STERILIZATION Definition: The freeing of an article from all living organisms, including viruses, bacteria , spores & fungi: Pathogenic & non-pathogenic.

Uses Sterilization is used for : Microbiology : Culture media, suspending fluids, reagents, containers & equipment Hospital : medical & surgical instruments , surgical operations, I.V. infusions, hypodermic injections , diagnostic aspirations.

Methods Physical or chemical: Physical Methods: Heat Filtration Irradiation (2) Chemical Methods: @ Strong disinfectants: * Formaldehyde * Ethylene oxide

Sterilization By Heat Moist Heat: @ Most effective and efficient. @ Kills organisms by denaturing their enzymes & proteins @ Spores are killed by exposure to moist heat at 121°C for 10-30 minutes. Dry Heat: @ Kills organisms by oxidative destruction of cell constituents. @ Spores killed at 160°C for 1 hour

Factors influencing Sterilization by Heat: Temperature & exposure time: Higher temperature, shorter time and vice versa. 2.No.of Vegetative cells + spores @ Plenty of organisms, make sterilization less efficient. @ So, clean before sterilizing

4. Nature of material containing organisms 3. Nature of organism: Vegetative cells & spores affect susceptibility of organism 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.

Holding 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.

Thermal 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.

Methods 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.

3. Hot-air oven: 4. Infra-red Radiation: @ Oven has a thermostat + fan to circulate hot air @ Works at 160˚C for 1 hour, to sterilize glassware, metals , swabs, oils, powder 4. 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.

Methods 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 a sterilization method. Temperature Below 100°C: @ This is the pasteurization process. @ Used for milk, vaccines & utensils.

Milk: @ 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)

Pasteurization is tested by :- @ Phosphatase test : testing for presence of phosphatase enzyme found in milk and destroyed by pasteurization. @ Methylene blue test : To indicate that bacteria present in milk is destroyed. Coxiella burneti is destroyed by flash method.

Vaccines: Inactivated in the vaccine water bath, at 60°C for 1 hr House articles: Utensils, clothing, bedding, are disinfected at 70°– 80°C for several minutes.

Temperature 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

Steaming at 100°C: @ This is done by the steamer (Koch Steamer) @ It uses steam of boiling water at 100°C and at atmospheric pressure.

@ Steamer is used in two ways:- Single exposure at 100°C for 90 minutes. @ Time includes heating-up time. @ Thermophilic & mesophilic spores will survive this treatment.

2. 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.

Temperature above 100˚C: @ Uses saturated steam - better than dry hot air because:- Lethal action of moist heat is more Quicker in : heating up exposed particles penetrating cotton stoppers, paper, wrappers, surgical linen & hollow apparatus

How Does it Act? @ Saturated steam when meets an article, it condenses to a small volume of water & liberates its latent heat to article surface. @ Avoid presence of air that prevents steam penetration in article

What 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.

Importance of Air Discharge: @ Air is removed from autoclave: a) Mixture of steam + air lower the temperature. b) Air hinders penetration of steam in the load. @ Air denser than steam, sinks down & makes a layer over load

@ Sealed bottles containing solutions are autoclaved although air is found in them – WHY? Because water found in these solutions is heated up to steam temp. and so performing same work as moist heat.

Types 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.

@ Autoclave is opened at right time because: # If opened still under pressure, an explosion occurs # If opened below atmospheric pressure, evaporation of aqueous materials occurs.

Simple Autoclave Deficiencies : Lacks control of air discharge (nothing to show that discharge is complete) 2. Lacks means of drying load after sterilization. @ Suitable for load wrapped in paper to prevent contamination.

2. Steam-Jacketed Autoclave: @ Has got an automatic air & condensate discharge. @ Load is dried up by steam circulating in jacket, & vacuum in autoclave.

3. 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

Advantages of high pre-vacuum sterilizers: Operation time is shortened. Damage to heat sensitive materials (sugars) is avoided.

Autoclave Control and Indicators: Automatic process control, * Advantages are:- @ Saving time of an operator. @ Safeguard against errors due to negligence.

2. Recording Thermometer, @ Draws graphic records of temperature changes inside chamber discharge channel. @ Helps to avoid errors in timing the holding period.

3. Thermocouple load temp. measurement: @ Thermocouple is inserted deeply inside load, its wire is carried to a potentiometer which reads temperature inside the load .

4. 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 .

b) 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 .

5. Spore indicators, @ Bacillus stearothermophilus spores are destroyed at 121˚C for 12 minutes. @Spores placed within load and cultured after sterilizing

Disadvantages 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

Sterilization 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

2. 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 resists radiation (has efficient DNA repair)

Sterilization 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.

Test 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.

Uses 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.

2. 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.

@ Membrane is 120 µm thick, placed in two layers. @ Bacteria are trapped in upper layer : better than Seitz filter Micro-filters: @ To filter small fluid volumes @ May be membrane, or asbestos @ May be syringe or centrifuge filter.