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STERILISATION - 1

STERILISATION - 1. DR SATHYA ANANDAM. Introduction. Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth. Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage.

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STERILISATION - 1

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  1. STERILISATION - 1 DR SATHYA ANANDAM

  2. Introduction • Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth. • Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage. • Low water content or low pH prevents microorganisms multiplying

  3. Introduction • In mid 1800s Semmelweiss and Lister developed aseptictechniques to prevent contamination of surgical wounds. • Nosocomial infections caused death in 10% of surgeries. • Up to 25% mothers delivering in hospitals died due to infection

  4. HAND WASHING- Semmelweis • Practices, emphasizes the importance of washing hands with chlorinated water in Obstetrics to reduce maternal mortality

  5. Beginning of Safe Operation Theatre Practices • 1867 –Dr.Joseph Lister first identifies airborne bacteria and used Carbolic acid spray in surgical areas • 1880 – Johnson and Johnson introduce antiseptic surgical dressings.

  6. Historical Aspects Changed the History • Lister era 1868 changed the concept of safe surgical procedures. • Carbolic Acid in decontamination caused Reduction of Hospital associated infections • Mortality reduced • Morbidity reduced.

  7. Importance • Laboratory work with pure cultures requires the use of apparatus and culture media that is sterile • Prevention of infection in patients requires the use of equipment, instruments, dressings and parenteral drugs that are free from living micro-organisms

  8. Definitions • Sterilization is the process of killing all the microorganisms in all its forms inclusive ofbacterial endospores in a preparation/article such as lab media, surgical instruments and equipment. • A sterile environment is free of life of every kind.

  9. Definitions Disinfection: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. • Usually involves the removal of vegetative or non-endosporeforming pathogens. May use physical or chemical methods. Fumigation: Process of disinfection by fumes of vaporized germicide

  10. Definitions Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination. Aseptic techniques are used to • prevent contamination during surgery. • prevent bacterial contamination in food industry

  11. Definitions ANTISEPSIS:Is the prevention of infection by inhibiting the growth of bacteria. ANTISEPTICS:Chemical disinfectants which can safely be applied to skin or mucus membranes to prevent infection. CONTAMINATION: Soiling of an article, food or water with microbes by contactis known as soiling. DECONTAMINATION: Soil-removing process which removes many of microbes

  12. Definitions • Disinfectant: Applied to inanimate objects. • Antiseptic: Applied to living tissue (antisepsis). • Degerming: Mechanical removal of most microbes in a limited area. • Sanitization: Use of chemical agent to reduce the microbes to minimize chances of disease transmission. Eg: Hot water & soap

  13. Definitions Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them Germicide: An agent that kills certain microorganisms. • Bactericide: An agent that kills bacteria. Most do not kill endospores. • Viricide: An agent that inactivates viruses. • Fungicide: An agent that kills fungi. • Sporicide: An agent that kills bacterial endospores & fungal spores.

  14. CLASSIFICATION: A. PHYSICAL AGENTS: 1. SUNLIGHT 2. DRYING 3. HEAT A. Dry Heat (a) Red heat. (b) Flaming. (c) Incineration. (d) Hot Air Oven.

  15. B. Moist Heat : (a) Temperature below 1000C. Pasteurization. Inspissation. (b) Temperature at 1000 C: Ex: Boiling (c) Steam under normal pressure. Ex: Tyndallization (d) Steam under pressure. Ex: Autoclave

  16. 2. FILTRATION. (A) Candle filters. (B) Asbestos pads (Seitz filter) (C) Sintered glass filter. (D) Collodion or membrane filters. (E) HEPA filters. 3.RADIATION. 4. ULTRASONIC AND SONIC VIBRATIONS.

  17. B)CHEMICAL AGENTS: • Alcohols • Aldehydes • Gases • Halogens. • Phenols • Dyes • Surface active agents. • Metallic salts.

  18. Mode of action • Sterilization and disinfection:Damage of DNAProtein denaturationDisruption of cell membrane or cell wallChemical antagonismRemoval of free sulfahydryl group

  19. Control of Microbial Growth: Heat: Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes. Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a liquid suspension will be killed in ten minutes.

  20. Control of Microbial Growth: • Thermal Death Time (TDT): Minimal length of time in which all bacteria will be killed at a given temperature. • Decimal Reduction Time (DRT): Time in minutes at which 90% of bacteria at a given temperature will be killed.

  21. Physical Methods

  22. SUNLIGHT • Spontaneous sterilisation in natural environment • Due to UV rays and heat rays

  23. Drying • Moisture is essential for growth of bacteria • Deleterious effect on many bacteria • Unreliable method • No effect on spores

  24. Dry Heat-methods • Dry Heat: Kills by protein and enzyme denaturation, oxidation & toxic effect of elevated levels of electrolytes. • Flaming glass slides, mouth of the culture bottles • Red heat inoculating loops and needles. Heat metal until it has a red glow • Incineration: Effective way to sterilize disposable items and biological waste.

  25. Red Heat: • Instant method of sterilization. • The articles to be sterilized are held in Bunsen flame till they become red hot. • Ex :Inoculating wires, Tips of forceps, Scalpels, Searing spatulas, Needles.

  26. 2. Flaming : • This method is of uncertain efficacy. • The articles has be passed a few times through the Bunsen flame but not allowed to get red hot. • Ex: Glass slides, Cover slips, Mouths of culture tubes

  27. FLAMING

  28. INCINERATION : • Excellent method for rapid destructionof Soiled dressings, Animal carcasses, Pathological specimens. • Effective way to sterilize disposable items • But Polystyrene materials emit clouds of dense black smoke. • Reduced to ashes by burning. • Temperature at which it works is 7000 C - 9000 C.

  29. Incineration is a process where by combustible materials are converted into non combustible residue or ash ;achieving reduction in weight or volume . consists of two chambers –primary& secondary . • Primary –temp of 750-8500c • Secondary – temp of 1000-1100 0c Chimney of incinerator –30 mts high combustion efficiency –99% .

  30. PRINCIPLE OF INCINERATOR

  31. INCINERATOR

  32. HOT AIR OVEN • Most widely used sterilizer by dry heat. • Electrically operated instrument. • Hot air is a bad conductor of heat and its penetrating power is low. • Fitted with fans for adequate air circulation in the chamber. • Holding time & temperature :1600 C for 2 hrs or 1800 C for 30 mts.

  33. Uses: • Glassware: Syringes, Petri dishes, test tubes, flasks, pipettes, etc. • Surgical instruments (forceps, scalpels, scissors). • Oily fluids( Liquid paraffin) • Chemical & dusting powders,sulphonamides • Swabs • Silicon rubber only (not other rubber material) • Fats, Grease

  34. Precautions: 1. Articles should be absolutely dry & wrapped in paper. 2. Not suitable for rubbers, fabrics, inflammable or volatile substances. 3. It should not be overloaded. 4. The oven should be allowed to cool for 2 hrs before opening the doors to prevent cracking of glassware.

  35. Hot air oven-steps • Decontaminate, clean, and dry the items • Wrap items using foil, double-layered cotton, craft paper or muslin fabric; • Put unwrapped items on a tray or shelf; • Place instruments and other items in a metal, lidded container • Place in the oven, and heat to the designated temperature.

  36. Hot air oven • 170 degrees C (340 degrees F) - 1 hour • 160 degrees C (320 degrees F) - 2 hours • 150 degrees C (300 degrees F) - 2.5 hours • 140 degrees C (285 degrees F) - 3 hours • Cutting instruments sterilized for 2 hours at 150oC • For oils, glycerol, dusting powder – 1hour at 150oC

  37. Dry heat can dull sharp instruments and needles, these items should not be sterilized at temperatures higher than 160 degrees C. • Leave items in the oven to cool before removing. • When they are cool, remove items using sterile pickups and use or store immediately.

  38. Sterilization control: 1. Spores of non-toxigenic strains of Cl.tetani. Paper strips impregnated with 106 spores are placed in envelops & inserted into suitable packs. Then inoculate into thioglycollate or cooked meat broth & incubate under strict anaerobic conditions for 5 days at 37oC . 2. Browne's tube (green spot): This is convenient for routine use. After 60 minutes at 160oC or 115 minutes at 150oC. 3. Thermocouples may also be used.

  39. Hot air oven

  40. Moist heat • More effective- high penetration power • Kills micro-organisms by coagulation and denaturing their enzymes and structural proteins. • A process in which water participates therefore necessary for all parts of the load to be in contact with water molecules or steam. • Sterilization requires moist heat at 1210c for 15minutes, usually using an autoclave.

  41. MOIST HEAT STERILISATION IS CARRIED OUT WITH FOLLOWING METHODS Killing effect – Denaturation and coagulation of protein. • Temp below 100oC: “Pasteurisation”, Inspissator. • Temperature at 100oC: Boiling. • Steam at atmospheric pressure: Koch/Arnold’s steamer. • Steam under pressure: Autoclave.

  42. MOIST HEAT TEMPERATURES BELOW 100oC • Pasteurization: Developed by Louis Pasteur to prevent spoilage of beverages. • Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.

  43. Classic Method of Pasteurization: Milk was exposed to 63oC for 30 min (holder method) followed by cooling quickly to 13oC or lower. • High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds (flash process) followed by cooling quickly to 13oC or lower.. • Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 sec and then cooled very quickly in a vacuum chamber.

  44. HTST Pasteurisation

  45. Advantage: • Milk can be stored at room temperature for several months. • All nonsporing pathogens destroyed by these methods (mycobacteria, brucellae, and salmonellae). Coxiellaburnetii survives holder method. • Serum or body fluids containing coagulable proteins sterilized by heating for 1 hour at 56oC in a water bath on several successive days. • Vaccines: Non sporing bacterial vaccines heat inactivated in special vaccine baths at 60oC for 1 hour.

  46. INSPISSATOR Working Principle: • Temperature below 1000 C by moist heat. • Chamber made up of copper plates fitted with a water jacket around it. • With a glass or wooden lid. • Thermostat fitted in it. • There are holes in water jacket through which water vapors comes out to keep the medium moist. • Screw capped Media tubes are kept in slanting position to prepare slopes.

  47. Sterilization temperature & Holding period : 80 - 850 C for 30 mts. for three successive days. Uses: Sterilization of Heat labilesubstances Serum containing medium Ex: Loeffler's serum slope Medium that containing egg protein Ex: Lowenstein - Jensen's medium.

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