Reactions of antigens with antibodies are called serological or humoral because the specific antibodies involved are always present in the blood serum.

Reactions between antibodies and antigens that occur in a living organism can be reproduced in the laboratory for diagnostic purposes.

Serological immune reactions entered the practice of diagnosing infectious diseases at the end of the 19th and beginning of the 20th centuries.

The use of immune reactions for diagnostic purposes is based on the specificity of the interaction of antigen with antibody.

Determination of the antigenic structure of microbes and their toxins made it possible to develop not only diagnosticums and therapeutic sera, but also diagnostic sera. Immune diagnostic sera are obtained by immunizing animals (for example, rabbits). These sera are used to identify microbes or exotoxins by antigenic structure using serological reactions (agglutination, precipitation, complement fixation, passive hemagglutination, etc.). Immune diagnostic sera treated with fluorochrome are used for rapid diagnosis of infectious diseases using the immune fluorescence method.

Using known antigens (diagnosticums), it is possible to determine the presence of antibodies in the blood serum of a patient or subject (serological diagnosis of infectious diseases).

The presence of specific immune sera (diagnostic) makes it possible to establish the species and type of microorganism (serological identification of the microbe by antigenic structure).

The external manifestation of the results of serological reactions depends on the conditions of its production and the physiological state of the antigen.

Corpuscular antigens give the phenomenon of agglutination, lysis, complement fixation, immobilization.

Soluble antigens give rise to the phenomenon of precipitation and neutralization.

In laboratory practice, agglutination, precipitation, neutralization, complement fixation, hemagglutination inhibition, etc. reactions are used for diagnostic purposes.

Agglutination reaction (RA)

Due to its specificity, ease of performance and demonstrativeness, the agglutination reaction has become widespread in microbiological practice for the diagnosis of many infectious diseases: typhoid fever and paratyphoid fever (Vidal reaction), typhus (Weigl reaction), etc.

The agglutination reaction is based on the specificity of the interaction of antibodies (agglutinins) with whole microbial or other cells (agglutinogens). As a result of this interaction, particles are formed - agglomerates that precipitate (agglutinate).

The agglutination reaction can involve both live and killed bacteria, spirochetes, fungi, protozoa, rickettsia, as well as red blood cells and other cells.

The reaction occurs in two phases: the first (invisible) is specific, the combination of antigen and antibodies, the second (visible) is nonspecific, the gluing of antigens, i.e. agglutinate formation.

An agglutinate is formed when one active center of a divalent antibody combines with the determinant group of an antigen.

The agglutination reaction, like any serological reaction, occurs in the presence of electrolytes.

Externally, the manifestation of a positive agglutination reaction has a twofold character. In flagellated microbes that have only somatic O-antigen, the microbial cells themselves adhere directly. This agglutination is called fine-grained. It occurs within 18 – 22 hours.

Flagellar microbes have two antigens - somatic O-antigen and flagellar H-antigen. If cells are glued together by flagella, large, loose flakes are formed and this agglutination reaction is called coarse-grained. It occurs within 2 – 4 hours.

The agglutination reaction can be performed both for the purpose of qualitative and quantitative determination of specific antibodies in the patient’s blood serum, and for the purpose of determining the species of the isolated pathogen.

The agglutination reaction can be performed both in an expanded version, which allows you to work with serum diluted to a diagnostic titer, and in the version of an indicative reaction, which allows, in principle, to detect specific antibodies or determine the species of the pathogen.

When performing a detailed agglutination reaction, in order to detect specific antibodies in the blood serum of the subject, the test serum is taken at a dilution of 1:50 or 1:100. This is due to the fact that normal antibodies may be present in very high concentrations in whole or slightly diluted serum, and then the reaction results may be inaccurate. The material being tested in this version of the reaction is the patient’s blood. Blood is taken on an empty stomach or no earlier than 6 hours after a meal (otherwise there may be droplets of fat in the blood serum, making it cloudy and unsuitable for research). The patient's blood serum is usually obtained in the second week of the disease, collecting 3–4 ml of blood sterilely from the cubital vein (by this time the maximum amount of specific antibodies is concentrated). A diagnosticum prepared from killed but not destroyed microbial cells of a specific species with a specific antigenic structure is used as a known antigen.

When performing a detailed agglutination reaction in order to determine the species and type of pathogen, the antigen is a live pathogen isolated from the material being studied. Antibodies contained in immune diagnostic serum are known.

Immune diagnostic serum is obtained from the blood of a vaccinated rabbit. Having determined the titer (the maximum dilution at which antibodies are detected), the diagnostic serum is poured into ampoules with the addition of a preservative. This serum is used for identification by the antigenic structure of the isolated pathogen.

When performing an indicative agglutination reaction on a glass slide, sera with a higher concentration of antibodies are used (in dilutions of no more than 1:10 or 1:20).

Using a Pasteur pipette, one drop of saline and serum is applied to the glass. Then a small amount of microbes is added to each drop in a loop and thoroughly mixed until a homogeneous suspension is obtained. After a few minutes, with a positive reaction, a noticeable crowding of microbes (granularity) appears in the serum drop, while a uniform turbidity remains in the control drop.

The approximate agglutination reaction is most often used to determine the species of microbes isolated from the test material. The obtained result allows us to approximately speed up the diagnosis of the disease. If the reaction is difficult to see with the naked eye, it can be observed under a microscope. In this case, it is called microagglutination.

The approximate agglutination reaction, which is performed with a drop of the patient’s blood and a known antigen, is called a blood drop.

Indirect or passive hemagglutination reaction (IPHA)

This reaction is superior in sensitivity to the agglutination reaction and is used in the diagnosis of infections caused by bacteria, rickettsia, protozoa and other microorganisms.

RPGA allows you to detect a small concentration of antibodies.

This reaction involves tannized sheep erythrocytes or human erythrocytes with group I blood, sensitized with antigens or antibodies.

If antibodies are detected in the test serum, then red blood cells sensitized with antigens are used (erythrocyte diagnosticum).

In some cases, if it is necessary to determine various antigens in the test material, erythrocytes sensitized with immune globulins are used.

The results of RPGA are taken into account by the nature of the erythrocyte sediment.

The result of a reaction is considered positive when red blood cells evenly cover the entire bottom of the test tube (an inverted umbrella).

In a negative reaction, red blood cells in the form of a small disk (button) are located in the center of the bottom of the test tube.

Precipitation reaction (RP)

In contrast to the agglutination reaction, the antigen for the precipitation reaction (precipitinogen) is soluble compounds, the size of the particles approaching the size of molecules.

These can be proteins, complexes of proteins with lipids and carbohydrates, microbial extracts, various lysates or filtrates of microbial cultures.

Antibodies that cause the precipitating property of immune serum are called precipitins, and the reaction product in the form of a precipitate is called precipitate.

Precipitating sera are obtained by artificially immunizing an animal with live or killed microbes, as well as a variety of lysates and extracts of microbial cells.

By artificial immunization, it is possible to obtain precipitating sera to any foreign protein of plant and animal origin, as well as to haptens when an animal is immunized with a full-fledged antigen containing this hapten.

The mechanism of the precipitation reaction is similar to the mechanism of the agglutination reaction. The effect of precipitating sera on antigen is similar to the effect of agglutinating sera. In both cases, under the influence of immune serum and electrolytes, the antigen particles suspended in the liquid become larger (a decrease in the degree of dispersion). However, for the agglutination reaction, the antigen is taken in the form of a homogeneous turbid microbial suspension (suspension), and for the precipitation reaction - in the form of a transparent colloidal solution.

The precipitation reaction is highly sensitive and allows the detection of negligible amounts of antigen.

The precipitation reaction is used in laboratory practice for the diagnosis of plague, tularemia, anthrax, meningitis and other diseases, as well as in forensic medical examination.

In sanitary practice, this reaction is used to determine the falsification of food products.

The precipitation reaction can be carried out not only in test tubes, but also in a gel, and for fine immunological studies of the antigen, the method of immunophoresis is used.

The agar gel precipitation reaction, or the diffuse precipitation method, allows for a detailed study of the composition of complex water-soluble antigenic mixtures. To carry out the reaction, a gel (semi-liquid or denser agar) is used. Each component that makes up the antigen diffuses towards the corresponding antibody at different speeds. Therefore, complexes of various antigens and corresponding antibodies are located in different areas of the gel, where they form precipitation lines. Each line corresponds to only one antigen-antibody complex. The precipitation reaction is usually carried out at room temperature.

The method of immunophoresis has become widespread in the study of the antigenic structure of microbial cells.

The antigen complex is placed in a well located in the center of an agar field poured onto the plate. An electric current is passed through the agar gel. The various antigens included in the complex move as a result of the action of the current, depending on their electrophoretic mobility. After electrophoresis is completed, specific immune serum is added to a trench located along the edge of the plate and placed in a humid chamber. Precipitation lines appear at the sites where the antigen-antibody complex is formed.

Exotoxin neutralization reaction with antitoxin (RN)

The reaction is based on the ability of the antitoxic serum to neutralize the effect of the exotoxin. It is used for titration of antitoxic serums and determination of exotoxin.

When titrating the serum, a certain dose of the corresponding toxin is added to different dilutions of the antitoxic serum. When the antigen is completely neutralized and there are no unspent antibodies, initial flocculation occurs.

The flocculation reaction can be used not only for the titration of serum (for example, diphtheria), but also for the titration of toxin and toxoid.

The reaction of toxin neutralization with antitoxin is of great practical importance as a method for determining the activity of antitoxic therapeutic serums. The antigen in this reaction is a true exotoxin.

The strength of the antitoxic serum is determined by conventional units of AE.

1 AE of diphtheria antitoxic serum is the amount that neutralizes 100 DLM of diphtheria exotoxin. 1 AE of botulinum serum is the amount that neutralizes 1000 DLM of botulinum toxin.

The neutralization reaction to determine the species or type of exotoxin (for the diagnosis of tetanus, botulism, diphtheria, etc.) can be carried out in vitro (according to Ramon), and when determining the toxigenicity of microbial cells - in a gel (according to Ouchterlony).

Lysis reaction (RL)

One of the protective properties of immune serum is its ability to dissolve microbes or cellular elements entering the body.

Specific antibodies that cause cell dissolution (lysis) are called lysines. Depending on the nature of the antigen, they can be bacteriolysins, cytolysins, spirochetolysins, hemolysins, etc.

Lysines exhibit their effect only in the presence of an additional factor – complement.

Complement, as a factor of nonspecific humoral immunity, is found in almost all body fluids, except for cerebrospinal fluid and fluid of the anterior chamber of the eye. A fairly high and constant content of complement is noted in human blood serum and a lot of it in the blood serum of guinea pigs. In other mammals, the content of complement in the blood serum is different.

Complement is a complex system of whey proteins. It is unstable and collapses at 55 degrees for 30 minutes. At room temperature, complement is destroyed within two hours. Very sensitive to prolonged shaking, acids and ultraviolet rays. However, complement is stored for a long time (up to six months) in a dried state at low temperatures.

Complement promotes the lysis of microbial cells and red blood cells.

A distinction is made between the reactions of bacteriolysis and hemolysis.

The essence of the bacteriolysis reaction is that when a specific immune serum combines with its corresponding homologous living microbial cells in the presence of complement, microbial lysis occurs.

The hemolysis reaction is that when erythrocytes are exposed to a specific serum that is immune to them (hemolytic) in the presence of complement, the dissolution of erythrocytes is observed, i.e. hemolysis.

The hemolysis reaction in laboratory practice is used to determine the complement range, as well as to take into account the results of the Bordet-Giangu and Wassermann diagnostic complement fixation reactions.

The complement titer is its smallest amount, which causes the lysis of red blood cells within 30 minutes in a hemolytic system in a volume of 2.5 ml. The lysis reaction, like all serological reactions, occurs in the presence of an electrolyte.

Complement fixation reaction (CFR)

This reaction is used in laboratory studies to detect antibodies in blood serum for various infections, as well as to identify the pathogen by its antigenic structure.

The complement fixation reaction is a complex serological reaction and is highly sensitive and specific.

A feature of this reaction is that the change in the antigen during its interaction with specific antibodies occurs only in the presence of complement. Complement is adsorbed only on the antibody-antigen complex. The antibody-antigen complex is formed only if there is affinity between the antigen and the antibody in the serum.

Adsorption of complement on the antigen-antibody complex can have different effects on the fate of the antigen depending on its characteristics.

Some of the antigens undergo sharp morphological changes under these conditions, up to dissolution (hemolysis, Isaev-Pfeiffer phenomenon, cytolytic effect). Others change the speed of movement (treponema immobilization). Still others die without sudden destructive changes (bactericidal or cytotoxic effect). Finally, adsorption of complement may not be accompanied by changes in the antigen that are easily observable (Bordet-Zhangou, Wasserman reactions).

According to the RSC mechanism, it occurs in two phases:
a) The first phase is the formation of the antigen-antibody complex and the adsorption of complement on this complex. The result of the phase is not visually visible.
b) The second phase is a change in the antigen under the influence of specific antibodies in the presence of complement. The result of the phase may be visible visually or not.

In the case when changes in the antigen remain inaccessible for visual observation, it is necessary to use a second system, which acts as an indicator, allowing one to assess the state of complement and draw a conclusion about the result of the reaction.

This indicator system is represented by components of the hemolysis reaction, which includes sheep erythrocytes and hemolytic serum, which contains specific antibodies to erythrocytes (hemolysins), but does not contain complement. This indicator system is added to the test tubes an hour after the main RSC is placed.

If the complement fixation reaction is positive, then an antibody-antigen complex is formed, which adsorbs complement on itself. Since complement is used in the amount necessary for only one reaction, and lysis of erythrocytes can only occur in the presence of complement, then when it is adsorbed on the antigen-antibody complex, lysis of erythrocytes in the hemolytic (indicator) system will not occur. If the complement fixation reaction is negative, the antigen-antibody complex is not formed, the complement remains free, and when a hemolytic system is added, erythrocyte lysis occurs.

Hemagglutination reaction (HRA)

In laboratory practice, two hemagglutination reactions that differ in their mechanism of action are used.

In one case, the hemagglutination reaction is classified as serological. In this reaction, red blood cells are agglutinated when interacting with appropriate antibodies (hemagglutinins). The reaction is widely used to determine blood group.

In another case, the hemagglutination reaction is not serological.

In it, the gluing of red blood cells is caused not by antibodies, but by special substances (hemagglutinins) formed by viruses. For example, the influenza virus agglutinates chicken red blood cells, and the polio virus agglutinates monkey red blood cells. This reaction makes it possible to judge the presence of a particular virus in the material under study.

The results of the reaction are taken into account by the location of the red blood cells. If the result is positive, the red blood cells are arranged loosely, lining the bottom of the tube in the form of an “inverted umbrella.” If the result is negative, the red blood cells settle to the bottom of the tube in a compact sediment (“button”).

Hemagglutination inhibition reaction (HAI)

This is a serological reaction in which specific antiviral antibodies, interacting with the virus (antigen), neutralize it and deprive it of the ability to agglutinate red blood cells, i.e. inhibit the hemagglutination reaction.

The high specificity of the agglutination inhibition reaction allows it to be used to determine the type and type of viruses or to identify specific antibodies in the test serum.

Immunofluorescence reaction (RIF)

The reaction is based on the fact that immune sera, to which fluorochromes are chemically attached, when interacting with the corresponding antigens, form a specific luminous complex, visible in a fluorescent microscope. Serums treated with fluorochromes are called luminescent.

The method is highly sensitive, simple, and does not require isolation of a pure culture, because microorganisms are detected directly in the test material. The result can be obtained 30 minutes after applying the luminescent serum to the preparation.

The immune fluorescence reaction is used for the rapid diagnosis of many infections.

In laboratory practice, two types of immunofluorescence reaction are used: direct and indirect.

The direct method is when the antigen is immediately treated with immune fluorescent serum.

The indirect method of immune fluorescence consists in the fact that the drug is initially treated with ordinary (non-fluorescent) immune diagnostic serum specific to the desired antigen. If the preparation contains an antigen specific to a given diagnostic serum, then an “antigen-antibody” complex is formed, which cannot be seen. If this preparation is additionally treated with luminescent serum containing specific antibodies to serum globulins in the “antigen-antibody” complex, the adsorption of luminescent antibodies onto the globulins of the diagnostic serum will occur and, as a result, the luminescent contours of the microbial cell can be seen in a luminescent microscope.

Immobilization reaction (RI)

The ability of immune serum to cause immobilization of motile microorganisms is associated with specific antibodies that manifest their effect in the presence of complement. Immobilizing antibodies have been found in syphilis, cholera and some other infectious diseases.

This served as the basis for the development of the treponema immobilization test, which in its sensitivity and specificity is superior to other serological tests used in the laboratory diagnosis of syphilis.

Virus neutralization reaction (VRN)

Antibodies that can neutralize the infectious properties of the virus are found in the blood serum of people who have been immunized or have had a viral disease. These antibodies are detected by mixing serum with the corresponding virus and then introducing this mixture into the body of susceptible laboratory animals or infecting a cell culture. Based on the survival of animals or the absence of the cytopathic effect of the virus, the neutralizing ability of antibodies is judged.

This reaction is widely used in virology to determine the type or type of virus and the titer of neutralizing antibodies.

Modern methods for diagnosing infectious diseases include the immunofluorescence method for detecting antigens and antibodies, radioimmunoenzyme immunoassay method, immunoblotting method, detection of antigens and antibodies using monoclonal antibodies, method of detecting antigens using polymerase chain reaction (PCR - diagnostics), etc.

Biochemical properties mostly typical for the genus Salmonella Distinctive features are: the absence of gas formation during fermentation of S. Typhi, the inability of S. Paratyphi A to produce hydrogen sulfide and decarboxylate lysine.

Epidemiology.Typhoid fever and paratyphoid fever are anthroponoses, i.e. cause disease only in humans. The source of infection is the patient or the bacteria carrier, who release the pathogen into the external environment with feces, urine, and saliva. The causative agents of these infections, like other salmonellae, are stable in the external environment and persist in soil and water. S. Typhi can become uncultivable. A favorable environment for their reproduction is food products (milk, sour cream, cottage cheese, minced meat, jelly). The pathogen is transmitted by water, which currently plays a significant role, as well as by nutritional and household contact routes. The infecting dose is approximately 1000 cells. The natural susceptibility of people to these infections is high.

Pathogenesis and clinical picture. Once in the small intestine, typhoid and paratyphoid pathogens invade the mucous membrane when

with the help of effector proteins TTSS-1, forming the primary focus of infection in Peyer's patches. It should be noted that in the submucosa the osmotic pressure is lower compared to the intestinal lumen. This promotes intensive synthesis of Vi-antigen, which increases the antiphagocytic activity of the pathogen and suppresses the release of proinflammatory tissue mediators by submucosal cells. The consequence of this is the lack of development of inflammatory diarrhea in the initial stages of infection and the intensive proliferation of microbes in macrophages, leading to inflammation of Peyer's patches and the development of lymphadenitis, resulting in a violation of the barrier function of the mesenteric lymph nodes and the penetration of salmonella into the blood, resulting in the development of bacteremia. This coincides with the end of the incubation period, which lasts 10-14 days. During bacteremia, which accompanies the entire febrile period, pathogens of typhoid and paratyphoid fever spread throughout the body through the bloodstream, settling in the reticuloendothelial elements of parenchymal organs: liver, spleen, lungs, as well as in the bone marrow, where they multiply in macrophages. From the Kupffer cells of the liver, salmonella enter the gallbladder through the bile ducts, into which they diffuse, into the gallbladder, where they also multiply. Accumulating in the gallbladder, salmonella cause inflammation and reinfect the small intestine with a flow of bile. The repeated introduction of Salmonella into Peyer's patches leads to the development of hyperergic inflammation in them according to the Arthus phenomenon, their necrosis and ulceration, which can lead to intestinal bleeding and perforation of the intestinal wall. The ability of typhoid and paratyphoid pathogens to persist and multiply in phagocytic cells when the latter are functionally insufficient leads to the formation of bacterial carriage. Salmonella can also persist for a long time in the gallbladder, excreted in feces for a long time, and contaminate the environment. By the end of the 2nd week of the disease, the pathogen begins to be excreted from the body in urine, sweat, and breast milk. Diarrhea begins at the end of the 2nd or beginning of the 3rd week of the disease, from this time the pathogens are sown from the feces.

Bacteria can do everything and a little more. They created our world - breathable air, fertile soil, minerals. Even the emergence of life on Earth is the result of such properties of bacteria as variability, the ability to carefully select and inherit genetic information aimed at preserving and developing the species.

A property is a distinctive feature, a characteristic feature of an object or object. Microbiology studies the properties of microorganisms - their structure, patterns of development, role in maintaining the natural balance and human economic activity.

When studying single-celled organisms, the first stage of identification is based on the general properties of bacteria inherent in all prokaryotes (nuclear-free cells):

• microscopic dimensions (not visible to the naked eye);
• enormous speed of metabolism and, as a consequence, growth and reproduction;
• rapid adaptation to changed living conditions;
• the ability to change in a short time with the transmission of heredity;

Another feature common to all unicellular organisms is wide distribution. Microorganisms exist everywhere - in water, air, earth, human and animal bodies. The boundary conditions of their habitat range from temperatures of hundreds of degrees and water pressure at a depth of several kilometers to rarefied air and subzero temperatures in the stratosphere. True, curious researchers have found a place on earth where it is not so easy to find bacteria - certain areas of the Atacama Desert (South America). This land has not seen rain for decades, perhaps hundreds, of years. Even the bacteria gave in - water is necessary for any form of protein life.

Identification of bacteria by species

Scientists divide bacteria by type, or rather, they are trying to do so. Presumably (well, science doesn’t know for sure!) there are millions of species of bacterial cells. But science can “recognize by sight” only a few tens of thousands, the characteristics of which have been well studied. For example, bifidobacteria and lactobacilli are necessary for digestion, the properties of lactic acid bacteria and yeast are used in industry, pathogenic microorganisms carry diseases or cause food poisoning by producing dangerous toxins, etc.

To identify the species of bacteria, you need to know their following properties:

• morphological (shape, cell structure);
• cultural (method of nutrition, reproduction conditions, i.e. growth factors of bacterial culture);
• tinctorial (a reaction to dyes that helps determine the degree of health hazard);
• biochemical (breakdown of nutrients, release of waste products, synthesis of enzymes, proteins, vitamins);
• antigenic (from the English antibody-generator - “antibody producer”), causing an immune response in the body.

Morphological properties are determined using microscopy (examined through a conventional or electron microscope). Cultural (biological) properties appear during the growth of crops on nutrient media. Identification by biochemical properties is needed to determine the cell’s relationship to oxygen (respiration method), its enzymatic and reducing (reductive) properties (reduction is the chemical process of taking away oxygen or replacing it with hydrogen). In addition, biochemical research studies the production of bacterial waste products (toxins) and their impact on the environment.

Analysis of all these properties together helps determine the type of bacterial cell. Such identification makes it possible to distinguish “good” bacteria that are beneficial from harmful pathogens with negative properties. Strictly speaking, this division is quite arbitrary. The same type of bacteria can have a positive or negative effect depending on the situation. For example, E. coli is part of the microflora of a healthy person and takes an active part in digestion. But once the population of these bacteria grows above the limit parameters, there is a danger of poisoning with toxins that are hazardous to health.

What do bacteria look like?

The appearance and parameters of the cell affect its properties - mobility, functional features, attachment to the surface. According to their form, microorganisms are divided into:

1. Cocci are spherical or round bacteria. They differ in the number of cells in the linkage:

• micrococci (single cell);
• diplococci (two cells connected to each other);
• tetracocci (four connected cells);
• streptococci (connected in length in the form of a chain);
• sarcinas (layers or packages of 8, 12, 16 or more pieces);
• staphylococci (the compound has the shape of a grape bunch).

2. Sticks are distinguished:

• according to the shape of the ends: flat (chopped off), rounded (hemisphere), sharp (cone), thickened;
• by the nature of the connection: single, pairs, chains (streptobacteria).

3. Spirals have a curved or spiral shape (strictly speaking, these bacteria are also classified as rod-shaped). They are distinguished by the shape and number of curls:

• vibrios – slightly curved;
• spirilla - one or more turns (up to four);
• more than four curls have borelli (from 4 to 12) and (Dr. Bykov’s favorite curse, the causative agents of syphilis) treponema (from 14 to 17 small coils);
• Leptospira resembles the Latin "S".

In addition, there are stars, cubes, C-shaped and other cell shapes. Moreover, the same type of bacteria, depending on the circumstances, can change shape, and significantly. For example, lactic acid bacteria are rods, but some members of the species can have the shape of a very short rod (almost a ball), while others extend in length, approaching filamentous cells. The length in this case depends on the composition of the medium, the presence and percentage of oxygen, and the method of cultivation (artificial cultivation) of microorganisms.

It’s a little easier with the size of single-celled organisms:

• the smallest (brucella);
• medium (bacteroid, Escherichia coli);
• large (bacillus, clostridia).

Structure of microorganisms

What all prokaryotes have in common is the absence of a nucleus; its role is played by a closed DNA molecule (nucleoid). The role of internal organs in a bacterial cell is performed by various inclusions, called by analogy organelles. This set is not the same for different types of bacteria, but there is a certain mandatory minimum present in every bacterium:

• nucleoid (analogous to the nucleus);
• cell wall (outer layer of varying thickness);
• cytoplasmic membrane (thin film between the internal semi-fluid medium and the cell wall);
• cytoplasm (internal semi-liquid substance in which organelles float);
• ribosomes (RNA molecules containing additional or reserve genetic information).

The first attempts to examine the structure of bacteria through a microscope revealed one important detail - bacterial cells are transparent, it is impossible to see them without additional preparation. Danish researcher Gram proposed a method that allows microorganisms to be stained using aniline dyes. It turned out that, depending on the structure of the outer shell, bacteria perceive the dye differently - some retain the pigment, others become discolored after the final washing of the prepared preparation with an alcohol-containing solution (washing is carried out in both cases, but only in one case it washes out the dye). Based on the thickness of their cell walls, bacteria are divided into two large groups:

• gram-positive (thick wall is stainable);
• gram-negative (the thin wall does not retain the dye).

These properties are important for identification - most often harmful (pathogenic) microorganisms are gram-negative. This division is especially useful for medical research. You can get quick results with a relatively simple laboratory test.

In addition to the main ones, microorganisms have additional structures that determine some important properties of the cell:

1. Capsule is a superficial (above the cell membrane) mucous layer formed as a reaction to the environment. That is, in comfortable conditions, the bacterium can easily do without a capsule, but at the slightest threat it protects itself with a soft shell, which provides additional safety.
2. Flagella are long (longer than the body of the bacterium) thread-like organs of movement. They work like a kind of engine, allowing the cell to move freely.
3. Pili are very small villi on the surface of the bacterium (thinner and shorter than flagella). The pili do not move the cell, but help it securely attach to the chosen location.
4. Spores are solid inclusions formed inside bacteria as a reaction to the threat of death (lack of water, aggressive environment). They allow the cell to survive difficult times (sometimes a bacterium can “sleep” for years or decades) and be reborn again. But spores are only a tool for survival, not reproduction.

There are also additional inclusions that give the bacteria different properties. Thus, chlorosomes are responsible for the production of oxygen from the energy of sunlight (photosynthesis); gas vacuoles give the cell buoyancy; lipids and volutin preserve food and energy reserves, etc.

Growth and reproduction

For accurate identification and industrial production, pure cultures of bacteria are required - a population grown from a single cell in laboratory conditions. And for this you need to know their biological properties - under what conditions and how microorganisms grow and reproduce. Growth is an increase in cell mass and all its structures, and reproduction is an increase in the number of cells in a colony.

Most bacteria reproduce by binary fission, i.e. the cell divides in two down the middle, forming two identical organisms. The budding method differs from binary fission only in form - a protrusion is formed on the surface of the cell, where half of the divided nuclear substitute (nucleoid) moves, then the protrusion grows and separates from the mother cell.

A more complex method is genetic recombination, which is similar to sexual reproduction. The essence of the method is that part of the DNA enters the cell from the outside (through contact of bacteria with each other, with the help of bacteriophages, or as a result of the absorption of genetic material from dead cells). As a result, this method produces two genetically modified cells that carry information from both “parents”. The properties of a modified cell may differ significantly from its predecessors. This method of reproduction allows bacteria to adapt to changing conditions, and perhaps it served as the basis for the emergence of intelligent life on the planet.

In addition, the recombinant propagation method facilitates genetic research. Bacteria change in a very short time and at the same time retain heredity. This makes it possible to follow several generations of a cell and evaluate positive and negative changes in its structure, behavior, and properties.

Features of cell respiration and nutrition

Depending on their relationship to oxygen, bacteria differ into:

1. Anaerobes are microorganisms that obtain energy in the absence of oxygen. There are obligate (strict) anaerobes, which do not tolerate oxygen, and facultative anaerobes (most pathogenic microbes), the main method of obtaining energy is the oxygen-free version, but they can also exist with access to oxygen.
2. Aerobes are cells that live only in an oxygen-containing environment. Strict aerobes require 20% oxygen in the atmosphere, microaerophiles are content with much less oxygen, but their basic method of respiration remains the same as that of aerobic cells.

Identification by breathing and feeding methods is important for creating comfortable conditions when growing bacterial cultures in artificial media and in biotechnologies.

Thanks to the multidirectional beneficial properties of bacteria, a closed cycle is obtained - autotrophs create organic substances using the energy of the sun or inorganic compounds, heterotrophs (saprophytes) decompose organic matter, returning chemical components to nature that are suitable for further use.

Enzymes and bacterial toxins (biochemical activity)

Microorganisms produce protein substances - enzymes (Latin “sourdough”) or enzymes (Greek “ferment”), which serve as catalysts (accelerators) in absolutely all biological processes (metabolism and energy). Moreover, each individual enzyme is responsible for only one process of converting one compound into another. Enzymes are divided into:

• endoenzymes are intracellular substances that take part in cell metabolism.
• exoenzymes are extracellular (released into the environment), they carry out digestion from outside the bacterial cell.

The properties of microorganisms to secrete certain enzymes are used to identify the type of single-celled organism, since this is a constant and unchanging feature inherent only to this type of cell. There are:

1. Saccharolytic properties of a cell are the ability to ferment (decompose) carbohydrates with the release of chemical energy. For example, during alcoholic fermentation, yeast enzymes decompose sugar into ethyl alcohol and carbon dioxide.
2. Proteolytic properties of microorganisms - fermentation of proteins and peptone (large protein fragments formed at the initial stage of digestion of milk and meat under the action of enzymes). Cells release proteolytic enzymes into the external environment, which break down proteins into intermediate products (peptones, amino acids) and/or into final breakdown products (hydrogen sulfide, ammonia). The absorption of proteins and blood clotting depend on proteolytic enzymes.

Biochemical identification makes it possible to distinguish between almost identical species of bacteria, the structure and appearance of which are indistinguishable from each other. For example, there are hundreds of species of pathogenic enterobacteria; the specific culprit of the disease can only be determined by studying its biochemical properties.

Harmful waste products of the cell (toxins) are extremely dangerous, but nevertheless important. When toxins enter the body, antibodies are produced that identify and neutralize foreign objects. Bacterial toxins cause disturbances in metabolic and other processes in the cell, this explains their high activity even with a small amount of toxin in the body. There are:

• exotoxins (released into the environment, very dangerous);
• endotoxins (structural components of the cell, released into the environment only after the death of the bacterium, less dangerous than exotoxins).

All toxins are dangerous, but exotoxins cause more harm. However, the ability of these toxins to induce the formation of antibodies (antigens) makes it possible to produce therapeutic and preventive serums against many diseases.

Some bacteria have hemolytic properties, that is, they secrete toxins that destroy red blood cells (hemolysins). In the natural process of red blood cell renewal, the hemolytic properties of cells are necessary, but they can become dangerous if the process develops pathologically.

Bacteria are ubiquitous and diverse. There are “good”, beneficial microorganisms, but there are also harmful, pathogenic microbes that cause diseases and release dangerous toxins. Man has learned to use the beneficial properties of microorganisms in biotechnology to improve the quality of life. Medicine actively (and sometimes effectively) fights pathogens. It is within the power of any person to protect himself from harmful bacteria (usual hygiene rules) and take the best from the diversity of the bacterial world.

Isolation of microorganisms from various materials and obtaining their cultures is widely used in laboratory practice for microbiological diagnosis of infectious diseases, in research work and in the microbiological production of vaccines, antibiotics and other biologically active products of microbial life.

Cultivation conditions also depend on the properties of the relevant microorganisms. Most pathogenic microbes are grown on nutrient media at a temperature of 37°C for 12 days. However, some of them require longer periods. For example, whooping cough bacteria - in 2-3 days, and mycobacterium tuberculosis - in 3-4 weeks.

To stimulate the processes of growth and reproduction of aerobic microbes, as well as to reduce the time required for their cultivation, the method of deep cultivation is used, which consists of continuous aeration and mixing of the nutrient medium. The deep method has found wide application in biotechnology.

For the cultivation of anaerobes, special methods are used, the essence of which is to remove air or replace it with inert gases in sealed thermostats - anaerobes. Anaerobes are grown on nutrient media containing reducing substances (glucose, sodium formic acid, etc.) that reduce the redox potential.

In diagnostic practice, pure cultures of bacteria that are isolated from the test material taken from a patient or environmental objects are of particular importance. For this purpose, artificial nutrient media are used, which are divided into basic, differential diagnostic and elective media of the most diverse composition. The choice of nutrient medium for isolating a pure culture is essential for bacteriological diagnostics.

In most cases, solid nutrient media are used, previously poured into Petri dishes. The test material is placed in a loop on the surface of the medium and rubbed with a spatula to obtain isolated colonies grown from a single cell. Reseeding an isolated colony on a slanted agar medium in a test tube results in a pure culture.

For identification, i.e. To determine the generic and species affiliation of an isolated crop, phenotypic characteristics are most often studied:

a) morphology of bacterial cells in stained smears or native preparations;

b) biochemical characteristics of the culture according to its ability to ferment carbohydrates (glucose, lactose, sucrose, maltose, mannitol, etc.), to form indole, ammonia and hydrogen sulfide, which are products of the proteolytic activity of bacteria.

For a more complete analysis, gas-liquid chromatography and other methods are used.

Along with bacteriological methods, immunological research methods, which are aimed at studying the antigenic structure of the isolated culture, are widely used to identify pure cultures. For this purpose, serological reactions are used: agglutanation, immunofluorescence precipitation, complement fixation, enzyme immunoassay, radioimmune methods, etc.

Methods for isolating pure culture

In order to isolate a pure culture of microorganisms, it is necessary to separate the numerous bacteria that are found in the material from one another. This can be achieved using methods that are based on two principles − mechanical And biological separation of bacteria.

Methods for isolating pure cultures based on the mechanical principle

Serial dilution method , proposed by L. Pasteur, was one of the very first, which was used for the mechanical separation of microorganisms. It consists of carrying out successive serial dilutions of material that contains microbes in a sterile liquid nutrient medium. This technique is quite painstaking and imperfect in operation, since it does not allow controlling the number of microbial cells that enter the test tubes during dilutions.

Doesn't have this drawback Koch method (plate dilution method ). R. Koch used solid nutrient media based on gelatin or agar-agar. Material with associations of different types of bacteria was diluted in several test tubes with melted and slightly cooled gelatin, the contents of which were later poured onto sterile glass plates. After the medium had gelled, it was cultivated at the optimal temperature. Isolated colonies of microorganisms formed in its thickness, which can easily be transferred to a fresh nutrient medium using a platinum loop to obtain a pure culture of bacteria.

Drigalski method is a more advanced method that is widely used in everyday microbiological practice. First, the material to be tested is applied to the surface of the medium in a Petri dish using a pipette or loop. Using a metal or glass spatula, thoroughly rub it into the medium. The cup is kept open during sowing and gently rotated to evenly distribute the material. Without sterilizing the spatula, apply it to the material in another Petri dish, and if necessary, in a third one. Only after this is the spatula dipped in a disinfectant solution or fried in a burner flame. On the surface of the medium in the first cup, we usually observe continuous growth of bacteria, in the second - dense growth, and in the third - growth in the form of isolated colonies.

Colonies using the Drigalsky method

Line seeding method Today it is most often used in microbiology laboratories. The material that contains microorganisms is collected with a bacteriological loop and applied to the surface of the nutrient medium near the edge of the dish. Remove excess material and apply it in parallel strokes from edge to edge of the cup. After a day of incubation of the crops at the optimal temperature, isolated colonies of microbes grow on the surface of the dish.

Stroke method

To obtain isolated colonies, you can use a swab used to collect the test material. Open the Petri dish with the nutrient medium slightly, insert a tampon into it and carefully rub the material into the surface of the dish, gradually returning the tampon and the dish.

Thus, a significant advantage of the Koch, Drygalski plate dilution and streak culture methods is that they create isolated colonies of microorganisms, which, when inoculated onto another nutrient medium, turn into a pure culture

Methods for isolating pure cultures based on biological principles

The biological principle of bacterial separation involves a focused search for methods that take into account the numerous characteristics of microbial cells. Among the most common methods are the following:

1. By type of breathing. All microorganisms according to the type of respiration are divided into two main groups: aerobic (Corynebacterium diphtheriae, Vibrio сholeraeetc) And anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringensand etc.). If the material from which anaerobic pathogens are to be isolated is preheated and then cultivated under anaerobic conditions, then these bacteria will grow.

2. By sporulation . It is known that some microbes (bacillus and clostridia) are capable of sporulation. Among them Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Bacillus subtilis, Bacillus cereus. The spores are resistant to environmental factors. Consequently, the material under study can be subjected to the action of a thermal factor, and then inoculatively transferred into a nutrient medium. After some time, exactly those bacteria that are capable of sporulation will grow on it.

3. Resistance of microbes to acids and alkalis. Some microbes (Mycobacterium tuberculosis, Mycobacterium bovis) As a result of the peculiarities of their chemical structure, they are resistant to acids. That is why the material that contains them, for example, sputum from tuberculosis, is pre-treated with an equal volume of 10% sulfuric acid solution and then sown on nutrient media. Foreign flora dies, and mycobacteria grow as a result of their resistance to acids.

Vibrio cholerae (Vibrio сholerae) , on the contrary, is a halophilic bacterium, therefore, to create optimal growth conditions, it is sown on media that contain alkali (1% alkaline peptone water). Within 4-6 hours, characteristic signs of growth appear on the surface of the medium in the form of a delicate bluish film.

4. Motility of bacteria. Some microbes (Proteus vulgaris) have a tendency to creeping growth and are able to quickly spread over the surface of some moist environment. To isolate such pathogens, they are inoculated into a droplet of condensation liquid, which is formed when a column of slanted agar is cooled. After 16-18 years they spread to the entire surface of the medium. If we take material from the top of the agar, we will have a pure culture of pathogens.

5. The sensitivity of microbes to the action of chemicals, antibiotics and other antimicrobial agents. As a result of the metabolic characteristics of bacteria, they may have different sensitivity to certain chemical factors. It is known that staphylococci, aerobic bacilli that form spores, are resistant to the action of 7.5–10% sodium chloride. That is why, to isolate these pathogens, selective nutrient media (yolk-salt agar, mannitol-salt agar) that contain this particular substance are used. Other bacteria practically do not grow at this concentration of sodium chloride.

6. Administration of certain antibiotics (nystatin) is used to inhibit the growth of fungi in material that is heavily contaminated with them. Conversely, adding the antibiotic penicillin to the medium promotes the growth of bacterial flora if fungi are to be isolated. The addition of furazolidone in certain concentrations to the nutrient medium creates selective conditions for the growth of corynebacteria and micrococci.

7. The ability of microorganisms to penetrate intact skin. Some pathogenic bacteria (Yersinia pestis) As a result of the presence of a large number of aggressive enzymes, they are able to penetrate through intact skin. To do this, the hair on the body of a laboratory animal is shaved and the test material, which contains a pathogen and a large amount of third-party microflora, is rubbed into this area. After some time, the animal is slaughtered, and microbes are isolated from the blood or internal organs.

8. Sensitivity of laboratory animals to pathogens of infectious diseases. Some animals exhibit high sensitivity to various microorganisms.

For example, with any method of administration Streptococcus pneumoniae white mice develop a generalized pneumococcal infection. A similar picture is observed when Guinea pigs are infected with tuberculosis pathogens. (Mycobacterium tuberculosis) .

In everyday practice, bacteriologists use such concepts as strain And pure culture microorganisms. A strain refers to microbes of the same species that are isolated from different sources, or from the same source, but at different times. A pure culture of bacteria is microorganisms of one species, descendants of one microbial cell, which grew on (in) a nutrient medium.

Isolation of pure culture aerobic microorganisms consists of a number of stages.

First day (Stage 1 of the study) Pathological material is collected into a sterile container (test tube, flask, bottle). It is studied - appearance, consistency, color, smell and other signs, a smear is prepared, painted and examined under a microscope. In some cases (acute gonorrhea, plague), at this stage it is possible to make a preliminary diagnosis, and in addition, select the media on which the material will be inoculated. Then it is carried out with a bacteriological loop (used most often), using a spatula - the Drigalsky method, and a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimal temperature (37 ° C) for 18-48 hours. The purpose of this stage is to obtain isolated colonies of microorganisms.

However, sometimes, in order to accumulate material, it is sown on liquid nutrient media.

On the second day (Stage 2 of the study) On the surface of a dense nutrient medium, microorganisms form continuous, dense growth or isolated colonies. The colony– these are accumulations of bacteria visible to the naked eye on the surface or in the thickness of the nutrient medium. As a rule, each colony is formed from the descendants of one microbial cell (clones), therefore their composition is quite homogeneous. The growth characteristics of bacteria on nutrient media are a manifestation of their cultural properties.

The plates are carefully examined and isolated colonies that have grown on the surface of the agar are studied. Pay attention to the size, shape, color, nature of the edges and surface of the colonies, their consistency and other characteristics. If necessary, examine the colonies under a magnifying glass, low or high magnification microscope. The structure of the colonies is examined in transmitted light at low microscope magnification. They can be hyaline, granular, filamentous or fibrous, which are characterized by the presence of intertwined filaments in the thickness of the colonies.

Characteristics of colonies is an important part of the work of a bacteriologist and laboratory assistant, because microorganisms of each species have their own special colonies.

On the third day (Stage 3 of the study) study the growth pattern of a pure culture of microorganisms and carry out its identification.

First, they pay attention to the characteristics of the growth of microorganisms on the medium and make a smear, staining it with the Gram method, in order to check the culture for purity. If bacteria of the same type of morphology, size and tinctorial (ability to stain) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, just by the appearance and characteristics of their growth, one can draw a conclusion about the type of pathogens isolated. Determining the type of bacteria by their morphological characteristics is called morphological identification. Determining the type of pathogens based on their cultural characteristics is called cultural identification.

However, these studies are not enough to make a definitive conclusion about the type of microbes isolated. Therefore, the biochemical properties of bacteria are studied. They are quite diverse.

Identification of bacteria.

Determining the type of pathogen by its biochemical properties is called biochemical identification.

In order to establish the species of bacteria, their antigenic structure is often studied, that is, identification is carried out by antigenic properties. Each microorganism contains different antigenic substances. In particular, representatives of the Enterobacteriaceae family (Escherichia, Salmonella, Shigela) contain envelope O-antigen, flagellar H-antigen and capsular K-antigen. They are heterogeneous in their chemical composition, therefore they exist in many variants. They can be determined using specific agglutinating sera. This determination of the type of bacteria is called serological identification.

Sometimes identification of bacteria is carried out by infecting laboratory animals with a pure culture and observing the changes that pathogens cause in the body (tuberculosis, botulism, tetanus, salmonellosis, etc.). This method is called identification by biological properties. The objects most often used are Guinea pigs, white mice and rats.

APPLICATIONS

(tables and diagrams)

Physiology of bacteria

Scheme 1. Physiology of bacteria.

reproduction

growing on nutrient media

Table 1. General table of bacterial physiology.

		Characteristic
		The process of acquiring energy and substances.
		A set of biochemical processes that result in the release of energy necessary for the life of microbial cells.
		Coordinated reproduction of all cellular components and structures, ultimately leading to an increase in cell mass
	Reproduction	Increasing the number of cells in a population
	Growing on nutrient media.	In laboratory conditions, microorganisms are grown on nutrient media, which must be sterile, transparent, moist, contain certain nutrients (proteins, carbohydrates, vitamins, microelements, etc.), have a certain buffering capacity, have an appropriate pH, and redox potential.

Table 1.1 Chemical composition and physiological functions of elements.

	composition element		Characteristics and role in cell physiology.
		The main component of a bacterial cell, accounting for about 80% of its mass. It is in a free or bound state with the structural elements of the cell. In spores, the amount of water decreases to 18.20%. Water is a solvent for many substances, and also plays a mechanical role in providing turgor. During plasmolysis—the loss of water by a cell in a hypertonic solution—protoplasm is detached from the cell membrane. Removing water from the cell and drying it out stop metabolic processes. Most microorganisms tolerate drying well. When there is a lack of water, microorganisms do not multiply. Drying in a vacuum from a frozen state (lyophilization) stops reproduction and promotes long-term preservation of microbial individuals.
		40 – 80% dry weight. They determine the most important biological properties of bacteria and usually consist of combinations of 20 amino acids. The bacteria contain diaminopimelic acid (DAP), which is absent in human and animal cells. Bacteria contain more than 2,000 different proteins, located in their structural components and involved in metabolic processes. Most proteins have enzymatic activity. Proteins of a bacterial cell determine the antigenicity and immunogenicity, virulence, and species of bacteria.
	composition element	Characteristics and role in cell physiology.
	Nucleic acids	They perform functions similar to nucleic acids of eukaryotic cells: the DNA molecule in the form of a chromosome is responsible for heredity, ribonucleic acids (information, or matrix, transport and ribosomal) are involved in protein biosynthesis.
	Carbohydrates	They are represented by simple substances (mono- and disaccharides) and complex compounds. Polysaccharides are often included in capsules. Some intracellular polysaccharides (starch, glycogen, etc.) are reserve nutrients.
		They are part of the cytoplasmic membrane and its derivatives, as well as the cell wall of bacteria, for example the outer membrane, where, in addition to the biomolecular layer of lipids, there is LPS. Lipids can act as reserve nutrients in the cytoplasm. Bacterial lipids are represented by phospholipids, fatty acids and glycerides. Mycobacterium tuberculosis contains the largest amount of lipids (up to 40%).
	Minerals	Found in the ash after cells are burned. Phosphorus, potassium, sodium, sulfur, iron, calcium, magnesium, as well as microelements (zinc, copper, cobalt, barium, manganese, etc.) are detected in large quantities. They are involved in the regulation of osmotic pressure, pH of the environment, redox potential , activate enzymes, are part of enzymes, vitamins and structural components of microbial cells.

Table 1.2. Nitrogenous bases.

Table 1.2.1 Enzymes

		Characteristic
	Definition	Specific and efficient protein catalysts present in all living cells.
		Enzymes reduce the activation energy, ensuring the occurrence of chemical reactions that without them could only take place at high temperature, excess pressure and other non-physiological conditions unacceptable for a living cell.
		Enzymes increase the rate of reaction by about 10 orders of magnitude, which reduces the half-life of any reaction from 300 years to one second.
		Enzymes “recognize” the substrate by the spatial arrangement of its molecule and the distribution of charges in it. A certain part of the enzymatic protein molecule, its catalytic center, is responsible for binding to the substrate. In this case, an intermediate enzyme-substrate complex is formed, which then decomposes to form the reaction product and free enzyme.
	Varieties	Regulatory (allosteric) enzymes perceive various metabolic signals and change their catalytic activity in accordance with them.	Effector enzymes are enzymes that catalyze certain reactions (more details in Table 1.2.2.)
	Functional activity	The functional activity of enzymes and the rate of enzymatic reactions depend on the conditions in which a given microorganism is located and, above all, on the temperature of the environment and its pH. For many pathogenic microorganisms, the optimal temperature is 37°C and pH 7.2-7.4.

CLASSES OF ENZYME:

microorganisms synthesize various enzymes belonging to all six known classes.

Table 1.2.2. Effector enzyme classes

	Enzyme class	Catalyzes:
	Oxidoreductases	Electron transfer
	Transferases	Transfer of various chemical groups
	Hydrolases	Transfer of functional groups to a water molecule
		Addition of double bond groups and reverse reactions
	Isomerases	Transfer of groups within a molecule to form isomeric forms
		Formation of C-C, C-S, C-O, C-N bonds due to condensation reactions associated with the breakdown of adenosine triphosphate (ATP)

Table 1.2.3. Types of enzymes according to formation in a bacterial cell

		Characteristic	Notes
	Inducible (adaptive) enzymes "substrate induction"	Enzymes whose concentration in the cell increases sharply in response to the appearance of an inducer substrate in the environment. Synthesized by a bacterial cell only if the substrate of this enzyme is present in the medium
	Repressible enzymes	The synthesis of these enzymes is inhibited as a result of excessive accumulation of the reaction product catalyzed by this enzyme.	An example of enzyme repression is the synthesis of tryptophan, which is formed from anthranilic acid with the participation of anthranilate synthetase.
	Constitutive enzymes	Enzymes synthesized regardless of environmental conditions	Glycolytic enzymes
	Multienzyme complexes	Intracellular enzymes combined structurally and functionally	Respiratory chain enzymes localized on the cytoplasmic membrane.

Table 1.2.4. Specific enzymes

	Enzymes	Bacteria identification
	Superoxide dismutase and catalase	All aerobes or facultative anaerobes possess superoxide dismutase and catalase, enzymes that protect the cell from toxic products of oxygen metabolism. Almost all obligate anaerobes do not synthesize these enzymes. Only one group of aerobic bacteria, lactic acid bacteria, are catalase-negative.
	Peroxidase	Lactic acid bacteria accumulate peroxidase, an enzyme that catalyzes the oxidation of organic compounds under the influence of H2O2 (reduced to water).
	Arginine dihydrolase	A diagnostic feature that allows one to distinguish saprophytic Pseudomonas species from phytopathogenic ones.
		Among the five main groups of the family Enterobacteriaceae, only two - Escherichiae and Erwiniae - do not synthesize urease.

Table 1.2.5. Application of bacterial enzymes in industrial microbiology.

	Enzymes	Application
	Amylase, cellulase, protease, lipase	To improve digestion, ready-made enzyme preparations are used, which facilitate the hydrolysis of starch, cellulose, protein and lipids, respectively.
	Yeast invertase	In the manufacture of sweets to prevent crystallization of sucrose
	Pectinase	Used to clarify fruit juices
	Clostridia collagenase and streptococcal streptokinase	Hydrolyze proteins, promote healing of wounds and burns
	Lytic enzymes of bacteria	They are secreted into the environment, act on the cell walls of pathogenic microorganisms and serve as an effective means of combating the latter, even if they are multi-resistant to antibiotics
	Ribonucleases, deoxyribonucleases, polymerases, DNA ligases and other enzymes that specifically modify nucleic acids	Used as a tool in bioorganic chemistry, genetic engineering and gene therapy

Table 1.2.6. Classification of enzymes by localization.

		Localization
	Endoenzymes	In the cytoplasm In the cytoplasmic membrane In the periplasmic space	They function only inside the cell. They catalyze reactions of biosynthesis and energy metabolism.
	Exoenzymes	Released into the environment.	They are released into the environment by the cell and catalyze reactions of hydrolysis of complex organic compounds into simpler ones that are available for assimilation by the microbial cell. These include hydrolytic enzymes, which play an extremely important role in the nutrition of microorganisms.

Table 1.2.7. Enzymes of pathogenic microbes (aggression enzymes)

	Enzymes
	Lecitovitellase Lecithinase	Destroys cell membranes	Inoculation of the test material on the ZhSA nutrient medium Result: a zone of turbidity around the colonies on the LSA.
	Hemolysin	Destroys red blood cells	Inoculation of the test material on a blood agar nutrient medium. Result: a complete zone of hemolysis around the colonies on blood agar.
	Coagulase-positive cultures	Causes blood plasma clotting	Inoculation of the test material on sterile citrated blood plasma. Result: plasma coagulation
	Coagulase-negative cultures	Mannitol production	Sowing mannitol on a nutrient medium under anaerobic conditions. Result: Appearance of colored colonies (in the color of the indicator)
	Enzymes		Formation of some enzymes in vitro
	Hyaluronidase	Hydrolyzes hyaluronic acid - the main component of connective tissue	Inoculation of the test material on a nutrient medium containing hyaluronic acid. Result: in test tubes containing hyaluronidase, no clot formation occurs.
	Neuraminidase	It splits off sialic (neuraminic) acid from various glycoproteins, glycolipids, polysaccharides, increasing the permeability of various tissues.	Detection: reaction for determining antibodies to neuraminidase (RINA) and others (immunodiffusion, immunoenzyme and radioimmune methods).

Table 1.2.8. Classification of enzymes according to biochemical properties.

	Enzymes		Detection
	Saccharolytic	Breakdown of sugars	Differential diagnostic media such as Hiss's environment, Olkenitsky's environment, Endo's environment, Levin's environment, Ploskirev's environment.
	Proteolytic	Protein breakdown	Microbes are inoculated by injection into a column of gelatin and after 3-5 days of incubation at room temperature, the nature of gelatin liquefaction is noted. Proteolytic activity is also determined by the formation of protein decomposition products: indole, hydrogen sulfide, ammonia. To determine them, microorganisms are inoculated into meat-peptone broth.
	Enzymes identified by final products	Formation of alkalis Acid formation Hydrogen sulfide formation Ammonia formation, etc.	To distinguish some types of bacteria from others based on their enzymatic activity, they are used. differential diagnostic environments

Scheme 1.2.8. Enzyme composition.

ENZYME COMPOSITION OF ANY MICROORGANISM:

Determined by its genome

Is a stable sign

Widely used for their identification

Determination of saccharolytic, proteolytic and other properties.

Table 1.3. Pigments

	Pigments	Synthesis by microorganism
	Fat-soluble carotenoid pigments that are red, orange, or yellow.	They form sarcina, mycobacterium tuberculosis, and some actinomycetes. These pigments protect them from UV rays.
	Black or brown pigments - melanins	Synthesized by obligate anaerobes Bacteroides niger and others. Insoluble in water and even strong acids
	A bright red pyrrole pigment called prodigiosin.	Formed by some serata
	Water-soluble phenosine pigment - pyocyanin.	Produced by Pseudomonas bacteria (Pseudomonas aeruginosa). In this case, a nutrient medium with a neutral or alkaline pH turns blue-green.

Table 1.4. Glowing and aroma-producing microorganisms

		Condition and characteristics
	Glow (luminescence)	Bacteria cause the glow of those substrates, such as fish scales, higher fungi, rotting trees, and food products, on the surface of which they multiply. Most luminescent bacteria are halophilic species that can reproduce at elevated salt concentrations. They live in seas and oceans and rarely in fresh water bodies. All luminescent bacteria are aerobes. The luminescence mechanism is associated with the release of energy during the biological oxidation of the substrate.
	Aroma formation	Some microorganisms produce volatile aromatic substances, for example, ethyl acetate and amyl acetate, which add flavor to wine, beer, lactic acid and other food products, and are therefore used in their production.

Table 2.1.1.Metabolism

		Definition
	Metabolism	The biochemical processes occurring in the cell are united by one word - metabolism (Greek metabole - transformation). This term is equivalent to the concept of “metabolism and energy”. There are two sides of metabolism: anabolism and catabolism.
		Anabolism is a set of biochemical reactions that carry out the synthesis of cell components, i.e. that side of metabolism, which is called constructive metabolism.	Catabolism is a set of reactions that provide the cell with energy necessary, in particular, for constructive exchange reactions. Therefore, catabolism is also defined as the energy metabolism of a cell.
	Amphibolism	Intermediate metabolism that converts low molecular weight fragments of nutrients into a series of organic acids and phosphorus esters is called

Scheme 2.1.1. Metabolism

METABOLISM –

a combination of two opposite but interacting processes: catabolism and anabolism

Anabolism= assimilation = plastic metabolism = constructive metabolism

Catabolism= dissimilation = energy metabolism = breakdown = providing energy to the cell

Synthesis (of cell components)

Enzymatic catabolic reactions that result in energy release, which accumulated in ATP molecules.

Biosynthesis of monomers:

amino acids nucleotides monosaccharides fatty acids

Biosynthesis of polymers:

proteins nucleic acids polysaccharides lipids

As a result of enzymatic anabolic reactions, the energy released in the process of catabolism is spent on the synthesis of macromolecules of organic compounds, from which biopolymers are then assembled - components of the microbial cell.

Energy is spent on the synthesis of cell components

Table 2.1.3. Metabolism and transformation of cell energy.

	Metabolism	Characteristic	Notes
		Metabolism ensures the dynamic balance inherent in a living organism as a system, in which synthesis and destruction, reproduction and death are mutually balanced.	Metabolism is the main sign of life
	Plastic exchange Synthesis of proteins, fats, carbohydrates.	This is a set of biological synthesis reactions.	From substances entering the cell from the outside, molecules similar to cell compounds are formed, that is, assimilation occurs.
	Energy exchange	The process is the opposite of synthesis. This is a set of splitting reactions.	When high-molecular compounds are broken down, the energy necessary for the biosynthesis reaction is released, that is, dissimilation occurs. When glucose is broken down, energy is released in stages with the participation of a number of enzymes.

Table 2.1.2. Difference in metabolism for identification.

Table 2.2 Anabolism (constructive metabolism)

Scheme 2.2.2. Biosynthesis of amino acids in prokaryotes.

Scheme 2.2.1. Biosynthesis of carbohydrates in microorganisms.

Figure 2.2.3. Lipid biosynthesis

Table 2.2.4. Stages of energy metabolism - Catabolism.

	Stages	Characteristic	Note
	Preparatory	Molecules of disaccharides and polysaccharides, proteins break down into small molecules - glucose, glycerol and fatty acids, amino acids. Large molecules of nucleic acids into nucleotides.	At this stage, a small amount of energy is released and dissipated as heat.
	Anoxic or incomplete or anaerobic or fermentation or dissimilation.	The substances formed at this stage undergo further breakdown with the participation of enzymes. For example: glucose breaks down into two molecules of lactic acid and two molecules of ATP.	ATP and H 3 PO 4 are involved in the breakdown of glucose. During the oxygen-free breakdown of glucose in the form of a chemical bond in the ATP molecule, 40% of the energy is retained, the rest is dissipated as heat. In all cases of the breakdown of one glucose molecule, two ATP molecules are formed.
	The stage of aerobic respiration or oxygen breakdown.	With oxygen access to the cell, the substances formed during the previous stage are oxidized (broken down) to final products CO 2 AndH 2 O.	The overall equation for aerobic respiration is:

Scheme 2.2.4. Fermentation.

Fermentative metabolism – characterized by the formation of ATP through phosphorylation of substrates.

First (oxidation) = splitting

Second (recovery)

Includes the conversion of glucose to pyruvic acid.

Includes hydrogen utilization to restore pyruvic acid.

Pathways for the formation of pyruvic acid from carbohydrates

Scheme 2.2.5. Pyruvic acid.

Glycolytic pathway (Embden-Meyerhof-Parnas pathway)

Entner-Doudoroff path

Pentose phosphate pathway

Table 2.2.5. Fermentation.

	Fermentation type	Representatives	Final product	Notes
	Lactic acid		Form lactic acid from pyruvate	In some cases (homoenzyme fermentation) only lactic acid is formed, in others also by-products.
	Formic acid	Enterobacteriaceae	Formic acid is one of the final products. (along with it - side effects)	Some types of enterobacteria break down formic acid to H 2 and CO 2/
	Butyric acid		Butyric acid and by-products	Some types of clostridia, along with butyric and other acids, form butanol, acetone, etc. (then it is called acetone-butyl fermentation).
	Propionic acid	Propionobacterium	Form propionic acid from pyruvate	Many bacteria, when fermenting carbohydrates, along with other products, form ethyl alcohol. However, it is not the main product.

Table 2.3.1. Protein synthesis system, ion exchange.

	Item name	Characteristic
	Ribosomal subunits 30S and 50S	In the case of bacterial 70S ribosomes, the 50S subunit contains 23S rRNA (~3000 nucleotides long) and the 30S subunit contains 16S rRNA (~1500 nucleotides long); In addition to the “long” rRNA, the large ribosomal subunit also contains one or two “short” rRNAs (5S rRNA of bacterial ribosomal subunits 50S or 5S and 5.8S rRNA of large ribosomal subunits of eukaryotes). (for more details, see Fig. 2.3.1.)
	Messenger RNA (mRNA)
	A complete set of twenty aminoacyl-tRNAs, the formation of which requires the corresponding amino acids, aminoacyl-tRNA synthetases, tRNA and ATP	This is an amino acid charged with energy and bound to tRNA, ready to be transported to the ribosome and included in the polypeptide synthesized on it.
	Transfer RNA (tRNA)	Ribonucleic acid, the function of which is to transport amino acids to the site of protein synthesis.
	Protein initiation factors	(in prokaryotes - IF-1, IF-2, IF-3) They got their name because they participate in the organization of the active complex (708 complex) of subunits 30S and 50S, mRNA and initiator aminoacyl-tRNA (in prokaryotes - formylmethionyl -tRNA), which “starts” (initiates) the work of ribosomes - the translation of mRNA.
	Protein elongation factors	(in prokaryotes - EF-Tu, EF-Ts, EF-G) Participate in the lengthening (elongation) of the synthesized polypeptide chain (peptidyl). Protein termination or release factors (RF) ensure codon-specific separation of the polypeptide from the ribosome and the end of protein synthesis.
	Item name	Characteristic
	Protein termination factors	(in prokaryotes - RF-1, RF-2, RF-3)
	Some other protein factors (associations, subunit dissociations, release, etc.).	Protein translation factors necessary for the functioning of the system
	Guanosine triphosphate (GTP)	To carry out translation, the participation of GTP is necessary. The requirement of the protein synthesizing system for GTP is very specific: it cannot be replaced by any of the other triphosphates. The cell spends more energy on protein biosynthesis than on the synthesis of any other biopolymer. The formation of each new peptide bond requires the cleavage of four high-energy bonds (ATP and GTP): two in order to load the tRNA molecule with an amino acid, and two more during elongation - one during aa-tRNA binding and the other during translocation.
	Inorganic cations in a certain concentration.	To maintain the pH of the system within physiological limits. Ammonium ions are used by some bacteria to synthesize amino acids, and potassium ions are used to bind tRNA to ribosomes. Iron and magnesium ions act as a cofactor in a number of enzymatic processes

Figure 2.3.1. Schematic representation of the structures of prokaryotic and eukaryotic ribosomes.

Table 2.3.2. Features of ion exchange in bacteria.

	Peculiarity	Characterized by:
	High osmotic pressure	Due to the significant intracellular concentration of potassium ions in bacteria, high osmotic pressure is maintained.
	Iron intake	For a number of pathogenic and opportunistic bacteria (Escherichia, Shigella, etc.), the consumption of iron in the host body is difficult due to its insolubility at neutral and slightly alkaline pH values	Siderophores – special substances that, by binding iron, make it soluble and transportable.
	Assimilation	Bacteria actively assimilate SO2/ and P034+ anions from the environment to synthesize compounds containing these elements (sulfur-containing amino acids, phospholipids, etc.).
		For the growth and reproduction of bacteria, mineral compounds are required - ions NH4+, K+, Mg2+, etc. (for more details, see Table 2.3.1.)

Table 2.3.3. Ion exchange

	Name of mineral compounds	Function
	NH 4 + (ammonium ions)	Used by some bacteria to synthesize amino acids
	K+ (potassium ions)	Used to bind tRNA to ribosomes Maintain high osmotic pressure
	Fe 2+ (iron ions)	Act as cofactors in a number of enzymatic processes Part of cytochromes and other hemoproteins
	Mg 2+ (magnesium ions)
	SO 4 2 - (sulfate anion)	Necessary for the synthesis of compounds containing these elements (sulfur-containing amino acids, phospholipids, etc.)
	PO 4 3- (phosphate anion)

Scheme 2.4.1. Energy metabolism.

To synthesize, bacteria need...

Nutrients

Table 2.4.1. Energy metabolism (biological oxidation).

	Process	Necessary:
	Synthesis of structural components of microbial cells and maintenance of vital processes	Sufficient amount of energy. This need is satisfied through biological oxidation, which results in the synthesis of ATP molecules.
	Energy (ATP)	Iron bacteria receive energy released during the direct oxidation of iron (Fe2+ to Fe3+), which is used to fix CO2; bacteria that metabolize sulfur provide themselves with energy through the oxidation of sulfur-containing compounds. However, the vast majority of prokaryotes obtain energy through dehydrogenation. Energy is also obtained during the breathing process (see the detailed table in the corresponding section).

Scheme 2.4. Biological oxidation in prokaryotes.

Breakdown of polymers into monomers

Carbohydrates

glycerol and fatty acids

amino acids

monosaccharides

Decomposition under oxygen-free conditions

Formation of intermediates

Oxidation under oxygen conditions to final products

Table 2.4.2. Energy metabolism.

	Concept	Characteristic
	The essence of energy metabolism	Providing the energy cells need to manifest life.
		The ATP molecule is synthesized as a result of the transfer of an electron from its primary donor to its final acceptor.
		Respiration is biological oxidation (breakdown). Depending on what is the final electron acceptor, they distinguish breath: Aerobic - in aerobic respiration, the final electron acceptor is molecular oxygen O 2. Anaerobic - the final electron acceptor is inorganic compounds: NO 3 -, SO 3 -, SO 4 2-
	Mobilizing energy	Energy is mobilized in oxidation and reduction reactions.
	Oxidation Reaction	The ability of a substance to donate electrons (oxidize)
	Recovery Response	The ability of a substance to gain electrons.
	Redox potential	The ability of a substance to donate (oxidize) or accept (recover) electrons. (quantitative expression)

Scheme 2.5. Synthesis.

carbohydrates

Table 2.5.1. Synthesis

Table 2.5.1. Synthesis

	Biosynthesis	Of what	Notes
	Biosynthesis of carbohydrates	Autotrophs synthesize glucose from CO 2 . Heterotrophs synthesize glucose from carbon-containing compounds.	Calvin cycle (see diagram 2.2.1.)
	Biosynthesis of amino acids	Most prokaryotes are able to synthesize all amino acids from: Pyruvate α-ketoglutorate fumorate	The energy source is ATP. Pyruvate is formed in the glycolytic cycle. Auxotrophic microorganisms consume ready-made microorganisms in the host’s body.
	Lipid biosynthesis	Lipids are synthesized from simpler compounds - metabolic products of proteins and carbohydrates	Acetyl transfer proteins play an important role. Auxotrophic microorganisms consume ready-made microorganisms in the host body or from nutrient media.

Table 2.5.2. The main stages of protein biosynthesis.

	Stages	Characteristic	Notes
	Transcription	The process of RNA synthesis on genes. This is the process of rewriting information from DNA - gene to mRNA - gene.	It is carried out using DNA-dependent RNA polymerase. The transfer of information about protein structure to ribosomes occurs using mRNA.
	Broadcast (transmission)	The process of self-protein biosynthesis. The process of deciphering the genetic code in mRNA and implementing it in the form of a polypeptide chain.	Because each codon contains three nucleotides, the same genetic text can be read in three different ways (starting at the first, second, and third nucleotides), that is, in three different reading frames.

Note to the table: The primary structure of each protein is the sequence of amino acids in it.

Scheme 2.5.2. Electron transfer chains from the primary donor of hydrogen (electrons) to its final acceptor O 2.

organic matter

(primary electron donor)

Flavoprotein (- 0.20)

Quinone (-0.07)

Cytochrome (+0.01)

Cytochrome C(+0.22)

Cytochrome A(+0.34)

final acceptor

Table 3.1. Classification of organisms by type of nutrition.

	Organogen element	Power types	Characteristic
	Carbon (C)	Autotrophs	The cells themselves synthesize all the carbon-containing components from CO 2 .
		Heterotrophs	They cannot satisfy their needs with CO 2; they use ready-made organic compounds.
		Saprophytes	The food source is dead organic substrates.
			The source of nutrition is living tissues of animals and plants.
		Prototrophs	Meet your needs with atmospheric and mineral nitrogen
		Auxotrophs	They require ready-made organic nitrogen compounds.
	Hydrogen (H)	The main source is H 2 O
	Oxygen (O)

Table 3.1.2. Conversion of energy

Table 3.1.3. Carbon Nutrition Methods

	Energy source	Electron donor	Carbon nutrition method
	Energy from sunlight	Inorganic compounds	Photolithoheterotrophs
		Organic compounds	Photoorganoheterotrophs
	Redox reactions	Inorganic compounds	Chemolithoheterotrophs
		Organic compounds	Chemoorganoheterotrophs

Table 3.2. Power Mechanisms:

	Mechanism	Conditions	Concentration gradient	Energy costs	Substrate specificity
	Passive diffusion	The concentration of nutrients in the environment exceeds the concentration in the cell.	By concentration gradient
	Facilitated diffusion	Permease proteins are involved.	By concentration gradient
	Active transport	Permease proteins are involved.
	Translocation of chemical groups	During the transfer process, chemical modification of nutrients occurs.	Against a concentration gradient

Table 3.3. Transport of nutrients from the bacterial cell.

	Name	Characteristic
	Phosphotransferase reaction	Occurs when the transported molecule is phosphorylated.
	Translational secretion	In this case, the synthesized molecules must have a specific leading sequence of amino acids in order to attach to the membrane and form a channel through which the protein molecules can escape into the environment. In this way, tetanus, diphtheria and other toxins are released from the cells of the corresponding bacteria.
	Membrane budding	Molecules formed in the cell are surrounded by a membrane vesicle, which is released into the environment.

Table 4. Growth.

	Concept	Definition of the concept.
		An irreversible increase in the amount of living matter, most often caused by cell division. If multicellular organisms usually experience an increase in body size, then in multicellular organisms the number of cells increases. But in bacteria, an increase in the number of cells and an increase in cell mass should also be noted.
	Factors influencing bacterial growth in vitro.	Culture media: Mycobacterium leprae is not capable of in vitro Temperature (increasing in range): Mesophilic bacteria (20-40 o C) Thermophilic bacteria (50-60 o C) Psychrophilic (0-10 o C)
	Bacterial growth assessment	Quantification of growth is usually carried out in liquid media where the growing bacteria form a homogeneous suspension. The increase in the number of cells is determined by determining the concentration of bacteria in 1 ml, or the increase in cell mass is determined in weight units per unit volume.

Growth factors

Amino acids

Vitamins

Nitrogenous bases

Table 4.1. Growth factors

		Growth factors	Characteristic	Function
	Amino acids			Many microorganisms, especially bacteria, need certain amino acids (one or more), since they cannot synthesize them on their own. Such microorganisms are called auxotrophic for those amino acids or other compounds that they are not able to synthesize.
	Purine bases and their derivatives		Nucleotides:	They are bacterial growth factors. Some types of mycoplasmas require nucleotides. Required for the construction of nucleic acids.
	Pyrimidine bases and their derivatives		Nucleotides
	Growth factors		Characteristic	Function
			Neutral lipids	Contains membrane lipids
			Phospholipids
			Fatty acid	They are components of phospholipids
			Glycolipids	In mycoplasmas they are part of the cytoplasmic membrane
	Vitamins (mostly group B)		Thiamine (B1)	Staphylococcus aureus, pneumococcus, Brucella
			Nicotinic acid (B3)	All types of rod-shaped bacteria
			Folic acid (B9)	Bifidobacteria and propionic acid
			Pantothenic acid (B5)	Some types of streptococci, tetanus bacilli
			Biotin (B7)	Yeast and nitrogen-fixing bacteria Rhizobium
			Hemes are components of cytochromes	Haemophilus influenzae bacteria, Mycobacterium tuberculosis

Table 5. Breathing.

	Name	Characteristic
		Biological oxidation (enzymatic reactions)
	Base	Respiration is based on redox reactions that occur with the formation of ATP, a universal accumulator of chemical energy.
	Processes	During breathing the following processes occur: Oxidation is the giving away of hydrogen or electrons by donors. Reduction is the addition of hydrogen or electrons to an acceptor.
	Aerobic respiration	The final acceptor of hydrogen or electrons is molecular oxygen.
	Anaerobic respiration	The hydrogen or electron acceptor is an inorganic compound - NO 3 -, SO 4 2-, SO 3 2-.
	Fermentation	Organic compounds are hydrogen or electron acceptors.

Table 5.1. Classification by breathing type.

	Bacteria	Characteristic	Notes
	Strict anaerobes	Energy exchange occurs without the participation of free oxygen. ATP synthesis during glucose consumption under anaerobic conditions (glycolysis) occurs due to phosphorylation of the substrate. Oxygen for anaerobes does not serve as the final electron acceptor. Moreover, molecular oxygen has a toxic effect on them	Strict anaerobes lack the enzyme catalase, so it accumulates in the presence of oxygen and has a bactericidal effect on them; Strict anaerobes lack a system for regulating redox potential (redox potential).
	Strict aerobes	They are able to obtain energy only through breathing and therefore necessarily need molecular oxygen. Organisms that obtain energy and form ATP using only oxidative phosphorylation of the substrate, where only molecular oxygen can act as an oxidizing agent. The growth of most aerobic bacteria stops at oxygen concentrations of 40-50% or higher.	Strict aerobes include, for example, representatives of the genus Pseudomonas
	Bacteria	Characteristic	Notes
	Facultative anaerobes	Grows in both the presence and absence of molecular oxygen Aerobic organisms most often contain three cytochromes, facultative anaerobes - one or two, obligate anaerobes do not contain cytochromes.	Facultative anaerobes include enterobacteria and many yeasts that can switch from respiration in the presence of 02 to fermentation in the absence of 02.
	Microaerophiles	A microorganism that, unlike strict anaerobes, requires for its growth the presence of oxygen in the atmosphere or nutrient medium, but in reduced concentrations compared to the oxygen content in ordinary air or in normal tissues of the host body (unlike aerobes, the growth of which requires normal oxygen content in the atmosphere or nutrient medium). Many microaerophiles are also capnophiles, meaning they require increased concentrations of carbon dioxide.	In the laboratory, such organisms can be easily cultured in a “candle jar.” A “candle jar” is a container into which a burning candle is placed before being sealed with an airtight lid. The candle flame will burn until it is extinguished from lack of oxygen, resulting in a carbon dioxide-rich, oxygen-depleted atmosphere in the jar.

Table 6. Reproduction characteristics.

Scheme 6. Dependence of generation duration on various factors.

Generation duration

Type of bacteria

Population

Temperature

Composition of the nutrient medium

Table 6.1. Bacterial reproduction phases.

	Phase	Characteristic
	Initial stationary phase	Lasts 1-2 hours. During this phase, the number of bacterial cells does not increase.
	Lag phase (phase of delayed reproduction)	It is characterized by the beginning of intensive cell growth, but the rate of their division remains low.
	Log phase (logarithmic)	Characterized by a maximum rate of cell reproduction and an exponential increase in the size of the bacterial population
	Negative acceleration phase	Characterized by less activity of bacterial cells and longer generation period. This occurs as a result of depletion of the nutrient medium, accumulation of metabolic products in it and oxygen deficiency.
	Stationary phase	It is characterized by a balance between the number of dead, newly formed and dormant cells.
	Death phase	Occurs at a constant speed and is replaced by UP-US phases of decreasing rate of cell death.

Scheme 7. Requirements for nutrient media.

Requirements

Viscosity

Humidity

Sterility

Nutritional value

Transparency

Isotonicity

Table 7. Reproduction of bacteria on nutrient media.

	Nutrient medium	Characteristic
	Solid culture media	On solid nutrient media, bacteria form colonies - clusters of cells.
		S- type(smooth – smooth and shiny) Round, with a smooth edge, smooth, convex.	R- type(rough – rough, unequal) Irregular in shape with jagged edges, rough, dented.
	Liquid culture media	Bottom growth (sediment) Surface growth (film) Diffuse growth (uniform cloudiness)

Table 7.1. Classification of nutrient media.

	Classification	Kinds	Examples
	By composition		MPA – meat-peptone agar MPB - meat-peptone broth PV – peptone water
			Blood agar JSA – yolk salt agar Hiss media
	By purpose	Basic
		Elective	Alkaline agar Alkaline peptone water
		Differential - diagnostic	Ploskireva
		Special	Wilson-Blair Kitta-Tarozzi Thioglycol broth Milk according to Tukaev
	By consistency		Blood agar Alkaline agar
		Semi-liquid	Semi-solid agar
	By origin	Natural
		Semi-synthetic
		Synthetic	Simmonson

Table 7.2. Principles of isolating pure cell culture.

Mechanical principle	Biological principle
1. Fractional dilutions of L. Pasteur 2. Plate dilutions of R. Koch 3. Surface crops of Drigalsky 4. Surface strokes	Take into account: a - type of breathing (Fortner method); b - mobility (Shukevich method); c - acid resistance; g - sporulation; d - temperature optimum; e - selective sensitivity of laboratory animals to bacteria

Table 7.2.1. Stages of isolating a pure cell culture.

	Stage	Characteristic
	Stage 1 of the study	Pathological material is collected. It is studied - appearance, consistency, color, smell and other signs, a smear is prepared, painted and examined under a microscope.
	Stage 2 of the study	On the surface of a dense nutrient medium, microorganisms form continuous, dense growth or isolated colonies. The colony– these are accumulations of bacteria visible to the naked eye on the surface or in the thickness of the nutrient medium. As a rule, each colony is formed from the descendants of one microbial cell (clones), therefore their composition is quite homogeneous. The growth characteristics of bacteria on nutrient media are a manifestation of their cultural properties.
	Stage 3 of the study	The growth pattern of a pure culture of microorganisms is studied and its identification is carried out.

Table 7.3. Identification of bacteria.

	Name	Characteristic
	Biochemical identification	Determining the type of pathogen by its biochemical properties
	Serological identification	In order to establish the species of bacteria, their antigenic structure is often studied, that is, identification is carried out by antigenic properties
	Identification by biological properties	Sometimes bacteria are identified by infecting laboratory animals with a pure culture and observing the changes that pathogens cause in the body.
	Cultural identification	Determining the type of pathogens based on their cultural characteristics
	Morphological identification	Determining the type of bacteria by their morphological characteristics

Which process is not related to the physiology of bacteria?

Reproduction

What substances make up 40–80% of the dry mass of a bacterial cell?

Carbohydrates

Nucleic acids

What classes of enzymes are synthesized by microorganisms?

Oxyreductases

All classes

Transferases

Enzymes whose concentration in the cell increases sharply in response to the appearance of an inducer substrate in the environment?

Iducible

Constitutional

Repressive

Multienzyme complexes

Pathogenicity enzyme secreted by Staphylococcus aureus?

Neuraminidase

Hyaluronidase

Lecithinase

Fibrinolysin

Do proteolytic enzymes have a function?

Protein breakdown

Breakdown of fats

Breakdown of carbohydrates

Formation of alkalis

Fermentation of enterobacteria?

Lactic acid

Formic acid

Propionic acid

Butyric acid

What mineral compounds are used to bind tRNA to ribosomes?

Biological oxidation is...?

1. Reproduction

2. Cell death

What substances themselves synthesize all the carbon-containing components of the cell from CO 2.

Prototrophs

Heterotrophs

Autotrophs

Saprophytes

Nutrient media vary:

By composition

By consistency

By purpose

For all of the above

The reproduction phase, which is characterized by a balance between the number of dead, newly formed and dormant cells?

2. Negative acceleration phase

   Stationary phase

The duration of generation depends on?

Age

Populations

All of the above

In order to establish the species identity of bacteria, their antigenic structure is often studied, that is, identification is carried out, which one?

Biological

Morphological

Serological

Biochemical

The Drigalski method of surface seeding is referred to as...?

Mechanical principles of pure culture isolation

Biological principles of isolating pure culture

Bibliography

1. Borisov L. B. Medical microbiology, virology, immunology: a textbook for honey. universities – M.: Medical Information Agency LLC, 2005.

2. Pozdeev O.K. Medical microbiology: a textbook for honey. universities – M.: GEOTAR-MED, 2005.

3. Korotyaev A.I., Babichev S.A. Medical microbiology, immunology and virology / textbook for medical professionals. universities – St. Petersburg: SpetsLit, 2000.

4. Vorobyov A. A., Bykov A. S., Pashkov E. P., Rybakova A. M. Microbiology: textbook. – M.: Medicine, 2003.

5. Medical microbiology, virology and immunology: textbook / ed. V. V. Zvereva, M. N. Boychenko. – M.: GEOTar-Media, 2014.

6. Guide to practical training in medical microbiology, virology and immunology / ed. V.V. Tetsa. – M.: Medicine, 2002.

Introduction 6

Composition of bacteria from the point of view of their physiology. 7

Metabolism 14

Nutrition (nutrient transport) 25

Breathing 31

Reproduction 34

Microbial communities 37

APPLICATIONS 49

References 105

IDENTIFICATION OF MICROBES(Late Lat. identificare to identify) - determination of the species or type of microbes. I. m. is the most important stage of microbiol, research necessary to determine the etiology of an infectious disease; it is of great importance for epidemiol, analysis of outbreaks of infectious diseases and carrying out effective measures to eliminate them. I. m. is also widely used for dignity. assessment of soil, air, water and food.

I. m. is carried out by studying the complex of morphological, cultural, biochemical, antigenic, pathogenic and other properties of a given culture, which makes it possible to establish its identity (identity) to typical representatives of a certain species (type) of microorganisms. For these studies, it is usually necessary to have a pure culture, since the presence of foreign microbes can lead to erroneous conclusions.

The choice of research methods for I. m. is largely determined by the source of isolation of the microbe (for example, material obtained from a patient, from a corpse or environmental objects).

Determination of the properties of microorganisms

There are no general I. m. schemes used in practice. For each group of microorganisms, identification is carried out on the basis of their biol characteristics. Thus, for the identification of viruses (see), the types of cell cultures in which their reproduction occurs, the nature of the cytopathic action, the formation of inclusions, the antigenic structure, in some cases the morphology of the viruses, as well as the pathogenicity of the viruses for experimental animals are important.

The proposal of some researchers deserves attention [Cowan and Steel (S. T. Cowan, K. I. Steel), 1961, 1965; Seeley and Van Demark (H. W. Seeley, V. I. Van Demark), 1972] use Gram staining as the starting point for identifying bacteria. At the first stage of differentiation of gram-positive bacteria, the authors take into account cell shape, acid resistance, spore formation, motility, production of catalase, oxidase, and relationship to glucose, and of gram-negative bacteria - cell shape, motility, production of catalase, oxidase, and relationship to glucose. At subsequent stages of research, using tables characterizing bacteria belonging to a particular genus, they find the key to identifying species, subspecies and types.

Morphological and tinctorial properties

The study of morphol and tinctorial signs of a microbe is usually only the initial stage of its identification. The morphology of microorganisms is studied by microscopy of fixed and stained preparations, as well as living unstained microorganisms in a hanging or crushed drop.

For long-term observation of living bacteria, special cameras are used (Peshkova, Fontbrune). Microscopic examination makes it possible to determine the shape, size and structure of microorganisms, their relative position, motility, number and distribution of flagella, shape and position of spores, as well as the formation of capsules. To study motility, young (no older than 6-8 hours) fast-growing broth cultures are taken. Flagella are more easily detected in young agar cultures, spores, on the contrary, in cultures grown for several days, and capsules in patol and exudates. For hanging drop microscopy, it is better to use a darkfield or phase contrast device. It should be taken into account that the shapes and sizes of microorganisms change depending on the characteristics of the strain, the age of the culture, the composition of the medium, the incubation temperature and other factors.

The tinctorial properties of microbes are determined by staining fixed preparations. Gram staining allows you to divide all bacteria into 2 groups: gram-negative and gram-positive (see Gram method). Ziehl-Neelsen staining makes it possible to differentiate acid-fast bacteria from non-acid-fast bacteria (see Ziehl-Neelsen method). Using special methods, individual elements of a bacterial cell are identified: nucleoid, protoplasm and inclusions (methods of Romanovsky-Giemsa, Feilgen, Robineau, etc.), metachromatic granules (see Neisser methods, etc.), flagella, capsules and spores. The method of fluorescent antibodies makes it possible to preliminary determine the type and even type of microbe (see Immunofluorescence).

In cases where the morphology of a microbe is specific, microscopic examination can presumptively identify it. In honey In microbiology, this kind of identification is justified only when it corresponds to the wedge and diagnosis. So, for example, acid-fast bacilli in the cerebrospinal fluid of a patient with wedge, symptoms of meningitis can be tentatively attributed to tuberculous mycobacteria. Gram-negative bipolar staining ovoid rods in the juice of lymph nodes of a patient with inguinal buboes in areas where plague is widespread can be considered presumably as plague bacteria.

Cultural properties indicate that a microbe belongs to a specific group and outline the direction of further research in order to finally identify it. They are determined by sowing the culture under study on nutrient media (agar, broth, injection into gelatin, etc.). Of the cultural characteristics of bacteria and fungi, the appearance and internal structure of the colonies formed when the culture is sown on solid nutrient media are important. If a microbe does not grow on regular meat peptone agar, then another medium that is optimal for it should be used. Colonies are usually examined after 24 hours of incubation at t° 37°, and then again at intervals of 1 - 3 days. When describing colonies, pay attention to their size, color (pigment formation), shape, profile, surface, edges, density. If bacteria tend to dissociate into phase variants (see Dissociation of bacteria), then they are separated by sieving on Petri dishes with a nutrient medium. When growing on liquid nutrient media, growth is near the bottom, growth in the form of a film, or uniform turbidity of the medium. In some cases, growth is studied on special media, such as Loeffler's serum, glycerin potatoes, media containing blood, etc. The cultural properties of the microbe are an essential addition to its morphological characteristics.

Resistance of microbes to various environmental factors

The resistance of microbes to various environmental factors is used in I. m., since in some cases microbes differ significantly in this characteristic. So, for example, non-spore-bearing bacteria and vegetative forms of spore-bearing bacteria are sensitive to temperature and to low concentrations of antiseptics. They die at a temperature of 60° within half an hour and in a 1% phenol solution within 1 hour. Acid-fast bacteria are temperature sensitive but relatively resistant to disinfectants; they die at a temperature of 60° within half an hour, but in the cold they resist antiseptics often for several hours. Bacterial spores are especially highly resistant (see Spores, bacteria). They die either from steam under pressure (at a temperature of 120° for half an hour) or from high concentrations of antiseptics, for example, under the influence of 5% phenol for several hours. Therefore, if the formation of spores by a microbe is suspected, temperature resistance tests are performed.

For certain types of bacteria, their resistance to certain antibiotics and chemotherapeutic drugs is indicative. So, for example, one of the tests that makes it possible to differentiate the classic cholera vibrio from the El-Tor vibrio, as well as Proteus mirabilis from other intestinal bacteria, is the ability of the El-Tor vibrio and Proteus mirabilis to grow in the presence of polymyxin B (50 units in 1 ml and higher).

Features of physiology and biochemical activity

When determining the biochemical activity of microbes, their relationship to oxygen, carbon dioxide and various substrates, the optimal growth temperature, hemolytic ability, as well as the influence of various substances on their growth, including bacterial growth factors, are taken into account (see). In relation to free oxygen, microbes are usually divided into strict aerobes (see), strict and facultative anaerobes (see). Therefore, to isolate and identify the pathogen, special methods and nutrient media are used that promote the growth of only aerobic, facultative aerobic or anaerobic representatives.

For most pathogenic microbes, the optimal cultivation temperature is 37° (see Bacteria).

The hemolytic activity of microbes is determined by growing them in blood agar plates or by adding various dilutions of a broth culture to a suspension of washed erythrocytes.

Study of the influence of various biol, substrates and chemicals on the growth of bacteria. compounds (blood, serum, glucose, nitrates, bile salts, vitamins, amino acids, etc.) is often important for differentiating this group of microorganisms.

For I. m., the characteristics of the enzymatic activity of microbes, detected on media containing sugars and alcohols, protein substrates and fats (lipolytic properties), are of great importance, which makes it possible to identify subtle differences between closely related microbes. It is also important to determine the reducing properties of bacteria and their ability to form indole, ammonia and hydrogen sulfide, and use citrates and tartrates (see Differential diagnostic media).

Antigenic structure and relationship to bacteriophage

The antigenic structure and relationship to the bacteriophage and bactericins are studied at the final stage of I. m. Identification of the antigenic structure of microbes is carried out using various serols, reactions, for example, the agglutination reaction (see), the complement fixation reaction (see), etc.

If, in an extensive agglutination reaction, the tested microbe agglutinates to the titer of the immune serum or half the titer, then in practice it can be considered to belong to the species (type) with which this serum is designated. For complete identification, the isolated pathogen must be agglutinated to titer with an immune serum prepared against the reference microbe: the test microbe must adsorb all agglutinins from this serum. On the other hand, the reference microbe must be agglutinated to titer by the serum prepared against the microbe under study, and also adsorb all agglutinins from this serum. In other words, there must be complete cross-agglutination and cross-adsorption between both sera and both microbes. The agglutination reaction is sometimes supplemented or replaced by the precipitation reaction (see), as well as the indirect hemagglutination reaction (with red blood cells loaded with antibodies). Serol, the method reveals subtle differences between related microbes. It is often the only available method for differentiating subspecies or types of a given species.

Agglutinating monoreceptor sera are widely used in laboratory practice for the identification of Salmonella, Shigella and other microbes. The use of the immunofluorescence method (see), which allows you to quickly (1 - 2 hours) carry out I. m., is also very effective.

A sensitive method of I. m. is typing the identifying culture with a bacteriophage (see). This method is used, for example, in the study of typhoid bacillus (see Vi-typhoid phages), since it allows one to recognize the phagotype within a species. Specific phages are used to differentiate Shigella, cholera vibrios from cholera-like ones, classical cholera vibrios from El Tor vibrios, plague bacillus from pseudotuberculosis bacteria and other bacteria.

To differentiate some bacteria within a species, the phenomenon of bacteriocinogeny is used (see), as well as testing the sensitivity of bacteria to bactericins of various types (colicins, vibriocins, pesticins, diphtheriocins, etc.). Colicinotyping has found wide application to determine whether an isolated Shigella culture belongs to a specific colicinotype.

Pathogenicity for animals

The pathogenicity of microbes is usually determined in experiments on white mice, guinea pigs and rabbits. Animals are infected subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, orally, intranasally or intracerebrally (see Biological Assay).

When studying pathogenic microorganisms, it is sometimes necessary to determine whether they produce exotoxins. For this purpose, a filtrate of a bacterial culture grown for a certain period of time in an appropriate liquid medium is tested on sensitive animals. Exotoxins of highly toxic bacteria (diphtheria bacillus, tetanus bacillus, botulinum bacillus, etc.) cause disease in animals with a characteristic clinical picture and their subsequent death with typical pathological anatomical changes. To detect some microbial exotoxins, cultures of tissues sensitive to them, as well as chicken embryos, are used. Neutralization of exotoxins with specific antitoxins plays a significant role in I. m.

Bibliography: Krasilnikov N.A. Key to bacteria and actinomycetes, M.-L., 1949, bibliogr.; Guide to microbiological diagnosis of infectious diseases, ed. K. I. Matveeva, M., 1973; Tim and kov V.D. and Goldfarb D.M. Fundamentals of experimental medical bacteriology, M., 1958, bibliogr.; Bergey's manual of determinative bacteriology, ed. by R. E. Buchanan a. N. E. Gibbons, Baltimore, 1975, bibliogr.; Cowan S. T. a. Steel K. J. Manual for the identification of medical bacteria, Cambridge, 1974; Identification methods for microbiology, ed. by W. M. Gibbs a. F. A. Skinner, v. 1-2, L.-N.Y., 1966-1968; International code of nomenclature of bacteria, ed. by S. P. Lapage a. o., Washington, 1975; M e u n e 1 1 G. G. a. M e y n e 1 1 E. Theory and practice in experimental bacteriology, Cambridge, 1970, bibliogr.; Nomura M. Colicins and related bacteriocins, Ann. Rev. Microbiol., v. 21, p. 257, 1967, bibliogr.; W i 1-s o n G. S. a. M i 1 e s A. A. Topley and Wilson’s principles of bacteriology and immunity, v. 1-2, L., 1964.

A. V. Ponomarev.