Introduction of Autoclave
Autoclave is the most common method of sterilization in the laboratory working on moist heat. Sterilization is the process of removing or destroying all microorganisms including viruses, bacteria and their spores from the article or surface without destroying its quality and quantity. It is a physical method of sterilization due to moist heat. Factors influencing sterilization by heat are nature of heat i.e dry or moist, temperature and time, number of microorganism, nature of microorganisms, type of microorganism and presence of organic material. Mode of action, moist heat kills microorganism by coagulating and denaturing their enzymes and their structural proteins. Heating in presence of water is preferred than dry heat because of its rapid killing and effective even at lower temperature than dry heat due to its latent heat.Temperature is above 100°C and sterilization by dry saturated steam under pressure. Most efficient method of sterilization is commonly called as autoclaving and the instrument used is as autoclave.
Fundamental structures of Autoclave
Essentially a modified pressure cooker may be horizontal or vertical. It contains double walled or Jacketed chamber made of stainless steel or gun metal with a supporting frame. In modern type of autoclave steam circulate within the jacket and is supplied under high pressure to the closed inner chamber where goods are kept for sterilization. One fifth part of the cylinder is filled with water and the materials to be sterilized are placed inside. The lid is closed securely with discharge tap on it open. A safety valve is present to permit the escape of steam from the chamber. It works on the principle of steam under pressure. It was invented by Charles Chamberland in 1879.
Principle of autoclaving
It utilizes the thermodynamics principle of water. Water boils when its vapor pressure equals that of surrounding atmosphere. When pressure inside the closed vessel increases, the temperature at which water boils also increases. Dry saturated steam at high pressure when strikes with the cooler surfaces of the articles in the autoclave ,it condenses into water and efficiently destroys all microorganisms along with thermal resistant bacterial spores. Condensation of steam into water has 3 effects:
1. It wets the micro organisms and provide the essential conditions for killing.
2. Liberates latent heat of steam and so rapidly heats up the items in the load .(Amount of heat liberated is 4 times greater than the heat available in same mass of boiling water at the same temperature and pressure.3. Causes significant contraction of steam , drawing more to the site.The cycle of condensation ,liberation of latent heat and drawing of fresh steam is repeated until the article is heated up to the sterilizing temperature.
Types of autoclaves
According to structure
According to the structures, autoclaves are of following types and they are-
Simple non jacketed autoclave, steam jacketed autoclave with automatic air and condensate discharge and high pre-vacuum sterilizers.
According to function
According to function, autoclaves are of following types and they are-Simple laboratory autoclave, transportable bench top autoclave, large simple autoclave, downward displacement laboratory autoclaves, media preparatory autoclaves and multi purpose laboratory autoclave
Sterilization cycle includes warming of the chamber, vacuum extraction, pre-steam penetration time, steam penetration time, holding time and cooling time.
Dynamics of sterilization
Thermal death time (TDT): It is the time in minutes required to kill all cells in a suspension at a given temperature. It is highly dependent on the inoculum size. Thermal death point(TDP): It is the temperature needed to kill all cells in suspension after fixed exposure time. D Value(decimal reduction time, DRT): It is the time in minute needed at a particular temperature to reduce the viable organisms by 90% i.e. to 10% or by 1 log 10. D value is independent of inoculum size and is inversely related to temperature.
Sterilization Times for Autoclave
Sterilization time is inversely proportional to the temperature at constant pressure. For examples 115°C, 10 lb/in 2 for 45 minutes; 121°C, 15 lb/in 2 for 15-20 minutes; 126°C, 20 lb/in 2 for 10 minutes and 134°C, 30 lb/in 2 for 3 minutes.
Common sterilization condition is at 121°C for 15-20 minutes at 15 lb/sq inch but it may vary as-10 lbs pressure for 10 minutes is for culture media. 15 lbs pressure is for 20 minutes is for infected material. 20 lbs,30 minutes is for dressing and towels where as 5 lbs-30 minutes is for gloves.
Automatic Process Control: It carries through the sterilization cycle according to a pre selected scheme for duration,temperature,and pressure of each stage. Recording Thermometer: Graphic record of temperature changes in chamber discharge channel avoiding errors in timing the holding period. Thermocouple: It is when kept inside the test article and attached to potentiometer, it indicates the temperature inside the test article during autoclaving. Chemical Indicators : Browne’s Sterilizer has red solution which turns green when heated at 115°C for 25 minutes (type 1),or 15 minutes (type 2). Store it at 20°C to avoid pre mature color change. Adhesive Tapes: Bowie-Dick autoclave tape test for steam penetration. Biological indicators
Organism : Bacillus stearothermophilus (NCTC 10003 ATCC 7953), a thermophile that requires to be cultivated 55-60°C. Its spores are killed at 121°C in 12 minutes. Preparation: culture grown aerobically on nutrient agar for 5 days is suspended in sterile water to a concentration of one million spores per mili liter. Small strips of filter paper soaked in the suspension are dried at room temp and packed in envelopes.
Procedure of autoclaving
Initially check the water level . Place the articles to be sterilized in the center of the largest or most densely packed items and some in the coolest part. Now switch on autoclave. Check the pressure and wait for proper time (If there is automated system, no need of these steps). After autoclaving, envelope is cut with a sterile scissors and strip transferred to a recovery medium, e.g. thioglycolate broth with strict precautions against contamination. Incubate tube for 7 days at 55°C and examined for growth. An unautoclaved spore strip is used as positive control and uninoculated tube of medium as negative control. Use results in terms of degree of heat resistance of preparation.
Uses of autoclave
It is widely used for following purposes and they are-sterilization of culture media, aqueous solutions, empty bottles and impervious containers, surgical instruments, wrapped dry goods and dressings, gowns and dressings, rubber goods , syringes etc.
Advantages: It is a very effective way of sterilization, quicker than hot air oven.
Disadvantage: Articles may trapped air,takes long time to cool.
Air must be completely removed until saturated steam is filled. Contents should be arranged loosely to ensure free circulation of steam. It shouldn’t allow to form supersaturated steam. Lid should be opened only after pressure gets down to normal. Follow manufacturers’ guidelines. Avoid standing directly in front of autoclave door when opening. Cool to below 80°C before opening.
Mechanism of bacterial spore resistance:
There is no any clear and proved concept but several theories are as follows-Spores have low water content and therefore essential factor in resistance.
-According to Warth (1985), stability of protein could be intrinsically ,or due to presence of substance ( might be Calcium-diplocolinate) that helps to stabilize or due to the removal of water. Several properties of spore i.e. thermo tolerance ,mineralization ,thermal adaptation might help in resistance. Small acid soluble spore proteins(SASPs) may also play some role.
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