Sterilization is the process of destruction or elimination of all forms of microorganisms by physical means (including heat, radiation, and filtration) and chemical agents (acids, alkalis, heavy metals, salts, halogens, etc). The equipment or material treated then becomes “Sterile”.
In medical laboratories, materials and equipment are sterilized for the following main purposes.
- In preparation for taking specimens, such as needles, syringes, test tubes, etc.
- To sterilize contaminated materials and equipment.
- To prepare apparatus used for bacteriological cultures, such as petridishes, Pasteur pipettes, and others.
Methods of sterilization techniques.
1. Physical method
Dry heat (hot air oven, flaming and red – hot) (2.) Moist heat (autoclave or steam under pressure and boiling (3.) Radiation
Hot Air oven
Owing to the low penetrating power of dry heat and great resistance of bacteria to it, higher temperatures are needed with a hot- air oven than with an autoclave. A temperature of 1800°c for 30 minutes will kill most resistant spores. The material to be sterilized is placed in an oven and the temperature is raised and maintained at 1800°c for 30 minutes.
The sterilized material should not be removed until the oven is cold. This is important particularly with pertidishes, as cold air will be sucked in to them, causing contamination, if they are removed before the oven is cold. This is due to the contraction of hot air as it cools. This method is used only for glass or metal articles such as test tubes, petridishes, all glass syringes, and instruments.
Metal spatula, glass slides, and cover slips may be sterilized by passing them through a Bunsen flame, without letting them become red hot. Alternatively they may be dipped in metylated spirit, and the alcohol burned off. This procedure should be repeated two or three times.
Moist heat may be applied by boiling water or steam. Boiling water is generally used for sterilizing instruments and syringes. These are boiled for 10 minutes in a water bath. This will kill all non-sporing organisms but certain spore forming organisms can resist the temperature of boiling water for 1-2 hours.
The addition of 2% sodium carbonate increases the disinfecting power of boiling water for 1-2 hours. Spores, which resist boiling water for 10 hours, have been killed within 30 minutes by the addition of sodium carbonate. Sodium carbonate also prevents the rusting of metal instruments. N.B: This method is unsuitable if instruments are to be stored in a sterile condition.
Steam under pressure (autoclave)
Autoclave is an instrument that operates by creating high temperature under steam pressure. Autoclaving is the most common, effective, reliable and practical method of sterilizing laboratory materials. The principle of autoclave is that steam is kept at a pressure of 15 pound (lb) per square inch to give a temperature of 1210°c, which will kill spores within 15 minutes. At this particular temperature, pressure and time, all forms of lives are destroyed.
Steam is more penetrating than hot air, and will give up its latent heat on striking a colder object; there by raising the temperature of the object rapidly. It is used to sterilize syringes, needles, glasswares, culture media, etc. For most purposes, the following cycles will ensure sterilization of correctly loaded autoclaves correctly loaded:
- Three minute holding time at 134°c
- Ten minute holding time at 126°c
- Fifteen minute holding at 121°c
- Twenty holding time at 115°c
Types of autoclaves
- Gravity displacement autoclaves
In gravity displacement autoclave, steam enters the chamber under pressure and displaces the heavier air downwards and through the valve in the chamber drain, fitted with a HEPA filter.
- Pre- vacuum autoclaves
These autoclave allow the removal of air from the chamber before steam is admitted. The exhaust air is evacuated through a valve fitted with a HEPA filter. At the end of the cycle, the steam is automatically exhausted. These autoclaves can operate at 134°c and the sterilization cycle can therefore be reduced to 3 minute. They cannot be used to process liquid because of the vacuum.
- Fuel heated pressure cooker autoclaves
Fuel heated pressure cooker autoclaves should be used if a gravity displacement autoclave is not available. They are loaded from the top and heated by gas or electricity. Steam is generated by heating water in the base of the vessel and air is displaced upwards through a relief vent. When all the air has been removed, the valve on the relief vent is closed and the heat is reduced.
The pressure and temperature rise until the safety valve operates at a preset level, which is the start of holding time. At the end of the cycle, the heat is turned off and the temperature allowed to fall to 800°c or below before the lid is opened.
- Radiation (ultra – violet ray)
UV radiation is lethal to certain microorganisms by inactivating the DNA of the organisms. It is effective and valuable in sterilization of air in a given room or place. E.g.: Pulmonary tuberculosis Laboratory