Pasteur oven. The effect of physical factors on microorganisms. Control number …
Sterilization
Sterilization is sterilization, i.e. complete release of objects environment from microorganisms and their spores.
Sterilization is carried out in various ways:
1) physical (impact high temperature, UV rays, use of bacterial filters);
2) chemical (use of various disinfectants, antiseptics);
3) biological (use of antibiotics).
In laboratory practice, physical methods of sterilization are usually used.
The possibility and feasibility of using one or another sterilization method is determined by the characteristics of the material to be sterilized, its physical and chemical properties.
Physical methods
Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, since both vegetative cells and microbial spores die. Typically, bacteriological loops, spatulas, pipettes, slides and cover glasses, and small instruments are calcined. Scissors and scalpels should not be sterilized by heating, since under the influence of fire the cutting surface becomes dull.
Sterilization with dry heat or hot air is carried out in Pasteur ovens (drying dry-heat ovens). The Pasteur oven is a double-walled cabinet made of heat-resistant materials - metal and asbestos. Heat the cabinet with gas burners or electric heating devices. Electrically heated cabinets are equipped with regulators that ensure required temperature. To control the temperature, there is a thermometer inserted into the hole in the top wall of the cabinet.
Liquids (nutrient media, isotonic sodium chloride solution, etc.), items made of rubber and synthetic materials cannot be sterilized with dry heat, since liquids boil and pour out, and rubber and synthetic materials melt.
Sterilization by boiling
Boiling is a sterilization method that guarantees sterility provided there are no spores in the sterilized material. Used for processing instrument syringes, glass and metal utensils rubber tubes, etc. Steam sterilization under pressure is carried out in an autoclave. This sterilization method is based on exposing the materials being sterilized to saturated water vapor at a pressure above atmospheric. As a result of such sterilization, both vegetative and spore forms of microorganisms die with a single treatment. An autoclave (Fig. 12) is a massive boiler, covered on the outside with a metal casing, hermetically sealed with a lid, which is tightly screwed to the boiler with hinged bolts.
The temperature and duration of autoclaving of nutrient media is determined by their composition specified in the recipe for preparing 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 that are 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 with rubber hoses, plugs, bacterial candles and filters are sterilized for 20 minutes at 120 ° C (1 atm).
Sterilization with flowing steam is carried out in a Koch apparatus. This method is used in cases where the object being sterilized changes at a temperature above 100° C. Nutrient media containing urea, carbohydrates, milk, potatoes, gelatin, etc. are sterilized with flowing steam.
The Koch apparatus (boiler) is a metal cylinder lined 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, 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 using gas or electricity. The sterilization time is counted from the moment of vigorous steam release at the edges of the lid and from the steam outlet. Sterilize for 30-60 minutes. At the end of sterilization, heating is stopped. Remove the bucket of material from the apparatus and leave it at room temperature until the next day. Warming 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, vegetative forms of microbes die, while spore forms are preserved. Within a day, the spores manage to germinate and turn into vegetative forms, which 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 a Koch apparatus does not require special control, since the sterility of the prepared nutrient media is an indicator of the correct operation of the device. You can also sterilize with flowing steam in an autoclave with the lid unscrewed and the outlet valve open.
Sterilization by ultraviolet irradiation
Sterilization with UV rays is carried out using special installations - bactericidal lamps. UV rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also spores. UV irradiation is used to sterilize air in hospitals, operating rooms, children's institutions, etc. In a microbiological laboratory, a box is treated with UV rays before work.
Chemical methods
This type of sterilization is used to a limited extent, and it serves mainly to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums).
Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 ° C (the preservatives evaporate).
To preserve vaccines and serums, merthiolate, boric acid, formaldehyde, etc. are used.
Biological sterilization
Biological sterilization is based on the use of antibiotics. This method is used for cultivating viruses.
B. Detailed technology for the production of cattle whey in the slaughterhouses of Lyon
Blood has already been drawn from 1,000 animals, the serum has been bottled and distributed free of charge to almost 20,000 children.
Thus it is shown that industrial production whey at the slaughterhouse in compliance with aseptic rules and sanitary regulations is possible.
The serotherapy enterprise does not provide additional guarantees - it does not have the right to perform an autopsy on the donor animal.
The technology used in our production may seem less aseptic than classic method. But it has a big advantage in speed, since the serum is completely produced on the day of blood collection.
If current circumstances have prompted us to produce the serum in a slaughterhouse, it is clear that this is a temporary measure, since hematogen and medicinal serums can only be produced in a specialized institute.
Animal selection. In Lyon, Dr. Guier, the chief veterinarian of the slaughterhouse, and Dr. Fontenay, the veterinary inspector, themselves select donors from among the livestock intended to provide our city with meat. The selected animal is branded on the right shoulder to facilitate further control.
After slaughter, the animals' organs are carefully checked. It is known that autopsy is the most reliable method of detecting tuberculosis.
Subsequent operations will show that the serum of the diseased animal was never consumed.
Taking blood from animals. In the blood collection room, the donor bull is securely held by an automatic yoke.
The veterinarian disinfects the skin with iodine at the level of the animal's neck and makes an incision in the area of the jugular vein with a scalpel. Venous puncture is performed with a trocar sterilized by prolonged boiling. Once blood appears, an autoclaved rubber hose is attached to the trocar to connect it directly and aseptically to the defibrinator.
The defibrinating technology and method for sterilizing the defibrinator are described below.
From each animal, 8-10 liters of blood are obtained, which is weighed on scales located under the apparatus.
Sterilization methods
To facilitate the sanitary control operation, each defibrinator has a label with the data of the donor animal.
It should be noted that blood sampling is performed aseptically due to the closed circuit of all components: trocar, rubber hose and defibrinator, which are pre-sterilized.
A label with the date of blood collection accompanies the collected blood from the moment of venous puncture until it is converted into serum and stored in refrigerators.
Blood defibration. In most serotherapy establishments, blood poured into glass vessels separates the serum under the pressure of the load. Under these conditions, blood first obtained from cattle contains little serum (approximately 10%).
Therefore, the serotherapy center in Lyon uses special technology, giving 50% of the whey, and also in a shorter time.
Dr. Merrier was able to develop this method partly after the findings he made at the Royal Institute in Rotterdam and at the Serotherapeutic Institute in Milan.
Once blood is obtained from these institutes, it is defibrinated in sterile machines that resemble churns.
Within 5 minutes, the blood is collected in a vessel protected from air. It is necessary to strictly observe the defibration time: if it is insufficient, coagulation can occur, and if it is too much, hemolysis can occur (due to the rupture of red blood cells). It is necessary to use a laboratory clock that allows you to mark exactly 15 minutes of defibrination.
At the top of Fig. 4 shows that the number of blood draws has reached 1000, which is recorded on the Center’s emblem.
Centrifugation.
Immediately after defibration, the devices are transferred to the laboratory, located a few meters from the blood collection room. The contents of each defibrinator are processed separately so that the serum of an animal that turns out to be sick can be removed.
Blood that does not clot after defibrination is passed through an Alfa Laval separator (a milk separator that we have adapted for the production of whey).
Under the influence of centrifugal force, the blood is divided into equal parts: the red part contains red balls, and the transparent part makes up the serum (fibrin remains on the defibrinator blades).
From one animal, 8-10 liters of blood or approximately 4-5 liters of serum are obtained, which is poured into a Pyrex bottle, sterilized at 180°C.
A defibrinator label is attached to the bottle, and a card is filled out under the same number to ensure sanitary control.
A special antiseptic is added to the serum, designed so that it is sufficiently active without disturbing the transparency and taste of the serum. For 1 liter of serum, also add 100 ml of a solution containing 1:1000 formaldehyde and 1:5000 syunuxol.
(Between preparations, the centrifuge is thoroughly disinfected with an antiseptic.)
In principle, the red fraction of the blood should be returned to the butchers to make blood sausage, but most often it remains unused, so syrup can be prepared from it using the technology described below.
Special cases of preparing blood syrup. The syrup has two advantages: it makes it possible to use the red part of the blood remaining after receiving the serum, and it has a pleasant taste that children like.
Due to the lack of glycerin, it is difficult to prepare syrup for long-term storage, but you can make a very active product using water and give it to children 2-3 tablespoons per day. Add 20% to the red part of the blood drinking water and store the syrup in glaciers while sanitary control is carried out.
Then add an equal part 100% sugar syrup (sugar can be obtained from the cards of the groups to whom the syrup is intended).
Lemon or orange extract is added to the syrup to neutralize the taste of blood and poured into 250 ml bottles.
Tyndallization of whey.
Immediately after centrifugation, that is, less than an hour after blood collection, the serum is lysed at 56°C for an hour.
To do this, it is lowered into a water bath with an automatically maintained temperature. Tyndallization at this temperature (at a higher temperature the whey coagulates) is necessary for partial sterilization of the whey, although the speed of its preparation is in itself a guarantee of asepsis.
It should be noted that each five-liter bottle is provided with the original defibrinator label, thus the numbering of the serum corresponds to the numbering of the donor animals.
Control card file. The card allows you to check at any time the origin of the donor animal, the stages of whey preparation, the bottling date, as well as the distribution of the whey.
Sanitary control. While the vessels are stored in refrigeration chamber, veterinarian inspectors are engaged in sanitary control of donor animals. After their slaughter, a thorough autopsy is carried out to reveal the slightest symptoms of tuberculosis.
If a disease is detected, the corresponding serum can be easily withdrawn. It is known that the blood of each animal is processed separately and the serum is contained in separate, numbered bottles.
Serotherapy center Public Committee childhood hygiene at Lyon slaughterhouses
Bovine whey in liters
Card and label
The above card and label prevents any confusion.
In Lyon, sanitary measures are especially strict, as the same veterinarians select the animals, draw blood and check the meat.
Sterilization of equipment. Animal serum is an excellent breeding ground for microbes and can only be partially sterilized. At temperatures above 56° they coagulate and become cloudy if a strong antiseptic is added. Therefore, during all serum production operations, maximum sterility is required; equipment must be decontaminated before use.
Individual defibrinators are sterilized in the following way: the night before blood collection, they are filled with an antiseptic solution, and a few hours before blood collection, they are emptied of antiseptic using a tap located in the lower part of the device. Centrifuges used to produce serum are also treated with an antiseptic, including between processing the contents of each defibrinator.
All glassware, including five-liter vessels for storing whey, is sterilized in an electric oven at a temperature of 180°C.
250 ml bottles for serum are also sterilized at 180°C. To simplify the operation, the dishes are located in boxes, which are used during bottling and distribution to the public.
flower delivery to Voskresensk
Sterilization is represented by physical, chemical, mechanical and biological methods and various methods.
The feasibility of using a particular sterilization method and its methods depends on the characteristics of the material to be sterilized, its physical and chemical properties.
The duration of sterilization depends on the object being sterilized, the sterilizing agent and its dose, the temperature and humidity of the environment.
Physical sterilization method
To the ways physical method sterilization includes drying, burning and calcination, boiling, pasteurization and tindization, hot air(dry heat), ultrasound, ultraviolet and radioactive radiation, high frequency current, sunlight.
The most common method of sterilizing items that can be exposed to high temperatures is sterilization with fire, hot air and saturated steam under pressure.
Fire is used to burn infected objects that do not represent any value (unnecessary papers, old wallpaper, rags, garbage), to disinfect the sputum of tuberculosis patients, the corpses of people and animals who died from particularly dangerous infections, as well as to burn and calcinate various objects .
Burning and calcination are widely used in microbiological practice for the disinfection of instruments, laboratory and pharmaceutical glassware.
Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, as vegetative cells, cysts and spores of microorganisms die.
Typically, loops, spatulas, pipettes, slides and coverslips, small instruments and other contaminated items are sterilized by calcination if they cannot be boiled. It is not recommended to sterilize scissors and scalpels by heating, as the cutting surface becomes dull when exposed to fire.
One of the simplest and most common methods of physical sterilization used in medical practice is hot air sterilization (dry heat). Dry heat sterilization is carried out in drying cabinets(Pasteur ovens). Dry hot air has a bactericidal, virusicidal, sporicidal effect and is used mainly for the sterilization of glass products (laboratory dishes - cups Petri flasks, pipettes, test tubes, etc.), as well as metal products that can be sterilized with steam under pressure.
In addition, dry heat is used to sterilize items made of porcelain and heat-resistant substances (talc, white clay), as well as mineral and vegetable oils, fats, petroleum jelly, lanolin, wax. The most effective mode for this sterilization method, which ensures the death of vegetative forms and spores, is a temperature of 160 - 180 degrees for 15 minutes.
You cannot sterilize food items, isotonic solution, or items made of rubber and synthetic materials with dry heat, as liquids boil and pour out, and rubber and synthetic materials melt.
Sterilization saturated steam under pressure - this is the most reliable and most often tried method of sterilizing dressings, water, some medicines, nutrient media, soft equipment, tools, as well as for the disinfection of waste contaminated material.
In surgical practice dressing, surgeons' gowns, and underwear for the person being operated on are disinfected using steam in autoclaves. Steam sterilization under pressure is carried out in special devices– autoclaves.
Autoclaving completely destroys all microorganisms and spores. The steam pressure sterilization method is based on heating the material with saturated water vapor under pressure above atmospheric pressure. The combined action of high temperature and steam makes this method particularly effective. In this case, both vegetative cells and microbial spores die.
Microbial spores die within 10 minutes under the influence of saturated water vapor, and vegetative forms die within 1 to 4 minutes.
The high bactericidal power of saturated steam is due to the fact that, under the influence of water vapor under pressure, the proteins of the microbial cell swell and coagulate, as a result of which the microbial cells die.
The bactericidal effect of saturated water vapor is enhanced by excess pressure.
Sterilization in an autoclave is carried out under different modes.
Thus, simple nutrient media (meat - peptone agar and meat - peptone broth) are sterilized for 20 minutes at 120 degrees (1 atm.). But with this mode it is impossible to sterilize media containing proteins, carbohydrates and other substances that are easily changed by heating.
Media with carbohydrates are sterilized in an autoclave at 0.5 atm. 10 – 15 minutes or fractionally flowing steam.
Using high temperature, you can destroy the most persistent forms of pathogenic microorganisms (including spore-forming ones) not only on the surface of the objects being disinfected, but also in their depth.
This is the great advantage of high temperature as a reliable means of sterilization. However, some items deteriorate under the influence of high temperatures and in these cases it is necessary to resort to other methods and means of disinfection.
Complete sterilization of materials and objects that do not allow the use of high temperature sterilization is achieved by repeated sterilization with water vapor in a Koch apparatus at a temperature not exceeding 100 degrees. This method is called fractional sterilization. It boils down to the fact that the remaining unkilled spore forms of microbes, after a day in a thermostat at 37 degrees, germinate into vegetative cells, the death of which occurs during subsequent sterilization of this object flowing steam.
Treatment with fluid steam is carried out three times for 30–40 minutes. Heating the material once at a temperature below 100 degrees is known as pasteurization. Pasteurization was proposed by Pasteur and is intended mainly to destroy mostly non-spore microorganisms. Pasteurization is carried out at 60 - 70 degrees for 15 to 30 minutes, at 80 degrees for 10 to 15 minutes.
In microbiological practice, pasteurization seed material often used to isolate pure cultures of spore-forming microorganisms and to determine the ability of microorganisms to form spores.
For liquids that lose taste and other valuable qualities when exposed to high temperatures (milk, berry and fruit juices, beer, nutrient media containing carbohydrates or urea, etc.), sterilization with flowing steam is carried out at 50 - 60 degrees for 15 - 33333330 minutes or at 70 - 80 degrees for 5 - 10 minutes. In this case, microbes of average resistance die, while more resistant microbes and spores are preserved.
Fractional 5 - 6-fold sterilization at 60 degrees for 1 hour is called tyndalization.
Many medical products made from polymer materials, do not withstand sterilization steam method according to generally accepted modes. For many products, due to the characteristics of the liquids they contain (preservatives, medicines and other products), it is impossible to sterilize using generally accepted methods and methods. For such products, individual sterilization regimes are developed to ensure reliable sterilization of objects.
Thus, sterilization of the rotor to separate blood into fractions is carried out with water vapor at a temperature of 120 degrees for 45 minutes.
Sterility of preservative containers is achieved at 110 degrees for 60 minutes.
Boiling is a sterilization method used to desterilize reusable syringes, surgical instruments, rubber tubes, glass and metal utensils.
Sterilization by boiling is carried out in sterilizers. Spore forms in boiling water die after 20 - 30 minutes. Boiling for 45 minutes is widely used to disinfect secretions and other infectious materials, linen, dishes, toys, and patient care items.
Hot water (60 - 100 degrees) with detergents used for washing and cleaning mechanical removal contaminants and microorganisms.
Most vegetative cells die at 70 degrees after 30 minutes.
Filtration sterilization is used in cases where substrates cannot withstand heating, in particular for media containing proteins, serums, some antibiotics, vitamins, and volatile substances. This technique is quite widely used for sterilizing a culture liquid, when it is necessary to free it from microbial cells, but to preserve all the metabolic products it contains unchanged.
The method involves filtering liquids through special filters that have finely porous partitions and therefore retain microbial cells.
The two most widely used types of filters are membrane filters and Seitz filters.
Membrane filters are prepared from collodion, acetate, cellulose and other materials.
Seitz filters are made from a mixture of asbestos and cellulose.
In addition, filters made from kaolin with an admixture are used for sterilization. quartz sand, from infusor soil and from other materials (“candles” by Chamberlan, Berkfeld).
Membrane and asbestos filters are designed for one-time use.
With ultraviolet irradiation, the bactericidal effect is provided by rays with a length of 200 - 450 nm, the source of which is bactericidal lamps.
Sterilization is carried out using bactericidal lamps ultraviolet rays air in medicinal preventive institutions, boxes of microbiological laboratories, at enterprises Food Industry, in boxes for the production of vaccines and serums, in operating rooms, manipulation rooms, children's institutions, etc.
Ultraviolet rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also their spores.
Sunlight causes the death of microorganisms as a result of the action ultraviolet irradiation and drying.
Drying with sunlight has a detrimental effect on many types of microorganisms, but its effect is superficial and therefore sunlight plays a supporting role in sterilization practice.
IN Lately in the treatment of wounds and burns, coatings made from synthetic and natural polymers are used in the form of gels.
Polymer antiseptic films are widely used for local treatment of wounds and burns. They contain such broad-spectrum antimicrobial agents as catapol, dioxidine, blue iodine, as well as sorbitol containing glutaraldehyde. To sterilize these films it is used ionizing radiation at a dose of 20.0 kGy. During the industrial production of polymer antiseptic films and sorbents, their sterility under this sterilization regime is fully ensured.
Radioactive radiation kills all types of microorganisms, both in vegetative and spore forms. It is widely used for sterilization in enterprises producing sterile products and sterile disposable medical devices, for disinfection Wastewater and raw materials of animal origin.
Mechanical method sterilization
Mechanical sterilization methods remove germs from the surface of objects. These include washing, shaking out, sweeping, wet wiping, airing, ventilation, vacuuming, washing.
Chemical sterilization method
Plastics are now increasingly used in medical practice.
They are used in dentistry, maxillofacial surgery, traumatology, orthopedics, and surgery. Most plastics cannot withstand the heat sterilization methods of steam under pressure and dry heat (dry heated air). The solutions of alcohol, diocide, and ternary solution used to sterilize such objects do not ensure the sterility of the products being processed.
Therefore, gas and radiation methods, as well as solutions of chemicals, are used to sterilize plastic products.
Introduction into practice of medical institutions large number products made from thermolabile materials contributes to the introduction of radiation, gas methods disinfection and sterilization with disinfectant solutions.
At chemical sterilization use gases and agents from various chemical groups (peroxide, phenolic, halogen-containing, aldehydes, alkalis and acids, surfactants, etc.). For everyday use, detergents, cleaning, bleaching and other preparations are produced that have an antimicrobial effect due to the introduction of various chemical substances.
These preparations are used for cleaning and disinfection of sanitary technical equipment, dishes, linen, etc.
Formaldehyde vapor (vaporform) can be used in medical institutions for sterilization metal products medical purposes (scalpels, needles, tweezers, probes, clamps, hooks, wire cutters, etc.).
Before sterilization with formaldehyde vapor, products must be subjected to pre-sterilization cleaning and thoroughly dried.
When sterilized in any way chemically the regulations for processing a particular object depend on the characteristics of the object being disinfected, the resistance of microbes, and the characteristics of the properties chemical preparation, ambient temperature, humidity and other factors.
Thus, the sterility of metal instruments is achieved by keeping them in a sealed chamber with steam for five hours at a temperature of at least 20 degrees and relative humidity 95 - 98%, at a temperature of 15 degrees, complete sterility of these objects is achieved only after 16 hours.
The sporicidal activity of glutaraldehyde depends on temperature. Its optimum action occurs at a temperature of 15 – 25 degrees. As the temperature rises, the sporicidal activity of this drug decreases.
Sterilization chemical method Use is somewhat limited. Most often, this method is used to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums). Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 degrees and the preservatives evaporate.
For the preservation of vaccines or serums, merthiolate is used, boric acid, formalin.
Biological method sterilization
Biological sterilization is based on the use of antibiotics.
This method is widely used in the cultivation of viruses.
Sterilization (from Latin sterilis - sterile) involves the complete inactivation of microbes on objects being processed.
Pasteur oven - dry heat sterilization.
There are three main methods of sterilization: heat, radiation, chemical.
Iodine.
Heat sterilization is based on the sensitivity of microbes to high temperature.
At 60 °C and the presence of water, denaturation of proteins occurs, including enzymes, as a result of which the vegetative forms of microbes die. Disputes containing very a large number of waters in a bound state and having dense shells are inactivated at 160-170 °C. For heat sterilization, dry heat and steam under pressure are mainly used.
Dry heat sterilization is carried out in dry heat ovens, or Pasteur ovens. The Pasteur oven is a tightly closed metal cabinet, heated by electricity and equipped with a thermometer.
Disinfection of the material in it occurs at 160-170 °C for 60-120 minutes. The disadvantage of this method is that only some sterilizable objects, such as laboratory glass, can withstand such high temperatures.
The most universal method of sterilization is steam treatment under pressure in autoclaves, in which dressings, linen, many instruments, culture media, solutions, infectious material, etc. are sterilized.
An autoclave is a metal cylinder with strong walls, hermetically sealed, consisting of a water-steam and sterilizing chamber. The device is equipped with a pressure gauge, thermometer and other monitoring devices. Increased pressure is created in the autoclave, which leads to an increase in the boiling point of water. So, at 0.5 atm the boiling point is 80 °C, at 1 atm - 100 °C, at 2 atm - 121 °C and at 3 atm - 136 °C.
Due to the fact that, in addition to high temperature, steam acts on microorganisms, spores die already at 120 ° C. The most common autoclave operating mode is 2 atm, 121 °C, 15-20 minutes. Sterilization time decreases with increasing atmospheric pressure, and therefore the boiling point. Microorganisms die in a few seconds, but the material is processed for a longer time, since, firstly, the temperature must be high inside the material being sterilized, and, secondly, there is a so-called safety field, designed for possible deviation from the specified parameters when autoclave operation.
Tags: body, growth, sterilization, enzyme
Sterilization with dry heat or hot air is carried out in Pasteur ovens (drying ovens). The Pasteur oven is a double-walled cabinet made of heat-resistant materials - metal and asbestos. Heat the cabinet using gas burners or electric heating devices. Electrically heated cabinets are equipped with regulators to ensure the required temperature. To control the temperature, there is a thermometer inserted into the hole in the top wall of the cabinet.
Dry heat is used to sterilize laboratory glassware. The dishes prepared for sterilization are loosely loaded into the oven to ensure uniform and reliable heating of the material being sterilized. Close the cabinet door tightly, turn on the heating device, bring the temperature to 160-165 ° C and sterilize at this temperature 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 cold air, entering the cabinet can cause cracks in hot cookware.
Sterilization in a Pasteur oven can be carried out at different temperatures and exposures (sterilization time) (Table 1).
Table 1. Sterilization mode
Liquids (nutrient media, isotonic sodium chloride solution, etc.), items made of rubber and synthetic materials cannot be sterilized with dry heat, since liquids boil and pour out, and rubber and synthetic materials melt.
To control sterilization in a Pasteur oven, silk threads are moistened in a culture of spore-forming bacteria, dried, placed in a sterile Petri dish and placed in a Pasteur oven. Sterilization is carried out at a temperature of 165° C for 1 hour (for control, some of the threads are left at room temperature). Then the sterilized and control threads are placed on the surface of the agar in a Petri dish or placed in test tubes with broth and incubated in a thermostat at 37° C for 2 days. At proper operation Pasteur oven in test tubes or dishes with nutrient media where sterilized threads were placed, there will be no growth, since bacterial spores will die, while bacterial spores on threads that were not sterilized (control) will germinate and growth will be noted on nutrient media .
To determine the temperature inside the Pasteur oven, you can use sucrose or granulated sugar, which caramelizes at a temperature of 165-170 ° C.
Preparing laboratory glassware for sterilization in a Pasteur oven. Before sterilization, laboratory glassware (Petri dishes, graduated and Pasteur pipettes, vials, flasks, test tubes) must be thoroughly washed, dried and wrapped in paper, otherwise after sterilization they may again become contaminated with air bacteria.
Petri dishes are wrapped in paper one or more pieces at a time or placed in special metal cases.
Cotton swabs are inserted into the upper ends of the pipettes to prevent the test material from entering the mouth. Graduated pipettes are wrapped in long strips of paper 4-5 cm wide. The volume of the wrapped pipette is marked on the paper. In pencil cases, graduated pipettes are sterilized without additional wrapping in paper.
Note. If the graduation on the pipettes is poorly visible, it is restored before sterilization. Oil paint is applied to the pipette and, without allowing the paint to dry, barium sulfate powder is rubbed into it using a cloth. After this, remove excess paint with a rag, which remains only in the graduation notches. Pipettes treated in this way should be rinsed.
The sharp ends of Pasteur pipettes are sealed in a burner flame and wrapped in paper, 3-5 pieces at a time. Pasteur pipettes must be wrapped carefully so as not to break off the sealed ends of the capillaries.
Vials, flasks, test tubes are closed with cotton-gauze stoppers. The cork should fit into the neck of the vessel 2/3 of its length, not too tight, but not loose either. A paper cap is placed over the stoppers on each vessel (except test tubes). Test tubes are tied together in groups of 5-50 and wrapped with paper.
Note. At high temperatures, the paper in which cups and pipettes are wrapped, and cotton wool turn yellow and may even become charred, so every new variety paper received by the laboratory should be tested at the accepted temperature conditions.
1. What is meant by the term sterilization?
2. How is sterilization carried out?
3. What is sterilized by calcination over fire?
4. Describe the structure and operating mode of the Pasteur oven.
5. What is sterilized in a Pasteur oven?
6. How are glassware prepared for sterilization?
7. Why can’t nutrient media and rubber objects be sterilized in a Pasteur oven?
Exercise
Prepare Petri dishes, graduated pipettes, Pasteur pipettes, test tubes, flasks and vials for sterilization.
Sterilization- infertility; destruction of pathogenic and non-pathogenic microorganisms in vegetative and spore forms in any material.
Preparing dishes for sterilization. Laboratory glassware must be cleanly washed and sterilized. For washing, use soap solutions or chemical detergents. New dishes are pre-boiled in a 1-2% solution of hydrochloric acid, to avoid subsequent leaching of the glass. Dishes washed in running water are rinsed with distilled water and dried.
Bacteriological tubes. Conical, matte flasks are closed with cotton-gauze stoppers, consisting of tightly twisted rolls of cotton wool, covered with a layer of gauze. Metal stoppers in the form of outer caps have also been developed for bacteriological test tubes. It should be taken into account that sterilizing cotton plugs at high temperatures leads to the release of substances from the cotton wool that inhibit the growth of some sensitive bacteria, such as Brucella.
When installing pipettes, insert a cotton swab into the upper end. Pasteur pipettes must have a sealed capillary. Each measuring pipette is wrapped in a long strip of paper 4-5 cm wide, starting from the spout, in a helical manner along its entire length. Pasteur pipettes are wrapped in paper, 10-20 pieces each, test tubes - 15-20 pieces each. It is better to store all types of pipettes before and after sterilization in special metal cases. The stoppers on the flasks are additionally covered with paper caps.
Before sterilization, clean, assembled Petri dishes are wrapped in paper, 3 to 4 pieces each. After sterilization, the paper protects sterile glassware from contamination by microflora.
Before sterilization, the dishes are placed in the drying cabinet not too tightly to ensure air circulation, and care is taken that the temperature does not exceed 180? C, since at a higher temperature the paper and cotton wool will char. After sterilization is completed, the drying cabinet is not opened until then. Until the temperature in it drops to 70-80? C, because sharp drop temperatures may cause glass to break.
If the dishes are intended for sterilization of nutrient media in them by autoclaving under a pressure of at least 1 atm, then they are not pre-sterilized. When sterilizing media with flowing steam or in an autoclave under a pressure of no more than 0.5 atm. Sterile containers must be used.
Sterilization with dry heated air. The method is used to sterilize clean glassware. For this purpose, a Pasteur oven is used - a special drying cabinet with double walls. The outside is lined with heat-proof material. At the top there is a thermometer. Between the heatproof lining and the inner metal body An automatic electric heating element is placed at the bottom. When the drying cabinet is turned on, the air inside it heats up. Once the set temperature is reached, the start time of sterilization is noted. Sterilization mode: at a temperature of 155-160? C - exposure for 2 hours, at 165-170? C - 1-1.5 hours, at 180? C - 1 hour. After the sterilization time, heating is stopped.
Autoclaving. This is steam sterilization under pressure combined with high temperature in a special apparatus - an autoclave. When saturated steam encounters a cooler object, the steam condenses into water, releasing a large amount of heat. In addition, the volume of steam is reduced, which facilitates its penetration into the internal parts of the material being sterilized. A prerequisite is the supply of truly saturated steam, so that its contact with a cold object leads to immediate condensation and heating. The industry produces vertical and horizontal autoclaves.
A vertical autoclave is a double-walled cylindrical metal cauldron, sealed with a lid. Water is poured between the walls through a special tap with a funnel to a certain level. The inner wall of the boiler is equipped with holes in the upper part and a tap in the lower part, through which, when the water is heated, steam displaces air from the boiler. A metal protective frame is placed on top of the autoclave, and between it and the autoclave itself there should be free space. The autoclave is heated by connecting to the electrical network.
The autoclave is loaded with the material to be sterilized, the lid and the tap through which water was poured are closed, and the bottom tap is temporarily left open. The heated water between the walls of the autoclave boils, the resulting steam rises up and passes through the upper holes of the inner wall into the boiler, pushing out the air through the lower open tap. When all the air is displaced and the steam begins to come out in an even stream, the lower valve is closed. As a result, the steam pressure inside the autoclave increases. The beginning of sterilization is considered the moment when the pressure reaches a given value (according to the pressure gauge). Heat is adjusted throughout sterilization, maintaining steam pressure at the same level. If the pressure inside the autoclave increases excessively, there is a safety valve through which the excess steam automatically escapes.
As the steam pressure increases, the temperature in the autoclave increases accordingly.
The pressure gauge shows the steam pressure without taking into account the ambient atmospheric pressure (760 mm Hg). After the sterilization time has expired, the autoclave is turned off. After cooling, when the pressure gauge reading is zero, open the valve to release steam.
A horizontal autoclave differs from a vertical autoclave in design, but its operating principle is the same.
virological sterilization pathological animal
Samples of forms to be filled out when sending pathological material to the laboratory
It is a double-walled metal cylinder covered on the outside with a metal casing. It is hermetically sealed with a massive lid using several screws. It is equipped with a pressure gauge with a safety valve and a steam valve.
Before sterilization, distilled water is poured into the autoclave through a funnel with a water-measuring glass up to the line indicated on the casing. The material for sterilization is loaded into the sterilizing chamber, closed tightly with a lid, screwed on and the heating source is turned on. In this case, the steam valve is left open. The steam generated during boiling passes between the walls of the autoclave and enters the chamber through the holes in the inner wall. When heating, air first comes out of the autoclave through the steam valve, and then steam. The release of a continuous stream of dry steam indicates complete displacement of air from the autoclave: the tap is closed, and from that moment the pressure in the autoclave begins to gradually increase, the needle on the pressure gauge rises. The beginning of sterilization is considered to be the moment when the pressure gauge needle reaches the desired pressure.
Fig.3
The pressure gauge reading corresponds to certain temperature steam in an autoclave: 0.50 MPa - 112 °C, 0.1 MPa - 120, 0.15 MPa - 127, 0.2 MPa - 134 °C.
Material in an autoclave is most often sterilized at 0.1 MPa for 20-30 minutes. At the end of sterilization, turn off the heating source (the pressure gauge needle gradually reaches zero). After this, open the steam valve and release the remaining steam. Then carefully unscrew the lid and open it. After complete cooling, remove the sterilized material.
An autoclave can be used to sterilize dishes, instruments, culture media (except for gelatin and media with carbohydrates), dressings, etc. When working, you must follow safety rules. Persons who have a certificate for the right to use an autoclave are allowed to work. The serviceability of the autoclave is checked by the boiler inspectorate.
The Koch apparatus (Fig. 4) is a metal cylinder lined on the outside with material (linoleum, asbestos) that does not conduct heat well. Water is poured into the bottom, and the sterilizing material is placed on top of the stand. The device is closed with a conical lid, which has holes for a thermometer and steam outlet. At the bottom there is a tap for draining water. Sterilization is carried out with flowing steam at 100 °C for 30-60 minutes. In this mode, vegetative cells of spore-forming and non-spore-forming forms of microbes die. Fractional sterilization (three times) for 30-60 minutes over three days with an interval of 18-20 hours allows you to create conditions for the germination of spores into vegetative cells and to get rid of them. In the time intervals between sterilization, the spores germinate and die during subsequent heating. The Koch apparatus sterilizes those materials that cannot withstand temperatures above 100 °C (gelatin, milk, carbohydrate media, etc.).
Protein media and blood serum that cannot tolerate temperatures of 100 °C are sterilized fractionally at 56-58 °C in a water bath.
Drying cabinet(Pasteur oven) (Fig. 5) is a metal double-walled cabinet covered with asbestos on top. The top wall has holes for a thermometer and ventilation. Heated air rises from below between the walls and through the upper opening enters the cabinet, where the material to be sterilized is placed on the shelves. Sterilization is carried out with dry heat at 150 °C for 2 hours, at 165-170 °C - 45 minutes, at 180 °C - 15 minutes. Glassware is sterilized in a Pasteur oven. After sterilization, the cabinet is disconnected from the heating source and opened only after complete cooling.
Bacterial filters used to sterilize liquids without heating. These include Chamberlant, Berkefeld candles and Seitz asbestos filters (plates).
Filter candles (Fig. 6) are hollow cylinders made of finely porous substances: kaolin with an admixture of quartz sand (Chamberlan candles) and infusor earth (Berkefeld candles). Chamberlant candles have various sizes pores through which microbes pass. Candles that allow large bacteria to pass through are designated by the letters L9, L1(bis), L3, medium ones - L5, L7, the smallest ones - L9, L11 , L13 Berkefeld candles are designated by porosity W, N, V(candles with brand U have the largest pores).
Seitz filters are asbestos plates of various sizes. When mounting the device for sterilization, the plate is placed on the mesh between metal discs(with a hole in the middle), which are pressed tightly together with screws. The mounted filter is inserted through a stopper into a flask with a side outlet (Bunsen flask) and a rubber tube, wrapped in paper and sterilized in an autoclave at 120 °C for 20-30 minutes.
To filter the material, create a vacuum in a Bunsen flask by connecting to it a rubber tube with a rarefied oil hand pump Komovsky or an electric vacuum pump.
Completing of the work. Microbes are cultivated at optimal temperature conditions. For this purpose, laboratories use air or water thermostats.
(Fig. 7) is metal cabinet with double walls, between which there is a layer of water or air. The outer part of the thermostat is covered with a material that conducts heat poorly (asbestos, linoleum).
Rice. 4, 5, 6.
Inside the thermostat there are shelves for placing seed material of grown microorganisms. A constant temperature in the thermostat is maintained using a thermostat, which is built into the top cover of the thermostat. The thermostat device is based on the principle of linear expansion of substances. Thermoregulators are an alloy of any two metals with different coefficients of thermal expansion (brass, zinc) or a metal “cushion” filled with alcohol, a mixture of alcohol and ether, mercury or other substances that change their volume at a certain temperature. When the thermostat heats above the established norm, the metals expand, the contacts open and further heat flow is automatically delayed. After the temperature drops it turns on electricity and the flow of heat resumes.
Sterilization is represented by physical, chemical, mechanical and biological methods and various methods. The feasibility of using a particular sterilization method and its methods depends on the characteristics of the material to be sterilized, its physical and chemical properties. The duration of sterilization depends on the object being sterilized, the sterilizing agent and its dose, the temperature and humidity of the environment.
Physical sterilization method Methods of the physical method of sterilization include drying, burning and calcination, boiling, pasteurization and tindalization, hot air (dry heat), ultrasound, ultraviolet and radioactive radiation, high frequency current, sunlight. The most common method of sterilizing items that can be exposed to high temperatures is sterilization with fire, hot air and saturated steam under pressure. Fire is used to burn infected objects that do not represent any value (unnecessary papers, old wallpaper, rags, garbage), to disinfect the sputum of tuberculosis patients, the corpses of people and animals who died from particularly dangerous infections, as well as to burn and calcinate various objects . Burning and calcination are widely used in microbiological practice for the disinfection of instruments, laboratory and pharmaceutical glassware. Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, as vegetative cells, cysts and spores of microorganisms die. Typically, loops, spatulas, pipettes, slides and coverslips, small instruments and other contaminated items are sterilized by calcination if they cannot be boiled. It is not recommended to sterilize scissors and scalpels by heating, as the cutting surface becomes dull when exposed to fire. One of the simplest and most common methods of physical sterilization used in medical practice is hot air sterilization (dry heat). Dry heat sterilization is carried out in drying ovens (Pasteur ovens). Dry hot air has a bactericidal, virusicidal, sporicidal effect and is used mainly for the sterilization of glass products (laboratory glassware - Petri dishes, flasks, pipettes, test tubes, etc.), as well as metal products that can be sterilized with steam under pressure. In addition, dry heat is used to sterilize items made of porcelain and heat-resistant substances (talc, white clay), as well as mineral and vegetable oils, fats, petroleum jelly, lanolin, and wax. The most effective mode for this sterilization method, which ensures the death of vegetative forms and spores, is a temperature of 160 - 180 degrees for 15 minutes. You cannot sterilize food items, isotonic solution, or items made of rubber and synthetic materials with dry heat, as liquids boil and pour out, and rubber and synthetic materials melt. Sterilization with saturated steam under pressure is the most reliable and most often used method for sterilizing dressings, water, some medicines, culture media, soft equipment, instruments, as well as for disinfecting waste contaminated material. In surgical practice, dressings, surgeons' gowns, and underwear for the operated patient are disinfected using steam in autoclaves. Steam sterilization under pressure is carried out in special devices - autoclaves. Autoclaving completely destroys all microorganisms and spores. The steam pressure sterilization method is based on heating the material with saturated water vapor under pressure above atmospheric pressure. The combined action of high temperature and steam makes this method particularly effective. In this case, both vegetative cells and microbial spores die. Microbial spores die within 10 minutes under the influence of saturated water vapor, and vegetative forms die within 1 to 4 minutes. The high bactericidal power of saturated steam is due to the fact that, under the influence of water vapor under pressure, the proteins of the microbial cell swell and coagulate, as a result of which the microbial cells die. The bactericidal effect of saturated water vapor is enhanced by excess pressure. Sterilization in an autoclave is carried out under different modes. Thus, simple nutrient media (meat - peptone agar and meat - peptone broth) are sterilized for 20 minutes at 120 degrees (1 atm.). But with this mode it is impossible to sterilize media containing proteins, carbohydrates and other substances that are easily changed by heating. Media with carbohydrates are sterilized in an autoclave at 0.5 atm. 10 – 15 minutes or fractionally flowing steam. Using high temperature, you can destroy the most persistent forms of pathogenic microorganisms (including spore-forming ones) not only on the surface of the objects being disinfected, but also in their depth. This is the great advantage of high temperature as a reliable means of sterilization. However, some items deteriorate under the influence of high temperatures and in these cases it is necessary to resort to other methods and means of disinfection. Complete sterilization of materials and objects that do not allow the use of high temperature sterilization is achieved by repeated sterilization with water vapor in a Koch apparatus at a temperature not exceeding 100 degrees. This method is called fractional sterilization. It boils down to the fact that the remaining unkilled spore forms of microbes, after a day in a thermostat at 37 degrees, germinate into vegetative cells, the death of which occurs during the subsequent sterilization of this object with flowing steam. Treatment with fluid steam is carried out three times for 30–40 minutes. Heating the material once at a temperature below 100 degrees is known as pasteurization. Pasteurization was proposed by Pasteur and is intended mainly to destroy mostly non-spore microorganisms. Pasteurization is carried out at 60 - 70 degrees for 15 to 30 minutes, at 80 degrees for 10 to 15 minutes. In microbiological practice, pasteurization of seed material is often used to isolate pure cultures of spore-forming microorganisms and to identify the ability of microorganisms to form spores. For liquids that lose taste and other valuable qualities when exposed to high temperatures (milk, berry and fruit juices, beer, nutrient media containing carbohydrates or urea, etc.), sterilization with flowing steam is carried out at 50 - 60 degrees for 15 - 33333330 minutes or at 70 - 80 degrees for 5 - 10 minutes. In this case, microbes of average resistance die, while more resistant microbes and spores are preserved. Fractional 5 - 6-fold sterilization at 60 degrees for 1 hour is called tyndalization. Many medical products made from polymer materials cannot withstand steam sterilization according to generally accepted regimes. For many products, due to the characteristics of the liquids they contain (preservatives, medicines and other products), it is impossible to sterilize using generally accepted methods and methods. For such products, individual sterilization regimes are developed to ensure reliable sterilization of objects. Thus, sterilization of the rotor to separate blood into fractions is carried out with water vapor at a temperature of 120 degrees for 45 minutes. Sterility of preservative containers is achieved at 110 degrees for 60 minutes. Boiling is a sterilization method used to desterilize reusable syringes, surgical instruments, rubber tubes, glass and metal utensils. Sterilization by boiling is carried out in sterilizers. Spore forms in boiling water die after 20 - 30 minutes. Boiling for 45 minutes is widely used to disinfect secretions and other infectious materials, linen, dishes, toys, and patient care items. Hot water (60 - 100 degrees) with detergents is used when washing and cleaning to mechanically remove dirt and microorganisms. Most vegetative cells die at 70 degrees after 30 minutes. Filtration sterilization is used in cases where substrates cannot withstand heating, in particular for media containing proteins, serums, some antibiotics, vitamins, and volatile substances. This technique is quite widely used for sterilizing a culture liquid, when it is necessary to free it from microbial cells, but to preserve all the metabolic products it contains unchanged. The method involves filtering liquids through special filters that have finely porous partitions and therefore retain microbial cells. The two most widely used types of filters are membrane filters and Seitz filters. Membrane filters are prepared from collodion, acetate, cellulose and other materials. Seitz filters are made from a mixture of asbestos and cellulose. In addition, filters made of kaolin with an admixture of quartz sand, infusor earth and other materials (“candles” by Chamberlan, Berkfeld) are used for sterilization. Membrane and asbestos filters are designed for one-time use. With ultraviolet irradiation, the bactericidal effect is provided by rays with a length of 200 - 450 nm, the source of which is bactericidal lamps. With the help of bactericidal lamps, air is sterilized with ultraviolet rays in medical and preventive institutions, boxes of microbiological laboratories, food industry enterprises, in boxes for the production of vaccines and serums, in operating rooms, manipulation rooms, children's institutions, etc. Ultraviolet rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also their spores. Sunlight causes the death of microorganisms as a result of ultraviolet irradiation and drying. Drying with sunlight has a detrimental effect on many types of microorganisms, but its effect is superficial and therefore sunlight plays a supporting role in sterilization practice. Recently, in the treatment of wounds and burns, coatings made of synthetic and natural polymers have been used in the form of gels. Polymer antiseptic films are widely used for local treatment of wounds and burns. They contain such broad-spectrum antimicrobial agents as catapol, dioxidine, blue iodine, as well as sorbitol containing glutaraldehyde. To sterilize these films, ionizing radiation is used at a dose of 20.0 kGy. During the industrial production of polymer antiseptic films and sorbents, their sterility under this sterilization regime is fully ensured. Radioactive radiation kills all types of microorganisms, both in vegetative and spore forms. It is widely used for sterilization in enterprises producing sterile products and sterile disposable medical devices, for the disinfection of wastewater and raw materials of animal origin.
Mechanical sterilization method Mechanical sterilization methods remove germs from the surface of objects. These include washing, shaking out, sweeping, wet wiping, airing, ventilation, vacuuming, washing.
Chemical sterilization method Plastics are now increasingly used in medical practice. They are used in dentistry, maxillofacial surgery, traumatology, orthopedics, and surgery. Most plastics cannot withstand the heat sterilization methods of steam under pressure and dry heat (dry heated air). The solutions of alcohol, diocide, and ternary solution used to sterilize such objects do not ensure the sterility of the products being processed. Therefore, gas and radiation methods, as well as solutions of chemicals, are used to sterilize plastic products. The introduction of a large number of products made of thermolabile materials into the practice of medical institutions contributes to the introduction of radiation and gas methods of disinfection and sterilization with disinfectant solutions. During chemical sterilization, gases and agents from various chemical groups are used (peroxide, phenolic, halogen-containing, aldehydes, alkalis and acids, surfactants, etc.). For everyday use, detergents, cleaning, bleaching and other preparations are produced that have an antimicrobial effect due to the introduction of various chemicals into their composition. These preparations are used for cleaning and disinfecting sanitary equipment, dishes, linen, etc. Formaldehyde steam (vapor form) can be used in medical institutions to sterilize metal medical products (scalpels, needles, tweezers, probes, clamps, hooks, wire cutters, etc. .). Before sterilization with formaldehyde vapor, products must be subjected to pre-sterilization cleaning and thoroughly dried. When sterilizing by any chemical method, the procedure for processing a particular object depends on the characteristics of the object being disinfected, the resistance of microbes, the characteristics of the properties of the chemical, ambient temperature, humidity and other factors. Thus, the sterility of metal instruments is achieved after five hours of storage in a sealed chamber with steam at a temperature of at least 20 degrees and a relative humidity of 95 - 98%; at a temperature of 15 degrees, complete sterility of these objects is achieved only after 16 hours. The sporicidal activity of glutaraldehyde depends on temperature. Its optimum action occurs at a temperature of 15 – 25 degrees. As the temperature rises, the sporicidal activity of this drug decreases. Chemical sterilization is used to a somewhat limited extent. Most often, this method is used to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums). Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 degrees and the preservatives evaporate. To preserve vaccines or serums, merthiolate, boric acid, and formalin are used.
Biological sterilization method Biological sterilization is based on the use of antibiotics. This method is widely used in the cultivation of viruses.
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