STERILIZATION.pptx

sterilization slide share

1. STERILIZATION

2. CONTENT ●Introduction ●History of sterilization ●Definition ▪ Types of sterilizaton − ●Physical method ●Chemical method ●Advantages ●Disadvantages ●Applications

3. Introduction Microorganisms cause contamination,infection and decay, it becomes necessary to remove or destroy them from materials or from areas. This is the object of Sterilization . Methods to remove or kill microorganism are known as Sterilization . The time requied for sterilization is inversely proportional to the temperature of exposure and can be expressed as Thermal death time .

4. History of Sterilization Denis P apin ( 1679 ) A French physicist, Denis Papin invented a pressure cooker that would trap boiling water,convert it into steam,& was found to cleanse objects by cooking them .

5. Charles Chamberland (1879 ) A French Microbiologist , created a new version of pressure cooker, called the AUTOCLAVE to be used in medical microbiology.

6. Louis pasteur (1860) 2 major contributions to the art of sterilization came in the 1860’s when- Louis pasteur wrote extinsively on how germ cause disease & the technique of pasteurization . Joseph Lister developed a technique that used Carbolic acid as a spray to disinfect instruement .

7. DEFINITION STERILIZATION Medium is made free of all microorganism either in the vegetative or spore form. DISINFECTION -Destruction or reduce the number of pathogens, -Capable of producing infections but not spores. ANTISEPTICS -Chemical disinfectants, -Which can safely be applied to living tissues, - Prevent infection by inhibiting the growth of bacteria. GERMICIDES -Substances that can kill bacteria.

8. TYPES OF STERILIZAION PHYSICAL METHOD Heat Radiation Filtration Sunlight Ozone CHEMICAL METHOD – sporocidal agent--- Ethylene oxide, Formaldehyde , Gutaraldehyde ,Hydrogen peroxide,peracitic acid , O- phtalic acid, plasma sterilization .

9. HEAT Sterilization- Most Reliable Commonly employed method. Factors influencing sterilization by heat are- Nature of heat Temperature & Time No. of microbes present Characterstics of the organism Type of material. DRY HEAT TYPE OF HEAT MOIST HEAT

10. DRY HEAT Kills the microorganism By denaturation of bacterial protein, O xidative damage & By toxic effect of elevated levels of electrolytes.

11. Red heat Inoculating wires or loops Tips of forceps and Needles are held in flame of a B unsen burner till they become red hot.

12. Flaming Glass slides, scalpels and mouths of culture tubes are passed through B unsen flame without allowing them to become red hot. Incineration Excellent method for destroying biomedical waste. A instrument named incinerator used for this purpose.

13. Materials are reduced to ashes by burning . Plastics such as PVC and polythene can be dealt with similarly. Guidelines on “Design and construction of biomedical waste incinerator” recommend various design feature of an incinerator as well as the air pollution control device

14. Hot air oven Most widely used Oven is electrically heated and is fitted with fan to ensure adequate and even distribution of HOT AIR in the chamber.

15. Principle of Hot air oven- The heat is absorbed by the surface of the item to be sterilized, which then penetrates to the centre , untill the entire items reaches the desired temperature. Temperature & Time- -150°C for 2 hours 30 min. -160°C for 2 hours . -170°C for 1 hour. - 180°C for 30 min.

16. USES- Sterilization of- (At 160°C for 2 hrs ) Glasswares like- Glass syringes, petridishes ,flasks, pipettes & test tubes. Surgical instruments- forceps, scissors, scalps etc. Parmaceuticals products- liquid paraffin, fats & grease etc. For oils, glycerols & dusting powder (British Pharmacopoeia recommends a holding time of 1 hr at 150°C)

17. Precautions- It should not be overloaded . Material should be arranged Allows free circulation of air. Glassware should be dry fitted with cotton plugs. Petridishes & pipettes should be wrapped in craft paper. Rubber material should not be kept inside the oven. Allowed to cool for 2 hrs before opening the door, since the glasswares may crack by sudden cooling.

18. Sterilization control- The spores of Bacillus subtilis subsp. niger (NCTC10075 or ATCC 9372) Are kept inside the oven. These spores should be destroyed if the sterilization is proper. Thermocouples may also be used. Browne’s tube with red colour is available, after proper sterilization a green colour is produced.(after 1 hr. at 160°C).

19. moist heat – kills the microorganism by denaturation and coagulation of proteins.

20. Temperature below 100°C Pasteurization of milk. Inspissation. Vaccine bath. Low temperature steam formaldehyde (LTSF) sterilization.

21. Pasteurization of milk Holder method- 63°C for 30 min. Flash method- 72°C for 20 min . Followed by cooling quickly to 13°C. All nonsporing pathogens such as m ycobacteria, b rucella and salmonellae are killed except Coxiella burnetii is relatively heat-resistant and may survive in holder method.

22. B. Inspissation Instrument used – Inspissator . Serum or egg medium- lowenstein jensen’s and loeffler’s serum slope are render by heating at 80-85°C temp . for half an hour daily on three consecutive days.

23. C. Vaccine bath Bacterial vaccines are heat inactivated in special vaccine bath at 60°C for 1 hour. Serum and body fluid containing coagulable proteins can be sterilized by heating for 1 hour at 56°C in a water bath or several successive days.

24. D. Ltsf - Low temperature steam formaldehyde sterilization Steam at atmospheric pressure 75°C Formaldehyde vapour is used. Bacillus stearothermophilus has been used as biological control to test the efficacy of LTSF sterilizers.

25. Temperature at 100°C 1.Boiling 2 . Steaming 3. Tyndallization

26. 1.boiling Boiling of the items in water at 100°C for 15 minutes may kill most of vegetative forms but not the spores. Not suitable for sterilzation of surgical instruments. When better methods are not available, boiling may be used for glass syringes and rubber stoppers . 2. Steaming Koch’s or Arnold’s steamer is used. Useful for those media which are decomposed At high temperature of autoclave. Articles are kept on a perforated tray exposed to steam 100° C at atmospheric pressure for 90 min.

27. 3. Tyndallization An exposure to 100°C for 20 minutes on three successive days is used. Also called Fractional sterilization . John Tyndall discovered tyndallization . May fail to kill spores of cetain anaerobes and thermophiles Principle- -1 st exposure kills all the vegetative bacteria, - A nd in the intervals between the heatings the remaining spores germinate into vegetative forms, - which are killed on subsequent heating. Uses T o sterilize gelatin and egg,serum or sugar containing media.

28. Temperature above 100°C Autoclave Physical method of sterilization under moist heat. Killed bacteria , viruses and even spores present in the material put inside of the vessel using steam under pressure. Similar to pressure cooker. Principle- water boils when its vapour pressure equals that of the surrounding atmosphere. So when the atmospheric pressure is raised , the boiling temperature is also raised.

29. Components of autoclave 1.A pressure chamber- large cylinder (vertical or horizontal) in which the materials to be sterilized are placed. made up of stainless steel and placed in a supporting case. A steam jacket (water compartment) 2.A lid- fastened by screw clamps and rendered air tight by an asbestos water. It bears -discharge tap for air and steam discharge. A pressure gauge. 3.An electrical heater- Heats the water to produce steam.

30. Procedure - Cylinder filled with sufficient water . Material to be sterilized is placed inside the pressure chamber. The lid is closed ,electrical heater is put on. The safety valve is adjusted to 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. Steam pressure rises inside and when it reaches the desired set level, the safety valve opens and excess steam escape out.

31. Holding period is counted from this point of time , which is about 15 min in most cases. After the holding period- Electrical heater is stopped and the allowed to cool till the pressure gauge indicates that the pressure inside is equal to the atmospheric pressure. Discharged tap opens slowly and air is allowed to enter the autoclave. The lid is now opened and the sterilized materials are removed. Sterilization conditions- -121°C for 15 min at pressure of 15 pound(psi). -126C for 10 min at pressure of 20 psi. -133C for 3 min at pressure of 30 psi.

32. Uses of autoclave- Sterilize culture media, rubber material, gowns, dressing, gloves etc. Useful for materials which cannot withstand the higher temperature of hot air oven. Precautions- It should not be used for waterproof material(oil and grease) Do not overfill the chamber. Material should not touch the sides or top of the chamber. Polyethylene trays should not be used.

33. Type of autoclave Gravity displacement type- Most common used in laboratories. ● Vertical type & ● Horizontal type. Positive pressure displacement type Negetive pressure displacement type

34. Sterilization control Biological indicator- Spore of Geobacillus stearothermophilus ( formely called Bacillus stearothermophilus )- -Are the best indicator because, -They are resistant to steaming -Their spores are killed in 12 min. at 121C. Chemical indicator- Autoclave tape - B rowne’s tube

35. TABLE- Indicators used for monitering the sterilization process of autoclave (steam sterilizer), ethylene oxide sterilizer and plasma sterilizers . 1.Physical or mechanical indicators:- distal displays of the serilizer equipment such as Temperature,time and pressure, etc. 2.Chemical indicators:- T o moniter satisfactory process of sterilization,uses chemical sensitive materials which undergo a color change if sterilization parameter is achieved TYPE-I ● Process indicator ●External pack indicator TYPE-II ●Called Bowei -dick test ,used only for steam sterilizers ●Done before the first load ●To check air removal, air leaks & steam penetration ●To check autoclave functioning well. TYPE-III Single parameter indicator, obsolete now.

36. TYPE-IV ●Internal pack indicator,used inside each pack. ●Designed to measure any two of the critical varibles :- time, steam quality & temp. TYPE –V ●Also an internal pack indicator ●measure all three variable: time,steam quality & temp. TYPE-VI ● Emulating indicator ● it is cycle specific. 3. Biological indicators- -Bacterial spores used to check effectiveness -Highly resistant spores Geobacillus stearothermophilus for autoclave Bacillus atrophaeus for ethylene oxide sterilizer & dry heat

37. Filtration Excellent way to remove the microbial population in solution For H eat labile materials like vaccine, antibiotics ,toxins , serum and sugar solution as well as for purification of air. Types of filtration- 1.Depth filter 2.Membrane filter

38. Depth filter Porous filter, Retain particles throughout the depth of the filter It composed of random mats of metallic ,polymeric These filters relay on the density and thickness of the filter to trap particles rather than pore size. TYPE- Candle filters , A sbestos filters, Sintered glass filters.

39. ADVANTAGE- -it can retain a large mass of particles -Flow rate of fluid is high -Low cost DISADVANTAGE- -Some of the particles still come out in the filter -Not suitable for filtration of solution containing bacteria

40. TYPES - 1.Candle filters- -Manufactured in Different grade of porosity -Used for purification of water for industrial & drinking purpose 2 Type- Unglazed ceramic (chamber land filter) -Diatomaceous earth filter ( berkfeld filters )

41. 2.Sintered glass filters Prepared by heat fusing finely powdered glass particles of graded sizes. They have low absorptive property and can be cleaned easily, but are brittle and expensive.

42. 3. Membrane filters Made up of cellulose acetate ,cellulose nitrate, polycarbonate, polyvinylidene fluoride,or other synthetic materials. Porous,Most widely used for bacterial filtration. Retain all the particles on the surface that are larger than their pore size. Pore size – diameter of 0.22 µm- Removes bacteria. Filters of 0.45µm- Retain coliform bacteria Filters of 0.8µm- Remove airborne microbes.

43. Filtration of liquid Done for following purposes:- To sterilized sera,sugar and antibiotics solutions Separation of toxins and bacteriophages from bacteria To obtain bacteria free filterate of clinical samples for virus isolation. Purification of water.

44. Filtration of air – deliver bacteria free air- HEPA filters- high efficiency particulate air filter It removes 99.97% of articles that have a size of 0.3 µ m

45. ULPA filter- ultra low particulate air It can remove from the air atleast 99.99% of dust, pollen,mould,bacteria and any air borne particles with a size of 0.12µm STERILIZATION CONTROL - By Brevundimonas diminuta and serratia marcescens .

46. RADIATION Radiation kills germs that can cause disease and neutralizes other harmful organisms. TYPES- IONIZING RADIATION- I t includes x-rays , gamma rays, cosmic rays . Mechanism- it cross breakage of DNA without temperature rise( cold sterilization ).

47. Uses- gamma radiation used for sterilization of disposable plastic supplies, such as- disposable rubber, infusion sets and catheters. Bone and tissue graft and irradiation of pores.

48. Advantages High penetration power Rapidity of action Temperature is not raised Sterilization control- E fficacy of ionising radiation is tested by using Bacillus pumilus .

49. 2.Non ionizing radiation- It includes infra red and UV radiations They are quiet lethal but do not penetrates glass,dirt films,water , hence there uses is restricted. UV rays – 250-300nm wavelength for 30min use for disinfection of clean surfaces in OT , laminar flow ,as well as water treatment.

50. Sunlight Direct sunlight is a natural method of sterilization of water in tank ,rivers ,and lakes . Direct sunlight has an active germicidal effect due to its content of ultraviolet and heat rays. Bacteria present in natural water sources are rapidly distroyed by exposer to sunlight.

51. Ozone sterilization Ozone has extremely great oxidative power and ready to react to germs, viruses and a host of microbes. Useful for disinfect the water and food It said to be 50% more powerful and act 3000 times faster than chlorine at 100 times the strength. “ Ozone(O3)kills microbes in high voltage electricity, when healthy bacterial cell exposed to ozone oxidative burst puncturing of the cell ,causing cell death”.

52. REFERENCES 1.Essential of microbiology by Apurba Sastry 2. C. P. B aveja 3. Ananthanarayan and P aniker’s

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