Sterilisation techniques.pptx

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1. Sterilisation techniques DR. GOURAV KUMAR Assistant professor Department of Radiation and Imaging Technology NIMS University, Jaipur Rajasthan

2. Introduction Microorganism are responsible for contamination and infection. They are all around. The aim of sterilisation is to remove or destroy them from materials or from surface.

3. Sterilisation It is a process by which an article surface or medium is made free of all microorganisms either in the vegetative or spore form.

4. Disinfection It means the destruction of all pathogens or organisms capable of producing infections but not necessarily spores. All organisms may not be killed but the number is reduced to a level that is no longer harmful to health.

5. Antiseptic These are chemical disinfectants which can safety be applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms.

6. Asepsis The technique by which the occurrence of infection into an uninfected tissue is prevented.

7. Uses of sterilisation Sterilisation of materials instruments used in surgical and diagnostic procedure. For media and reagent used in the microbiology laboratory. In food and drug manufacturing to ensure safety from contamination organisms. Food and drug manufacture. Vaccine preparation.

8. Method of Sterilisation Physical method Chemical method

9. Physical method Sunlight Heat – 1.dry heat, 2. moist heat Ozone Filtration Radiation

10. Chemical Methods Alcohol Aldehydes Phenols Halogens Oxidising agents Salt Surface active agent Vapour disinfection Dyes

11. Physical Methods Sunlight Heat – dry heat and moist heat.

12. Basic principle of Heat sterilisation Dry heat kills the organism by denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated levels of electrolytes. However the possibility of DNA damage is also incriminated as one of the mechanisms of inactivation's of microbes. Moist heat kills the microorganisms by denaturation and coagulation of proteins.

13. Sunlight Sunlight has bacterial effect due to a combination of the effect of ultraviolet rays and heat rays. It is the natural method of sterilization in tanks, river and lakes. e.g. Typhoid bacteria exposed to the sun were killed in two hours where as control the kept in the dark were still alive after six days.

14. Dry heat sterilisation The following procedures are used for sterilisation by dry heat. Red heat Flaming Incineration Hot air oven

15. Red heat Incubating wire or loops tips of forceps and needles are held in the flame of a Bunsen burner till they become red hot.

16. Flaming Glass slides, scalpels and mouth of culture tubes are passed through Bunsen flame without allowing them to become red hot.

17. Incineration By this method infective material is reduced to ashes by burning instrument named incineration may be used for this purpose. Soiled dressings, animal carcasses, bedding and pathogical materials are dealt with this method.

18. Hot air oven It is the most widely used method of sterilisation by dry heat. The oven 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 that maintains the chamber air at a chosen temperature.

19. Temperature and time 160°C for two hours is required for sterilisation (previously it was 160°C for one hour). However, alternative temperatures and holding time include 170°C for one hr and 180°C for 30 min.

20. Uses It is used for sterilisation of – Glassware's like glass syringes, Petri dishes, flasks, pipettes and test tubes. Surgical instruments like scalpels, scissors, forceps. Chemicals such as liquid paraffin, fats sulphonamides powders etc.

21. Precautions It should not be overloaded. The material should be arranged in a manner which allows free circulation of air. Material to be sterilised should be perfectly dry. Test tubes, flasks etc. should be fitted with cotton plugs. Petri dishes and pipettes should be wrapped in paper. Rubber materials (except silicone rubber) or any inflammable material should not be kept inside the oven. The oven must be allowed to cool for two hrs before opening the doors since the glassware's may crack by sudden cooling.

22. Sterilisation control The spores of bacillus subtilis subsp. Niger (NCTC 10075 or ATCC 9372) are kept inside the oven. These spores should be destroyed if the sterilisation is proper. Thermocouples may also be used. Browne's tube with green spot is available. After proper sterilisation a green spot is produced ( after two hrs at 160°C).

23. Moist Heat Sterilisation This method of sterilisation may be used at different temperature as follows. 1. At a temperature below 100°C 2. At a temperature of 100°C 3. At a temperature above 100°C

24. At Temperature below 100°C Pasteurisation of milk – two type of method 1. holder method (63°C for 30 min.) 2. flash method ( 72°C for 20 sec. followed by cooling quickly to 13°C or lower) are used. all non sporing pathogens such as mycobacteria, brucellae and salmonellae are killed except Coxiella burnetiid which being relatively heat resistant may survive in holder method.

25. Inspissation Some serum or egg media such as Lowenstein – Jensen and Loeffler’s serum are rendered sterile by heating at 80-85°C temperature for half hour daily on three consecutive days. This process of sterilisation is called inspissation. The instrument used is called inspissator.

26. Vaccine bath Bacterial vaccines are sterilized in special vaccine baths at 60°C for one hour. Serum or body fluids can be sterilized by heating for one hour at 56°C in a water bath on several successive days.

27. Low temperature steam formaldehyde sterilization Items which cannot withstands the temperature of 100°C may be sterilized by a method known as low temperature steam formaldehyde sterilization. In this method steam at sub atmospheric pressure at the temperature of 75°C with formaldehyde vapour is used. Bacillus stearothermophilus has been used as biological control to test the efficacy of LTSF sterilizers.

28. At a temperature of 100°C Boiling – Boiling for 10 to 30 min. may kill most of the vegetative forms. But many spores withstand boiling for a considerable time. When better methods are not available boiling may be used for glass syringes and rubber stoppers. It is not recommended for the sterilization of instruments used for surgical procedures.

29. Tyndallisation Steam at 100°C for 20 min. on three successive days is used. This is known as tyndallisation or intermittent sterilization. The principle is that the first exposer kills all the vegetative forms and in the intervals between the heatings the remaining spores germinate into vegetative forms which are killed on subsequent heating. It is used for sterilization of egg serum or sugar containing media which are damaged at higher temperature of autoclave. The instrument commonly used is Koch's or Arnolds steam steriliser.

30. Steam steriliser at 100°C for 90 min. Kochs or Arnolds Steam steriliser is usually used for 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 at atmospheric pressure for 90 min. Most of the vegetative forms are killed by this method except thermophiles.

31. At temperature above 100°C Water boils when its vapour pressure equal that of the surrounding atmosphere. When the atmospheric pressure is raised then 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. This principle has been applied in autoclave and pressure cooker.

32. FILTRATION This method of sterilisation is useful for substances which get damaged by heat process eg. Sera, sugars, antibiotic solution etc.

33. Uses of filtration To sterilise sera sugars and antibiotic solutions Separation of toxic and bacterio phage's from bacteria. To obtain bacteria free filtrates of clinical samples for virus isolation. Sterilisation of hydatid fluid. Purification of water

34. Limitation The pore size of filter is around 0.75micro meter in diameter which retains bacteria but allows viruses and mycoplasma to pass through filter, therefore filtered preparations are not safe for clinical use.

35. Type of filter Candle filter Membrane filter Air filters Syringe filter

36. Candle filter These have been used widely for purification of water for industrial and drinking purposes. These filters are usually made in the form of hollow candles. The liquid to be filtered is passed through the candle. They are available in different grades of porosity.

37. Membrane filters Membrane filters are made up of cellulose easters. These are routinely used in water analysis sterility testing and for the preparation of solutions for parenteral use. Nitrocellulose membrane filters are widely used. Membrane filters are available in pore sizes of 0.015 to 12 micro meter. The 0.22micrp meter filter is the most commonly used because the pore size is smaller than that of bacteria. For bacterial counts of water a known amount of water is filtered through the membrane filter disc.

38. The upper side of filter disc is placed on a culture medium and incubated. The colonies develop on the medium. Viable counts can be calculated by counting the colonies.

39. Air filters These filters are used to deliver clean bacteria free air to a cubicle or a room. High efficiency particular air filters are used in air filtration in laminar air flow system in microbiology in laboratories. HEPA filters can remove particles of 0.3micro meter.

40. Syringe filters Syringes fitted with membrane of different diameters are available. For sterilisation the fluid is forced through the disc by pressing the piston of the syringe.

41. Ozone Sterilisation Ozone is a substance with a strong bactericidal effect that destroys microorganisms such as viruses and bacteria. Decontamination and disinfection by ozone is also used in sterilization work to prevent viral infections as an effective method.

42. Radiation sterilization Two type of radiation are used for sterilization – ionizing and non – ionizing . Ionizing radiations – ionizing radiation include – gamma rays, x-rays and cosmic rays. they have high penetrating power . They are highly lethal to all cells including bacteria. They damage DNA by various mechanisms. Gamma radiations from a cobalt 60 source are commercially used for sterilization of disposable items such as plastic syringes, swabs, culture plates, cannula catheters etc.

43. This method is also knowns as cold sterilization because there is no appreciable increase in temperature. The advantages of this method include speed high penetrating power and the absence of heat. Bacillus pumilis has been used for testing the efficacy of ionizing radiations. High energy electron radiation is not used widely in medicine.

44. Non-ionizing radiations – these include infrared and ultraviolet radiations. Infrared is used for rapid mass sterilization of syringes and catheters. Ultraviolet radiation with wavelength of 240 to 280nm has marked bactericidal activity. It acts by denaturation of bacterial protein and interference with DNA replication. UV radiation is used for disinfecting enclosed areas such as bacteriological laboratory inoculation theatres. Most vegetative bacteria are susceptible to UV radiation but spores are highly resistant. Susceptibility of viruses is variable.

45. Alcohols – Ethyl alcohol and isopropyl alcohol are the most frequently used, They act by denaturation bacterial proteins they rapidly kill bacteria including tubercle bacilli but they have no sporicidal or activity . However human immunodeficiency virus is susceptible to 70% ethyl alcohol and 35% isopropyl alcohol in the absence of organic matter. They are used mainly as skin antiseptics. Chemical Method

46. To be effective they should be used at a concentration of 60-70 % in water. Isopropyl alcohol is preferred to ethyl alcohol as it is a better fat solvent more bactericidal and less volatile. Methyl alcohol is effective against fungal spores and is used for treating cabinets affected by them. Methyl alcohol vapour is toxic and inflammable.

47. Aldehydes Formaldehyde – It is markedly bactericidal, sporicidal and virucidal, It is used both as aqueous solution and in gaseous form. A 10% aqueous solution of formalin is routinely used. It is active against amino group in the protein molecule. Uses preservation of tissue for histological examination . To sterilise bacterial vaccines To prepare toxoid from toxin.

48. Reference Baveja, CP. 2015 Sterilisation in textbook of microbiology Fifth Edition, Arya Publication, New Delhi, 28 – 34.