Sterilization (physical methods)

A brief description of the various physical methods of sterilisation of materials used in health care field.

1. Sterilization Presented by Dr. Swapnaneel Pradhan

2. Introduction Microorganisms are ubiquitous. They are found in the surroundings, on inanimate objects and on the surface of the human body. Since they cause contamination , infection and decay , it becomes necessary to remove and/or destroy them. This is the objective of sterilization. Sterilization is defined as the process by which an article , surface or medium is freed of all living microorganisms either in the vegetative or spore state.

3. Important terminology Sterilization Steri1ization is a process by which all Living microorganisms, including viable spores, are either destroyed or removed from all articles body surface or medium. Disinfection It refers to a process that destroys or removes most if not all pathogenic organisms but not bacterial spores. Asepsis It is a process where the chemical agents (called antiseptics) are applied on to the body surfaces (skin), which kill or inhibit the microorganisms present on skin. Decontamination ( or Sanitization) It is the process of rendering an article or area free of contaminants, including all chemical, radioactive and other hazardous materials from an object or body surface.

4. Sterilization It results in reduction of at least 10 6 log colony-forming units of microorganisms and their spores. It can be achieved by a physical agent or a chemical agent (called chemical sterilant).

5. Disinfection It results in reduction of at least 10 3 log colon y -forming units of microorganism, but not spores. The primary goal in disinfection is to destroy potential pathogens, but it also substantially reduces the total microbial population. A g e n t s : Disinfectants can also be achieved by a physical agent or a chemical agent (called disinfectant) and they are normally used only on inanimate objects, nor on body surfaces.

6. Asepsis Antiseptics are agents that can be safely applied on the skin or mucous membrane to prevent infection by inhibiting the growth of bacteria. They prevent the entry of pathogen s into sterile ti s sues and thus prevents infection or sepsis. Howe v er, they are generally not as toxic as disinfectants as they must not destroy too much of host tissue.

7. Decontamination I t results in reduction of at lea s t 1 log c o lony-forming unit of most of the microorganisms, but not spores. Examples include manual or mechanical cleaning by soap and detergents to eliminate debris or organic matter from the medical devices.

8. The agent used for sterilization/disinfection can also be named with suffix based on whether they kill or inhibit the microorganisms. Suffix ' cide ' is used for the agents that can kill microorganisms. Examples include bactericide, virucide and fungicide. Suffix ' static ' is used for the agents that do not kill; but inhibit the microbial growth. If these agents are removed, growth will resume- for example, bacteriostatic and fungistatic. Although these agents have been described in terms of their effects on pathogens , it should be noted that they also kill or inhibit the growth of nonpathogens as well .

9. Factors Influencing Efficacy of Sterilant/Disinfectant Organ i sm l oa d: L arger microbial population requires a longer time to die than a smaller one. Na tu r e of o r gan i sms : It greatly influences the efficacy of the disinfectants or sterilizing agents. Concentration of the chemical agent or the temperature of heat sterilization. Na tur e of th e Ster il an t / disinfectan t s u c h as it s : Microbicidal ability Rapidity of action Ability to act in presence of organic matter R esidual activity Duration of exposure : More is the exposure time to sterilant / disinfectant , better is the efficacy. Temperature : An increase in the temperature at which a chemical acts often enhances its activity. A lower concentration of disinfectant or sterilizing agent can be used at a higher temperature. Local e n v ironm e nt : The microorganisms to be controlled are not isolated but surrounded by environmental factors that may either offer protection or aid in their destruction, e.g. pH, organic matter, biofilm etc.

10. Classification of sterilization/disinfection methods Physical Methods Drying Heat Dry Heat Flaming Incineration Hot air oven Moist heat Temperature below 100°C, e.g. pasteurization, water bath and inspissation Temperature at 100°C, e.g. boiling, steaming and tyndalllization Temperature above 100°C, e.g. autoclave Filtration: Depth filters and membrane filters Radiation Ionizing radiation: y rays, X-rays and cosmic rays Non ionizing radiation: Ultraviolet (UV) and infrared rays Ultrasonic vibration Chemical Methods Alcohol: Ethyl alcohol, isopropyl alcohol Aldehydes: Formaldehyde, glutaraldehyde, Orthophthaladehyde Phenolic: compounds: Cresol, Lysol chlorhexidine, chloroxylenol, hexachlorophene Halogens: Chlorine, Iodine, iodophors Oxidizing agents: Hydrogen peroxide, peracetic acid Salts: Mercuric chloride, copper salts Surface active agents: Quaternary ammonium compounds and soaps Dyes: Aniline dyes and acridine dyes Gas sterilization: low temperature steam formaldehyde Ethylene oxide (ETO} Betapropiolactone (BPL) Plasma sterilization

11. Physical Agents of sterilization

12. Drying Moisture is essential for the growth of bacteria. 7 0-80 % of the weight of the bacterial cell is due to water. Drying, therefore has a deleterious effect on many bacteria. Microwave drying machine Both drying and sunlight are not reliable. They do not affect many microbes, including spores.

13. Heat Heat is the most reliable and commonly employed method of sterilization/ disinfection. It should be considered as the method of choice unless contraindicated. two types of heat are used, dry heat and moist heat . Mechanism of action heat : Dry heat kills the organisms by charring , denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated levels of electrolytes. Moist heat kills the microorganisms by denaturation and coagulation of proteins. Materials containing organic substances require more time for sterilization/ disinfection.

14. Dry Heat

15. Flaming Items are held in the flame of a Bunsen burner either for a long time or short time. longer time exposure in flame till they become red hot : this is done for inoculating wires or loops, tips of forceps, etc. For shorter period of time without allowing the items to become red hot : this is done for fragile items, e.g. mouth of test lubes.

16. Incineration Incineration is used for the disposal of biomedical waste materials. It burns (sterilizes) the anatomical waste and microbiology waste by providing a very high temperature 870 - 1,200°C and thereby converting the waste into ash, flue gas and heat.

17. Hot Air Oven Pr eca ution s : The following precautions should be taken while using hot air oven. Overloading of hot air oven should be avoided. T he material should be arranged in a manner so that free circulation of air is maintained. Material to be sterilized should be dried completely. Cotton plugs should be used to close the mouths of test tubes, flasks, etc. Paper wrapping of the items should be done. Any inflammable material like rubber (except silicone rubber) should not be kept inside the o ven. the oven must be allowed to cool for two hours before opening the doors, since the glassware may crack by sudden cooling. St e rilization control: the effectiveness of the sterilization done by hot air oven can be monitored by: Biological indicator : Spores (10 6 ) of nontoxigenic strains of Clostridium tetani or Bacillus subtilis subspecies niger are used to check the effectiveness of sterilization by hot air oven. These spores should be destroyed if the sterilization is done properly. Thermocouples : It is a temperature measuring device that records the temperature by a potentiometer. Browne 's tube : It contains a heat sensitive red dye which turns green after being exposed to certain temperature for a definite period of lime. It was invented by Albert Browne in 1 930. Hot air oven is the most widely used method of sterilization by dry heat. It is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber. It is also fitted with a thermostat which maintains the chamber air at a chosen temperature. Temperature : A holding temperature and time of 160°C for 2 hours is required for sterilization in hot air oven. Materials sterilized : Hot air oven is the best method for sterilization of: Glassware like glass syringes, petri dishes, flasks, pipettes and test tubes. Surgical instruments like scalpels, forceps, etc. Chemicals such as liquid paraffin, fats, glycerol, and glove powder, etc.

18. Moist Heat

19. At temperature below 100°C

20. Pasteurization It is used for control of microorganisms from beverages like fruit and vegetables, juices, beer, and dairy products, such as milk. Two methods are available : Holder method (63 ° C for 30 minutes) and Flash method (72 ° C for 20 seconds followed by rapid cooling to 13 °C or lower). All nonsporing pathogens, including mycobacteria , brucellae and salmonellae are killed except Coxiella burnetii which being relatively heat resistant may survive in Holder method.

21. Water Bath It is used for disinfection of serum , body fluids and vaccines . • Bacterial vaccines are disinfected at 60 ° C for 1 hour. • Serum or heat labile body fluids can be disinfected at 56 ° C for one hour.

22. Ins pissation (fractional sterilization) It is a process of heating an article on 3 successive days at 80-85°C for 30 minutes by a special instrument called inspissator. Working principle : In inspissator, the first exposure kills all the vegetative forms, and in the intervals between the heating; the remaining spores germinate into vegetative forms which are then killed on subsequent heating. Uses : Inspissation is useful for sterilization of egg and serum based media which generally get destroyed at higher temperatures: Egg based media : e.g. Lowenstein - Jensen medium and Dorset ' s egg medium. Serum based media: e.g. Loeffler ' s serum slope.

23. At temperature 100°C

24. Boiling Boiling of the items in water for 15 minutes may kill most of the vegetative forms but not the spores, hence not suitable for sterilization of surgical instruments. T hough boiling is a simple, easily available option to most people, however, boiling can be hazardous and not effective; hence should not be used if better methods are feasible.

25. Steaming Koch ' s or Arnold ' s steam sterilizer are useful for those media which are decomposed at high temperature of autoclave. The articles are kept on a perforated tray through which steam can pass. They are exposed to steam (100 °C ) at atmospheric pressure for 90 minutes. Most of the vegetative forms are killed by this method except thermophiles and spores.

26. Tyndallization (intermittent sterilization) N amed after John Tyndall, it involves steaming at 100°C for 20 minutes for 3 consecutive days . The principle is similar to that of inspissation , except that here, the temperature provided is 100°C , instead of 80°C . It is used for sterilization of gelatin and egg, serum or sugar containing media, which are damaged at higher temperature of autoclave.

27. At temperatures above 100°C

28. Autoclave Principle of Autoclave Autoclave functions similar to a pressure cooker and follows the general laws of gas: Water boils when its vapor pressure equals that of the surrounding atmosphere. So, when the atmospheric pressure is raised, the boiling temperature is also raised. At normal pressure, water boils at 100°C but when pressure inside a closed vessel increases, the temperature at which water boils also increases.

29. Components of Autoclave Autoclave comprises of three parts: a pressure chamber, a lid and an electrical heater. Pressure chamber consists of- It is a large cylinder (vertical or horizontal) in which the materials to be sterilized are placed. It is made up of gunmetal or stainless steel and placed in a supporting iron case A steam jacket (water compartment) The lid is fastened by screw clamps and rendered air tight by an asbestose washer. The lid bears thee following: A discharge tap for air and steam discharge A pressure gauge (sets the pressure at a particular level) A safety valve (to remove the excess steam) An electrical heater is attached to the Jacket; that heats the water to produce steam.

30. Procedure The cylinder is filled with sufficient water and the material to be sterilized is placed inside the pressure chamber. The lid is closed and the electrical heater is put on. The safety valve is adjusted to the required pressure. · After the water boils, the steam and air mixture is allowed to escape through the discharge tap till all the air has been displaced. This can be tested by passing the steam-air mixture liberated from the discharge tap into a pail of water through a connecting rubber tube. When the air bubbles stop coming in the pail, it indicates that all the air has been displaced by steam. The discharge tap is then closed. The steam pressure rises inside and when it reaches the desired set level (e.g. 15 lbs per square inch in most cases), t he safety valve opens and excess steam escapes out. T he holding period is counted from this point of time, which is about 15 minutes in most cases. After the holding period, the electrical heater is stopped and the autoclave is allowed to cool till the pressure gauge indicates that t he pressure inside is equal to the atmospheric pressure. T he discharge tap is opened slowly and air is allowed to enter the autoclave. The lid is now opened and the sterilized materials are removed.

31. Sterilization Conditions 121°C for 15 minutes at pressure of 15 pounds per square inch (psi): This is the most commonly used sterilization condition for autoclave. 126°C for 10 minutes at pressure of 20 psi 133°C for 3 minutes at pressure of 30 psi

32. Uses of Autoclave Autoclave is particularly useful for media containing water that cannot be sterilized by dry heat. It is the method of choice for sterilizing the following: Surgical instruments Culture media Autoclavable plastic containers P lastic tubes and pipette tips Solutions and water Biohazardous waste Glassware (autoclave resistible)

33. Types of Autoclave There are different types of autoclaves available: • Gravity displacement type autoclave: It is the most common type used in laboratories. They are available in various sizes and dimensions. • Vertical type (small volume capacity) • Horizontal autoclave (Large volume capacity) • Positive pressure displacement type autoclave • Negative pressure (vacuum) displacement type.

34. Disadvantages of Gravity displacement type: The method of ai r d isch a r g e i s i n efficient , and it is difficult to decide when the discharge is complete. Materials remain m o ist after removal from the a u toclave . Advantages of vacuum displacement type: They u sually have automatic cycle control. The air i s drawn out , whereby steam penetrates faster and the tim e required for sterilization is shorter. Onc e the sterilization is over, post - cycle vacuum can be programmed for quick drying .

35. Precautions Autoclave should not be used for sterilizing waterproof materials, such as oil and grease or dry materials, such as glove powder. Materials are loaded in such a way that it allows efficient steam penetration (do not overfill the chamber). Material should not touch the sides or top of the chamber. The clean items and the wastes should be autoclaved separately. Polyethylene trays should not be used as they may melt and cause damage to the autoclave.

36. Sterilization Control Biological indicator: Spores of Geobacillus stearothermophilus (formerly called Bacillus stearothermophilus ) are the best indicator, because they are resistant to steaming. Their spores are killed in 12minutes at 121°C. Other indicators such as: Browne's tube, and thermocouple. Autoclave tapes.

37. Filtration

38. Filtration Filtration is an excellent way to remove the microbial population from solutions of heat labile materials like vaccine, antibiotics, toxin, serum and sugar solution as well as for purification of air.

39. Depth Filters They are porous filters that retain particles throughout the depth of the filter, rather than just on the surface. Depth fillers are composed of random mats of metallic, polymeric, or inorganic materials. These fillers rely on the density and thickness of the filter to trap particles rather than the pore size. For example: Candle filters made up of diatomaceous earth ( Berkefeld filters), unglazed porcelain (Chamberland fillers) Asbestos filters (Seitz and Sterimat filters) Sintered glass filters. Advantages They can retain a large mass of particles before becoming clogged Flow rate of the fluid is high Low cost Disadvantage As some of die particles still come out in the filtrate; hence they are not suitable for filtration of solution containing bacteria. Uses Depth fillers are commonly used when the fluid to be filtered contains a high load of particles, e.g. industrial applications, such as filtration of food, beverages and chemicals.

40. Membrane Filters They are the most widely used filters for bacterial filtration. They are porous; retain all the particles on the surface that are larger than their pore size. Membrane filters are made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials. Pore size : Most commonly used membrane fillers have an average pore diameter of 0.22 μm which removes most of the bacteria; allowing the viruses to pass through Filters of 0.45 μm are used to retain coliform bacteria in water microbiology 0.8 μm filters are used to remove airborne microorganisms in clean rooms and for the production of bacteria free gases. The sterilization control of membrane filters includes Brevundimonas diminuta and Serratia marcescens.

41. Filtration of Water To sterilize sera, sugar and antibiotic solutions. Separation of toxins and bacteriophages from bacteria. To obtain bacteria free filtrates of clinical samples for virus isolation. Purification of water- when water samples pass through, the filter discs retain the organisms which can then be cultured, e.g. testing of water samples for Vibrio cholerae or typhoid bacilli .

42. Filtration of Air Air fillers are used to deliver bacteria-free air. Air can be filtered by various methods: Surgical masks (that allow air in but keep microorganisms out) are the simplest examples. There are two important air filters That are used in biological safety cabinets and laminar airflow systems: HEPA fillers (High-efficiency par1icula1e air fillers) : HEPA filter removes 99.97% of particles that have a size of 0.3 μm or more. ULPA filters (Ultra-low particulate/ penetration air) : An ULPA filter can remove from the air at least 99.999% of dust, pollen, mold, bacteria and any airborne particles with a size of 0.12μm or larger. HEPA ULPA

43. RADIATION Ionizing radiation Non-ionizing radiation

44. Ionizing Radiation Advantages of ionizing radiation- high penetrating power, rapidity of action, temperature is not raised It destroys bacterial endospores and vegetative cells, both eukaryotic and prokaryotic. Ionizing radiations include, X-rays, gamma rays (from Cobalt 60 source) , and cosmic rays. Mechanism : It causes breakage of DNA without temperature rise (hence this method is also called as cold sterilization). Disadvantages of ionizing radiation- Not always effective against viruses Uses : Gamma radiation is used in the sterilization/disinfection of- Disposable plastic supplies, such as disposable rubber or plastic syringes, infusion sets and catheters. Catgut sutures, bone and tissue grafts and adhesive dressings as well as antibiotics and hormones. Irradiation of food (permitted in some countries) Sterilization/disinfection control : Bacillus pumilus .

45. Non-ionizing Radiation Examples of non-ionizing radiation include infrared and ultraviolet radiations. They are quite lethal but do not penetrate glass, dirt films, water; hence their use is restricted. The recommended dose is 250-300 nm wavelength, given for 30 minutes. It is used for disinfection of clean surfaces in operation thearers , laminar flow hoods as well as for water treatment. Because UV radiation burns the skin and damages eyes, hence the area should be closed and UV lamps must be switched off immediately after use.

46. High-frequency ultrasonic and sonic sound waves disrupt bacterial cells; but this method is not reliable, hence is not used now a days. Ultrasound (Ultra sonic) Waves

47. Recent Advancements in Physical Sterilizations Pulsed light sterilization Ultra high pressure sterilization Hydroclave Plasma Sterilization (STERRAD)

48. Pulsed light sterilization A non thermal sterilization method that destroys microorganisms using brief pulses of white light generated by Xenon lamps.

49. Advantages Total DNA destruction Quick No chemicals used Safe and easy to use Minimum space required Disadvantage Its is a surface treatment. Only areas receiving light are decontaminated. Applications Sterilization of cups, caps, lids and packaging materials. Sterilization of food packaging.

50. Ultra high pressure sterilization Also called high hydrostatic pressure sterilization (HHP)/ pascalisation . It is a cold sterilization Technique in which, products already sealed in its final package are introduced into a vessel and subjected to high level of isostatic pressure (200 – 600MPa) transmitted by water, for few seconds to few minutes.

51. Advantages Characteristics of fresh product are retained. Destroys pathogens Extends product self life. Avoids or reduces the need of food preservatives. Environment friendly. Disadvantage For inactivation of bacterial endospores, synergistic action of very high pressure (600MPa) and Temperature (>60°C) is required. Applications Mainly used in food sterilization technology.

52. Hydroclave Sterilizes the waste utilizing steam, similar to an autoclave, but with much faster and much more even heat penetration. It hydrolyses the organic components of the waste and dehydrates the waste. It substantially reduces the weight and volume of waste.

53. Advantages Guaranteed high level of sterilization. Automatic operation and operator skill does not effect sterility. No infectious or harmful emissions. It is very economic to use. Dry waste regardless of original water content. Applications The Hydroclave can sterilize: Bagged waste in ordinary bags Sharp containers and needles. Metal objects, plastic, liquids etc.

54. Gas Plasma Sterilization (STERRAD) Plasma is defined as an ionized gas with equal number of + ve and – ve ions. It is the 4 th state of matter. Gas plasma is generated in an enclosed chamber under deep vacuum using radio or microwaves to excite gas molecules (hydrogen peroxide) to produce ionized gas particles.

55. Mechanism of action Free radical interaction UV/VUV radiation: It causes formation of thymine dimers in DNA, inhibiting bacterial replication. Breakage of DNA strands. Volatilization: It is able to vaporize microbiological matter, causing physical destruction of spores. Applications STERRAD System enables sterilization of surgical instruments, rigid and flexible endoscopes, cameras, catheters etc.

56. Advantages Process occurs at room temperature and hence no associated thermal dangers. Byproducts are generally water and oxygen which are harmless to the environment. Fast treatment time (1 min or less) Disadvantage Weak penetration of Plasma Complications arise in: Presence of organic residue Packaging material Complex geometries Bulk sterilization High power consumption Can corrode certain materials Due to its small size, absence of heating and fast sterilization times it has the potential of becoming an integral part of modern Dental Clinics.

57. Thank you