Pasteur oven. Sterilization of laboratory glassware. Drying cabinet and hot air sterilization

The effect of temperature on microorganisms.

Temperature - important factor, affecting the life of microorganisms. For microorganisms, there are minimum, optimal and maximum temperatures. Optimal– the temperature at which the most intensive proliferation of microbes occurs. Minimum– temperature below which microorganisms do not exhibit vital activity. Maximum– the temperature above which the death of microorganisms occurs.

In relation to temperature, 3 groups of microorganisms are distinguished:

2. Mesophiles. Optimum – 30-37°С. Minimum – 15-20°C. Maximum – 43-45°C. They live in the bodies of warm-blooded animals. These include most pathogenic and opportunistic microorganisms.

3. Thermophiles. Optimum – 50-60°C. Minimum - 45°C. Maximum - 75°С. They live in hot springs and participate in the processes of self-heating of manure and grain. They are not able to reproduce in the body of warm-blooded animals, so they have no medical significance.

Favorable action optimal temperature used in growing microorganisms for the purpose of laboratory diagnostics, preparation of vaccines and other drugs.

Braking action low temperatures used for storage products and cultures of microorganisms in a refrigerator. Low temperature stops putrefactive and fermentation processes. The mechanism of action of low temperatures is the inhibition of metabolic processes in the cell and the transition to a state of suspended animation.

Detrimental effect high temperature (above maximum) used for sterilization . Mechanism actions – denaturation of protein (enzymes), damage to ribosomes, disruption of the osmotic barrier. Psychrophiles and mesophiles are most sensitive to high temperatures. special sustainability show disputes bacteria.

The effect of radiant energy and ultrasound on microorganisms.

There are non-ionizing (ultraviolet and infrared rays of sunlight) and ionizing radiation (g-rays and high-energy electrons).

Ionizing radiation has a powerful penetrating effect and damages the cellular genome. Mechanism damaging effect: ionization macromolecules, which is accompanied by the development of mutations or cell death. Moreover, lethal doses for microorganisms are higher than for animals and plants.

Mechanism damaging effect UV rays: formation of thymine dimers in a DNA molecule , which stops cell division and is the main cause of their death. The damaging effect of UV rays is more pronounced for microorganisms than for animals and plants.

Ultrasound(sound waves 20 thousand Hz) has a bactericidal effect. Mechanism: education in the cytoplasm of the cell cavitation cavities , which are filled with liquid vapor and a pressure of up to 10 thousand atm arises in them. This leads to the formation of highly reactive hydroxyl radicals, destruction of cellular structures and depolymerization of organelles, denaturation of molecules.

Ionizing radiation, UV rays and ultrasound are used for sterilization.

Effect of drying on microorganisms.

Water is necessary for the normal functioning of microorganisms. A decrease in environmental humidity leads to the transition of cells to a state of rest, and then to death. Mechanism detrimental effects of drying: dehydration of the cytoplasm and denaturation of proteins.

Pathogenic microorganisms are more sensitive to drying: pathogens of gonorrhea, meningitis, typhoid fever, dysentery, syphilis, etc. Bacterial spores, protozoan cysts, bacteria protected by sputum mucus (tuberculosis bacilli) are more resistant.

In practice drying is used for canning meat, fish, vegetables, fruits, when preparing medicinal herbs.

Drying from frozen state under vacuum – lyophilization or freeze drying. She's being used for crop conservation microorganisms that in this state for years (10-20 years) do not lose their viability and do not change their properties. Microorganisms are in a state of suspended animation. Lyophilization is used in the production of drugs from living microorganisms: eubiotics, phages, live vaccines against tuberculosis, plague, tularemia, brucellosis, influenza, etc.

The effect of chemical factors on microorganisms.

Chemicals affect microorganisms in different ways. This depends on the nature, concentration and time of action of the chemicals. They can stimulate growth(used as energy sources), provide microbicidal, microbostatic, mutagenic effect or may be indifferent to vital processes

For example: a 0.5-2% glucose solution is a source of nutrition for microbes, and a 20-40% solution has an inhibitory effect.

For microorganisms it is necessary optimal pH value of the environment. For most symbionts and pathogens of human diseases - a neutral, slightly alkaline or slightly acidic environment. As the pH increases, it often shifts to the acidic side, and the growth of microorganisms stops. And then death comes. Mechanism: denaturation of enzymes by hydroxyl ions, disruption of the osmotic barrier of the cell membrane.

Chemicals that have antimicrobial effect, used for disinfection, sterilization and preservation.

The effect of biological factors on microorganisms.

Biological factors– these are various forms of influence of microbes on each other, as well as the effect of immune factors (lysozyme, antibodies, inhibitors, phagocytosis) on microorganisms during their stay in the macroorganism. Coexistence of various organisms - symbiosis. The following are distinguished: forms symbiosis.

Mutualism– a form of cohabitation where both partners receive mutual benefits (for example, nodule bacteria and legumes).

Antagonism- a form of relationship when one organism causes harm (even death) to another organism with its metabolic products (acids, antibiotics, bacteriocins), due to better adaptability to environmental conditions, through direct destruction (for example, normal intestinal microflora and pathogens of intestinal infections).

Metabiosis– a form of cohabitation when one organism continues the process caused by another (uses its waste products) and frees the environment from these products. Therefore, conditions are created for further development (nitrifying and ammonifying bacteria).

Satellism– one of the cohabitants stimulates the growth of the other (for example, yeast and sarcina produce substances that promote the growth of other, more nutrient-demanding bacteria).

Commensalism– one organism lives at the expense of another (benefits) without causing harm to it (for example, E. coli and the human body).

Predation– antagonistic relationships between organisms, when one captures, absorbs and digests another (for example, intestinal amoeba feeds on intestinal bacteria).

Sterilization.

Sterilization is the process of complete destruction of all viable forms of microbes in an object, including spores.

There are 3 groups of sterilization methods: physical, chemical and physico-chemical. Physical methods: sterilization by high temperature, UV irradiation, ionizing irradiation, ultrasound, filtration through sterile filters. Chemical methods– use of chemicals, as well as gas sterilization. Physico-chemical methods– joint use of physical and chemical methods. For example, high temperature and antiseptics.

High temperature sterilization .

This method includes: 1) dry heat sterilization; 2) steam sterilization under pressure; 3) flowing steam sterilization; 4) tindialization and pasteurization; 5) calcination; 6) boiling.

Dry heat sterilization.

The method is based on the bactericidal effect of air heated to 165-170°C for 45 minutes.

Equipment: dry heat oven (Pasteur oven). A Pasteur oven is a metal cabinet with double walls, lined on the outside with a material that does not conduct heat well (asbestos). Heated air circulates in the space between the walls and exits through special openings. When working, it is necessary to strictly monitor the required temperature and sterilization time. If the temperature is higher, then charring of cotton plugs and paper in which the dishes are wrapped will occur, and at a lower temperature, longer sterilization is required. After sterilization is completed, the cabinet is opened only after it has cooled, otherwise the glassware may crack due to a sudden change in temperature.

a) glass, metal, porcelain items, dishes, wrapped in paper and closed with cotton-gauze stoppers to maintain sterility (165-170°C, 45 min);

b) heat-resistant powdered medicines - talc, white clay, zinc oxide (180-200°C, 30-60 min);

c) mineral and vegetable oils, fats, lanolin, petroleum jelly, wax (180-200°C, 20-40 min).

Steam sterilization under pressure.

The most effective and widely used method in microbiological and clinical practice.

The method is based on the hydrolyzing effect of steam under pressure on the proteins of the microbial cell. The combined action of high temperature and steam provides high efficiency this sterilization, which kills the most persistent spore bacteria.

Equipment – ​​autoclave. The autoclave consists of 2 metal cylinders inserted into each other with a hermetically sealed lid screwed in with screws. The outer boiler is a water-steam chamber, the inner boiler is a sterilization chamber. There is a pressure gauge, steam release valve, safety valve, and water meter glass. At the top of the sterilization chamber there is a hole through which steam passes from the water-steam chamber. The pressure gauge is used to determine the pressure in the sterilization chamber. There is a certain relationship between pressure and temperature: 0.5 atm - 112°C, 1-01.1 atm - 119-121°C, 2 atm - 134°C. Safety valve – to protect against excessive pressure. When the pressure rises above the set value, the valve opens and releases excess steam. Operating procedure. Water is poured into the autoclave, the level of which is monitored using a water meter glass. The material is placed into the sterilization chamber and the lid is screwed on tightly. The steam valve is open. Turn on the heating. After the water boils, the tap is closed only when all the air has been displaced (steam flows in a continuous strong dry stream). If the tap is closed earlier, the pressure gauge readings will not correspond to the desired temperature. After closing the tap, the pressure in the boiler gradually increases. The beginning of sterilization is the moment when the pressure gauge needle shows the set pressure. After the sterilization period has expired, stop heating and cool the autoclave until the pressure gauge needle returns to 0. If you release steam earlier, the liquid may boil due to a rapid change in pressure and push out the plugs (sterility is impaired). When the pressure gauge needle returns to 0, carefully open the steam release valve, release the steam and then remove the objects to be sterilized. If the steam is not released after the needle returns to 0, water may condense and wet the plugs and the material being sterilized (sterility will be impaired).

Material and sterilization mode:

a) glass, metal, china, linen, rubber and cork stoppers, products made of rubber, cellulose, wood, dressings (cotton wool, gauze) (119 - 121 ° C, 20-40 min));

b) physiological solution, solutions for injections, eye drops, distilled water, simple nutrient media - MPB, MPA (119-121°C, 20-40 min);

c) mineral and vegetable oils in hermetically sealed vessels (119-121°C, 120 min);

Sterilization with flowing steam.

The method is based on the bactericidal effect of steam (100°C) against only vegetative cells.

Equipment– an autoclave with an unscrewed lid or Koch apparatus.

Koch apparatus - This is a metal cylinder with a double bottom, the space in which is 2/3 filled with water. The lid has holes for a thermometer and for steam to escape. The outer wall is lined with a material that conducts heat poorly (linoleum, asbestos). The start of sterilization is the time from the boiling of water and the entry of steam into the sterilization chamber.

Material and sterilization mode. This method sterilizes the material which cannot withstand temperatures above 100°C: nutrient media with vitamins, carbohydrates (Hiss, Endo, Ploskirev, Levin media), gelatin, milk.

At 100°C, spores do not die, so sterilization is carried out several times - fractional sterilization - 20-30 minutes daily for 3 days.

In the intervals between sterilizations, the material is kept at room temperature in order for spores to germinate into vegetative forms. They will die upon subsequent heating at 100°C.

Tyndallization and pasteurization.

Tyndalization - method of fractional sterilization at temperatures below 100°C. It is used to sterilize objects, which cannot withstand 100°C: serum, ascitic fluid, vitamins . Tyndallization is carried out in a water bath at 56°C for 1 hour for 5-6 days.

Pasteurization - partial sterilization (spores are not killed), which is carried out at a relatively low temperature once. Pasteurization is carried out at 70-80°C, 5-10 minutes or at 50-60°C, 15-30 minutes. Pasteurization is used for objects that lose their quality at high temperatures. Pasteurization, for example, use For some food products: milk, wine, beer . This does not damage their commercial value, but the spores remain viable, so these products must be stored refrigerated.

In laboratory practice, when working with microorganisms, it is necessary to constantly take measures to ensure that dishes, culture media, and metal instruments used during work do not contain microbes. For this purpose, the following sterilization methods are used:

Sterilization superheated steam under pressure;

Sterilization with flowing steam;

Hot air sterilization;

Disinfection.

The best way to sterilize is to treat various objects with superheated steam in special devices - autoclaves.

Autoclave and superheated steam sterilization under pressure

Autoclaves are metal (steel, cast iron or copper) boilers with double walls and a massive lid that is hermetically sealed with bolts and a rubber gasket. Depending on the heating system, autoclaves are steam, electric and fire (Fig. 42). Producing high pressure and superheated steam at a temperature of 115-120°C in an autoclave allows you to destroy both vegetative cells and microbial spores within 20-30 minutes.

In an autoclave, all those items that do not deteriorate at high temperatures are sterilized: various liquids (water, nutrient media that do not contain carbohydrate components), glassware, metal instruments, cotton wool, gauze, paper, etc.

In cases where some substances do not withstand the normal sterilization regime in an autoclave (in particular, nutrient media containing sugars), they are sterilized in an autoclave at a temperature of 112 ° C for 20 minutes, as well as under milder conditions.

At high pressures of superheated steam in the autoclave, the heating temperature increases accordingly; This causes caramelization of sugars added to some culture media, making the media unsuitable for determining the physiological properties of microbes.

The pressure inside the autoclave is measured using a pressure gauge mounted in the lid or body of the autoclave. If the pressure increases excessively, a safety valve is automatically activated, located, depending on the design, either on the lid or on the side surface of the autoclave wall. A jet of steam coming out of the safety valve with a whistle warns of the need to stop heating. If heating is not stopped, the autoclave may explode.

A thermometer is sometimes placed in a special pocket on the lid of the autoclave, with which the sterilization temperature is measured. There is a stand inside the autoclave, under which water is poured through a tube with a funnel located outside the autoclave. In addition, autoclaves are equipped with a tap for releasing steam and air and a tap for pouring out water.

The rules for handling an autoclave during sterilization are as follows: the device is filled with water through a funnel and tube, the level of which should be below the stand. Items to be sterilized are placed in special metal bottles and loaded into an autoclave. The lid of the autoclave is screwed on.

By opening the tap to allow steam and air to escape, heating begins. As soon as the water boils, the resulting steam begins to displace air from the autoclave. The steam outlet valve is kept open until dry steam flows out of it in a continuous stream. This indicates complete removal of air from the autoclave. Then the tap is closed. Steam, which accumulates with further heating in greater and greater quantities, increases the pressure in the autoclave, and at the same time the temperature. When working with an autoclave, you can be guided by the table of relationships between steam pressure and its temperature (Table 4).

After the pressure gauge needle reaches the required indicator pressure (the temperature in the autoclave will correspond to the accepted sterilization temperature), adjust the heating of the autoclave so that the pressure remains at the same level for the required time. At the end of sterilization, stop heating. When the temperature in the autoclave drops and the pressure gauge needle drops to zero (the pressure in the autoclave equals atmospheric pressure), carefully open the steam release valve, release the steam and, opening the lid of the autoclave, remove the material. It is impossible to open the steam release valve prematurely, before the pressure in the autoclave drops. A sharp decrease in pressure in the autoclave chamber will cause violent boiling of liquids heated to higher temperatures than 100 °C, i.e. above the boiling point at normal atmospheric pressure. Violently boiling liquids will wet or even push cotton plugs out of the vessels - the work will be done in vain. Nutrient media will deteriorate, since microflora coming from the air easily develops on wet plugs, penetrates inside and infects the media. In addition, opening an autoclave with increased pressure is dangerous for the worker.

However, as soon as the pressure gauge needle stops at zero, the autoclave must be opened immediately, otherwise condensation will begin to flow onto the plugs and also cause them to become wet. To avoid wetting the plugs with condensation water, they are covered with paper before sterilization.

Materials placed in an autoclave will be reliably sterilized in 20-30 minutes if the temperature is maintained at 120°C, which corresponds to a pressure of 2 atm (19.61 * 10000 N/m2) or on a pressure gauge 1 atm above normal. An autoclave can also be successfully used to sterilize media with flowing steam; in this case, the lid of the autoclave is not screwed on.

Koch boiler and flowing steam sterilization

The Koch boiler is a cylinder made of galvanized sheet or copper with double walls and a conical helmet-shaped lid (Fig. 43). There is a hole in the middle of the lid for a thermometer. The outside of the Koch boiler is covered with a layer of heat-insulating material: asbestos, linoleum, etc.

A partition (stand) is placed inside the boiler, dividing the internal space of the boiler into two sections: upper and lower. The lower section is filled with water, the level of which is determined by the water meter glass: water should not cover the upper part of the stand. The items to be sterilized are placed in the upper section of the boiler in lattice buckets placed one on top of the other. Having closed the boiler with a lid, they begin to heat the water in it. The beginning of sterilization is considered the moment when the thermometer shows 98-100°C. In the absence of a thermometer, the beginning of sterilization is considered to be the moment when steam begins to vigorously escape from the hole in the lid of the boiler. Thus, the objects to be sterilized will always be in a stream of flowing steam while the boiler is operating.

The method of sterilization with flowing steam, due to its simplicity and availability, is widely used in laboratory practice. Flowing steam is used to sterilize mainly nutrient media whose properties change when heated above 100°C: protein, carbohydrate and gelatin. For these environments, the flowing steam sterilization method is most suitable.

The disadvantage of the flowing steam sterilization method is its duration, since to completely sterilize the medium it is necessary to carry out repeated heating in a boiler for a certain time - from 20 minutes to 1.5 hours (on average 30-45 minutes) depending on the volume of liquid at intervals of 24 hours. It is recommended to keep the entire period of time between heating the medium in a thermostat at 25-30 °C.

A single heating in a Koch boiler causes the death of only vegetative microbial cells, but spores can survive. When the sterilized nutrient medium is kept in favorable conditions (at room temperature, or even better in a thermostat), some of the remaining spores will germinate and turn into vegetative cells by the next day. Repeated heating will cause the death of these newly developed cells. Finally, a third heating after a day of keeping the medium in a thermostat will ensure complete sterility. This method is called fractional sterilization. IN practical work Instead of sterilization with flowing steam in a Koch boiler, conventional sterilization in autoclaves is often used at 112 °C with a back pressure of 0.5 atm for 15-20 minutes.

Drying cabinet and hot air sterilization

In laboratory practice, to sterilize microbiological glassware, it is necessary to have a drying cabinet or the so-called Pasteur oven. The design principle of the drying cabinet and the Pasteur oven are the same. Stoves are made only rectangular shape, and drying cabinets can have not only a rectangular, but also a cylindrical shape (Fig. 44 and 45). In these devices, sterilization is carried out with hot air (dry heat) at a temperature of 160 °C for 1 hour or at a temperature of 150 °C for 2 hours.

Both Pasteur ovens and drying ovens are hollow inside devices with double walls, with double doors that close tightly. On the outside, they are covered with a layer of asbestos for thermal insulation. Hot air circulates between the walls, which is heated either by electric coils or gas burners. Inside the cabinet there are several (usually two or three) holey shelves. There are two openings at the top of the cabinet: one is for the thermometer, and the other is for ventilation. Electric drying cabinets are the most convenient.

The latest design drying cabinets have four heating stages, which can be activated by a special regulator placed on the side wall of the cabinet. The desired degree of heating is achieved by turning on one, two, three or all four electric spirals, and the sequence of turning on the spirals can be any.

In addition to glassware, you can sterilize gauze and cotton wool in drying cabinets, although it is still better to process them in an autoclave, since they turn yellow at a temperature of 160 °C. Rubber products cannot be sterilized in a drying cabinet, as they cannot withstand high temperatures - they become brittle and deteriorate. Liquids boil at 150-160 °C and change their chemical composition.

To avoid subsequent contamination of sterilized items by microbes from the air, they are wrapped in paper before sterilization. Petri dishes are wrapped in paper, 2 pieces each, so that there are no gaps in the wrapping. Glass tubes and pipettes are also wrapped in paper, first each separately, and then in packs of 10-20 pieces. The wrapping of tubes and pipettes must be extremely careful to completely protect them outer surface from communication with air. Before sterilization with hot air, flasks, test tubes, bottles are closed with cotton stoppers and paper caps.

Do not allow the temperature in the drying cabinet to rise above 170 °C, since at this temperature the cotton plugs turn brown, and the paper wrappers become brittle and even charred. The beginning of sterilization is considered the moment when the thermometer shows 150-160 °C. After the required time for sterilization has passed, heating is stopped. To protect dishes from cracking, you only need to sterilize dry dishes and open the cabinet after sterilization only when the temperature in it drops to 50-70 ° C. Small laboratory items, such as platinum loops, needles, tweezers, scissors, etc., can be sterilized by simply calcining them on the flame of a gas burner (or alcohol lamp).

Disinfection

Disinfection in a microbiological laboratory has to be resorted to very often. The most commonly used disinfectants are the following: 3-5% solution of carbolic acid and solutions of other higher phenols, 50-70% solution of ethyl alcohol, butyl alcohol of the same concentration, 4% solution of formalin, 1-2% solutions of chloroform and toluene, 0.5% solution of chloramine, etc.

In microbiological laboratories of canning factories, table surfaces, dishes, floors, and walls of premises are disinfected. To disinfect table surfaces, you can use not only ethyl alcohol solutions, but also carbolic acid solutions.

Disinfection of sewage systems in canneries and other food enterprises is carried out with a 5-10% solution of bleach. To disinfect recyclable glass containers, canneries use chlorine water containing at least 100 mg of active chlorine per liter. To prepare such a solution take bleach, mix it with a small amount of water until a thick, milky mass is obtained. This mixture is added to water, stirred thoroughly and left for a day. Bleached lime reacts to form calcium oxide hydrate - Ca(OH)2 - and active chlorine. Ca(OH)2 settles to the bottom, the solution above the sediment after clarification turns out to be transparent and greenish in color. Soaking the container in this solution lasts 10 minutes. After chlorination, the container must be thoroughly washed in running water.

The life of microorganisms is closely dependent on environmental conditions. All environmental factors that influence microorganisms can be divided into three groups: physical, chemical and biological, the beneficial or detrimental effect of which depends both on the nature of the factor itself and on the properties of the microorganism.

Physical factors

From physical factors greatest influence The development of microorganisms is influenced by temperature, drying, radiant energy, and ultrasound.

Temperature. The life activity of each microorganism is limited by certain temperature limits. This temperature dependence is usually expressed by three main points: minimum - the temperature below which the reproduction of microbial cells stops; optimum - the best temperature for the growth and development of microorganisms; maximum - the temperature above which the vital activity of cells weakens or stops. Optimal temperature usually corresponds to the temperature conditions of the natural habitat.

All microorganisms in relation to temperature are divided into psychrophiles, mesophiles and thermophiles.

Psychrophiles (from the Greek psychros - cold, phileo - love), or cold-loving microorganisms, grow at relatively low temperatures: minimum temperature- 0° C, optimal - 10-20° C, maximum - 30° C. This group includes microorganisms living in northern seas and oceans, soil, wastewater. This also includes luminous and iron bacteria, as well as microbes, causing damage products in the cold (below 0° C).

Mesophiles (from the Greek mesos - middle) are the most extensive group, including most saprophytes and all pathogenic microorganisms. The optimal temperature for them is 28-37° C, the minimum is 10° C, the maximum is 45° C.

Thermophiles (from the Greek termos - heat, heat), or heat-loving microorganisms, develop at temperatures above 55 ° C, the temperature minimum for them is 30 ° C, the optimum is 50-60 ° C, and the maximum is 70-75 ° C. They meet in hot mineral springs, surface layer of soil, self-heating substrates (manure, hay, grain), intestines of humans and animals. Among thermophiles there are many spore forms.

High and low temperatures have different effects on microorganisms. Some are more sensitive to high temperatures. Moreover, the higher the temperature beyond the maximum, the faster the death of microbial cells occurs, which is due to the denaturation (coagulation) of cell proteins.

Vegetative forms of mesophilic bacteria die at a temperature of 60° C for 30-60 minutes, and at 80-100° C - after 1-2 minutes. Bacterial spores are much more resistant to high temperatures. For example, spores of anthrax bacilli can withstand boiling for 10-20 minutes, and spores of clostridium botulism - 6 hours. All microorganisms, including spores, die at a temperature of 165-170 ° C for an hour (in a dry-heat oven) or when exposed to steam under pressure 1 atm (in an autoclave) for 30 minutes.

The effect of high temperatures on microorganisms is the basis of sterilization - the complete release of various objects from microorganisms and their spores (see below).

Many microorganisms are extremely resistant to low temperatures. Salmonella typhus and Vibrio cholerae survive for a long time in ice. Some microorganisms remain viable at liquid air temperatures (-190°C), and bacterial spores can withstand temperatures down to -250°C.

Only certain types of pathogenic bacteria are sensitive to low temperatures (for example, Bordetella pertussis and parapertussis, Neisseria meningococcus, etc.). These properties of microorganisms are taken into account in laboratory diagnostics and when transporting the material under study, it is delivered to the laboratory protected from refrigeration.

The action of low temperatures stops putrefactive and fermentation processes, which is widely used to preserve food in refrigeration units, cellars, and glaciers. At temperatures below 0° C, microbes fall into a state of suspended animation - metabolic processes slow down and reproduction stops. However, in the presence of appropriate temperature conditions and a nutrient medium, the vital functions of microbial cells are restored. This property of microorganisms is used in laboratory practice to preserve microbial cultures at low temperatures. Rapid changes in high and low temperatures (freezing and thawing) also have a detrimental effect on microorganisms - this leads to rupture of cell membranes.

Drying. Water is necessary for the normal functioning of microorganisms. Drying leads to dehydration of the cytoplasm, disruption of the integrity of the cytoplasmic membrane, as a result of which the nutrition of microbial cells is disrupted and their death occurs.

The timing of the death of different types of microorganisms under the influence of drying differs significantly. For example, pathogenic Neisseria (meningococci, gonococci), Leptospira, Treponema pallidum and others die when dried after a few minutes. Vibrio cholerae can withstand drying for 2 days, Salmonella typhoid - 70 days, and Mycobacterium tuberculosis - 90 days. But the dried sputum of tuberculosis patients, in which the pathogens are protected by a dry protein cover, remains infectious for 10 months.

Spores are particularly resistant to drying, as well as to other environmental influences. Spores of anthrax bacilli retain the ability to germinate for 10 years, and spores of mold fungi for up to 20 years.

The unfavorable effect of drying on microorganisms has long been used for preserving vegetables, fruits, meat, fish and medicinal herbs. At the same time, having found ourselves in conditions high humidity, such products quickly deteriorate due to the restoration of microbial activity.

The freeze-drying method is widely used for storing cultures of microorganisms, vaccines and other biological preparations. The essence of the method is that microorganisms or preparations are first frozen and then dried under vacuum conditions. In this case, microbial cells enter a state of suspended animation and retain their biological properties for several months or years.

Radiant Energy. In nature, microorganisms are constantly exposed to solar radiation. Direct sunlight causes the death of many microorganisms within a few hours, with the exception of photosynthetic bacteria (green and purple sulfur bacteria). The harmful effects of sunlight are caused by the activity of ultraviolet rays (UV rays). They inactivate cell enzymes and damage DNA. Pathogenic bacteria are more sensitive to the action of UV rays than saprophytes. Therefore, it is better to store microbial cultures in the laboratory in the dark. In this regard, Buchner's experience is demonstrative.

A abundant culture of bacteria is inoculated into a Petri dish with a thin layer of agar. Letters cut out of black paper are glued onto the outer surface of the seeded cup, forming, for example, the word “typhus”. The cup, with its bottom facing up, is irradiated by direct sunlight for 1 hour. Then the papers are removed, and the cup is placed in a thermostat at 37° C for a day. Bacterial growth is observed only in those places of the agar that were protected from UV rays by stickers letters. The rest of the agar remains transparent, i.e. there is no growth of microorganisms (Fig. 11).

The importance of sunlight as a natural factor in improving the health of the external environment is great. It frees the air, water of natural reservoirs, and upper layers of soil from pathogenic bacteria.

The bactericidal (bacteria-destroying) effect of UV rays is used to sterilize the air closed premises(operating rooms, dressing rooms, boxes, etc.), as well as water and milk. The source of these rays are ultraviolet radiation lamps and bactericidal lamps.

Other types of radiant energy - X-rays, α-, β-, γ-rays have a detrimental effect on microorganisms only in large doses, on the order of 440-280 J/kg. The death of microbes is caused by the destruction of nuclear structures and cellular DNA. Low doses of radiation stimulate the growth of microbial cells. Microorganisms are much more resistant to radioactive radiation than higher organisms. Thionic bacteria are known to live in uranium ore deposits. Bacteria were found in water nuclear reactors at a concentration of ionizing radiation of 20-30 kJ/kg.

Bactericidal effect ionizing radiation used for preserving some food products, sterilizing biological preparations (serums, vaccines, etc.), while the properties of the sterilized material do not change.

IN last years The radiation method is used to sterilize disposable products - polystyrene pipettes, Petri dishes, wells for serological reactions, syringes, as well as suture material - catgut, etc.

Ultrasound causes significant damage to microbial cells. Under the influence of ultrasound, gases located in the liquid environment of the cytoplasm are activated, and high pressure arises inside the cell (up to 10,000 atm). This leads to rupture of the cell membrane and cell death. Ultrasound is used to sterilize food products (milk, fruit juices) and drinking water.

High pressure. Bacteria and especially their spores are resistant to mechanical pressure. In nature, bacteria are found that live in seas and oceans at a depth of 1000-10000 m under pressure from 100 to 900 atm. Some types of bacteria can withstand pressures of up to 3000-5000 atm, and bacterial spores - even 20,000 atm.

Chemical factors

The effect of chemicals on microorganisms varies depending on the nature of the chemical compound, its concentration, and the duration of exposure to microbial cells. Depending on the concentration, a chemical substance can be a source of nutrition or have an inhibitory effect on the vital activity of microorganisms. For example, a 0.5-2% glucose solution stimulates the growth of microbes, and 20-40% glucose solutions inhibit the proliferation of microbial cells.

Many chemical compounds, which have a detrimental effect on microorganisms, are used in medical practice as disinfectants and antiseptics.

Chemicals used for disinfection are called disinfectants. Disinfection refers to measures aimed at destroying pathogenic microorganisms in various environmental objects. Disinfectants include halide compounds, phenols and their derivatives, salts of heavy metals, some acids, alkalis, alcohols, etc. They cause the death of microbial cells, acting in optimal concentrations for a certain time. Many disinfectants have a harmful effect on the tissues of the macroorganism.

Antiseptics are chemicals that can cause the death of microorganisms or inhibit their growth and reproduction. They are used for therapeutic purposes (chemotherapy), as well as for the disinfection of wounds, skin, and human mucous membranes. Hydrogen peroxide, alcohol solutions of iodine, brilliant green, solutions of potassium permanganate, etc. have antiseptic properties. Some antiseptic substances (acetic, sulfurous, benzoic acid, etc.) in doses that are harmless to humans are used for food preservation.

According to the mechanism of action, chemical substances with antimicrobial activity can be divided into several groups.

1. Surfactants (fatty acids, soaps and other detergents) cause a decrease in surface tension, which leads to disruption of the functioning of the cell wall and cytoplasmic membrane of microorganisms.

2. Phenol, cresol and their derivatives cause coagulation of microbial proteins. They are used to disinfect infectious material in microbiological practice and infectious diseases hospitals.

3. Oxidizing agents, interacting with microbial proteins, disrupt the activity of enzymes and cause protein denaturation. Active oxidizing agents are chlorine and ozone, which are used to disinfect drinking water. Chlorine derivatives (bleach, chloramine) are widely used for disinfection purposes. Hydrogen peroxide, potassium permanganate, iodine, etc. have oxidizing properties.

4. Formaldehyde is used in the form of a 40% solution (formalin) for disinfection. It kills vegetative and spore forms of microorganisms. Formalin blocks the amino groups of microbial cell proteins and causes their denaturation.

5. Salts of heavy metals (mercury, lead, zinc, gold, etc.) coagulate the proteins of the microbial cell, thereby causing their death. A number of metals (silver, gold, mercury, etc.) have a bactericidal effect on microorganisms in negligible concentrations. This property is called oligodynamic action (from the Latin oligos - small, dinamys - strength). It has been proven that water in silver vessels does not rot due to the bactericidal effect of silver ions. To prevent blenorrhea * in newborns, a 1% solution of silver nitrate was used for a long time. Colloidal solutions of organic silver compounds (protargol, collargol) are also used as local antiseptics.

* (Blennorea is an inflammation of the conjunctiva of the eye caused by gonococci.)

Mercury preparations have a strong antimicrobial effect. Since ancient times, mercury bichloride, or mercuric chloride (at a dilution of 1:1000), has been used for disinfection. However, she has toxic effect on the tissue of the macroorganism and its use is limited.

6. Dyes (diamond green, rivanol, etc.) have the property of inhibiting the growth of bacteria. Solutions of a number of dyes are used as antiseptics, and are also added to some nutrient media to inhibit the growth of accompanying microflora.

The destructive effect of a number of physical and chemical factors on microorganisms forms the basis of aseptic and antiseptic methods, widely used in medical and sanitary practice.

Asepsis is a system of preventive measures that prevent microbial contamination of an object (wounds, surgical field, cultures of microorganisms, etc.) based on physical methods.

Antiseptics is a set of measures aimed at destroying microorganisms in a wound, the whole body or on environmental objects, using various disinfecting chemicals.

Biological factors

In natural habitats, microorganisms do not exist in isolation, but are in complex relationships, which come down mainly to symbiosis, metabiosis and antagonism.

Symbiosis is the cohabitation of organisms of different species, bringing them mutual benefit. At the same time, together they develop better than each of them separately.

Symbiotic relationships exist between nodule bacteria and leguminous plants, between filamentous fungi and blue-green algae (lichens): The symbiosis of lactic acid bacteria and alcoholic yeast is used to prepare some lactic acid products (kefir, koumiss).

Metabiosis is a type of relationship in which metabolic products of one type of microorganisms create the necessary conditions for the development of others. For example, putrefactive microorganisms that break down protein substances contribute to the accumulation of ammonium compounds in the environment and create favorable conditions for the growth and development of nitrifying bacteria. And the development of anaerobes in well-aerated soil would be impossible without aerobes that absorb free oxygen.

Metabiotic relationships are widespread among soil microorganisms and underlie the cycle of substances in nature.

Antagonism is a form of relationship in which one microorganism inhibits the development of another or can cause its complete death. Antagonistic relationships have developed among microorganisms in the struggle for existence. Everywhere they live, there is a constant struggle between them for food sources, air oxygen, and habitat. Thus, most pathogenic bacteria, having entered the external environment (soil, water) with the secretions of patients, cannot withstand long-term competition with numerous saprophytes and die relatively quickly.

Antagonism can be caused by the direct influence of microorganisms on each other or by the action of their metabolic products. For example, protozoa devour bacteria, and phages lyse them. The intestines of newborns are colonized by lactic acid bacteria Bifidobacterium bifidum. By releasing lactic acid, they suppress the growth of putrefactive bacteria and thereby protect the still fragile organism of infants from intestinal disorders. Some microorganisms in the process of life produce various substances that have a detrimental effect on bacteria and other microbes. These substances include antibiotics (see "Antibiotics").

Control questions

1. What physical factors influence the life activity of microorganisms?

2. What substances are classified as disinfectants and how do they differ in their mechanism of action on microorganisms?

3. List what relationships exist between microorganisms?

Sterilization

Sterilization is sterilization, i.e. complete liberation of environmental objects from microorganisms and their spores.

Sterilization is carried out in various ways:

1) physical (exposure to 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.

Dry heat sterilization

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 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, the cold air entering the cabinet may cause cracks in the hot cookware.

Sterilization in a Pasteur oven can be carried out at different temperatures and exposures (sterilization time) (Table 1).

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. With proper operation of the Pasteur oven, there will be no growth in test tubes or dishes with nutrient media in which sterilized threads were placed, since bacterial spores will die, while bacterial spores on threads that were not sterilized (control) will germinate on nutrient media growth will be noted.

To determine the temperature inside the Pasteur oven, you can use sucrose or granulated sugar, which caramelizes at a temperature of 165-170 ° C.

Preparation laboratory glassware to 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.

Control questions

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 by boiling

Boiling is a sterilization method that guarantees sterility provided there are no spores in the sterilized material. Used for processing syringes, instruments, glass and metal utensils, rubber tubes, etc.

Sterilization by boiling is usually carried out in a sterilizer - a rectangular metal box with a tight-fitting lid. The material to be sterilized is placed on the mesh available 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 heated. The beginning of sterilization is considered to be the moment of boiling of water, the boiling time is 15-30 minutes. At the end of sterilization, the mesh with instruments is removed by the side handles with special hooks, and the instruments in it are taken with sterile tweezers or forceps, which are boiled along with the rest of the instruments.

Steam sterilization is carried out in two ways: 1) steam under pressure; 2) flowing steam.

Pressure steam sterilization produced 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. Another, smaller diameter, which is called a sterilization chamber, is inserted into the outer boiler. Objects to be sterilized are placed in this chamber. Between both boilers there is a free space called the water-steam chamber. Water is poured into this chamber through a funnel fixed on the outside to a certain level marked on a special water-measuring tube. When water is boiled in a water-steam chamber, steam is produced. The sterilization chamber is equipped with an outlet cock with a safety valve to allow steam to escape when the pressure increases above the required level. A pressure gauge is used to determine the pressure created in the sterilization chamber.

Rice. 12. Autoclave diagram. M - pressure gauge; PC - safety valve; B - funnel for water; K 2 - tap for water release; K 3 - valve for steam release

Normal Atmosphere pressure(760 mmHg) is taken as zero. There is a certain relationship between the pressure gauge readings and temperature (Table 2).

Currently, there are autoclaves with automatic control of the operating mode. In addition to the usual pressure gauge, they are equipped with an electric contact pressure gauge, which prevents the pressure from increasing above a predetermined value and thereby ensures the constancy of the desired temperature in the autoclave.

Steam under pressure sterilizes various nutrient media (except those containing native proteins), liquids (isotonic sodium chloride solution, water, etc.); devices, especially those with rubber parts.

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).

Attention! Infected material is also neutralized in autoclaves. 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 , forming disputes.

Only specially trained persons are allowed to work with the autoclave, who must strictly and accurately follow the rules specified in the instructions supplied with the device.

Autoclaving technique. 1. Before work, check the serviceability of all parts and the grinding of the taps.

2. Water (distilled or boiled to prevent scale formation) is poured through a funnel mounted outside the boiler to the top mark of the water meter glass. The tap under the funnel is closed.

3. The material to be sterilized is placed in the sterilization chamber on a special mesh. Items should not be loaded too tightly, as steam must pass freely between them, otherwise they will not heat up to the required temperature and may remain unsterile.

4. The rubber gasket on the lid is rubbed with chalk for better sealing.

5. The lid is closed and bolted to the autoclave body, and the bolts are screwed in pairs crosswise.

6. Open the outlet valve connecting the sterilization chamber with the outside air all the way, and begin to heat the autoclave. The autoclave is usually heated using 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 internal boiler (see Fig. 12), enters the sterilization chamber and exits through the open outlet valve. First, the steam escapes along with the air in the autoclave. It is necessary that all air is forced out of the autoclave, otherwise the pressure gauge reading will not correspond to the temperature in the autoclave.

The appearance of a continuous strong stream of steam indicates complete removal of air from the autoclave; After this, the outlet valve is closed and the pressure inside the autoclave begins to gradually increase.

7. The beginning of sterilization is considered the moment when the pressure gauge readings reach the specified value. Heating is adjusted so that the pressure in the autoclave does not change over a certain period of time.

8. After the sterilization time has expired, the heating of the autoclave is stopped, and the steam is released through the outlet valve. When the pressure gauge needle drops to zero, open the lid. To avoid burns from steam remaining in the autoclave, the lid should be opened towards you.

The temperature level in the autoclave, i.e. the correctness of the pressure gauge readings, can be checked. To do this, use various substances that have a certain melting point: antipyrine (113° C), resorcinol and sulfur (119° C), benzoic acid (120° C). One of these substances is mixed with a negligible amount of dye (muchsine or methylene blue) and poured into a glass tube, which is sealed and placed in vertical position between the material being sterilized. If the temperature is sufficient, the substance will melt and turn 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. Lack of growth indicates proper operation of the device.

Sterilization with flowing steam produced in the 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 indicator proper operation The device ensures the sterility of the prepared culture media. You can also sterilize with flowing steam in an autoclave with the lid unscrewed and the outlet valve open.

Control questions

1. What nutrient media are steam sterilized?

2. What is a sterilizer and how does it work?

3. Why should distilled water be used when sterilizing by boiling?

4. Describe the structure and operating mode of the autoclave.

5. What is sterilized in an autoclave?

6. What serves as a control for proper sterilization during autoclaving?

7. What is flowing steam sterilization?

8. Describe the structure of the Koch apparatus.

9. What is the purpose of fractional sterilization?

Exercise

Fill the form.

Fractional sterilization can also be carried out in a Koch coagulant.

Koch's coagulant is used to coagulate whey and egg culture media, and simultaneously with the compaction of the medium, it is sterilized.

Koch's coagulant is a flat, double-walled metal box covered on the outside thermal insulation material. Water is poured into the space between the walls through a special hole located in the upper part of the outer wall. The hole is closed with a stopper into which a thermometer is inserted. The device is closed with two lids: glass and metal. Through the glass lid you can observe the coagulation process. Test tubes with media are placed on the bottom of the coagulator in an inclined position.

The coagulator is heated 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 sterilization method is named after Tyndall, who proposed it.)

Pasteurization- sterilization at 65-70 ° C for 1 hour, proposed by Pasteur to destroy non-spore forms of microbes. Milk, wine, beer, fruit juices and other products are pasteurized. Milk is pasteurized to remove lactic acid and pathogenic bacteria (brucella, mycobacterium tuberculosis, shigella, salmonella, staphylococcus, etc.). When pasteurizing beer, fruit juices, and wine, microorganisms that cause various types of fermentation die. Pasteurized foods are best kept refrigerated.

Control questions

1. What is the purpose and structure of the Koch coagulator?

2. What are the methods of sterilization in a clotting machine?

3. What is tyndalization?

4. What is pasteurization?

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.

Control questions

1. What properties do ultraviolet rays have?

2. In what cases is sterilization using ultraviolet radiation used?

Mechanical sterilization using bacterial filters

Filtration sterilization is used in cases where the objects being sterilized change when heated. Filtration is carried out using bacterial filters made from various fine-porous materials. The pores of filters must be small enough (up to 1 micron) to ensure mechanical retention of bacteria, therefore some authors refer to filtration as mechanical methods sterilization.

The filtration method is used to sterilize nutrient media containing protein, serum, and some antibiotics, and also to separate bacteria from viruses, phages and exotoxins.

In microbiological practice, Seitz asbestos filters, membrane filters and Chamberlant and Berkefeld filters (candles) are used.

Seitz filters are discs made from a mixture of asbestos and cellulose. Their thickness is 3-5 mm, diameter 35-140 mm. The domestic industry produces filters of two brands: “F” (filtering) - retaining suspended particles but allowing bacteria to pass through; "SF" (sterilizing) - with smaller pores, retaining bacteria, but allowing viruses through. Crumpled asbestos plates, as well as plates with breaks and cracks, are unsuitable for work.

Membrane filters are made from nitrocellulose. They are white discs with a thickness of 0.1 mm and a diameter of 35 mm. Depending on the pore size, they are designated No. 1, 2, 3, 4 and 5 (Table 3).

Filter No. 1 is most suitable for sterilization. In addition to those listed, they also produce a so-called pre-filter, designed to free the filtered liquid from large particles contained in it.

Chamberlant and Berkefeld filters (candles) are hollow cylinders, closed at one end. Chamberlant candles are made from kaolin mixed with sand and quartz. They are standardized by pore size and designated L 1, L 2, L 3 ... L 13. Berkefeld filters (candles) are prepared from infusor soil; according to the size of their pores they are designated V, N, W, which corresponds to a pore diameter of 3-4, 4-7, 8-12 microns.

Work with bacterial filters is carried out as follows. The filter must be secured in a special holder, which is inserted into the filter receiver. The receiver is usually a Bunsen flask. The holders, in most cases made of stainless steel, consist of two parts: the upper, shaped like a cylinder without a bottom, and the lower, a supporting part ending in a tube. Seitz filters with the rough surface up are placed on a metal mesh and tightly clamped with screws between the top and bottom holder. The mounted filter is secured in a rubber stopper inserted into the neck of a Bunsen flask. A cotton swab is inserted into the outlet tube of the flask, which is connected to the vacuum pump. The prepared installation is wrapped in paper and sterilized in an autoclave under a pressure of 1 atm for 20-30 minutes. The entire assembled device is also called a Seitz filter (Fig. 13).

Immediately before filtration, the outlet end of the Bunsen flask is connected by a rubber tube to an oil or water jet pump. The junctions of the various parts are filled with paraffin to create a tight seal. The filtered liquid is poured into the cylinder of the apparatus and the pump is turned on, creating a vacuum in the receiver. As a result of the resulting pressure difference, the filtered liquid passes through the pores of the filter into the receiver, and the microbes remain on the surface of the filter.

Before use, membrane filters are sterilized by boiling in distilled water. To prevent filters from curling, they are first placed in distilled water, heated to a temperature of 50-60 ° C, and boiled over low heat for 30 minutes, changing the water 2-3 times. The filter holder and receiver are sterilized in advance, and the device is mounted under aseptic conditions. To avoid tearing the membrane filter on the metal mesh, place mugs of sterile filter paper under it. Then, using sterile tweezers with smooth tips, take the membrane filter from the sterilizer and place it on the support grid with the shiny surface down.

Candles (Chamberlant) sterilized in an autoclave are connected through a rubber tube to a receiver and lowered into a vessel (usually a cylinder) with a filtered liquid. Filtration occurs using a vacuum pump. A sterile filtrate enters the receiver, and bacteria are retained by the pores of the candle.

Membrane and asbestos filters are designed for single use. After use, candles are boiled in tap water, then calcined in a muffle furnace.

Before subsequent use, candles are checked for integrity. The candle is lowered into a vessel with water and air is passed through. If air bubbles appear on the surface of the candle, it means that cracks have formed in the candle and it is unusable.

Control questions

1. What is the filter sterilization method? What is sterilized using this method?

2. What bacterial filters do you know? How is the filtering device installed, what conditions must be observed?

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.

Control questions

1. What is chemical sterilization and when is it used?

2. What is biological sterilization?

The main methods of sterilization are presented in table. 4.

1 (Sterilization is incomplete: spores remain in the sterilized material.)

2 (Sterilization is incomplete: viruses remain in the sterilized material.)

Disinfection

In microbiological practice, various disinfectants are used: 3-5% phenol solutions, 5-10% Lysol solutions, 1-5% chloramine solutions, 3-6% hydrogen peroxide solutions, 1-5% formaldehyde solutions, mercuric chloride solutions in dilution 1: 1000 (0.1%), 70° alcohol, etc.

Spent pathological material (pus, feces, urine, sputum, blood, cerebrospinal fluid) is disinfected before draining it into the sewer. Disinfection is carried out with dry bleach or 3-5% chloramine solution.

Pipettes (graduated and Pasteur), glass spatulas, slides and coverslips contaminated with pathological material or cultures of microorganisms are immersed in glass jars with a 3% solution of phenol or hydrogen peroxide for a day.

After finishing work with infectious material, the laboratory technician must treat it with a disinfectant solution. workplace and hands. The surface of the work table is wiped with a piece of cotton wool moistened with a 3% phenol solution. Hands are disinfected with a 1% chloramine solution. To do this, moisten a cotton ball or gauze with a disinfectant solution and wipe the left hand, then the right, and then wash your hands with warm water and soap.

The choice of disinfectant, its concentration and duration of exposure (exposure) depend on biological properties microbe and the environment in which the disinfectant will come into contact with pathogenic microorganisms. For example, mercuric chloride, phenol, and alcohols are unsuitable for disinfecting protein substrates (pus, blood, sputum), since under their influence protein coagulation occurs, and the coagulated protein protects microorganisms from the effects of disinfectants.

When disinfecting material infected with spore forms of microorganisms, a 5% solution of chloramine, 1-2.5% solutions of activated chloramine, 5-10% solutions of formalin and other substances are used.

Disinfection, which is carried out throughout the day during work, is called current, and at the end of work - final.

Disinfectants and instructions for preparing working solutions from them. Chloride of lime is a white, lumpy powder with a pungent odor of chlorine; it does not completely dissolve in water. The bactericidal effect depends on the content of active chlorine, the amount of which ranges from 28 to 36%. Chlorine containing less than 25% active chlorine is unsuitable for disinfection.

If stored improperly, bleach decomposes and loses some of its active chlorine. Decomposition is promoted by heat, moisture, sunlight, therefore, bleach should be stored in a dry, dark place, in a tightly closed container.

Dry bleach is used to disinfect human and animal secretions (at the rate of 200 g per 1 liter of feces and 10 g per 1 liter of urine).

Preparation of the original 10% clarified bleach solution. Take 1 kg of dry bleach, place it in an enamel bucket and grind it. Then pour cold water to a volume of 10 liters, mix well, cover with a lid and leave for a day in a cool place. After this, the resulting 10% clarified solution is carefully drained and filtered through several layers of gauze or filtered through a thick cloth. Store in dark glass bottles, closed with a wooden stopper, in a cool place for no more than 10 days. Working solutions of the required concentration are prepared from the stock solution immediately before use. The amount of basic solution required for preparing 0.2-10% clarified bleach solutions is given in table. 5.

The concentration of clarified bleach solutions from 0.2 to 10% is selected depending on the nature of the object being disinfected and the resistance of the pathogen.

Chloramine - crystalline substance white or yellowish in color, contains 24-28% active chlorine. It dissolves well in water at room temperature, so solutions are prepared immediately before disinfection. Use 0.2-10% chloramine solutions. The relationship between the percentage concentration of the solution and the amount of chloramine in grams per 1 and 10 liters is given in table. 6.

Dissolve chloramine in glass or enamel dishes. When storing chloramine solutions in dark glass containers with a ground-in stopper, their activity persists for up to 15 days.

Activated chloramine. The disinfecting properties of chloramine are enhanced by adding an activator to it in a ratio of 1:1 or 1:2. Ammonium compounds are used as an activator - ammonium chloride, sulfate, ammonium nitrate. Activated chloramine is used in concentrations of 0.5, 1 and 2.5%. They are prepared immediately before use. Chloramine and ammonium salt are weighed separately. First, chloramine is dissolved in water, and then an activator is added.

The advantage of activated chloramine solutions over conventional ones is that the addition of an activator accelerates the release of active chlorine. Therefore, the drug has a detrimental effect not only on vegetative forms of microorganisms, but also on their spores. Activated chloramine is used in lower concentrations and with less exposure.

Phenol (carbolic acid) is a colorless, needle-shaped crystal with a pungent, characteristic odor. When exposed to light, air and moisture, the crystals acquire a crimson-red color. Store in closed banks made of dark glass and in a place protected from light.

Phenol is soluble in water, alcohol, ether, and fatty oils. Possessing great hygroscopicity, it absorbs moisture from the environment and becomes liquid. Liquid carbolic acid contains 90% crystalline phenol and 10% water.

Use 3-5% aqueous solutions of carbolic acid prepared from crystalline phenol and liquid carbolic acid according to the scheme given in table. 7. The activity of phenol increases when it is dissolved in hot water(40-50° C).

Attention! Crystalline phenol or liquid carbolic acid, if it gets on the skin, can cause irritation, and in high concentrations - severe burns. Therefore, carbolic acid must be handled with great care. When making solutions, you should wear rubber gloves or, in extreme cases, lubricate your hands with Vaseline.

If carbolic acid gets on your skin, wash it off immediately with warm water and soap or 40° ethyl alcohol.

Note. To prepare disinfectant solutions of phenol, it is more convenient and safer to use liquid carbolic acid.

Control questions

1. What disinfectants are used in microbiological practice?

2. Describe the appearance and basic properties of bleach, chloramine, phenol.

3. What solutions of disinfectants are used to disinfect material infected with spore forms of microorganisms?

Exercise

Prepare 2 liters of 5% working solution of clarified bleach; 500 ml of 3% chloramine solution, 300 ml of 1% activated chloramine solution.

Attention! Before you start preparing solutions, make calculations.

Carrying out stage 1 of the bacteriological method for isolating aerobes:

We prepare a fixed preparation from the material under study, stain using the Gram method, microscopy, and identify the detected microorganisms by morphotinctorial properties.

We sow the material under study onto half a dish with a dense nutrient medium using the “stroke with platform” method (we apply the material to the surface of the dense nutrient medium in a Petri dish in a limited area, and then distribute it by sowing in frequent parallel streaks)

Let's sign the cups, indicating the date of sowing, and place them upside down in a thermostat at a temperature of 37° for 18-24 hours.

Steam sterilizer (autoclave) - steam sterilization under pressure.

The most reliable and universal sterilization method in medical and microbiological practice is steam sterilization under pressure. It is produced in an autoclave, in which the objects to be sterilized are heated saturated steam under pressure above atmospheric. There is the following relationship between the pressure gauge readings and the saturated steam temperature

Zero pressure is considered normal atmospheric pressure (760 mm Hg).

Sterilization is achieved only when the autoclave is in full working order and properly operated by specially trained personnel. Therefore, constant monitoring of the sterilization regime is necessary, which is carried out physical (maximum thermometer, etc.), biological (biotest with spores of test cultures of microorganisms) and chemical (chemical tests, type indicators IP) ways

Control of the sterilization regime of autoclaves is carried out chemically each time the autoclave is loaded Chemical test - glass tube with chemical, having a certain melting point, antipyrine, resorcinol - 110±2°, benzoic acid - 120±2°, benzamide - 126+1°, urea, nicotinamide, D (+)-mannose - 132+2°. An aniline dye (magenta, gentsnan violet, etc.) is introduced into the chemical tests, which evenly colors the substance when it melts. Currently, indicators of the IS type (Vinar, Russia) are more often used, which are a strip of paper with a layer of indicator mixture applied to it and are intended for operational visual monitoring of not only temperature, but also the time of sterilization (IS-120, IS-132) The sterilization regime is monitored quarterly using a biotest with spores of the test culture Bacillus stearotermophilus BKM B-718

Pasteur oven- dry heat sterilization.

Glass, metal and rubber products based on silicone rubber are sterilized in a Pasteur oven. Sterilization mode: 160°C - 150 min; 180°C - 60 min Control of the sterilization mode during each cycle is carried out using sterilization indicators IS-160, IS-180; quarterly - using a biotest with spores of the Bacillus licheniformis test culture

Sterilization Table 1.

Disinfection Table 2.

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 quartz sand(Chamberlant candles) and infusorial 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 a mesh between metal disks (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, a vacuum is created in a Bunsen flask by connecting to it a rubber tube with a Komovsky rarefaction oil hand pump 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 a 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, the electric current is turned on and the flow of heat resumes.