Changes in temperature, osmotic pressure, irradiation, drying and other physical factors cause a significant disruption of metabolic processes in the cytoplasm of the cell, which can lead to its death.

Temperature. It is of great importance in the life of bacteria. Depending on the intensity and exposure (time) of exposure, the temperature factor can stimulate growth or, conversely, cause irreversible fatal changes in the microbial cell. For each type of microorganism there is a certain temperature range of growth, in which there are: optimal temperature, the most favorable for the growth and reproduction of microbes, maximum and minimum temperatures, above and below which the development of microorganisms stops. The optimal temperature usually corresponds to the temperature conditions of the natural habitat.

All microorganisms in relation to temperature are divided into three groups, within which the boundaries of the temperature range vary.
Psychrophiles (from the Greek psychros - cold) have adapted in the process of evolution to life at low temperatures. The optimal temperature for their development is 10-20°C, maximum 30°C and minimum 0°C. These are mainly saprophytic microbes of the northern seas, soil, and iron bacteria.

Mesophiles (from the Greek mesos - average) develop in the range of 20-45 ° C; The optimal temperature for them is 30-37°C. This broad group includes all pathogenic microbes.

Thermophiles (from the Greek termos - warm), growing at temperatures above 55°C, develop at an optimal temperature of 50-60°C. The minimum temperature for their development is 25°C, and the maximum is 70-80°C. Microbes of this group are found in soil, manure, and hot spring water. Among them there are many spore forms.
Both high and low temperatures can have an adverse effect on microorganisms. Significantly more sensitives mi crabs to hightemperatures. An increase in temperature beyond the maximum for their life activity causes an acceleration of biochemical reactions in the cell, disruption of the permeability of cell membranes, and damage to heat-sensitive enzymes. This entails disruption of vital metabolic processes in the cell, coagulation (denaturation) of cell proteins and its death. The death of most vegetative forms of bacteria occurs at 60°C on average after 30 minutes, at 70°C after 10-15 minutes, and at 80-100°C after 1 minute. Bacterial spores are much more resistant to high temperatures, for example, spores of the causative agent of tetanus can withstand boiling for up to 3 hours, and botulism for up to 6 hours. The death of spores when using moist heat (autoclave) occurs at 110-120 ° C after 20-30 minutes, and dry heat (Pasteur oven) at 180°C for 45 minutes. The action of high temperatures is the basis for sterilization - the desterorization of various materials and objects.

Microorganisms are extremely resistant to low temperatures. At temperatures below 0°C, they fall into a state of suspended animation, in which all vital processes of the cell are inhibited and its reproduction stops. Many bacteria remain alive in liquid hydrogen at a temperature of -253°C for hours. Vibrio cholerae and E. coli can survive in ice for a long time. Diphtheria pathogens tolerate freezing for 3 months, plague pathogens - up to 1 year. Viruses and bacteria that form spores are especially resistant to low temperatures; pathogenic bacteria such as gonococci, meningococci, spirochete pallidum, and rickettsia are less resistant. Repeated and rapid freezing and thawing, which leads to rupture of cell membranes and loss of cell contents, have a detrimental effect on microbes. The inhibitory effect of low temperature on the growth and reproduction of microorganisms is used when preserving food products in cellars, refrigerators, and frozen.

Drying, or dehydration, in vegetative forms of bacteria in most cases causes cell death, since it requires water for normal functioning. When the humidity of the substrate in which microorganisms multiply is below 30%, the development of most of them stops. The timing of the death of various microbes under the influence of drying varies widely: Vibrio cholera can withstand drying for up to 2 days, Shigella - 7 days, pathogens of diphtheria - 30 days, typhoid fever - 70 days, staphylococci and mycobacterium tuberculosis - 90 days, and lactic acid bacteria and yeast - several years. Bacterial spores are very resistant to drying. The dehydration method after preliminary freezing is widely used for the preservation of standard cultures of microorganisms (bacteria, viruses, etc.), immune sera and vaccine preparations. Such drugs cancan be stored for a long time. The essence of the method is that bacterial cultures in ampoules are quickly frozen at a temperature of -78°C in vessels with compacted carbon dioxide, and then dried in an airless space (vacuum, freeze drying). The culture ampoules are then sealed.

The unfavorable effect of drying on the growth and reproduction of microorganisms is used in the production and preservation of dry products. However, such products, when exposed to high humidity conditions, quickly deteriorate due to the restoration of microbial activity.

Effect of irradiation. The vital activity of microorganisms can be influenced by both radiant energy and sound irradiation.

Sunlight has a detrimental effect on all microorganisms, with the exception of green and purple sulfur bacteria. Direct sunlight kills most germs within a few hours. Pathogenic bacteria are more sensitive to light than saprophytes. The hygienic value of light as a natural disinfectant is very great. It frees the air and external environment from pathogenic bacteria. The most powerful bactericidal (bacteria-destroying) effect is exerted by rays with a short wavelength—ultraviolet. They are used to sterilize operating rooms, bacteriological laboratories and other premises, as well as water and milk. The source of these rays are mercury-quartz and bactericidal-violet lamps. Other types of radiant energy - X-rays, gamma rays - cause the death of microbes only when exposed in large doses. They are used to sterilize bacteriological preparations and some food products. The taste properties of the food do not change. During the action of radiant energy, cellular DNA is destroyed.

Sound irradiation: ordinary sound rays have virtually no harmful effect on microorganisms, unlike ultrasonic ones. Ultrasound rays cause significant damage to the cell, in which its outer shell ruptures and the cytoplasm is released. It is believed that gases dissolved in the liquid medium of the cytoplasm are activated by ultrasound, high pressure arises inside the cell and it mechanically ruptures.

Effect of pressure (mechanical, gas, osmotic).
Bacteria, especially spore-bearing ones, are very resistant to mechanical pressure. A pressure of 600 atm for 24 hours does not affect the anthrax pathogen, and at 20,000 atm for 45 minutes it is not completely destroyed. Non-spore-bearing bacteria are more sensitive to high pressure: Vibrio cholerae can withstand pressure of 3000 atm, but its mobility and ability to reproduce are partially reduced. Corynebacteria diphtheria, streptococci, neisseria, typhoid pathogens are resistant to pressure of 5000 atm for 45 minutes, but sensitive to 6000 atm. Viruses and bacteriophages are inactivated at a pressure of 5000-6000 atm, and bacterial toxins (tetanus and diphtheria) are weakened at a pressure of 12,000-15,000 atm. The mechanism of action of high mechanical pressure is the result of physical and chemical changes in the liquid: a decrease in its volume, an increase in viscosity, and the rate of chemical reactions.

The pressure of gases dissolved in the nutrient medium affects microorganisms depending on the nature of the gas and the type of metabolic process in the cell. Hydrogen at a pressure of 120 atm in 24 hours causes the death of 10-40% of E. coli cells, carbon dioxide at a pressure of 50 atm kills vegetative forms in 90 minutes, and nitrogen even at 120 atm does not have a pronounced effect on microbes.

Osmotic pressure is of great importance for the life of microorganisms. Based on their tolerance to various concentrations of mineral salts, bacteria are divided into two large groups: halophilic, which can develop in an environment with a high content of salts, especially sodium chloride, and non-halophilic, whose vital activity is possible with a sodium chloride content of 0.5-2%. The optimal sodium chloride content for most pathogenic microorganisms is a medium with 0.5% of this substance.

The destructive effect of concentrated solutions of salts and sugar on microorganisms is used when canning a number of products: fish, meat, vegetables, fruits. The content of 15-30% sodium chloride in the solution ensures the death of vegetative forms and suppresses sporulation. The sensitivity of microorganisms to the presence of sodium chloride in the environment is different: the causative agents of botulism cease their vital activity in a 6% solution, yeast - in 14%, and some halophiles can multiply in 20-30% solutions of sodium chloride.

Mechanical shaking. A moderate frequency of shaking (1-60 per minute) provides good aeration of the nutrient medium and creates favorable conditions for the growth of aerobes. Sharp and rapid shaking inhibits development, and when exposed for a long time, causes changes in cellular proteins and even complete destruction of cells. Strong mechanical shaking of bacteria in contact with inert dense particles (glass beads, quartz) has a direct harmful effect on the cells—the bacteria are destroyed. This method of mechanical disintegration is used to destroy microbial biomass when obtaining various antigens from them.

Lecture No. 10

Dictionary

RAW MATERIALS – raw materials intended for further processing. Medicinal raw materials.

MAZE – monitor grazing livestock and domestic animals; noun Grazing.

CORK - close tightly, plug up.

FAD - wither. Flowers fade .

Dwarf – the plant is unnaturally small in stature.

POISON – poisonous substance .

WASH – wash away, wash away, noun. Flush .

SHOCK – severe impairment of body functions due to physical injury ;

WIGGLE ( set in motion) - rock slightly.

FAST ≠ SLOW.

Influence of environmental factors on microorganisms. Sterilization. Methods and equipment. Sterilization quality control. The concept of disinfection, asepsis and antiseptics.

Microorganisms are influenced by physical, chemical and biological environmental factors. Physical factors: temperature, radiant energy, drying, ultrasound, pressure, filtration. Chemical factors: reaction of the environment (pH), substances of different nature and concentration. Biological factors– this is the relationship of microorganisms with each other and with the macroorganism, the influence of enzymes and antibiotics.

Environmental factors can affect microorganisms beneficial effect(growth stimulation) and bad influence: microbicidal action (destructive) and microbostatic action (growth suppression), as well as mutagenic action.

The effect of temperature on microorganisms.

Temperature is an important factor influencing the life activity 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 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 ensures the high efficiency of 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, porcelain dishes, 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 so that the spores 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.

Environmental factors constantly influence the life activity of microorganisms. Under favorable conditions, rapid growth and reproduction of microbes is observed. In conditions unfavorable for life, development slows down, and then their death may occur. Environmental factors that influence microorganisms are divided into physical, chemical and biological.

Physical factors. Physical environmental factors affecting the life activity of microorganisms include temperature, humidity, light, etc.

Effect of temperature. Microorganisms can tolerate significant temperature fluctuations. For the normal functioning of a microbial cell, a certain temperature is required. There are three temperature points: optimal, minimum and maximum, at which their vital activity can manifest itself in varying intensities. The optimal temperature is the one at which microorganisms grow and develop most intensively. The minimum temperature is the lowest at which microbial growth is still possible. Below this temperature, microorganisms reduce their biochemical activity, but do not die, but go into an anabiotic state, i.e. a state of hidden life, reminiscent of the winter torpor of many cold-blooded animals (frogs, snakes, lizards). Maximum is the highest temperature at which the growth and development of a microbe is still possible. Above the maximum temperature point, the microbe dies.

Depending on the temperature to which microorganisms have adapted in the process of long evolution, they are divided into psychrophiles, mesophiles and thermophiles.

Psychrophiles (cold-loving) are able to develop at low temperatures. The optimal temperature for them is 15-20 °C, minimum 0-10, maximum 30-35 °C. This group includes some representatives of coccal microflora, mold fungi, iron bacteria, etc., which cause spoilage of food when stored in refrigerators.

Mesophiles are a group of microorganisms that develop at average temperatures. The optimal temperature for them is 30-37 °C, minimum 10, maximum 43-50 °C. This group includes many molds, yeasts, putrefactive and all pathogenic microorganisms.

Thermophiles (heat-loving) are microbes that develop at relatively high temperatures. The optimal temperature for them is 50-60 °C, minimum 35, maximum 75-85 °C. Thermophiles are the main causative agents of spoilage of canned meat and meat and vegetables; they take part in the self-heating of silage, wet grain, hay, cotton, flour, etc. Some thermophilic microbes (spore sticks) remain vital at temperatures above 85 °C.

Microorganisms are very resistant to cooling and freezing. Some types of bacteria and molds can withstand the temperature of liquid air (- 190 ° C) and liquid hydrogen (- 253 ° C). Viruses are very resistant to low temperatures. At low temperatures, a number of changes still occur that can lead to the death of the microbe. The rate of death of microbes during freezing depends on the type of microbe, freezing temperature, frequency of freezing and thawing, type and duration of frozen product storage, etc.

A high temperature that causes the death of a microbial cell is called lethal. The destructive effect of high temperature is caused by damage to the colloidal state of plasma, denaturation of protein followed by its coagulation, as well as disruption of enzymatic systems. Most non-spore microbes die in a humid environment at a temperature of 60-70 °C in 15-30 minutes, at a temperature of 85 °C in 3-5 minutes and at a temperature of 100 °C instantly. Bacillus spores are very resistant to high temperatures. Spores of some microorganisms can withstand boiling from several minutes to several hours.

Influence of humidity. The minimum humidity required for the life of bacteria is 30%, for mold fungi - 15%. Different types of microorganisms are not equally sensitive to drying, which causes loss of water, resulting in cell death. Non-spore-forming microbes are most sensitive to drying. The spores are highly resistant to drying, remaining in a dried state for several years. Drying is used as one of the methods for preserving perishable products. In the meat industry, the drying method is widely used for preserving meat, sausages, meat and bone meal, etc.

Freeze drying (drying at low temperature and vacuum) promotes long-term preservation of microorganisms. This method is used in industry for the production of dry vaccines (live), canning meat and endocrine raw materials, preparing organ preparations and starter cultures for fermented milk products.

The influence of light. Direct sunlight, especially ultraviolet rays, have a bactericidal effect. The microbial cell of vegetative forms dies in sunlight after a few minutes. Scattered light does not have such a destructive effect on microbes, but with prolonged exposure it can gradually inhibit their growth and development.

Ultraviolet irradiation is used in meat industry enterprises to disinfect air, equipment surfaces and various objects using bactericidal lamps.

Effect of radiation. Microorganisms are more resistant to the effects of x-rays and gamma rays; the lethal dose for them is hundreds and thousands of times greater than for animals. X-ray and gamma radiation in small doses and with short exposure have a stimulating effect on the growth and reproduction of microbes. Large doses of X-rays inactivate enzymes, slow growth and prevent the proliferation of microbes.

The influence of ultrasonic waves. Ultrasonic waves have significant mechanical energy that can inactivate enzymes, toxins, and destroy microbial cells. The lethal effect on bacteria and viruses begins to manifest itself when the environment is sounded with an oscillation frequency of about 100 thousand Hz. Ultrasound can be used for sterilization and pasteurization of products, cleaning and disinfection of equipment, containers, and wastewater.

Effect of pressure. Microorganisms are resistant to high pressure. Microbes were found at the bottom of deep seas and oceans, where pressure reaches more than 90 MPa (900 kgf/cm2), some yeasts and molds can withstand pressure of 300 MPa (3000 kgf/cm2).

Chemical factors. A microbial cell reacts to the smallest amount of a chemical in the environment. So, if a capillary filled with a solution of peptone (a substance nutritious for microbes) is lowered into a drop of water containing mobile bacteria, then after a while you can notice an accumulation of microorganisms at the opening of the capillary. This is the so-called positive chemotaxis - bacteria move towards the substance that attracts them. If the capillary is filled with alkali or acid, then the bacteria move away from the substance that is toxic to them, diffusing into the water, i.e. negative chemotaxis is observed.

The effect of chemicals on microorganisms does not manifest itself to the same extent. As a rule, low concentrations not only do not cause the death of microbes, but even stimulate their growth and development.

Large concentrations of chemicals act on microorganisms bacteriostatically or bactericidally, causing their death. Chemicals that cause the death of microorganisms are called disinfectants. The effectiveness of chemicals depends on the chemical nature of the substance, its concentration, temperature, environmental reaction, type of microorganism, etc. Substances used to destroy microbes must be in a dissolved state. The more easily a substance is adsorbed by a microbial cell, the stronger its effect. Chemical substances, depending on their effect on the microbial cell, can be divided into the following groups:

substances that damage only the cell wall and do not change the internal structure of the microbe (soaps, fatty acids);

substances that cause damage to the membrane and cellular proteins (phenol, cresol and their derivatives);

substances that cause denaturation of proteins (formaldehyde - 40% formaldehyde solution);

substances that cause inactivation of enzymes (salts of heavy metals - salts of mercury, copper, silver, etc.).

The most sensitive to chemicals are microbes that do not form spores, vegetative forms. Spore forms are quite resistant to various chemicals. To destroy them, it is necessary to prepare hot solutions of high concentrations of chemicals. Thus, spores of anthrax bacillus die in a 5% phenol solution in only 14 days, while vegetative forms of this pathogen die from such a concentration in a few seconds.

When choosing disinfectants to kill germs, the type of microorganism must be taken into account. For example, viruses are very sensitive to alkalis, the causative agent of anthrax is to chlorine and formaldehyde, and the causative agents of tuberculosis are resistant to acids and alkalis.

The reaction of the environment (pH is an indicator of the concentration of hydrogen ions) affects the growth and development of microorganisms. The vital activity of various types of microbes is possible only at a certain pH. Most microorganisms develop in a slightly alkaline environment (pH 7.2-7.6); yeasts and molds are better cultivated at pH 3-6. By changing the reaction of the environment, it is possible to regulate the intensity of development and biochemical activity of microbes. When the pH decreases to 5, putrefactive bacteria do not develop, while with such a reaction the enzymatic activity of yeast is most active.

Biological factors. In the process of life, microorganisms are in various relationships with each other and with other organisms. These relationships, in the process of long evolution, developed in accordance with the general biological law of symbiosis (cohabitation) of living beings. In nature, relationships between microbes and other organisms exist in the form of various forms of symbiosis, metabiosis and antagonism.

Commensalism is a form of symbiosis in which one organism lives and develops at the expense of another without harming it. For example, E. coli, some types of staphylococci, streptococci and other microbes live on the surface or in the cavities of humans and animals.

Mutualism is a cohabitation in which both organisms receive mutual benefits without causing harm to each other, for example, the cohabitation of nodule bacteria with leguminous plants.

Metabiosis is a relationship between microorganisms in which, in the process of sequential development of some microbes, favorable conditions are created for the life of others.

Antagonism is a relationship between microbes in which the coexistence of microbial species is impossible, i.e. one type of microbe interferes with the growth of another, delaying its development, or causes complete death.



Introduction……………………………………………………………..………….….2

1) The influence of physical factors on microorganisms…………………..………3

1.1Radiations……………………………………………………..………………………3

1.2Ultrasound…………………………………….....………………………4

2) Ionizing radiation…………………………..…….…………………….5

2.1 Practical use of ionizing radiation………......7

3) Conclusion………………………………………………………...……..………8

References………………….……………………………..………….9

Introduction

All existing microorganisms live in continuous interaction with the external environment in which they are located, and therefore are exposed to various influences. In some cases they can promote better development, in others they can suppress their vital functions. It must be remembered that variability and rapid change of generations allows one to adapt to different living conditions. Therefore, new signs are quickly established.

Being in the process of development in close interaction with the environment, microorganisms can not only change under its influence, but can change the environment in accordance with their characteristics. So, during the process of respiration, microbes release metabolic products, which in turn change the chemical composition of the environment, therefore the reaction of the environment and the content of various chemicals change.

All factors influencing the development of microbes are divided into:

· Physical

· Chemical

· Biological

Below we will take a closer look at each of the factors.

1) The influence of physical factors on microorganisms

Temperature in relation to temperature conditions, microorganisms are divided into thermophilic, psychrophilic and mesophilic.

· Thermophilic species . The optimal growth zone is 50-60°C, the upper growth inhibition zone is 75°C. Thermophiles live in hot springs and participate in the processes of self-heating of manure, grain, and hay.

· Psychrophilic species (cold-loving) grow in the temperature range of 0-10°C, the maximum growth inhibition zone is 20-30°C. These include most saprophytes that live in soil, fresh and sea water. But there are some species, for example, Yersinia, psychrophilic variants of Klebsiella, pseudomonads, that cause diseases in humans.

· Mesophilic species grow best within 20-40°C; maximum 43-45°C, minimum 15-20°C. They can survive in the environment, but usually do not reproduce. These include most pathogenic and opportunistic microorganisms.

1.1 Radiation

Sunlight has a detrimental effect on microorganisms, with the exception of phototrophic species. Short-wave UV rays have the greatest microbicidal effect. Radiation energy is used for disinfection, as well as for sterilization of thermolabile materials.

Ultra-violet rays(primarily short-wavelength, i.e. with a wavelength of 250-270 nm) act on nucleic acids. The microbicidal effect is based on the rupture of hydrogen bonds and the formation of thymidine dimers in the DNA molecule, leading to the appearance of non-viable mutants. The use of ultraviolet radiation for sterilization is limited by its low permeability and high absorption activity of water and glass.

X-ray And g-radiation V large doses also causes the death of microbes. Irradiation causes the formation of free radicals that destroy nucleic acids and proteins, followed by the death of microbial cells. Used for sterilization of bacteriological preparations and plastic products.

Microwave radiation used for rapid re-sterilization of long-term stored media. The sterilizing effect is achieved by quickly raising the temperature.

1.2Ultrasound.

Certain frequencies of ultrasound, when exposed artificially, can cause depolymerization of the organelles of microbial cells; under the influence of ultrasound, gases located in the liquid medium 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.

Pressure.

Bacteria are relatively little sensitive to changes in hydrostatic pressure. Increasing the pressure to a certain limit does not affect the growth rate of ordinary terrestrial bacteria, but eventually begins to interfere with normal growth and division. Some types of bacteria can withstand pressures of up to 3,000 - 5,000 atm, and

bacterial spores - even 20,000 atm.

In conditions of deep vacuum, the substrate dries out and life is impossible.

Filtration.

To remove microorganisms, various materials are used (fine-porous glass, cellulose, koalin); they provide effective elimination of microorganisms from liquids and gases. Filtration is used to sterilize temperature-sensitive liquids, separate microbes and their metabolites (exotoxins, enzymes), and also to isolate viruses.

2) Ionizing radiation

Streams of photons or particles, the interaction of which with a medium leads to the ionization of its atoms or molecules. There are photon (electromagnetic) and corpuscular

Toward photonic I.I. include vacuum UV and characteristic X-rays, as well as radiation arising from radioactive decay and other nuclear reactions (mainly g-radiation) and when charged particles are decelerated into an electric or magnetic field - bremsstrahlung X-rays, synchrotron radiation.

To corpuscular I.I. include fluxes of a- and b-particles, accelerated ions and electrons, neutrons, fission fragments of heavy nuclei, etc.

Mechanisms of action of ionizing radiation on living organisms

The processes of interaction of ionizing radiation with matter in living organisms lead to a specific biological effect, resulting in damage to the organism. In the process of this damaging action, three stages can be roughly distinguished:

b. the effect of radiation on cells;

c. the effect of radiation on the whole organism.

The primary act of this action is the excitation and ionization of molecules, as a result of which free radicals arise (direct action of radiation) or the chemical transformation (radiolysis) of water begins, the products of which (OH radical, hydrogen peroxide - H 2 O 2, etc.) enter into chemical reaction with molecules of a biological system.

Primary ionization processes do not cause major disturbances in living tissues. The damaging effect of radiation is apparently associated with secondary reactions in which bonds within complex organic molecules are broken, for example SH groups in proteins, chromophore groups of nitrogenous bases in DNA, unsaturated bonds in lipids, etc.

The effect of ionizing radiation on cells is due to the interaction of free radicals with molecules of proteins, nucleic acids and lipids, when, as a result of all these processes, organic peroxides are formed and transient oxidation reactions occur. As a result of peroxidation, many altered molecules accumulate, as a result of which the initial radiation effect is greatly enhanced. All this is reflected primarily in the structure of biological membranes, their sorption properties change and permeability increases (including membranes of lysosomes and mitochondria). Changes in lysosome membranes lead to the release and activation of DNase, RNase, cathepsins, phosphatase, mucopolysaccharide hydrolysis enzymes and a number of other enzymes.

The released hydrolytic enzymes can, by simple diffusion, reach any cell organelle into which they easily penetrate due to increased membrane permeability. Under the influence of these enzymes, further decomposition of the macromolecular components of the cell occurs, including nucleic acids and proteins. The uncoupling of oxidative phosphorylation as a result of the release of a number of enzymes from mitochondria, in turn, leads to inhibition of ATP synthesis, and hence to disruption of protein biosynthesis.

Thus, the basis of radiation damage to cells is a violation of the ultrastructures of cellular organelles and associated metabolic changes. In addition, ionizing radiation causes the formation in the tissues of the body of a whole complex of toxic products that enhance the radiation effect - the so-called radiotoxins. Among them, the most active are lipid oxidation products - peroxides, epoxides, aldehydes and ketones. Formed immediately after irradiation, lipid radiotoxins stimulate the formation of other biologically active substances - quinones, choline, histamine and cause increased breakdown of proteins. When administered to non-irradiated animals, lipid radiotoxins have effects reminiscent of radiation injury. Ionizing radiation has the greatest effect on the cell nucleus, inhibiting mitotic activity.

Medical Faculty

Faculty of Pediatrics

DEPARTMENT OF MICROBIOLOGY TSMA

Lesson No. 7

EFFECT OF PHYSICAL AND CHEMICAL FACTORS ON MICROORGANISMS

Purpose of the lesson:

study the effect of physical and chemical factors on microbes; the concepts of “asepsis” and “antiseptics”; sterilization methods and equipment.

THE STUDENT SHOULD KNOW:

Effect on microorganisms of high and low temperatures and pressure. The concept of "sterilization".

The concepts of “asepsis” and “antiseptics”

Sterilization methods, equipment.

Effect of drying factors on microorganisms. Freeze drying.

The action of light, ultrasound, radiant energy, ionizing radiation.

The effect of chemical factors on microbes. Disinfectants and antiseptic substances.

THE STUDENT SHOULD BE ABLE TO:

prepare dishes for sterilization in a dry-heat oven and autoclave;

evaluate the results of monitoring the sterility of the autoclave and dry-heat oven;

evaluate the results of determining the sensitivity of microbes to antimicrobial substances (disinfectants, antiseptics).

STUDENT MUST HAVE REPRESENTATION

about the toxicity index when using antiseptics; about the asepsis regime in the manufacture of medicines; about chemical preservatives of blood, biological products, live vaccines.

Guidelines

Work No. 1. Methods and mode of sterilization of various materials

Target: study methods of sterilization of various materials.

Develop and enter into a notebook the table “Methods and mode of sterilization of various materials.”

Given: table.

METHODS AND REGIME FOR STERILIZATION OF VARIOUS MATERIALS

Sterilization method	Equipment	Temperature	Time (min)	Material
Boiling
Calcination
Autoclaving
Dry heat
Pasteurization
Tyndalization
Filtration
Freeze drying
Radiant Energy
Ionizing radiation

Work No. 2. Monitoring the effectiveness of sterilization

Target: evaluate the quality of the autoclave. Explain the mechanism of sterilization.

Result:

Work No. 3. Determination of the sensitivity of microorganisms to antiseptics

Target: assess the sensitivity of microbial cells to antiseptics. Explain the mechanism of action of the antiseptic in each specific case. Sketch. Draw a conclusion.

Given: experiment No. 2 (inoculation of E. coli with added antiseptics - iodine, methylene blue, carbolic acid, chloramine); table “Classification of antiseptics by mechanism of action” (see guidelines).

Result:

Theoretical information

Influence of physical factors on microorganisms

Temperature is the most significant factor influencing the life activity of microbes. The temperature required for the growth and reproduction of bacteria of the same species varies widely. There are temperature optimum, minimum and maximum.

Temperature optimum corresponds to the physiological norm of this type of microbe, in which reproduction occurs quickly and intensively. For most pathogenic and opportunistic microbes temperature optimum corresponds to 37 0 WITH.

Temperature minimum corresponds to the temperature at which a given type of microbe does not show vital activity.

Temperature maximum– the temperature at which growth and reproduction stops, all metabolic processes decrease and death may occur.

Depending on the temperature optimal for life, 3 groups of microorganisms are distinguished:

1) psychrophilic, cold-loving, multiplying at temperatures below 20 0 C (Yersinia, psychrophilic variants of Klebsiella, pseudomonads that cause human diseases. Reproducing in food products, they are more virulent at low temperatures);

2) thermophilic, the optimal development of which lies within 55 0 C (they do not reproduce in the body of warm-blooded animals and have no medical significance);

3) mesophilic, actively reproduce at temperatures of 20-40 0 C, the optimum development temperature for them is 37 0 C (bacteria pathogenic for humans).

Microorganisms withstand low temperatures well. This is the basis for the long-term preservation of bacteria in a frozen state. However, below the temperature minimum, the damaging effect of low temperatures appears, caused by the rupture of the cell membrane by ice crystals and the suspension of metabolic processes.

Low temperature stops putrefactive and fermentation processes. This underlies the preservation of substrates (in particular food products) by cold.

The destructive effect of high temperature (above the temperature maximum for each group) is used in sterilization. Sterilization– sterilization is the process of killing on or in products or removing from an object microorganisms of all types at all stages of development, including spores (thermal and chemical methods and means). To kill vegetative forms of bacteria, a temperature of 60 0 C for 20-30 minutes is sufficient; spores die at 170 0 C or at a temperature of 120 0 C in steam under pressure (in an autoclave).

Asepsis– a set of measures aimed at preventing the possibility of microorganisms entering the wound, tissues, organs, and body cavities of the patient during surgical operations, dressings, instrumental examinations, as well as to prevent microbial and other contamination when obtaining sterile products at all stages of the technological process.

Antiseptics– a set of therapeutic and preventive measures aimed at destroying microorganisms that can cause an infectious process in damaged or intact areas of the skin or mucous membranes.

Disinfection– disinfection of environmental objects: destruction of pathogenic microorganisms for humans and animals using chemicals that have an antimicrobial effect.

The growth and reproduction of microbes occurs in the presence of water, which is necessary for the passive and active transport of nutrients into the cytoplasm of the cell. A decrease in humidity (drying) leads to the transition of the cell to the resting stage and then to death. The least resistant to drying are pathogenic microorganisms - meningococci, gonococci, treponema, whooping cough bacteria, orthomyxo-, paramyxo- and herpes viruses. Mycobacterium tuberculosis, variola virus, salmonella, actinomycetes, and fungi are resistant to drying. Bacterial spores are particularly resistant to drying. Resistance to desiccation increases if microbes are pre-frozen. To preserve the viability and stability of the properties of microorganisms for production purposes, the method is used freeze drying- drying from frozen state under deep vacuum.

During the lyophilization process, the following is carried out: 1) preliminary freezing of the material at t -40 0 - -45 0 C in alcohol baths for 30-40 minutes; 2) drying is carried out from a frozen state in a vacuum in sublimation devices for 24-28 hours.

The drying process has 2 phases: sublimation of ice at temperatures below 0°C and desorption - removal of part of the free and bound water at temperatures above 0°C.

Lyophilization is used to obtain dry preparations when protein denaturation does not occur and the structure of the material does not change (antisera, vaccines, dry bacterial mass). In laboratory conditions, lyophilized microbial cultures are preserved for 10-20 years, and the culture remains pure and does not undergo mutations.

Calcination produced in the flame of an alcohol lamp or gas burner. This method is used to sterilize bacteriological loops, dissecting needles, tweezers and some other instruments.

Boiling used for sterilization of syringes, small surgical instruments, slides, cover glasses, etc. Sterilization is carried out in sterilizers, into which water is poured and brought to a boil. To eliminate hardness and increase the boiling point, add 1-2% sodium bicarbonate to the water. Tools are usually boiled for 30 minutes. This method does not provide complete sterilization, since bacterial spores are not killed.

Pasteurization- sterilization at 65-70°C for 1 hour to destroy non-spore microorganisms (milk is freed from Brucella, Mycobacterium tuberculosis, Shigella, Salmonella, Staphylococcus) Stored in the cold

Tyndalization- fractional sterilization of materials at 56-58 0 C for 1 hour for 5-6 days in a row. It is used for sterilization of substances that are easily destroyed at high temperatures (blood serum, vitamins, etc.).

Action radiant energy to microorganisms. Sunlight, especially its ultraviolet and infrared spectra, have a detrimental effect on vegetative forms of microbes within a few minutes.

Infrared radiation is used to sterilize objects, which is achieved through thermal exposure at a temperature of 300 0 C for 30 minutes. Infrared rays affect free radical processes, as a result of which chemical bonds in the molecules of the microbial cell are disrupted.

To disinfect the air in medical institutions and pharmacies, mercury-quartz and mercury-uviol lamps, which are a source of ultraviolet rays, are widely used. When exposed to UV rays with a wavelength of 254 nm at a dose of 1.5-5 μW t/s per 1 cm 2 with a 30-minute exposure, all vegetative forms of bacteria die. The damaging effects of UV radiation are caused by damage to the DNA of microbial cells, leading to mutations and death.

Ionizing radiation has a powerful penetrating and damaging effect on the cellular genome of microbes. To sterilize disposable instruments (needles, syringes), gamma radiation is used, the source of which is the radioactive isotopes 60 Co and 137 Cs in a dose of 1.5-2 MN.rad. This method also sterilizes blood transfusion systems and suture material. The effect of ultrasound at certain frequencies on microorganisms causes depolymerization of cell organelles and denaturation of their constituent molecules as a result of local heating or increased pressure. Sterilization of objects with ultrasound is carried out at industrial enterprises, since the source of ultrasound is powerful generators. Liquid media are subjected to sterilization, in which not only vegetative forms are killed, but also spores.

Sterilization by filtration- release from microbes of material that cannot be subjected to heating (blood serum, a number of drugs). Filters with very small pores that do not allow microbes to pass through are used: porcelain (Chamberlain filter), kaolin, asbestos plates (Seitz filter). Filtration occurs under increased pressure, the liquid is forced through the pores of the filter into the receiver, or a vacuum of air is created in the receiver and the liquid is sucked into it through the filter. A pressure or vacuum pump is connected to the filter device. The device is sterilized in an autoclave.