Presentation on theme: "STERILIZATION Definition:"— Presentation transcript:
1STERILIZATION Definition: The freeing of an article from all living organisms, including viruses, bacteria , spores & fungi: Pathogenic & non-pathogenic.
2Uses Sterilization is used for : Microbiology : Culture media, suspending fluids, reagents, containers & equipmentHospital : medical & surgical instruments , surgical operations, I.V. infusions, hypodermic injections , diagnostic aspirations.
3Methods Physical or chemical: Physical Methods: Heat Filtration Irradiation(2) Chemical Methods:@ Strong disinfectants:* Formaldehyde* Ethylene oxide
4Sterilization By Heat Moist Heat: @ Most effective and efficient. @ Kills organisms by denaturing their enzymes &proteins@ Spores are killed by exposure to moist heatat 121°C for minutes.Dry Heat:@ Kills organisms by oxidative destruction ofcell constituents.@ Spores killed at 160°C for 1 hour
5Factors influencing Sterilization by Heat: Temperature & exposure time:Higher temperature, shorter time andvice versa.2.No.of Vegetative cells + spores@ Plenty of organisms, make sterilization less efficient.@ So, clean before sterilizing
64. Nature of material containing organisms 3. Nature of organism:Vegetative cells & spores affect susceptibility oforganism to heat.4. Nature of material containing organisms@ Efficiency of sterilization is reduced by presence of organic matter that protect bacteria from lethal action of heat@ Presence of a disinfectant enhances the kill.@ Spores best killed in acidity or in alkalinity.
7Holding Time: Thermal Death Point: Time required for killing organism. (Not including heating-up time).Thermal Death Point:Lowest temperature to give complete killing in aqueous suspension within 10 min. at standard conditions.
8Thermal Death Time:Shortest time for complete killing at a stated temperature under standard conditions.Decimal Reduction Time (D-value):Time in minutes required to achieve a ten fold reduction in viability of a bacterial population at a given temperature under standard conditions.
9Methods of Sterilization By Dry Heat: Red Heat:Article is held on flame until red hot (inoculating wires, forceps, spatulas)2.Flaming:Burning an article in spirit or gas flame (scalpels, needles)Method will not produce sterilization.
103. Hot-air oven: 4. Infra-red Radiation: @ Oven has a thermostat + fan to circulate hot air@ Works at 160˚C for 1 hour, to sterilizeglassware, metals , swabs, oils, powder4. Infra-red Radiation:@ Given by an electrically heated element@ To sterilize glass syringes at 160°C & surgical instruments above 200°C in vacuum chamber in which N2 is passed during cooling time to avoid oxidation.
11Methods of Sterilization by Moist Heat: @ Employed at a temp. below 100°C, at 100˚C,and above 100°C.@ First two are disinfection methods, third is asterilization method.Temperature Below 100°C:@ This is the pasteurization process.@ Used for milk, vaccines & utensils.
12Milk: @ Pasteurized by 2 methods:- Holder method: at 63°–66°C for 30 min.Flash method: at 72°C for 20 seconds.@ Both methods destroy only milk-borne organisms (Myco., Brucella, Salmonella)
13Pasteurization is tested by :- @ Phosphatase test : testing for presence ofphosphatase enzyme found in milk anddestroyed by pasteurization.@ Methylene blue test : To indicate thatbacteria present in milk is destroyed.Coxiella burneti is destroyed by flash method.
14Vaccines:Inactivated in the vaccine water bath, at 60°C for 1 hrHouse articles:Utensils, clothing, bedding, are disinfected at 70°– 80°C for several minutes.
15Temperature at 100°C:Boiling at 100°C:@ Kills non-sporing bacteria within 5–10 min.@ Used to disinfect blades, syringes,@ Dry up articles on removing from boiler (sterilizer) to prevent contamination
16Steaming at 100°C:@ This is done by the steamer(Koch Steamer)@ It uses steam of boiling water at 100°C and at atmospheric pressure.
17@ Steamer is used in two ways:- Single exposure at 100°C for 90 minutes.@ Time includes heating-up time.@ Thermophilic & mesophilicspores will survive this treatment.
182. Tyndallization:Exposure at 100°C for 20 – 45 minutes for 3 successive days.@ Used for sterilizing sugars & gelatin@ First steaming kills vegetative bacteria and spores germinating following day are killed by subsequent heating and so on.@ Draw-back of tyndallization : spores not germinating in medium sterilized + thermophilic & anaerobic bacteria will escape killing.
19Temperature above 100˚C:@ Uses saturated steam - better than dry hot air because:-Lethal action of moist heat is moreQuicker in :heating up exposed particlespenetrating cotton stoppers, paper,wrappers, surgical linen & hollow apparatus
20How Does it Act?@ Saturated steam when meets anarticle, it condenses to a smallvolume of water & liberates itslatent heat to article surface.@ Avoid presence of air thatprevents steam penetration in article
21What is the apparatus Used? @ Autoclave : provides sterilization by dry saturated steam (steam at point of condensing to water)@ Steam is under pressure higher than atmospheric.
22Importance of Air Discharge: @ Air is removed from autoclave:a) Mixture of steam + air lower thetemperature.b) Air hinders penetration of steam inthe load.@ Air denser than steam, sinks down & makes a layer over load
23@ Sealed bottles containing solutions are autoclaved although air isfound in them – WHY?Because water found in these solutions is heated up to steam temp. and so performing same work as moist heat.
24Types of Autoclaves: Simple Autoclave: @ Pressure-cooker type, simple, not jacketed, & used in laboratory.@ Consists of a cylinder for water to be heated, articles placed on a tray@ Has got a discharge valve, + pressure & temperature gauges.
25@ Autoclave is opened at right time because:# If opened still under pressure, anexplosion occurs# If opened below atmospheric pressure, evaporation of aqueous materials occurs.
26Simple Autoclave Deficiencies : Lacks control of air discharge(nothing to show that discharge is complete)2. Lacks means of drying loadafter sterilization.@ Suitable for load wrapped in paper to prevent contamination.
272. Steam-Jacketed Autoclave: @ Has got an automatic air & condensate discharge.@ Load is dried up by steam circulating in jacket, & vacuum in autoclave.
283. High Pre-Vacuum Sterilizers: @ Have electric pumps creating a vacuum area in chamber.@ First a vacuum is drawn, steam is admitted to chamber, load is heated very rapidly.@ Temp. used 135˚C for 3 min. at 30 lb pressure & load dried by exhaustion of chamber
29Advantages of high pre-vacuum sterilizers: Operation time is shortened.Damage to heat sensitive materials (sugars) is avoided.
30Autoclave Control and Indicators: Automatic process control,* Advantages are:-@ Saving time of an operator.@ Safeguard against errors due to negligence.
312. Recording Thermometer, @ Draws graphic records of temperature changes inside chamber discharge channel.@ Helps to avoid errors in timing the holding period.
323. Thermocouple load temp. measurement:@ Thermocouple is inserted deeply inside load, its wire is carried to a potentiometer which reads temperature inside the load .
334. Chemical indicators:Browne’s Tubes, containing a red fluid that turns green on heating:* at 115˚C for 25 minutes (Type 1)* at 115˚C for 15 minutes (Type 2)* at 160˚ C for 60 minutes (Type 3)@ Tubes are stored below 20˚C to avoid change of color .@ Satisfactory for routine work .
34b) Bowies-Dick tape,@ Used to test efficiency of high pre-vacuum sterilizers & high-pressure autoclaves@ An adhesive tape is applied around load in the shape of (X).@ After sterilization, tape changes color all over .
355. Spore indicators,@ Bacillus stearothermophilus spores are destroyed at 121˚C for 12 minutes.@Spores placed within load and cultured after sterilizing
36Disadvantages of Chemical and Spore Indicators: @ Will not give a perfect efficiency of sterilization:( heating may be inadequate in a site away from of indicator)* To solve problem, autoclave is correctly operated & controlled by a thermometer & not by pressure gauge alone
37Sterilization by Radiation Ultra-violet Radiation:@ U.V. rays induce thymine diamers in cell DNA, to destroy bacterial cell .@ Produced by mercury vapor lamps, used to sterilize plastics
382. Ionizing Radiation:@ High-speed electrons, X-ray, gamma rays, using cobalt 60@ Produces free DNA radicals that destroy bacteria .Indicator for Radiation:Micrococcus radiodurans that resistsradiation (has efficient DNA repair)
39Sterilization by Filtration @ Used to sterilize toxins, serum, antibiotics@ Uses a filter of pore less than 0.75 µm for bacteria, & much smaller for viruses.@ Some bacterial filters allow viruses, and Mycoplasma to pass through.
40Test of Filter: @ Retain Serratia marcescens from broth culture. Types of Filters:Asbestos pad (Seitz) filter:@ Consists of an upper cylinder and a lower funnel with an asbestos pad in between.
41Uses of asbestos filter pads : Rapid clarification of fluids.Clarifying viscous fluids.General sterilization.4. Removing pyrogenes.5. Removing small organisms.6. Sterilizing serum.Disadvantage:Asbestos pads absorb some of filtrate.
422. Sintered glass filters ; Made up of ground glass, fused to make glass granules adhere together .3. Cellulose membrane filters:@ Less absorptive, has got a greater rate of filtration than Seitz filter.@ Used to separate viruses.* 2 types of membrane filters:a) Cellulose nitrate( Gradocol membrane) .b) Cellulose acetate: commonly used now.
43@ Membrane is 120 µm thick, placed in two layers. @ Bacteria are trapped in upper layer : better than Seitz filterMicro-filters:@ To filter small fluid volumes@ May be membrane, or asbestos@ May be syringe or centrifuge filter.