Steam sterilization under pressure, or autoclaving. Physical Sterilization Methods Dry Heat Sterilization Modes
Firing is currently not used in the surgical clinic for the sterilization of instruments. The method can be used at home when it is impossible to use others. Firing metal tools carried out with an open flame. Usually on metal tray put the tool, pour a small amount of ethyl alcohol and set fire to it. Boiling long time was the main method for sterilizing instruments, but has rarely been used recently, since this method achieves a temperature of only 100 degrees C, which is not enough to kill spore-bearing bacteria. Tools are boiled in special electric sterilizers of various capacities. Opened instruments (disassembled syringes) are placed on a grid and immersed in distilled water (sodium bicarbonate can be added - up to 2% solution). The usual sterilization time is 30 minutes from the moment of boiling. After the end of sterilization, the mesh with the instruments is taken out with the help of special sterile handles.
Steam sterilization under pressure (autoclaving)
With this method of sterilization, the active agent is hot steam. Sterilization with just flowing steam is not currently used, since the temperature of the steam at normal conditions(100 degrees C) is not enough to kill all germs. In an autoclave (pressurized steam sterilization apparatus), it is possible to heat water at high blood pressure. This raises the boiling point of water and, accordingly, the temperature of the steam to 132.9 degrees C (at a pressure of 2 atmospheres). Surgical instruments, dressings, underwear and other materials are loaded into the autoclave in special metal boxes - Schimmelbusch biks. Bixes have side holes that are opened before sterilization. The bix lid is tightly closed. After loading the biks, the autoclave is closed with a sealed lid and necessary manipulations to start its work in a certain mode. The operation of the autoclave is controlled by the indicators of a pressure gauge and a thermometer.
There are three main sterilization modes:
- - at a pressure of 1.1 atmospheres - 1 hour,
- - at a pressure of 1.5 atmospheres - 45 minutes,
- - at a pressure of 2 atmospheres - 30 minutes.
At the end of sterilization, the bixes remain for some time in a hot autoclave to dry with the door slightly ajar. When removing the biks from the autoclave, the holes in the walls of the biks are closed and the date of sterilization is noted (usually on a piece of oilcloth attached to the biks). A closed bix keeps the items in it sterile for 72 hours.
Boiling is a sterilization method that guarantees sterilization provided that there are no spores in the sterilized material. Used for processing syringes of instruments, glass and metal utensils rubber tubes etc.
Boiling sterilization is usually carried out in a sterilizer - a metal box rectangular shape with tight-fitting lid. The material to be sterilized is placed on the mesh in the sterilizer and filled with water. To increase the boiling point and eliminate water hardness, add 1-2% sodium bicarbonate (it is better to use distilled water). The sterilizer is closed with a lid and warmed up. The beginning of sterilization is considered the moment of boiling water, boiling time is 15-30 minutes. At the end of sterilization, the mesh with tools is removed by the side handles with special hooks, and the tools in it are taken with sterile tweezers or forceps, which are boiled along with the rest of the tools.
Steam sterilization is carried out in two ways: 1) steam under pressure; 2) flowing steam.
Steam pressure sterilization produced in an autoclave. This method of sterilization is based on the effect of saturated water vapor on the sterilized materials at a pressure above atmospheric. As a result of such sterilization, both vegetative and spore forms of microorganisms die during a single treatment.
Autoclave (Fig. 12) - a massive boiler, covered with a metal casing on the outside, hermetically sealed with a lid, which is tightly screwed to the boiler with hinged bolts. Another, smaller diameter, which is called the sterilization chamber, is inserted into the outer boiler. Items to be sterilized are placed in this chamber. Between both boilers there is free space called the water vapor chamber. Water is poured into this chamber through a funnel fixed on the outside to a certain level, marked on a special water-metering tube. When water is boiled in a steam chamber, steam is produced. The sterilization chamber is equipped with an outlet cock with a safety valve for steam to escape when the pressure rises above the required level. A manometer is used to determine the pressure created in the sterilization chamber.
Rice. 12. Scheme of the autoclave. M - pressure gauge; PC - safety valve; B - funnel for water; K 2 - tap for water release; K 3 - tap for steam release
Normal Atmosphere pressure(760 mm Hg) are taken as zero. There is a certain relationship between the pressure gauge readings and temperature (Table 2).
Table 2. Autoclave operation mode
Autoclaves with automatic mode control are now available. In addition to the usual pressure gauge, they are equipped with an electrocontact pressure gauge, which prevents the pressure from increasing above the set value and thereby ensures the constancy of the desired temperature in the autoclave.
Various nutrient media (except those containing native proteins), liquids (isotonic sodium chloride solution, water, etc.) are sterilized with steam under pressure; appliances, especially those with rubber parts.
The temperature and duration of autoclaving of nutrient media is determined by their composition, specified in the recipe for the preparation of the nutrient medium. For example, simple media (meat-peptone agar, meat-peptone broth) are sterilized for 20 minutes at 120 ° C (1 atm). However, at this temperature it is impossible to sterilize media containing native proteins, carbohydrates and other substances easily changed by heating. Media with carbohydrates are sterilized fractionally at 100°C or in an autoclave at 112°C (0.5 atm) for 10-15 minutes. Various liquids, devices having rubber hoses, stoppers, bacterial candles and filters are sterilized for 20 minutes at 120 ° C (1 atm).
Attention! In autoclaves, the infected material is also neutralized. Cups and test tubes containing cultures of microorganisms are placed in special metal buckets or tanks with holes in the lid for steam penetration and sterilized in an autoclave at 126 ° C (1.5 atm) for 1 hour. Instruments are sterilized in the same way after working with bacteria that generate controversy.
Only specially trained persons are allowed to work with the autoclave, who must strictly and accurately follow the rules specified in the instructions attached to the device.
Autoclaving technique. 1. Before work, check the serviceability of all parts and the lapping of taps.
2. Through a funnel fixed on the outside of the boiler, water (distilled or boiled) is poured up to the upper mark of the water gauge glass so that scale does not form. The faucet under the funnel is closed.
3. The material to be sterilized is placed on a special mesh in the sterilization chamber. Items should not be loaded too tightly, as steam must pass freely between them, otherwise they will not heat up to the correct temperature and may remain unsterile.
4. rubber gasket on the lid is rubbed with chalk for better sealing.
5. The lid is closed and bolted to the body of the autoclave, and the bolts are twisted in pairs crosswise.
6. Fully open the exhaust cock connecting the sterilization chamber to the outside air, and begin to heat the autoclave. The autoclave is usually heated by gas or electricity.
When the autoclave is heated, the water boils, the resulting steam rises between the walls of the boilers and through special holes in the wall of the inner boiler (see Fig. 12), enters the sterilization chamber and exits through the open outlet cock. First, the steam escapes along with the air that was in the autoclave. It is essential that all air is expelled from the autoclave, otherwise the pressure gauge reading will not correspond to the temperature in the autoclave.
The appearance of a continuous strong jet of steam indicates the complete removal of air from the autoclave; after that, the outlet cock is closed and the pressure inside the autoclave begins to rise gradually.
7. The beginning of sterilization is considered the moment when the pressure gauge readings reach the specified value. Heating is regulated so that the pressure in the autoclave does not change for a certain time.
8. After the sterilization time has elapsed, the heating of the autoclave is stopped, the steam is released through the outlet cock. When the pressure gauge needle drops to zero, open the lid. To avoid burns from the steam remaining in the autoclave, open the lid towards you.
The temperature level in the autoclave, i.e. the correctness of the pressure gauge, can be checked. To do this, use various substances that have certain point melting: antipyrine (113 ° C), resorcinol and sulfur (119 ° C), benzoic acid (120 ° C). One of these substances is mixed with a negligible amount of dye (magenta or methylene blue) and poured into a glass tube, which is sealed and placed in vertical position between the material to be sterilized. If the temperature is sufficient, the substance will melt and turn into the color of the corresponding dye.
To check the effectiveness of sterilization, a test tube with a known spore culture is placed in the autoclave. After autoclaving, the tube is transferred to a thermostat for 24-48 hours, the absence or presence of growth is noted. The absence of growth indicates the correct operation of the device.
Steam Sterilization produced in the Koch apparatus. This method is used in cases where the object to be sterilized changes at a temperature above 100 ° C. Fluid steam sterilizes nutrient media containing urea, carbohydrates, milk, potatoes, gelatin, etc.
The Koch apparatus (boiler) is a metal cylinder sheathed on the outside (to reduce heat transfer) with felt or asbestos. The cylinder is closed with a conical lid with a hole for steam to escape. Inside the cylinder there is a stand, up to the level of which water is poured. A bucket with a hole is placed on the stand, into which the material to be sterilized is placed. The Koch apparatus is heated with gas or electricity. The sterilization time is counted from the moment of vigorous release of steam at the edges of the lid and from the steam outlet. Sterilize for 30-60 minutes. At the end of sterilization, the heating is stopped. Remove a bucket of material from the apparatus and leave it at room temperature before next day. Warming up is carried out for 3 days in a row at a temperature of 100 ° C for 30-60 minutes. This method is called fractional sterilization. During the first heating, the vegetative forms of microbes die, while the spore ones remain. During the day, the spores have time to germinate and turn into vegetative forms that die on the second day of sterilization. Since it is possible that some of the spores did not have time to germinate, the material is kept for another 24 hours, and then a third sterilization is carried out. Sterilization with flowing steam in the Koch apparatus does not require special control, since the indicator correct operation The sterility of the prepared culture media serves the device. Steam sterilization can also be done in an autoclave with the lid unscrewed and the outlet cock open.
1. What culture media can be steam sterilized?
2. What is a sterilizer and how does it work?
3. Why should distilled water be used for boiling sterilization?
4. Describe the device and mode of operation of the autoclave.
5. What is sterilized in an autoclave?
6. What controls the correct sterilization in autoclaving?
7. What is steam sterilization?
8. Describe the device of the Koch apparatus.
9. What is the purpose of fractional sterilization?
Exercise
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Fractional sterilization can also be carried out in a Koch coiler.
The Koch Coagulator is used to coagulate whey and egg nutrient media, and simultaneously with the compaction of the medium, it is sterilized.
Koch coiler is a flat double-walled metal box, covered on the outside heat-insulating material. In the space between the walls through a special hole located in the upper part outer wall pouring water. The hole is closed with a stopper into which a thermometer is inserted. Close the device with two covers: glass and metal. Through glass lid you can watch the clotting process. Test tubes with media are placed on the bottom of the coiler in an inclined position.
The heating of the coiler is carried out using gas or electricity. The media are sterilized once at a temperature of 90 ° C for 1 hour or fractionally - 3 days in a row at 80 ° C for 1 hour.
Tyndallization* - fractional sterilization at low temperatures- used for substances that are easily destroyed and denatured at a temperature of 60 ° C (for example, protein liquids). The material to be sterilized is heated in a water bath or in special devices with thermostats at a temperature of 56-58 ° C for an hour for 5 days in a row.
* (The method of sterilization is named after Tyndall, who proposed it.)
Pasteurization- sterilization at 65-70 ° C for 1 hour, proposed by Pasteur for the destruction of non-spore forms of microbes. Pasteurize milk, wine, beer, fruit juices and other products. Milk is pasteurized in order to get rid of lactic acid and pathogenic bacteria (Brucella, Mycobacterium tuberculosis, Shigella, Salmonella, Staphylococcus, etc.). When pasteurizing beer, fruit juices, wine, microorganisms that cause different kinds fermentation. Pasteurized foods are best kept refrigerated.
test questions
1. What is the purpose and device of the Koch coiler?
2. What are the methods of sterilization in the coiler?
3. What is tyndalization?
4. What is pasteurization?
Before sterilization, laboratory glassware must be thoroughly washed, dried, and wrapped in paper. Cups are wrapped in paper one or more pieces. Cotton swabs are inserted into the upper ends of the pipettes to prevent material from being sucked in. Graduated pipettes are wrapped in long strips of paper 5 cm wide. The volume of the wrapped pipette is marked on the paper. In cases, pipettes are sterilized without additional wrapping in paper.
The sharp ends of the Pasteur pipettes are sealed in a burner flame and wrapped in paper, 3-5 pieces each.
Vials, flasks, test tubes are closed with cotton-gauze stoppers. The cork should enter the neck of the vessel by 2/3 of the length, not too tight, but not loose. A paper cap is put on top of the corks on the vessel. The test tubes are tied up in 5-50 pieces and wrapped over with paper.
Operating mode of the oven
Sterilization by dry heat is carried out in dry heat cabinets (Pasteur oven). Dry heat sterilizes laboratory glassware. It is loosely loaded into the furnace so that the material is evenly heated. The cabinet door is tightly closed, the electric heater is turned on and the temperature is brought to 160-165 0 C and sterilized for 1 hour. At the end of sterilization, turn off the heating, but do not open the cabinet door until the oven has cooled down (otherwise the cold air will cause cracks on the dishes). Sterilization mode: 160°С - 60 min, 180°С - 15 min, 200°С - 5 min. Liquids, culture media, rubber and synthetic materials must not be sterilized by dry heat.
Steam sterilizationunder pressure dressings, surgical underwear, surgical instruments, nutrient media, laboratory glassware, infected material, injection solutions are subjected to. The material is placed in containers (bixes). At the bottom of the bix, fabric pads are placed that absorb moisture after sterilization. The sterility of the material is maintained for 3 days. Infected material in cups and test tubes is sterilized in metal containers with a lid.
Steam sterilization under pressure is carried out in an autoclave. With a single treatment, both vegetative and spore forms of bacteria die. Nutrient media are sterilized with steam under pressure, except for media containing native proteins, liquids, devices with rubber parts. Simple media (MPA, MPB) are sterilized for 20 minutes at 120°C (1 atm). Media containing native proteins and carbohydrates cannot be sterilized at this temperature, since these are substances that easily change from heating. Environments with carbohydrates are sterilized fractionally at 100 0 C or in an autoclave at 112 0 C (5 atm.) for 10-15 minutes. Various liquids, devices with rubber hoses, plugs, bacterial candles and filters are sterilized at 120 0 C (1 atm.) for 20 minutes.
Infected material (in test tubes, cups) is placed in special metal buckets or tanks with holes for steam penetration and sterilized at 126 0 C (1.5 atm.) for 1 hour. Instruments are also sterilized after working with spore bacteria.
There are 2 sterilization modes:
Flowing steam in an autoclave or in a Koch apparatus with an unscrewed lid and an open exhaust valve, when the antibacterial effect of steam is manifested in relation to vegetative forms. This is how media with vitamins and carbohydrates, urea, milk, potatoes and gelatin is sterilized. For complete deposition, fractional sterilization is used (at 100 0 C) for 20-30 minutes for 3 days in a row. It also kills controversy.
Steam pressure sterilization is the most effective method deposition. Dressings, linen are sterilized at 1 atm. 15-20 min, infected material at 1.5-2 atm for 20-25 min.
Steam sterilization. Steam sterilization.
Saturated steam under pressure has high biocidal properties, does not damage most sterilized materials, and does not need to be released from the sterilizing agent. For S.p. are characterized by reliability, availability, safety for personnel, economy, high degree automation. The sterilizing effect of steam is associated with the heating of the object in the process of condensation of steam into water on the surface and inside the object. The condensation effect is optimal for saturated steam, when it contains only 3% of the liquid phase. superheated steam, containing less than 2% of the liquid phase, may burn the object; wet steam(more than 3% of the liquid phase) strongly moisturizes objects. To obtain a sterilizing effect, it is also necessary that air is completely removed from the working chamber, otherwise it may interfere with the contact of steam with the surface of the object and leave it unsterile. There are 2 types of steam sterilizers. The first one is weight displacement sterilizers; in them the flow of steam penetrates into upper part chamber and expels air from it through the drain tube. The steam pressure and temperature increase and reach the set value. The timer is turned on, which maintains a constant temperature until the end of the exposure. After that, the steam is removed through the drain from the chamber; the chamber jacket remains hot and its dry heat dries the objects to be sterilized. The lid (door) opens when the chamber temperature drops to 90 °C. Otherwise, liquid containers may explode. The second type is called the pre-vacuum steam sterilizer. In it, the vacuum system in 2 cycles draws most of the air from the chamber and objects to be sterilized through the drain, after which the chamber and its jacket are filled with pressurized steam. Pressure and constant temperature are maintained automatically. After the end of the sterilization period, the chamber is emptied of steam and the objects are dried with vacuum system, filtered air is admitted into the chamber. The sterilizer is being unloaded. Depending on the objects to be sterilized, the steam temperature in steam sterilizers is set from 110 to 138 ° C, steam pressure from 0.4 to 2.5 atm, exposure - from 15 to 60 minutes. Almost all products made of metal, glass, heat-resistant plastic, rubber, linen, dressing and suture material, nutrient media, including liquid ones, are sterilized with steam. medications. S.p. - the preferred method of sterilization. The exceptions are heat-labile materials, large-sized objects, complex multi-component devices such as endoscopes.
(Source: Glossary of Microbiology Terms)
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When using this method of sterilization, the effect is combined high temperature and humidity. If dry heat causes mainly pyrogenetic destruction of microorganisms, then damp heat- protein coagulation, requiring the participation of water. In practice, sterilization wet steam is carried out at a temperature of 50-150 ° C and is carried out in the following ways.
Boiling. This method sterilizes rubber objects, surgical (non-disposable) instruments, glassware. It is not recommended to use boiling for sterilization of injection solutions, since in terms of efficiency it is significantly inferior to steam sterilization and often leads to a decrease in the therapeutic effect of the drug.
Steam sterilization. Fluid is called saturated water vapor (without admixture of air), having a pressure of 760 mm Hg. Art. and a temperature of 100 °C. Sterilization with flowing steam is carried out in a steam sterilizer or autoclave for 15-60 minutes, depending on the volume of the solution. This is the most common method for sterilizing injectable solutions in pharmacies.
Steam sterilization under pressure, or autoclaving. Implemented in various designs autoclaves. The autoclave is a hermetically sealed container, consisting of a thick-walled sterilization chamber and a casing.
The autoclave has a safety valve that allows steam to escape at excess pressure, and a pressure gauge. Each autoclave must have instructions for use and care. The object to be sterilized is placed inside the steam chamber. The steam chamber is heated. First, the autoclave is heated with the tap open until the steam go strong continuous jet and will not displace the air in the autoclave, which significantly reduces the thermal conductivity of water vapor (with a content of 5% air in water vapor, it decreases by 50%). During the heating of the autoclave after closing the valve, it is necessary to monitor the pressure, in parallel with the increase of which the temperature of the steam increases. The relationship between temperature and vapor pressure is expressed as follows: 1 atm - 100 °C; 1.5 atm - 112.7 ° C; 2 atm - 119.6 ° C; 3 atm - 132.9 ° С; 5 atm - 151.1 ° C. Autoclaving is the most reliable sterilization method. Typically, sterilization in an autoclave is carried out at 120 °C for 5-30 minutes, depending on the volume of the solution. This guarantees a sufficiently complete sterilization, regardless of the type of microorganism. In this way, dishes, filters, instruments, aqueous solutions of high-temperature-resistant medicinal substances, dressings are sterilized.
Fractional sterilization. In fractional sterilization, an object (usually an aqueous solution) is heated with fluid steam at 100°C for 30 minutes, then the solution is kept at room temperature for 24 hours, after which it is sterilized again under the same conditions (30 minutes at 100°C). The described cycle is repeated 3-5 times. During the first heating, vegetative forms of microoranisms die, during subsequent heating, newly appeared forms and spores.
Pasteurization. This is a single heating of the object at a temperature of 6 °C for 1 hour or at a temperature of 70-80 °C for 30 minutes. Pasteurization allows you to destroy the vegetative forms of microorganisms (except theophilic), but not spores.
Tindalization (fractional pasteurization). During tyndalization, the object is heated at a temperature of 60-65 °C for 1 hour daily for 5 days or at a temperature of 70-80 °C for 3 days. This is a reliable and gentle way to sterilize thermolabile medicinal substances. However, due to its duration, it is of little use for pharmacies and is currently almost never used. These are the main thermal processes used in pharmaceutical practice.