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Sterilization by Heat Rohit Jain • Heat is the most reliable and commonly employed method of sterilization. • Method of choice unless contraindicated. • Generally there are two types: dry heat and moist heat • Dry heat kills the organisms by denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated levels of electrolytes. – Dna may also be damaged/destroyed • Moist heat kills the microorganisms by denaturation and coagulation of proteins. Factors Influencing Sterilization by Heat • Dry or Moist Heat • Temperature and Duration – time required for sterilization is inversely proportional to temperature. • Specific characteristics of microorganisms and spores present Factors Influencing Sterilization by Heat – bacteria spores are killed by moist heat at 121⁰c for 15min – Most vegitative bacteria, fungi, and viruses are killed in 30 min at 65⁰c by moist heat • Type of material – A high content of organic material tend to protect vegitative bacteria and spores against destruction by heat. Therefore materials containing organic substances require a longer time to sterilize effectively. Dry Heat • • • • Red Heat Flaming Incineration Hot Air Oven • Red Heat: wires, loops, tips of forceps, and needles are held in the open flame of a bunsen burner until they are red hot. This sterilizes them. • Flaming: Glass slides, scalples, and mouths of culture tubes are passed through the flame of a bunsen burner, without becoming red hot. • Incineration: infective material is reduced to sterile ash by burning in an incinerator. Soiled dressings, animal carcasses, bedding and pathological materials are dealt with using this method. • Hot Air Oven: most widely used method for sterilization by dry heat. Oven is heated electronically and is well ventilated. Hot Air Oven • Sterilization takes heating at 160⁰c for two hours – Or at 170⁰c for 1 hour, or at 180⁰c for 30 min • Used for sterilization of: – Glass materials- syringes, petridishes, flasks, pipettes, and test tubes – Surgical intruments- scalples, scissors, forceps – Chemicals- liquid paraffin, fats, sulphonamides powders Precautions for Hot Air Ovens • • • • Do not overload oven Materials to be placed to maximize air circulation Materials should be perfectly dry Test tubes, flasks and such should be fitted with cotton plugs • Petridishes and pipettes should be wrapped in paper • No rubber or flammable materials (except silicone rubber) • Wait for two hours to open doors after sterilization to cool Sterilization control • Spores of bacillus subtilis subsp. Niger are kept inside oven. These spores should be destroyed if the sterilization is proper. • Browne’s tube with green spot is used. After proper sterilization a green color is produced. Moist Heat • Can be used at different temperatures: – Below 100⁰c – At 100⁰c – Above 100⁰c Moist Heat below 100⁰c • Used for pasteurization of milk. Two methods are available: – Holder method • 63⁰c for 30 min – Flash method • 72⁰c for 20 seconds followed by quickly cooling to 17⁰c – All nonsporing pathogens such as mycobacteria, brucellae and salmonellae are killed except coxiella burnetii, which can withstand high temperatures. Moist Heat below 100⁰c • Used for inspissation- some serum or egg media are sterilized by heating at 80⁰c for 30 min daily on three consecutive days. – Examples are Lowenstien-Jensen’s serum and Loeffler’s serum • Used in vacccine bath- bacterial vaccines are sterilized in special vaccine baths at 60⁰c for 1 hour. Serum or body fluids can be sterilized by heating for 1 hour at 56⁰c in a water bath on several successive days • Used in low temperature steam formaldehyde sterilization (LTSF) – Materials which cannot withstand 100⁰c are sterilized using5 LTSF. Steam at subatmospheric pressure at 75⁰c with formaldehde vapor is used. Moist Heat at 100⁰c • Boiling for 30 min kills most vegitative forms but spores remain. May be used for glass syringes and rubber stoppers if better methods are not available. Not recomemeded for surgical equipment. • Tyndallisation- steam at 100⁰c for 20 min on three successive days. Also known as intermittent sterilization. First time kills vegitative bacteria, then it cools and spores become active, and then are killed again the next day Moist Heat above 100⁰c • Steam hotter than 100⁰c kills better than dry heat. • Bacteria are more susceptible to moist heat as bacterial protein coagulates rapidly. • Sterilization is done using an autoclave. • An Autoclave is a modified pressure cooker. It has a stainless steel cylinder in a supporting iron case. The lid is airtight but has a discharge tap for air and steam. The lid also has a pressure gauge and a safety valve. It is heated by electricity. The steam circulates within the jacket and is supplied under high pressure to the inner chamber where materials are kept for sterilization Autoclave • Used to sterilize culture media, rubber material, gowns, dressing, gloves etc. • Particularly useful for materials which cannot withstand the higher temperature of hot air ovens • Used for all glass syringes, but hot air oven is more effective.
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