The following points highlight the top four techniques of sterilisation which are employed to sterilize different materials needed for microbiological work. The techniques are: 1. Sterilisation by Heat 2. Sterilisation by Chemical Treatment 3. Sterilisation by Radiation 4. Sterilisation by Filtration.
Technique # 1. Sterilisation by Heat:
Heat is the most widely used lethal agent for sterilisation. Objects may be sterilised by dry heat, applied in an oven in an atmosphere of air or by moist heat, provided by wet steam.
Of the two methods, sterilisation by dry heat requires a much greater duration and intensity. Heat conduction is less rapid in air than in steam. Dry heat is used principally to sterilize glassware or other heat stable solid materials. But steam must be used for heat sterilisation of aqueous solutions.
Steam sterilisation is usually carried out in a metal vessel known as autoclave, which can be filled with steam at a pressure greater than atmospheric pressure. Sterilisation can thus be achieved at temperatures considerably above the boiling point of water; laboratory autoclaves are commonly operated at steam pressure of 15 lb/in2, above atmospheric pressure, which corresponds to a temperature of 120°C.
The autoclave is a cylindrical metal vessel having double walls around all parts except the front to withstand the high steam pressure. The base of the inner cylinder is concave, which contains water. The vessel is heated by immersion heater.
There is a strong lid covering the cylinder which can be tightly screwed. The lid is provided with pressure gauze, safety valve, a steam-outlet and metal handles. Inside the vessel cavity, there is a wire basket with triple stand where the materials are packed before the start of sterilisation (Fig. 1.1).
At the start of the operation, all the air present in the chamber must be expelled and replaced by steam; this is achieved by the use of steam trap, which remains open as long as air passes through it but closes when the atmosphere consists of only steam.
If some air remains in the sterilisation chamber, the partial pressure of steam will be lower than indicated on the pressure gauge, and the temperature will be correspondingly lower. For this reason an autoclave should always be equipped with both a temperature & pressure gauge. After adjustment of pressure (151bs/in2) inside the autoclave, the pressure is maintained for 15 minutes and then the autoclave is cooled.
Hot Air Oven:
It is an electrically operated oven used for sterilisation of glassware viz. petridishes, flasks, tubes, pipettes etc. The apparatus consists of a large, rectangular, copper-base and covered with asbestos sheets. It is also provided with a door and erected on a four-legged stand.
The roof is provided with a hole through which a thermometer is fitted inside for recording of temperature. The oven has two or three shelves. The oven is heated by electrically operated heater, fitted at the base of the instrument. There is a regulator of heater to control the inside temperature (Fig. 1.2).
Before sterilisation, the glassware are dried properly and wrapped in brown paper and then exposed to hot air inside the oven. After loading of glassware, the oven is switched on 160°C.
The temperature will increase slowly up to the desired point where it will remain steady. Then at 160°C the oven is kept for an hour. This is the appropriate temperature for sterilisation of glassware. Then gradually the temperature is brought down and thereafter sterilisation is complete.
Red Hot Heat:
This procedure is generally followed during working inside the culture room with inoculating needle. The inoculating needle is flamed to make it red hot. At that high temperature of the flame, all the organisms present on the needle are killed.
Technique # 2. Sterilisation by Chemical Treatment:
Many of the substances used in preparing culture media are too heat liable to be sterilised by autoclaving. For such substances, a reliable method of chemical sterilisation is extremely useful.
The essential requirement for a chemical sterilisation agent is that it should be volatile as well as toxic, so that it can be readily eliminated from the object sterilised after treatment. Alcohol, Dettol, Lysol, ethylene oxide and various other volatile chemicals are used for this purpose.
Technique # 3. Sterilisation by Radiation:
In recent years, various kinds of ionizing and non-ionizing radiation techniques have been employed for sterilisation of culture room, equipment’s etc. Exposure to UV radiation for a period of 45 min., is made. UV radiation is made by the use of suitable UV lamp.
Technique # 4. Sterilisation by Filtration:
The principal laboratory method used to sterilize solutions of heat liable materials is filtration through filters capable of retaining microorganisms. The pore size of the filters used for filtration is less than 0.75m, which can retain small microorganisms.
Since the diameter of the pores is very small, a suction or a pressure is essential during filtration. However, it is never possible to be certain that filtration procedures, which render a solution bacterium free, will also free it of viruses. There are different types of bacteriological filters used in microbiology.
A short description of some of them is given below:
1. Pasteur Chamberland Candle Filter:
This type of filter is made of unglazed porcelain. There are different types of this filter depending on their pore size. The grades are L1, L1a, L2, L3, L5, L7, L11, and L13. L1 is the coarser and L13 is the finest in the order. These filters are used for removing organisms from fluid to obtain bacterial toxin.
2. Berkefeld Filter:
This is made up of diatomaceous earth pressed in the shape of a candle. On the basis of porosity they are variously classified into V (veal) N (normal), W (dense) etc.
3. Seitz Filter:
It consists of a disc of asbestos material through which the fluid is passed during filtration. The disc is inserted into a metal holder before use. After use, the disc is discarded and a fresh one is fitted.
4. Sintered Glass Filter:
This is made up of finely ground glass which is subsequently fused to make the small particles adhere to each other. This filter is sterilised after use, avoiding temperature extremes.
5. Cellulose Membrane Filter:
There are two types of cellulose filters-Ciradocol membranes (older type) are made up of cellulose nitrate and the modern type is composed of cellulose. Diagrams of some bacterial filters are given in Fig. 1.3.