STERILIZATION AND DISINFECTION.pptx

Sterilization
It is defined as the process by which an article, surface or medium is freed of all living microorganisms either in vegetative or spore state.
Disinfection
It is destruction or removal of all pathogenic organisms or organisms capable of producing infections but not necessarily spores.

1. STERILIZATION AND DISINFECTION in dentistry Dr Purva Pihulkar Assistant Professor Dr HSRSM Dental College and Hospital

2. INTRODUCTION Sterilization It is defined as the process by which an article, surface or medium is freed of all living microorganisms either in vegetative or spore state. Disinfection It is destruction or removal of all pathogenic organisms or organisms capable of producing infections but not necessarily spores.

3. Disinfectant Products used to kill microorganisms on nonliving objects or surfaces. Antiseptic They are chemical disinfectants that can be safely applied on skin or mucous membrane and are used to prevent infection by inhibiting the growth of bacteria.

4. METHODS OF STERILIZATION 1. Physical methods Sunlight Heat -Dry -Moist Radiation Filtration 2. Chemical Methods

5. 1) Sunlight The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By killing bacteria suspended in water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes.

6. Semple and grieg showed that in india , typhoid bacilli exposed to sun on pieces of white drill cloth were killed in 2 hours, whereas contols kept in dark were alive after 6 days. Bacteria suspended in water are readily destroyed by exposure to sunlight.

7. 2) Heat Heat is considered to be most reliable method of sterilization of articles. It is of types A) dry heat B) moist heat

8. Dry heat sterilization The temperature required is usually higher than 356° F or 180 °C.  Thus Dry heat acts by protein denaturation and oxidative destruction. Articles that are heavily contaminated require higher temperature or prolonged exposure. More the number of microorganisms, higher the temperature or longer the duration required.

9. Moist heat sterilization Moist heat  penetrates materials much more rapidly than dry  heat  because water molecules conduct  heat  better than air. Thus moist heat is superior to dry heat in action. Lower temperatures and a shorter exposure time are therefore required than for dry  heat . Mode of Action:  Moist heat kills microorganism  by denaturing their proteins.

10. Dry Heat Sterilization Following procedures are used for sterilization of dry heat:- Flaming Incineration Hot air oven

11. 1) Flaming In flaming, Glass slides, scalpels,tip of forceps, inoculating loops and mouths of culture tubes are passed through bunsen flame. inoculating loops carrying infective material may be dipped in disinfectant before flaming to prevent spattering.

12. 2) Incineration This is a method of destroying contaminated material by burning them in incinerator. Articles such as soiled dressings; animal carcasses, pathological material and bedding etc should be subjected to incineration.

13. This technique results in the loss of the article, hence is suitable only for those articles that have to be disposed. Burning of plastic emits dense smoke, and hence they should not be incinerated.

14. 3) Hot Air Oven This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high temperature (160 º C) for duration of one hour in an electrically heated oven. Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved by a fan. The oven should be fitted with a thermostat control, temperature indicator, meshed shelves and must have adequate insulation.

15. Principle of HOT AIR OVEN Sterilization by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, then passes towards the centre of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place.

16. Sterilization process: Articles to be sterilized must be perfectly dry before placing them inside to avoid breakage. Articles must be placed at sufficient distance so as to allow free circulation of air in between. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool. Articles such as petri dishes and pipettes may be arranged inside metal canisters and then placed. Individual glass articles must be wrapped in kraft paper or aluminum foils.

17. Sterilization cycle: Different temperature-time relations for holding time are 60 minutes at 160 º C 40 minutes at 170 º C 20 minutes at 180 º C.

18. Hot air oven used for sterilization of Glass wares like glass syringes, petridishes , flasks,pipettes and test tubes. Surgical instruments like scalpels, scissors,forceps,mouth mirrors,probes . Chemical like liquid paraffin,fats , sulphonamides powders.

19. Sterilization control: ƒ Physical: Temperature chart recorder and thermocouple. ƒ Chemical: Browne’s tube (green spot, color changes from red to green)

20. Biological: Clostridium tetani on paper strips are placed inside envelopes and then placed inside the hot air oven. Upon completion of sterilization cycle, the strips are removed and inoculated into thioglycollate broth or cooked meat medium and incubated at 37oC for 3-5 days. Proper sterilization should kill the spores and there should not be any growth

21. Advantages: It is an effective method of sterilization of heat stable articles. The articles remain dry after sterilization. This is the only method of sterilizing oils and powders. Disadvantages: ƒ Since air is poor conductor of heat, hot air has poor penetration. ƒ Cotton wool and paper may get slightly charred. ƒ Glasses may become smoky. ƒ Takes longer time compared to autoclave

22. Precautions It should not be overloaded Rubber materials or inflammable materials should not be kept inside oven. Oven must allowed to cool for two hours before opening the doors,since the glasswares may crack by sudden cooling.

23. Moist heat sterilization Used at temperatures as follows:- At a temperature below 100 º C At a temperature of 100 º C At a temperature above 100 º C

24. 1) At a temperature below 100 º C Pasteurisation It uses heat at temperatures sufficient to inactivate harmful organism in milk. when heat-treated milk become denatured and stop the enzymes from functioning. This helps to stop pathogen growth by stopping the functionality of the cell.  Holder method 63 for 30 min Flash method 72 for 20 sec

25. B) Inspissation This is a technique disinfect egg and serum containing media. The medium containing serum or egg are placed in an inspissator and heated at 80-85 º C for 30 minutes on three successive days. Proteins in the serum will coagulate at higher temperature. On the first day, the vegetative bacteria would die and those spores that germinate by next day are then killed the following day.

26. C) Vaccine bath -The contaminating bacteria in a vaccine preparation can be inactivated by heating in a water bath at 60 º C for one hour. -Only vegetative bacteria are killed and spores survive.

27. 2) At a temperature of 100 C a) Boiling -Boiling water (100 º C) kills most vegetative bacteria and viruses immediately. -When absolute sterility is not required, certain metal articles and glasswares can be disinfected by placing them in boiling water for 10-20 minutes.

28. 2) Tyndallization -steam at 100 for 20 min on 3 successive days is used. -The principle is that 1 st exposure kills vegatative forms in intervals between heatings the remaining spores germinate into vegetative forms which are killed on subsequent heatings . -used for sterilization of culture media.

29. 3) At a temperature above 100 °C Autoclave Standard sterilization method in hospitals. The Autoclave works under the same principle as the pressure cooker where water boils at increased atmospheric pressure.

30. The autoclave is a double walled chamber in which air is replaced by pure saturated steam under pressure. The air in the chamber is evacuated and filled with saturated steam. The chamber is closed tightly the steam keeps on filling into it and the pressure gradually increases.

31. Autoclave

32. The items to be sterilized get completely surrounded by saturated steam (moist heat) which on contact with the surface of material to be sterilized condenses to release its latent heat of condensation which adds to already raised temperature of steam so that eventually all the microorganisms in what ever form are killed.

33. The usual temperature achieved is 121 °C at a pressure of 15 ppsi . at exposure time of only 15-20 mins . By increasing the temperature, the time for sterilizing is further reduced.

34. Uses To sterilize culture media,rubber material, gowns, dressing, gloves. Surgical instruments like forceps, scapels,mouth mirrors,probes,etc . use the  power of steam to kill bacteria, spores and germs resistant to boiling water and powerful detergents.

35. Precautions The air must be allowed to escape from chamber as temperature of air steam mixture is lower than that of pure steam. Materials should be arranged in such a manner as to ensure free circulation of steam inside chamber.

36. Advantage: Very effective way of sterilization, quicker than hot air oven. Disadvantages: Drenching and wetting or articles may occur, trapped air may reduce the efficacy, takes long time to coo

37. Glass bead sterilizer It is mainly used for sterilizing the working ends of endodontic files and reamers by placing them in a container containing glass beads heated upto approximately 225°C (437°F) Glass bead sterilizer works on the principle of intense dry heat. intense dry heat damages vegetative and spore forms of bacteria.

38. Glass beads should be less than 1mm in size because larger beads are not effective in transferring heat due to large spaces between the beads. The instruments to be sterilized are immersed into heated up glass beads and left for period of time. Glass bead sterilizer has disadvantage that beads which are less than 1 mm in diameter get struck in the instruments when they are introduced into the root canal.

39. Filtration Filtration does not kill microbes, it separates them out. Filtration  allows for the exclusion of organisms based upon size. Various applications of filtration include removing bacteria from ingredients of culture media, preparing suspensions of viruses and separating toxins from culture, counting bacteria.

40. Types of filters Earthenware candles These filters are made up of diatomaceous earth or porcelain. They are usually baked into the shape of candle. Water poured into the upper basin, where it  filters through the  candle  and collects in the lower container. it physically traps microorganisms and other suspended contaminants in its pores.

41. 2) Asbestos disc filters( seitz filter) These filters are made from chrysotile type of asbestos, chemically composed of magnesium silicate. It consists of disc, which is to be used only once. This disc absorbs the contaminants and sterilize the liquid.

42. 3) Sintered glass filter These are made from finely ground glass that are fused sufficiently to make small particles adhere to each other. They are usually available in the form of disc fused into a glass funnel. This funnel separates the contaminants and sterilize the solution. They are washed in running water in reverse direction and cleaned with warm concentrated H2SO4 and sterilized by autoclaving.

43. 4) Membrane filters These filters are composed of cellulose diacetate . These membranes have a pore diameter ranging from 0.015 µm to 12 µm. The membrane separation process is based on the presence of  semi  permeable membranes. The principle is quite simple: the membrane acts as a very specific filter that will let  water  flow through, while it catches suspended solids and other substances.

44. 3. Radiation: - Non Ionising Radiations Rays of wavelength longer than the visible light are non-ionizing. Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this wavelength that the absorption by the microorganisms is at its maximum, which results in the germicidal effect. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation are inactivated within seconds.

45. Since UV rays don’t kill spores, they are considered to be of use in surface disinfection. UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. Disadvantages of using uv rays include low penetrative power, limited life of the uv bulb, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't penetrate glass, paper or plastic.

46. - Ionizing radiation X- rays,gamma rays and cosmic rays are highly lethal to DNA and other vital constituents. They have high penetrative power. Used mainly in industrial facilities e.g. sterilization of disposable plastic syringes, gloves, specimens containers,Petri Dishes, oils,greases,fabrics and metal foils.

47. Instruments for sterilization are classified as follows:

48. Endodontic Instrument Sterilization: Files must be sterilized before use. ANSI/ADA Specification 28 recommends: Scrub the instruments with soap and warm water. Rinse thoroughly with distilled water. Allow to air dry.

49. 5) Place the instruments, unwrapped, in the autoclave tray. 6) Use fresh distilled water. 7) Steam Autoclave at 136° C (plus or minus 2° C) for 20 minutes. 8) All rotary files are single patient use instruments. 9) Recommended File Disposal: Place used files in a Biohazard Sharps container.

50. ULTRASONIC TIPS STERILIZATION: Maintain a temperature of at least 136°C (277°F) for a minimum of 20 minutes per USP recommendations.

51. OBTURATORS: Disinfect the obturator in a 5.25% sodium hypochlorite solution (bleach) for one minute. Rinse the obturator in 70% alcohol. Dry the obturator for several seconds on a clean work surface to allow evaporation of excess alcohol.

52. Handpieces Wipe with an alcohol gauze. Clean and lubricate handpieces with a cleaner/ lubricant after each use and before autoclaving. This will ensure proper operation and a long service life for your handpiece . Do not use a cleaner/ lubricant after autoclaving, only use before sterilization. Follow the instructions provided with the handpiece purchased for complete maintenance instructions.

53. Motors The entire motor and cord assembly supplied with your unit must be surface sterilized by autoclave. sterilize at 132º C (270º F) for 3 minutes. Before autoclaving the motor and cord assembly, always install the autoclaving plug into the motor and motor connector. Motor/ handpiece holding cradle is also fully autoclavable .

54. Recommended Procedure for disinfecting the alginate impression Rinse the impression thoroughly under running tap water, shake the impression to remove excess water. Dip the impression in a 1:10 solution of sodium hypochlorite for several seconds to ensure maximum contact of undercut with the disinfectant. Wrap the impression in gauze soaked in 1:10 sodium hypochlorite, place in a plastic bag and seal for 10 minutes.

55. Remove the impression and rinse thoroughly under running tap water. Dental casts from impressions may harbor infectious microorganisms or the wax bite records from the patients may cause dental casts to become infected. Dental casts may be disinfected by adding disinfectant like iodophor or neutral gluteraldehyde to dry gypsum during the mixing process. Spraying the dental casts with iodophor or chlorine products may be recommended.

56. Precautions for HIV patients Special precautions for dental care include the use of gloves, suction tubing and reservoirs when working inside the mouth and sterilization or disinfection of instruments and other contaminated objects. Masks and protective eyewear should be used particularly when the spattering of blood is likely. Disposable cups for mouth washing are advisable. Otherwise, glassware should always be disinfected adequately or autoclaved after each use.

57. Needles and other sharp instruments or materials should be placed in a puncture-proof container immediately after use and should preferably be incinerated. Liquid wastes such as bulk blood, suction fluids, excretions and secretions should be carefully poured down a drain connected to an adequately treated sewage system, or disposed of in a pit latrine. Solid wastes, such as dressings and laboratory and pathology wastes, should be considered as infectious and treated by incineration, burning or autoclaving. Other solid wastes, such as excreta, may be disposed of in a hygienically controlled sanitary landfill or pit latrine.

58. In mega check up camps, instruments are sterilized using disinfectants like 2% glutaraldehyde,6% hydrogen peroxide and 99.9% ethyl alcohol. Latter they are autoclaved. Sometimes, instead of mouth mirrors ice cream sticks can be used to examine the oral cavity.

59. An ideal disinfectant or antiseptic has the following characteristics: 1.     Ideally, the disinfectant should have a wide spectrum of antimicrobial activity. 2.     It should act in the presence of organic matter. 3.     It should not be toxic to human or corrosive. 4.     It should be stable upon storage and should not undergo any chemical change.

60. 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.

61. Alcohols Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein. Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol Application: 70% ethyl alcohol (spirit) is used as antiseptic on skin.

62. Isopropyl alcohol is used to disinfect surfaces. It disinfects clinical thermometers. Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods. Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

63. 2) Aldehydes Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic acids. Examples: Formaldehyde, Gluteraldehyde Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes , bronchoscopes, centrifuges, anasethetic equipments etc.

64. 3) Phenols Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes. Examples: 5% phenol, 1-5% Cresol, 5% Lysol, hexachlorophene, chlorhexidine , chloroxylenol ( Dettol ) Applications: They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses.

65. 4) Halogens Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal . Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores ) Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. For hand washing iodophores are diluted in 50% alcohol.

66. 5) Oxidising agents Hydrogen peroxide Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA. Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators.

67. 7) Surface active agents Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. They disrupt membrane resulting in leakage of cell constituents. Examples: These are soaps or detergents. Detergents can be anionic or cationic.

68. 8) Dyes Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids. Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes are acriflavin and aminacrine . A related dye, ethidium bromide, is also germicidal. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action.

69. Applications: They may be used topically as antiseptics to treat mild burns. They are used as paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain selective media

70. 9) Gases Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl -, amino-, carboxyl- and hydroxyl- groups. Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is flammable. Application: It is a highly effective chemisterilant , capable of killing spores rapidly. It requires presence of humidity.

71. It is used to sterilize heat labile articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine, respiratory and dental equipments. Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and carcinogenic.

72. Conclusion “Prevention is better than cure” a proverb well suited to sterilization. Thorough understanding of the application of sterilization will help ensure safety from the invisible but deadly world of microbial pathogens. Hence utilization of proper sterilization, disinfectants and aseptic procedures help us achieve the safety of our professional demands.

73. References Textbook of microbiology Anantnarayan Textbook of microbiology C P Baveja Sterilization and Disinfection - Sridhar Rao P.N Sterilization Protocols in Dentistry – A Review Lakshya Rani et al /J. Pharm. Sci . & Res . Vol. 8(6), 2016, 558-564 Endodontic Sterilization Guide Infection Control in Orthodontics Dr Shilpa Kalra , Dr Tulika Tripathi , Dr Priyank Rai Journal of Orthodontics and Endodontics 2015 Vol. 1