65 to 75°C for 10 | Serum, body denaturation min fluids and vaccines (b) At 100°C Protein Water bath 100°C for 10-20 Glass, metal denaturation min and rubber tems (c) Above 100°C Protein Autoclave 121°C for 1S min | Culture media, denaturation | surgical equipments, ——— $e L _| solution | Radiation sterilization (a) Ionizing Destruction of Cobalt 60 (x-rays, 2.5 Mrad DNA gamma rays) food, antibiotics, hormones sutures, plastic | srringess. canulas | (b) Non-tontzing Damage to UV lamps, UV units 250-260 nm Hospital DNA wavelength for operating 30 min. rooms, laboratory, | Gaseous sterilization — words. (a) Formaldehyde Oenaturation Formaldehyde =| 150 gm KMnO, in | Hospital | of protein and 280 mi formalin _ rooms, aseptic | nucleic acid for 1,000 cu. ftof area room (b) Ethylene oxide — Denaturation Ethylene oxide 44 mo/lit for 24 Medical and of protein and hrs OR 88 mg/lit_ biological enzyme for 2 hrs. Preparations, catgut, plastic | saapments, Filtration antibio! oe paperaion of fee Filter and ous size of filter | Sterilizing from liquids 35 um (HA) or | liquids, 0.22 ym (GS) enzymes, vaccines‘Pharmaceutical Microblology (B.Pharm. Sem.) 7.15 ___Sterilzation, (b) By using disinfectants or antimicrobial agents: Chemical agents most commonly used as disinfectants and antiseptics are phenols, alcohols, halogens, dyes, aldehydes etc. These chemical agents and its mode of action, properties, structures and applications are given in Chapter No. 10. (Please refer Chapter No. 10, Disinfectants). 7.3 STERILIZATION CRITERIA The bioburden: It is necessary to know the initial number of microorganisms present in a given product or associated with a given material for selection of parameters for any method intended to kill microorganisms. This initial number is called ‘bioburden' or ‘bioload’. For example, it has been the practice to choose the time and temperature relationship for steam sterilization to ensure that a large number of the most resistant pathogens would be killed. The time and temperature chosen in such a steam sterilization process is also greatly in excess of the treatment necessary to kill the small number of heat-sensitive contaminants likely to be present in pharmaceutical solutions. Sensitivity of microorganisms: Microorganisms show their varying degrees of resistance to heat, radiation and chemicals. The vegetative forms of bacteria and fungi are most sensitive. They are about a hundred to thousand times more sensitive to ionisation and UV-radiation than the bacterial spores. The thermophilic bacteria, smaller viruses and mould spores are killed at temperatures between 70 to 90°C, while bacterial spores may be destroyed at 90 to 120°C temperatures. Ideally, the sterilising treatment to be used should be decided for each specific load with prior knowledge of the type and numbers of contaminating microorganisms present. This is indeed the situation for official sterilization methods which must be capable of general application and modem pharmacopoeial recommendations are derived from a careful analysis of experimental data on bacterial spore survival following treatments with heat, ionizing radiation or gas. Death rates or survivor curve: Death in a microbial population is determined by assessing the reduction in the number of viable microorganisms resulting from contact with a given destructive force. This can be Fepresented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Fig. 7.8). The death of a Population n cells exposed to heat, radiation or toxic chemicals is often found to follow first order inetics,lization Pharmaceutical Microb! 8.Pharm. Sem. 7.16 Steril If No is the initial number of microorganisms and N, is the number of microorganisms surviving after time 't’, the death rate constant (inactivation constant) ‘K’ can be calculated as follows: N, = Noe* InN, = InNo—Kt " Kt logioN, = logioNo-3 393 Kt 2303 = !09:0No—logio Ny 2303 (No t logio N, nx " ° 102) 20(4) 30(6)40(8) Time in minutes at 118°C (or radiation dose, x10" rad) Fig. 7.8: Microbial survivor curve The determination of death rate provides the facility to compare the resistance of the same microorganisms at different temperatures or to compare the resistance of different microorganisms to the same lethal agent. e.g. temperature, radiation, chemicals etc. Death rates may also be used to give a quantitative measure of the effect of environmental factors such as pH, osmolarity and the presence of various chemicals on the sterilization process. Terms used for expressions of resistance: (il) D-vatue or decimal reduction time: Time in minutes at any defined temperature to destroy 90% viable microorganisms is called D-value (Fig. 7.9 a). It usually has a subscript showing the temperature at which it was measured, @.g. Dis% OF Dy21%. It is one of the functions to indicate the efficiency of sterilization process.0 10 20 0 40 50 04 1 10 100 Time (minutes) D- value (minutes, log scale) (a) (0) Fig. 7.9: Calculation of (a) D-value and (b) Z-value (il) Z-value or thermal destruction value: The Z-value is the number of degrees of temperature change required to produce a 10-fold change in D-value (Fig. 7.9 b). This relates the heat resistance of a microorganism to changes in temperature. Bacterial spores have a Z-value in the range 10 to 15°C while most non-sporing organisms have Z-values of 4 to 6°C. (iii) F-value: In the food industry a ‘Unit of lethality’ has been devised, called the F-value. This has been defined as the equivalent in minutes of 121°C of all heat considered with respect to its capacity to destroy spores or vegetative cells of a particular organism. The F-value can be used to calculate the probable number of survivors remaining in a load as: F = D(logN,-log N) where D No D-value at 121°C of the organism Initial population number/unit volume N_ = Final population number/unit volume (iv) Quo - value or temperature: coefficient: It is the increase in the reaction or killing rate brought about ture of 10°C, ie. The values are approximately 14 for moist heat processes, 4 for dry heat and 2 for many chemical changes. The Qio - value is employed more frequently for chemical disinfection than for heat sterilization. This also gives a measure of the relative resistance of different microorganisms and describes the change in the death rate over a 10°C change in temperature.Pharmaceutical Microbiology (8.Pharm. Sem. 11) 7.18 Sterilization (v) Inactivation factor: The inactivation factor is the degree to which the viable population of organisms is reduced by the treatment applied and is obtained by dividing the initial viable count by the final viable count. This can be expressed in terms of the D-value for the organism. where t is the holding time at sterilizing temperature and D is D-value for a marker organism at that temperature. This is the simplest method of calculating the probability of achieving sterility for any given initial survival level. 7.4 STERILITY INDICATORS It is essential that strict controls are carried out on products to be labelled ‘sterile’. Such controls must then ensure, the absence of viable microorganisms from these products. There are basically two types of controls: 1. Controls on the process of sterilization ie. sterilization monitors or sterilization indicators. 2. Sterility testing of the products. Monitoring of the sterilization process can be achieved by the use of physical, chemical or biological indicators of the sterilization performance. 1. Physical indicators: (@) ~~ Moist heat: A master process record (MPR) is prepared as part of the validation procedure for a particular autoclave and for each specified product and load configuration. This may then be used as a reference for the process record obtained from a single thermocouple placed in a strategic part of each load (batch process record, BPR). The MPR should be checked at annual intervals and whenever significant changes occur in the BPR when compared with the MPR. Microprocessor — controlled sterilization cycles are now a part of modem autoclaves. Pressure is measured by pressure gauges OF through pressure transducers. (i) Dry heat: In dry heat sterilization processes, temperature record chart is made of each sterilization cycle and is compared against a master temperature record. (ii) Radio sterilization: A plastic dosimeter gives an accurate measure of the radiation dose absorbed and is considered to be the best technique currently available for the radio sterilisation process. (iv) Gaseous methods: For gaseous sterilization procedures, elevated temperatures are monitored for each sterilization cycle by temperature probes and routine leakPharmaceutical Microb! (B.Pharm. Sem. 7.19 Sterilization tests are performed to ensure gas-tight seals. Gas concentration is measured independently of pressure rise, often by reference to the weight of gas used. Pressure and humidity measurements are recorded. (v) Filtration: Bubble point pressure test is a technique employed for determining the pore size of filters and may also be used to check the integrity of certain types of filter devices immediately after use. The principle of the test is that the filter is soaked in an appropriate fluid and pressure is applied to the filter. The Pressure difference when the first bubble of air breaks away from the filter is equivalent to the maximum pore size. When the air pressure is further increased slowly, there is general eruption of bubbles over the entire surface. The Pressure difference is equivalent to the mean pore size. 2. Chemical Indicators: Chemical monitoring of a sterilization Process is based on the ability of heat, steam, sterilant gases and ionizing radiation to alter the chemical or Physical characteristics of a variety of chemical substances, (i) Browne's tubes: The most commonly used chemical indicators for heat Processes are Browne's tubes. These are small sealed tubes containing a reaction mixture and an indicator. Exposure to high temperature completes the reaction producing a change in the colour of the indicator (Table 7.5). All four types change from red through yellow brown to green, the latter colour only being achieved after a Specified time at the given temperature. Table 7.5: Types of Browne's tubes Browne's Method of sterilization | Temperature | Colour of indicator tube eg Type I Moist heat | 126 Black spot Type I High vacuum moist heat | 130 or more Yellow spot Type I Dry heat 160 Green spot Type IV Dry heat infra-red conveyor oven 180 Blue spot (ii) Witness tubes: Witness tubes consist of single crystalline substances of known melting point contained in glass tubes e.g. sulphur (115°Q), succinic anhydride (120°C), benzoic acid (121°C) ete. A dye may be included to show more clearly that the crystals have melted. Such a device only indicates that a certain temperature has been reached. Exposure time can be calculated by Putting the crystals in one end of an ‘hour-glass' tube, the volume of the crystals an i constriction of the tube being adjusted so that the time for b r transfer of the melt is the same as that required for the Sterilisation at the requir. ‘ed temperature.Pharmaceutical Microb! [B.Pharm. Sem. 7.20 Sterilization (iii) Heat-sensitive tape: Heat-sensitive tape is used quantitatively in the Bowie-Dick test. This is a test to determine that all air has been removed from dressings and that subsequent steam penetration has been even and rapid. The tape is placed suitably wrapped at the centre of a test pack. All the bars on the tape should change colour to demonstrate full penetration of the steam. {lv) Royce sachet: The Royce sachet is a chemical indicator for ethylene oxide sterilization. This consists of a polythene sachet containing magnesium chloride, HCI and a bromophenol blue indicator. A given concentration-time exposure to ethylene oxide results in the formation of ethylene chlorohydrin and a colour change from yellow to purple. {v) Chemical dosimeters: Chemical dosimeters give an accurate measure of the radiation dose absorbed and are considered to be the best technique currently available for controlling radiation sterilization. Qualitative indicators made of radiosensitive chemicals impregnated in plastic are also available. The indicator changes from yellow to red during irradiation. 3. Blological indicators: Biological indicators consist of a suitable organism deposited on a carrier and are distributed throughout the sterilizer load. At the end of the sterilization process, the units are recovered and cultured to determine the presence or absence of survivors. The biological indicator measures sterilization processes directly and is able to integrate all sterilization parameters. The selected organism should possess high and reproducible resistance to the sterilizing agent, should be genetically stable, readily characterizable and non-pathogenic. The viability of the organisms, the storage conditions before use and the incubation and culture conditions after sterilization must be standardized for the results. The organisms used as biological indicators are usually resistant bacterial spores (Table 7.6). Table 7.6: Biological indicators for monitoring sterilization processes Sterilization process __ Species D-value Autoclave at 121°C Bacillus stearothermophilus = 1.5 min Clostridium sporogenes 0.8 min Dry heat at 160°C Bacillus subtilis var. niger 5-10 min Ethylene oxide at 600 mg/lit. Bacillus subtilis var. niger 2.5 min (Temperature - 54°C, 60% - relative humidity) Tonizing radiation Bacillus pumilus 3 kGy (0.3 M rad) Membrane filter (0.45 ym pore size) Serratia marcescens Membrane filter (0.22 ym pore size) Pseudomonas diminuta