Sterilization

This powerpoint describes about sterilization which is a basic technique applied by life science members who are performing microbiological, molecular biology, genetic engineering, recombinant DNA technology, molecular genetics techniques and also this process in performed in health care sectors to prevail aseptic conditions,

1. STERILIZATION DR.N.C.J.PACKIA LEKSHMI NICHE

2. INTRODUCTION Sterilization is making a substance free from all micro organisms both in vegetative and sporing states. Spore is a reproductive structure that is adapted for dispersal and surviving for extended periods of time in unfavourable conditions. Spores form part of the lifecycles of many bacteria, plants, algae , fungi and some protozoa.

3. Terms in Sterilization Disinfection : The destruction or removal of all pathogenic organisms capable of giving rise to infection. Disinfection does not affect spore state organisms. Antisepsis : The term is used to indicate the prevention of infection, usually by inhibiting the growth of bacteria in wounds or tissues. This is done by the antiseptics : Chemicals or disinfectants which can be safely applied on skin or mucous membrane to prevent infection by inhibiting the growth of bacteria. Bactericidal agents / germicides : Those which able to kill bacteria.

4. Cont… Bacteriostatic agents : Only prevent multiplication of bacteria, but they remain alive. Cleaning : Important preparatory step before sterilization or disinfection, by removing soil and other dirt. Decontamination : The process of rendering an article or area free of contaminants, including microbial, chemical, radioactive and other hazards.

5. Methods of Sterilization Physical Chemical Physical agents : Sunlight Drying Heat - Dry heat: flaming, incineration, hot air, Moist heat: pasteurization, boiling, steam under pressure. Filtration: candles, asbestos pads, membranes Radiation Ultrasonic and sonic vibrations.

6. Physical methods of sterilization Sunlight : Action primarily due to UV rays however, effects vary due to places Eg : In tropical country, the germicidal effect is better than 4 seasoned countries. Bacteria in water are readily destroyed by sunlight. Drying : Moisture is essential for growth of bacteria. Drying in air has deleterious effect on many bacteria. However, spores are unaffected. Therefore, it is not really unreliable.

7. Heat : Most reliable method of sterilization and should be the method of choice. Dry Heat & Moist Heat The factors influencing sterilization by heat: Nature of heat-dry or moist Temperature and time Number of microorganisms present Characteristics of organisms –species, strain, sporing capacity Type of material from which organism have to be eliminated. Killing effect is due to protein denaturation , oxidative damage and toxic effect of elevated level of electrolytes. Killing effect of moist heat due to denaturation and coagulation

8. Thermal Death Time:TDT “Minimum time required to kill a suspension of organisms at a predetermined temperature in a specified environment. Thermal death time is inversely proportional to temperature. TDT is increased in presence of organic substance, proteins, nucleic acid, starch, gelatin , sugar , fats, oils.

9. Dry heat: Flaming : Heating over fire, till they become red hot. Flaming is done to inoculation loops and straight-wires in microbiology labs for streaking. Leaving the loop in the flame of a Bunsen burner or alcohol burner until it glows red ensures that any infectious agent is inactivated. Instruments like: Point of Forceps, Spatulas, Inoculating loops and Wires. (Inoculating loop is better dipped in disinfectant first before flaming to prevent spattering)  A variation on flaming is to dip the object in a 70% or more concentrated solution of ethanol, then briefly touch the object to a Bunsen burner flame. The ethanol will ignite and burn off rapidly, leaving less residue than a gas flame.

10. Incineration : It is a process that involves the combustion of organic substances contained in waste materials. Items: contaminated cloth, animal carcasses and pathological material. PVC, polythene can be dealt. (However, polystyrene will emit black smoke. Hence should be autoclaved in appropriate container.) 

11. Hot air oven Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 C (122 to 572 F) . There is a thermostat controlling the temperature. This is the most widely used method of sterilization by dry heat. Items: glassware, forceps, scissors, scalpels, all-glass syringes, swabs, liquid paraffin, dusting powder, fats, grease. (Materials should be properly arranged to allow free circulation of air )

12. They were originally developed by Pasteur. Generally, they use a thermostat to control the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminium trays and may have an on/off rocker switch, as well as indicators and controls for temperature and holding time. The capacities of these ovens vary. Power supply needs vary from country to country, depending on the voltage and frequency (hertz) used. Temperature sensitive tapes or biological indicators using bacterial spores can be used as controls, to test for the efficacy of the device during use.

13. Precautions Glass wares should be dry. Oven should not be over loaded. Articles are to be arranged in a manner to allow free circular of air. Door of the Oven should be opened after it cools down (2Hours).  The standard settings for a hot air oven are: 1.5 to 2 hours at 160 °C (320 °F) 6 to 12 minutes at 190 °C (374 °F)

14. Advantages & Disadvantages They do not require water and there is not much pressure build up within the oven, unlike an autoclave, making them safer to work with. Suitable to be use in a laboratory environment. They are much smaller than autoclaves but can still be as effective. As they use dry heat instead of moist heat, some organisms like prions , may not be killed by them every time.

15. Moist heat Sterilization sterilization techniques that uses hot air that is heavily laden with water vapor and where this moisture plays the most important role in the sterilization. Heating an article is one of the earliest forms of sterilization practiced. The various procedures used to perform moist heat sterilization process cause destruction of micro-organisms by denaturation of macromolecules.

16. Methods Moist heat can be categorized into 3 groups: Temperature below 100° C Temperature at 100° C Temperature above 100° C

17. Temperature below 100 o C Pasteurization is a process in which water and certain packaged and non-packaged foods (such as milk and fruit juice) are treated with mild heat, usually to less than 100°C (212 °F), to eliminate pathogens and extend shelf life. The process is intended to destroy or deactivate organisms and enzymes that contribute to spoilage or risk of disease, including vegetative bacteria, but not bacterial spores. Since pasteurization is not sterilization, and does not kill spores, a second "double" pasteurization will extend the quality by killing spores that have germinated.

18. Cont… The process was named after the French scientist Louis Pasteur, whose research in the 1880s demonstrated that thermal processing would inactivate unwanted microorganisms in wine. Spoilage enzymes are also inactivated during pasteurization. Today, pasteurization is used widely in the dairy industry and other food processing industries to achieve food preservation and food safety.

19. Pasteurization Process In acidic foods (pH <4.6), such as fruit juice and beer, the heat treatments are designed to inactivate enzymes (pectin methylesterase and polygalacturonase in fruit juices) and destroy spoilage microbes (yeast and lactobacillus). Due to the low pH of acidic foods, pathogens are unable to grow. The shelf-life is thereby extended several weeks. In less acidic foods (pH >4.6), such as milk and liquid eggs, the heat treatments are designed to destroy pathogens and spoilage organisms (yeast and molds). Not all spoilage organisms are destroyed under pasteurization parameters, thus subsequent refrigeration is necessary.

20. Technique Food can be pasteurized in two ways: either before or after being packaged into containers. When food is packaged in glass, hot water is used to lower the risk of thermal shock. Plastics and metals are also used to package foods, and these are generally pasteurized with steam or hot water since the risk of thermal shock is low. Most liquid foods are pasteurized using continuous systems that have a heating zone, hold tube, and cooling zone, after which the product is filled into the package. Plate heat exchangers are used for low-viscosity products such as animal milks, nut milks and juices. A plate heat exchanger is composed of many thin vertical stainless steel plates which separate the liquid from the heating or cooling medium. Scraped surface heat exchangers contain an inner rotating shaft in the tube, and serve to scrape highly viscous material which might accumulate on the wall of the tube .

21. Methods of Pasteurization Holding period: 63° C , 30 minutes (holder method ) 72° C , 15-20 minutes flash method - followed by cooling quickly to 130°c or lower. Target: all nonsporing pathogens Eg : Mycobacteria , Brucellae , Salmonella. Coxiellaburnetti , relatively heat resistant, may survive the holder method. More than 100 o C for 2 seconds – ultra pasteurization

22. Temperature at 100° C Boiling Not recommended for sterilising but used for disinfection. sterilization may be promoted by addition of 2% sodium bicarbonate to the water. Holding period: 10-30 minutes. Steam at atmospheric pressure ( 100° C ) Used to sterilize culture media. This is an Inexpensive method Holding period: 100° C , 20 minutes on three succesive days (intermittent sterilization). Principle: first exposure kills vegetative bacteria and then the next exposure will kill vegetative bacteria that matures from the spore. 

23. Temperature above 100 o C Steam under pressure Autoclave/steam sterilizer--: autoclave is a device that uses steam to sterilize equipment and other objects. This means that all bacteria, viruses, fungi, and spores are inactivated. However, prions may not be destroyed by autoclaving at the typical 134° C for 3 minutes or 121° C for 15 minutes. Principle: Water boils when its vapour pressure equals the surrounding atmosphere. Thus, when pressure inside closed vessels increases, the temperature at which water boils increases too. Holding period: varies. Temperature: between 108° C and 147° C . Items: dressings, instruments, laboratory ware, media and pharmaceutical products.

24. Types of steam sterilizer Laboratory autoclaves Hospital dressings sterilizers Bowl and instrument sterilizers Rapid cooling sterilizers domestic pressure cooker can be used as a steriliser . Recommended temperature and duration: Temperature( o c ) Duration(min) 121 o C 15 lbs for 15 minutes

25. Horizontal autoclave Vertical Autoclave

26. Tyndallization Tyndallization is a process dating from the nineteenth century for sterilizing substances, usually food, named after its inventor, scientist John Tyndall, that can be used to kill heat-resistant endospores . Although considered old-fashioned, it is still occasionally used. A simple and effective sterilizing method commonly used today is autoclaving: heating the substance being sterilized to 121 °C for 15 minutes in a pressured system. If autoclaving is not possible because of lack of equipment, or the need to sterilize something that will not withstand the higher temperature, unpressurized heating for a prolonged period at a temperature of up to 100 °C, the boiling point of water, may be used. The heat will kill the bacterial cells; however, bacterial spores capable of later germinating into bacterial cells may survive. Tyndallization can be used to destroy the spores .

27. Cont… Tyndallization essentially consists of heating the substance to boiling point (or just a little below boiling point) and holding it there for 15 minutes, three days in succession. After each heating, the resting period will allow spores that have survived to germinate into bacterial cells; these cells will be killed by the next day's heating. During the resting periods the substance being sterilized is kept in a moist environment at a warm room temperature, conducive to germination of the spores. When the environment is favourable for bacteria, it is conducive to the germination of cells from spores, and spores do not form from cells in this environment (see bacterial spores). The Tyndallization process is usually effective in practice. But it is not considered totally reliable—some spores may survive and later germinate and multiply. It is not often used today, but is used for sterilizing some things that cannot withstand pressurized heating, such as plant seeds.

28. Filtration Fluids that would be damaged by heat, irradiation or chemical sterilization, such as drug solution, can be sterilized by microfiltration using membrane filters. This method is commonly used for heat labile pharmaceuticals and protein solutions in medicinal drug processing. A microfilter with pore size of usually 0.22 µm will usually effectively remove microorganisms. Some staphylococcal species have, however, been shown to be flexible enough to pass through 0.22 µm filters. In the processing of biologics, viruses must be removed or inactivated, requiring the use of nanofilters with a smaller pore size (20–50 nm) are used. Smaller pore sizes lower the flow rate, so in order to achieve higher total throughput or to avoid premature blockage, pre-filters might be used to protect small pore membrane filters. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems also reduce particulate accumulation and blockage.

29. Cont… Membrane filters used in production processes are commonly made from materials such as mixed cellulose ester or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre-sterilized disposable units in sealed packaging or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use and sometimes before use. The nondestructive integrity test assures the filter is undamaged and is a regulatory requirement. Typically, terminal pharmaceutical sterile filtration is performed inside of a cleanroom to prevent contamination.

30. Radiation Sterilization can be achieved using electromagnetic radiation, such as electron beams, X-rays, gamma rays, or irradiation by subatomic particles. Electromagnetic or particulate radiation can be energetic enough to ionize atoms or molecules (ionizing radiation), or less energetic (non-ionizing radiation).

31. Non Ionizing radiation Ultraviolet light irradiation (UV, from a germicidal lamp) is useful for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt (which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

32. Ionizing Radiation Gamma radiation is very penetrating, and is commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. It is emitted by a radioisotope, usually cobalt-60 (60Co) or caesium-137 (137Cs), which have photon energies of up to 1.3 and 0.66 MeV , respectively. Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or X-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. Because electrons carry a charge, electron beams are less penetrating than both gamma and X-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.

33. Cont… High-energy X-rays allow irradiation of large packages and pallet loads of medical devices. They are sufficiently penetrating to treat multiple pallet loads of low-density packages with very good dose uniformity ratios. X-ray sterilization does not require chemical or radioactive material: high-energy X-rays are generated at high intensity by an X-ray generator that does not require shielding when not in use. X-rays are generated by bombarding a dense material (target) such as tantalum or tungsten with high-energy electrons, in a process known as bremsstrahlung conversion.

34. Cont… Irradiation with X-rays, gamma rays, or electrons does not make materials radioactive, because the energy used is too low. Generally an energy of at least 10 MeV is needed to induce radioactivity in a material. Neutrons and very high-energy particles can make materials radioactive, but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.

35. Sonication Ultrasonic cleaning is a process that uses ultrasound (usually from 20–40 kHz) to agitate a fluid. The ultrasound can be used with just water, but use of a solvent appropriate for the item to be cleaned and the type of soiling present enhances the effect. Cleaning normally lasts between three and six minutes, but can also exceed 20 minutes, depending on the object to be cleaned. Ultrasonic cleaners are used to clean many different types of objects, including jewelry, lenses and other optical parts, watches, dental and surgical instruments, tools, coins, fountain pens, golf clubs, fishing reels, window blinds, firearms, car fuel injectors, musical instruments, gramophone records, industrial parts and electronic equipment.

36. Fumigation Fumigation is a process of gaseous sterilisation which is used for killing of micro-organisms and prevention of microbial growth in air, surface of wall or floor. It is generally used in the pharmaceuticals, operation theatres, hospitals, hotels and offices and wherever required. Method of fumigation completely fills an area with gaseous fumigants to suffocate or poison the microbes & pests within.

37. Widely used fumigants include: • Formaldehyde • phosphine • 1,3-dichloropropene • chloropicrin • methyl isocyanate • hydrogen cyanide • sulfuryl fluoride • Iodoform • Methyl bromide Fumigation equipment

38. Formaldehyde A gas at room temperature, formaldehyde is colorless and has a characteristic pungent, irritating odor. When dissolved in water, formaldehyde forms a hydrate methanediol . A saturated water solution, that contains about 40% formaldehyde by volume or 37% by mass, is called "100% formalin". A typical commercial grade formalin may contain 10– 12% methanol polymerization. added to suppress oxidation

39. Cont… Formaldehyde at approximately 5% in a solution with water is used as a fumigant and disinfectant in hospitals. Formoldehyde kills microbes by alkylating the amino acids and sulfydral group of proteins and purine bases. In order to be effective, the gas has to dissolve in the film of moisture surrounding the bacteria, for this reason relative humidities in the order of 75% RH and temperature above 22°C.

40. Methods Electric Boiler Fumigation Method : For Each 1000 cu.ft 500ml of formaldehyde added in 1000ml of water in an electric boiler. Switch on the boiler, leave the room and seal the door. After 45 minutes switch off the boiler without entering in to the room. Potassium Permanganate Method : For every 1000 cubic feet add 450gm of Potassium permanganate (KMnO4) to 500 ml of formaldehyde. Take about 5 to 8 bowels with equally divided parts of formaldehyde and add equally divided KMnO4 to each bowel. This will cause auto boiling and generate fume.

41. Neutralisation After the initiation of formaldehyde vapour immediately leave the room and seal it for at least 48 hours Neutralise residual formalin gas with Ammonia by exposing 250ml of Ammonia per litre of Formaldehyde used. Place the Ammonia solution in the centre of the room and leave it for 3 hours to neutralise formalin vapour .

42. Phosphine Phosphine is the compound with the chemical formula PH3. It is a colorless, flammable, toxic gas. Pure phosphine is odorless, but technical grade samples have a highly unpleasant odor. Phosphine -producing materials have become the predominant fumigants used for the fumigation of OT & bulk-stored grain throughout the world. It is available in solid formulations phosphide or magnesium phosphide of aluminum. When exposed to heat and moisture the formulations release phosphine , a highly toxic gas. The time required for release of phosphine will vary with temperature and formulation. Phosphine is very toxic to all forms of animal life, hence exposure of human beings even to small amounts should be avoided. Phosphine can be produced via tablets or phosphine producing equipment. Phosphine is generaly used for fumigating huge quantity of food grain than operation theatre

43. Methyl Bromide Methyl Bromide is most widely used fumigant for quarantine purposes It is a preferred fumigant because of its good penetrating ability, rapid action, high toxicity to a broad spectrum of micro organisms and pests. The effectiveness of methyl bromide is based on the following: • Dosage of the fumigant • Duration of exposure • Temperature

44. Preparation for fumigation First the area intended to be fumigated is usually covered to create a sealed environment. The fumigant is released into the space to be fumigated. The space is held for a set period while the fumigant gas percolates through the space and acts on and kills any infestation. The space is neutralised & ventilated so that the poisonous gases are allowed to escape from the space, and render it safe for humans to enter. Windows, doors should be closed. AC and AHU should be switched off before starting fumigation. Area under fumigation, do not enter status label should be displayed on either side of the entrance. Fumigants are irritating and toxic to human eye, nose and throat. Therefore use of nose mask and goggles while doing fumigation is advisable.

45. Under certain conditions formaldehyde can react with hydrochloric acid and chlorine containing disinfectants such as hypochlorites to form chloromethyl a potential lung carcinogen. HCl and chlorine containing disinfectants must therefore be removed from areas before fumigation. Check levels of residual fumigant in the room with suitable air monitoring equipment( formaldameter or air sampling tubes). The procedure is best carried out overnight. After a period of not less than 12 hours the room must be well ventilated. OT is usable only when the level of formaldehyde is less than 2ppm.

46. Personal care while Fumigation Adequate care must be taken by wearing cap, mask,foot cover, spectacles. Formaldehyde is irritant to eye & nose; and it has been recognized as potential carcinogen. Immediately leave the room after the initiation of fumigants. So the fumigating employee must be provided with personal protective equipment.

47. Advantages of Fumigation Fumigants are toxic to all forms of life. Therefore, it is possible to control all life stages of Micro organisms. Fumigation is often the quickest way of controlling an infestation, saving time and money. Fumigants can reach where sprays, dusts, aerosols etc cannot reach. Reduced residue problems in treated areas. Fumigants are used where standards call for "zero Microbial tolerance" in products or living environments.

48. Disadvantages of Fumigation Generally Formaldehyde is used for OT fumigation. Based on the available evidence in OSHA’s record on Formaldehyde, it determines formaldehyde as is genotoxic , showing properties of both a cancer initiator and promoter. In humans formaldehyde exposure has been associated with cancers of the lung, nasopharynx and nasal passages. Symptoms of excessive exposure include respiratory irritation, itchy eyes, runny or stuffy nose, sore throat and headache.