Sterilization is the killing or removal of all microorganisms, including bacterial spores which are highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the absence of all microorganisms.
Disinfection is the killing of many, but not all microorganisms. It is a process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed. Some organisms and bacterial spores may survive.
Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on inanimate objects.
Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes.
Uses of Sterilization
1. Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. are to be sterilized.
2. Sterilization in Microbiological works like preparation of culture media, reagents and equipments where a sterile condition is to be maintained.
CLASSIFICATION OF METHODS
Sterilization and disinfection are done by :
(A). Physical Agents
1. Heat
2. Radiation
3. Filtration
(B). Chemical Agents
In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores.
STERILIZATION BY HEAT
Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating above 100°C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and sterilization by autoclaving are the two most common method used in the laboratory.
Types of Heat :
A. Sterilization by moist heat
B. Sterilization by dry heat
A. Sterilization by Moist Heat
Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane.
(I). Sterilization at 100°C
1. Boiling. Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It leads to disinfection.
2. Steaming. Steam (100°C) is more effective than dry heat at the same temperature as: (a) Bacteria are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more sterilizing power as more heat is given up during condensation.
Steam Sterilizer. It works at 100°C under normal atmospheric pressure i.e. without extra pressure. It is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100°C.
It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is a small opening on the roof of the instrument for the escape of steam. Sterilization is done by two methods :
(a) Single Exposure for 11/2 hours. It leads to disinfection.
(b) Tyndallization (Fractional Sterilization). Heat labile media like those containing sugar, milk, gelatin can be sterilized by this method. Steaming at 100°C is done in steam sterilizer for 20 minutes followed by incubation at 37°C overnight. This procedure is repeated for another 2 successive days. That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria during incubation and are destroyed during steaming on second and third day. It leads to sterilization.
II. Sterilization above 100°C: Autoclaving
Autoclaving is one of the most common methods of sterilization. Principle: In this method sterilization is done by steam under pressure. Steaming at temperature higher than 100°C is used in autoclaving. The temperature of boiling depends on the surrounding atmospheric pressure. A higher temperature of steaming is obtained by employing a higher pressure. When the autoclave is closed and made air-tight, and water starts boiling, the inside pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C temperatures is obtained. This is kept for 15 minutes for sterilization to kill spores. It works like a pressure cooker.
'Sterilization holding time' is the time for which the entire load in the autoclave requires to be exposed.
Autoclave is a metallic cylindrical vessel. On the lid, there are : (1) A gauge for indicating the pressure, (2) A safety valve, which can be set to blow off at any desired pressure, and (3) A stopcock to release the pressure. It is provided with a perforated diaphragm. Water is placed below the diaphragm and heated from below by electricity, gas or stove. Working of Autoclave. (a) Place materials inside, (b) Close the lid. Leave stopcock open, (c) Set the safety valve at the desired pressure, (d) Heat the autoclave. Air is forced out and eventually steam ensures out through the tap, (e) close the tap. The inside pressure now rises until it reaches the set level (i.e. 15 Win), when the safety valve opens and the excess steam escapes, (f) Keep it for 15 minutes (holding time), (g) Stop heating, (h) Cool the autoclave below 100°C, (i) Open the stopcock slowly to allow air to enter the autoclave.
III. Sterilization below 100°C
1. Pasteurization. Pasteurization is heating of milk to such temperature and for such a period of time so as to kill pathogenic bacteria that may be present in milk without changing colour, flavour and nutritive value of the milk. Mycobacterium bovis, Salmonella species, Escherichia coli and Brucella species may be present in milk. It does not sterilize the milk as many living organisms including spores are not destroyed..
Methods of Pasteurization
(i) Flash Method. It is "high temperature- short time method". Heating is done at 72°C for 15 seconds.
(ii) Holding Method. Heating is done between 63°C and 66°C for 30 minutes.
2. Inspissation. Inspissation is done between 75°C to 80°C. Inspissation means stiffening of protein without coagulation as the temperature is below coagulation temperature. Media containing serum or egg is sterilized by heating for 3 successive days. It is done in 'Serum Inspissator'.
B. Sterilization by Dry Heat
Mechanisms. (1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte (in absence of water).
Dry heat at 160°C (holding temperature for one hour is required to kill the most resistant spores). The articles remain dry. It is unsuitable for clothing which may be spoiled.
1. Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen burner or spirit lamp flame. Temperature is above 100°C. It leads to sterilization.
2. Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel. Temperature is not high to cause sterilization.
3. Sterilization by Hot Air
Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares, swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For sterilization, a temperature of 160°C is maintained (holding) for one hour. Spores are killed at this temperature. It leads to sterilization.
Hot Air Oven is an apparatus with double metallic walls and a door. There is an air space between these walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the bottom becomes hot and passes between the two walls from below upwards, and then passes in the inner chamber through the holes on Me top of the apparatus. A thermostat is fitted to maintain a constant temperature of 160°C.
LECTURER IN CHARGE: MRS ALBERT
BLOGGER: HOC TEMIYEMI
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