Introduction:
Definition: Sterilization is defined as complete removal of microorganisms from an object, surface or a product.
Moist heat sterilization uses application of heat in the form of steam or hot water.
It is further divided into three types,
1) Temperature below 100°C (Pasteurization)
2) Temperature @ 100°C (Tyndallization)
3) Temperature above 100°C (Autoclave)
Principle:
The killing of microorganisms by heat is a function of the time-temperature combination used.
If the temperature is increased then the time required for killing all the bacteria will be decreased.
Mechanism of killing the bacteria:
The vital constituents of cells such as proteins (enzymes) and nucleic acids are denatured by coagulation.
Biological indicator:
Spores of Bacillus stearothermophilus or
Spores of Clostridium sporogenes.
Pasteurization:
Pasteur developed the procedure of gentle heating (Pasteurization) to prevent spoilage of beers, wines by undesired contaminating microbes.
This process has no pharmaceutical application.
This is used for processing of milk in order to kill the pathogenic bacteria without affecting the taste and nutritional value of the milk.
Following two methods are used:
Holding method:
The milk is heated in tanks at 62°C for 30 mins while it is gently stirred and the steam is flown on the surface to disperse the foam.
High temperature short time method (H.T.S.T. / Flash method):
In this process the milk is rapidly raised to 71.6°C held at this temperature for at least 20 seconds and quickly cooled.
The common milk borne pathogens (Mycobacterium tuberculosis, M. bovis, Brucella abortus) do not form spores and are reliably sterilized by this procedure. In addition to this, total bacterial count is generally reduced by 97 - 99%.
Therefore in a true sense this technique is not an absolute sterilization process.
Tyndallization:
Also called Fractional Sterilization.
Developed by John Tyndall.
Sterilization by the fractional method is achieved by heating @ 100°C for 20 mins for three continuous days.
During the first day’s exposure, steam kills virtually all organisms except bacterial spores, and it stimulates spores to germinate to vegetative cells.
During overnight incubation the cells multiply and are killed on the second day.
Again, the material is cooled and the few remaining spores germinate, only to be killed on the third day.
Although the method usually results in sterilization, occasionally it fails.
The method also requires that spores be in a suitable medium for germination, such as a broth.
Temperature above 100°C : Autoclaving:
Heat is used in the form of saturated steam under pressure.
Equipment used for the process is called “Autoclave” and the process as “Autoclaving”.
Principle:
Mechanism of killing of microorganisms:
Bacterial death by moist heat is due to denaturation by coagulation of essential proteins (enzymes) and other cell constituents.
Conditions:
The USP XXI and BP 1988 recommended the following condition:
Pressure:15 lb / square inch (psi)
Temperature: 121°C
Time: 15 minutes
Biological indicator:
Spores of Bacillus stearothermophilus or
Spores of Clostridium sporogenes.
Principles of sterilization by steam under pressure:
Pressure itself has no sterilizing power.
Steam is used under pressure as a means of achieving an elevated temperature.
Steam production:
This may be achieved in two ways:
1.In a small scale, steam may be generated from water within the sterilizer and since water is present this steam is known as ‘wet saturated steam’.
2.For large scale sterilizers dry saturated steam may be piped from a separate boiler.
Penetration of steam:
Steam flows quickly into every article in the load ( and into porous articles).
This is due to its condensation creating a low-pressure region into which more steam flows.
Rapid heating:
The saturated steam heats the load rapidly due to the release of its considerable amount of latent heat.
Moist heat:
The condensate produced on cooling hydrates the microorganisms and thus helps in coagulating microbial proteins.
No residual toxicity:
The product is free from toxic contamination
Types of Autoclave:
Pressure cooker type/ Laboratory bench autoclaves (N-type) (Vertical)
Gravity displacement type autoclave (Horizontal)
Positive pressure displacement type (B-type)
Negative pressure displacement type (S-type)
The design and operation of Pressure Cooker Type autoclave:
A portable autoclave is an elaborate pressure cooker.
It is a hollow cylindrical vessel fitted with a lid that can be tightly secured in a position by nut-bolts or screws.
The body is made up of aluminium or steel or gunmetal.
The lid is provided with a steam vent, a safety valve and a pressure or temperature gauge.
It is heated electrically or gas operated.
The electrical element is fitted at the bottom of the autoclave.
First water is added so that the heating coil is fully immersed in water.
Then the materials to be sterilized are placed over a perforated platform.
Heater is switched on.
Initially the steam-vent (outlet) is opened. The displaced air is first removed, then the vent is closed.
The pressure will then rise to 15 lb/sq inch (psi) and as a result the temperature inside will rise to 121°C.
In this condition the autoclave is kept for 15 minutes, then the heater is switched off.
When the pressure inside and outside the autoclave equals, the steam-vent is opened and the lid is removed to take out the sterilized articles.
Precautions while handling the autoclave:
Autoclaves should not be used to sterilize water-proof or water-resistant substances like oil or powders.
The autoclave should not be overcrowded.
To ensure sufficient penetration, articles should be wrapped in something that allows penetration by steam, and materials like aluminum foils should not be used.
The items placed inside the chamber should not touch the sides or top of the chamber.
The wastes and clean items should be autoclaved separately.
Attempts to open the lid when the autoclave is working should never be made.
Liquid components should never be autoclaved in sealed containers.
The liquid inside the containers should only be filled 2/3rd of the total volume to prevent the spilling of the liquid.
Plastic or polyethylene trays or containers should not be used as they might melt and damage the autoclave.
Besides, never autoclave flammable, reactive, corrosive, toxic or radioactive materials, household bleach, or paraffin-embedded tissue.
Application of autoclave:
It is used to sterilize anything, which is not injured by steam and high temperature of sterilization.
1.Aqueous parenteral solutions e.g. distilled water, saline solutions.
2.Aqueous liquid media e.g. liquid media with or without carbohydrate and gelatin.
3.Surgical dressings and fabrics.
4.Plastic and rubber closures.
5.Metal instruments.
6.Glass apparatus and containers.