Laboratory work with pure cultures requires the use of apparatus and culture media that is sterile 2. Prevention of infection in patients requires the use of equipment, instruments, dressings and parenteral drugs that are free from living microorganisms General definitions: Sterilization is the process of killing all of the microorganisms in all its forms in a preparation/article such as lab media, surgical instruments and equipment. A sterile environment is free of life of every kind. Disinfection is the process of destroying pathogenic organisms on the surface, but does not involve the tissues. It is a process which reduces the number of contaminating micro-organisms, to a level which is no longer harmful to health .This usually involves the application of chemicals. Handscrubbing prior to surgery is an example. Antisepsis is used to describe disinfection applied to living tissue such as a wound. Septic is the presence of pathogenic organisms in living tissues, such as an infected surgical site. Aseptic is the absence of pathogenic microbes. Bactericidal agents effectively kill bacteria, and no growth is seen after the removal of the agent. Bacteriostatic agents inhibit bacterial multiplication; growth is seen after the agent is removed. Pasteurization is the rapid heating and cooling of milk and other liquid products to kill pathogens, such as Mycobacterium, Brucella and Listeria. Tyndallisation is intermittent heating.

Mode of action of sterilization and disinfection: Damage of DNA Protein denaturation

Disruption of cell membrane or wall Chemical antagonism Removal of free sulfa hydral group Sterlization in practice, has a probability of failure. An article may be regarded as sterile if it can be demonstrated that there is a probability of less than 1 in a million of there being viable micro-organisms on it. Physical and chemical sterilization may be used. 5 main methods for sterilization: 1. Heat 2. Ionizing radiation 3. Filtration 4. Sterilant gases 5. Sterilant liquids

HEAT Moist heat is more effective than dry heat because it kills micro-organisms by coagulatin and denaturing their enzymes and structural proteins, a process in which water participates. It is therefore necessary for all parts of the load to be in contact with water molecules or steam. Sterilization requires moist heat at 121C for 15 minutes, usually using an autoclave. Steam is non-toxic and non-corrosive, but for it to be effective, it must hold all the water that it can carry in the form of transparent vapour. There are sterilizers for porous load(dressings, wrapped instruments and wrapped gowns and drapes); sterilizers for fluids in sealed containers;sterilizers for unwrapped instruments; and laboratory sterilizers (culture media, glassware and lab equipment). Thermometers and pressure gauges are recorded for every load. Indicator tapes may be used. Before, Bacillus stearothermophilus was used to test autoclaves. It can withstand 121c for 12 mins.

Dry heat kills micro-organisms by the destructive oxidation of essential cell constituents. Killing of the most resistant spores requires a temperature of 160C for 2 hours. This is an efficient method for sterilizationand disposal of contaminated materials (pathologic waste,surgical dressings, sharp needles and other clinical waste). Red heat is a form of dry heat, such as sterilizing inoculating wires using the flame of a Bunsen burner, avoiding splattering. Flaming is used for scalpels and the necks of flasks and test tubes. Hot air sterilizers are used to process materials which can withstand high temperatures, but will be affected by steam. (powders, microsurgical instruments)

IONIZING RADIATION Ionizing radiation, including x-rays and gamma rays are lethal to all cells. Bacterial species difer intheir sensitivity to ionizing radiation, and spores are generally more resistant. This is used in the large-scale sterilization of plastic syringes and catheters. It uses a linear accelerator or cobalt-60.

GASEOUS PROCESSES Ethylene oxide is a highly penetrative, non-corrosive, microbicidal gas which is used for the sterilization of single-use, heat –sensitive medical devices like prosthetic heart valves. Materials are exposed to a gas concentration of 700-1000mg/L at 4560C, 70% humidity, for 2 hours. Formaldehyde and low-temperature steam may be used for the reprocessing of heat-sensitive equipments.

FILTRATION Fluids, including bacterial cultures,can be rendered free of bacteria by passage through filters with a pore size of less than 0.45 m.Most viruses and mycoplasmas can pass through fileters with a pore size as low as 0.22 m. This method is used in : 1. Separation of toxins and other soluble products of bacterial growth 2. Preparation of thermolabile parenteral solutions (antibiotics) 3. Certain blood products

__________ DISINFECTION Moist heat is the method of first choice. Washing of laundry or eating utensils in water at 70-80C for a few minutes will kill most non-spore forming micro-organisms. Steam at 73C for thermolabile reusable equipment is also used. Exposure to boiling water for 20 minute achieves disinfection, and may be used for emergencies. Ultraviolet radiation using mercury lamps at 240-280nm may be used for treatment of air, water and lab cabinets. Gases such as formaldehyde may be used for complex heat –sensitive equipment like anaesthetic machines and incubators. Filtration of air removes micro-organisms from critical sites such as the operating room, and for the labs handling pathogenic organisms. A high-efficiency particulate air (HEPA) filter achieves 9.997% arrestance to particles of 0.5 m, and can produce a sterile air. Disinfection by Chemicals 1. Alcohols – isoprophyl, ethanol; these have optimal bactericidal activity at 7090% concentration. They have limited activity against mycobacteria and spores; effective against viruses. Due to their volatile nature, they are recommened as rapidly drying disinfectants for skn and surfaces(trolley tops, thermometers). They are less effective in the presence of blood, and other protein products. 2. Aldehydes- glutaraldehydes; broad spectrum action against bacteria, fungi and viruses, but acts slowly against spores; often used for endoscopes. It is an irritant to the eyes, skin and mcosa, hence needs to be handled in a wellventilated environment. 3. Biguanides-chlorhexidine; good for the skin and mucous membranes; low irritancy and toxicity; may be combined with alcohol or a detergent for handwashing. 4. Hypochlorites-broad spectrum, chlorine releasing disinfectant of choice for viruses, including Hepatitis B. 5. Iodine-may cause staining and hypersensitivity; the iodophors and povidoneiodine are less irritant and less staining. Good for preoperative preparation of the skin. 6. Phenolics-environment disinfectants for hospitals and labs; broad spectrum activity.

7. Surface active agents-anionic, cationic and non ionic detergents have antimicrobial activity except for mycobacteria and spores.

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