Sterilization methods.pptx

CSSD, STERILIZATION

1. Sterilization methods.

2. Hot-Air Oven Hot- air oven is the most widely used methods for Sterilization, which works on the principle of Dry Heat Sterilization. Temperature : holding temperature at 160 C for 2 hours for effective sterilization. Construction : - Consists of an insulated double walled cabinet heated by electricity or heating coils situated at bottom and is constructed to withstand a high temperature. - Walls are made of Stainless steel or Aluminum to prevent heat conduction. - There is a Motor and Fan fitted either at the bottom or back to circulate hot air inside the chamber

3. This hot air increases the temperature inside the chamber thus sterilizing it. A thermostat regulates the temperature the temperature at the desired level and a thermometer is fitted for recording the temperature. The shelves within the hot air oven are perforated to allow proper air circulation.

4. Materials sterilized : Glassware like glass syringes, petri dishes, flasks, pipettes and test tubes. Surgical instruments like scalpels, forceps. Etc. Chemical such as liquid paraffin, fats, Glycerol, oil, and glove powder, etc ,.

5. Precautions : Overloading of hot air oven should be avoided. The 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 silicon rubber) should not be kept inside the oven. The oven must be allowed to cool for two hours before opening the doors, since the glassware may crack by sudden cooling.

6. Sterilization control: The effective sterilization done by hot air oven can be monitored by, 1. Physical method : Temperature monitoring . 2. Chemical method : Browne’s tube (green spot) is used. A green colour is produced after 60 min at 160 C or 115 min at 150 C, suggesting complete sterilization. 3. Biological method : Heat-resistant spores of a non-toxigenic strain of Clostridium tetani or Bacillus subtilis are used to indicate efficiency of dry heat sterilization .

7. Browne’s tube

8. Autoclave Principle : Steam under produces moist heat of very high temperature. stem has higher penetrating capacity than dry heat . - S team lies in the latent heat liberated when it condenses on it increases its water content with ultimate hydrolysis and break down of the proteins and enzymes of microbes. - High temperature combined with high moisture is one of the most effective sterilization agent. - Acts by coagulation and denaturation of bacterial cell wall.

9. Temperature : 121 C for 15 min at 15 lbs. Construction : I t is made of double walled metallic vessel made of thick stainless steel or copper. - one end of which has an opening meant for keeping the materials in to the vessels for sterilization. - the lid or door is provided with pressure gauze to measure the steam pressue . - At the top there will be a safety valve to permit the escape of steam from chamber if it gets too high – to prevent explosion of the sterilizing chamber. - there are valves to hold the steam and stem outlet. - there is an exhaust valve below to let steam escape from bottom.

10. Precautions : The autoclave should not be overloaded , the material should be arranged in such that every part of the material should be exposed to the steam. Air inside the chamber should completely evacuated by opening the outlet to escape sir along with steam and then valve is closed. Sterilization time must be counted not from autoclave before switching on but the time after achieving the required pressure. Ensure there is sufficient water in an autoclave before switching on. The lid should be closed tightly. At the end of sterilization period, allow the steam pressure drop to zero and only then open the lid. During sterilization the recommended steam pressure should not be exceeded on any account .

11. Uses of Autoclave It is particularly used for media containing water that cannot be sterilized by dry heat. Surgical instruments Culture media Autoclavable plastic containers Plastic tubes and pipette tips. Solutions and water Bio hazardous waste Glassware.

12. Types of Autoclaves. Gravity displacement type autoclave : it is the most common type used in laboratories. They are available in various sizes. 1. Vertical type. ( small volume capacity) 2.Horizontal type (large volume capacity). Positive pressure displacement type autoclave. Negative pressure(vacuum) displacement.

13. Vertical autoclave.

14. Horizontal autoclave.

15. Sterilization control : Biological method : spores of Geobacillus sterothermophilus incarnated in a strip- are the best indicators, because they are resistant to steaming. the spores are killed in 12 min at 121 C. Chemical method : chemical indicators, autoclave tapes and thermocouples are used.

16. Sterilization by Filtration. Is an effective method of sterilization to remove microorganism from liquids and air. Filtration is used to prepare liquid that cannot withstand heat like serum, vaccines ,sugar solutions, drugs, enzymes etc. important in purification of water. Types of Filters : 1. Depth Filters. 2. Membrane filters.

17. Difference b/w Depth filter and M embrane filter.

18. Depth filters. Depth Filters consists of Fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter. The solution containing microorganism is sucked through this layer under vacuum, and microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of the filter material.

19. Advantage of depth filters . – they can retain a large mass of particles before becoming clogged, flow rate of fluid is high, low cost. Disadvantage : Some of the particles still come out of the filtrate so not suitable to filter the solutions containing Bacteria. Types of Depth Filters. 1. Candle filters 2. Asbestos filters 3. Sintered glass filters. Candle Filters : Manufactured in different pore sizes.prepared by mixing with asdectos and organic matter like plaster of paris (POP) in different proportion depending upon the pore size. It is of two types. 1. Diatomatious earth material( Berkefeld filters) 2. Unglazed porcelain material ( Chamberland filters)

20. Berkefeld and chamberland filters.

21. Seitz Filter/ S terimat filters : Consists of a compressed filter sheet of asbestos filter. The fiber are 2-6 mm thick. - It contains washed asbestos fibers, cotton liner and other filter material. - It is very soft and can be easily damaged. -The filter disc are always fitted to filter assembly for filtration purposes. - T he filter disc is fitted to a funnel like structure through which the liquid to be filtered is sucked through vacuum or negative pressure.

22. Sintered glass filter : prepared by heat-fusing finely powdered glass particles of graded size. I t is sealed into body of funnel and thoroughly washed with sulphuric acid which will oxidizes any organic matter if present, and followed by washing with distilled water. These may be classified into Extra Course, Course, Medium. Fine & Ultrafine .

23. Membrane Filters Replaced by depth filters. Menbrane filters are made up of Cellulose acetate , Cellulose nitrate and other synthetic materials . These filters are porous membranes, over 0.1 mm thick, and Average P ore Diameter(APF) of 0.22um which removes most of the bacteria. Are routinely used in water purification and analysis, sterilization and sterility testing.

24. Mode of operation of membrane filters. The apparatus consist of a funnel with a membrane filter attached to its base. It is then fitted on to a vacuum flask. The liquid to be filtered is poured into the funnel. The vacuum are negative pressure is applied to force the liquid through the membrane.

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