Lecture: Antigens. Antigenic structure of a bacterial cell. human antigens. Serological reactions Methods for the final identification of bacteria by antigenic composition

09.09.2020

Bacterial antigens:

group-specific (found in different species of the same genus or family)

species-specific (in different representatives of the same species);

type-specific (determine serological variants - serovars, antigenovars within one species).

Depending on the localization in the bacterial cell, K-, H-, O-antigens are distinguished (denoted by letters of the Latin alphabet).

O-AG - lipopolysaccharide of the cell wall of gram-negative bacteria. It consists of a polysaccharide chain (actually O-Ag) and lipid A.

The polysaccharide is thermostable (withstands boiling for 1-2 hours), chemically stable (withstands treatment with formalin and ethanol). Pure O-AG is weakly immunogenic. It shows structural variability and distinguishes many serovariants of bacteria of the same species. For example, each group of Salmonella is characterized by the presence of a certain O-AG (polysaccharide) - in group A

This is factor 2, group B has factor 4, and so on. In R-forms of bacteria, O-AG loses side chains

polysaccharide and type specificity.

Lipid A - contains glucosamine and fatty acids. It has strong adjuvant, non-specific immunostimulatory activity and toxicity. In general, LPS is an endotoxin. Already in small doses, it causes fever due to the activation of macrophages and the release of IL1, TNF and other cytokines, degranulocyte degranulation, and platelet aggregation. It can bind to any cells in the body, but especially to macrophages. In large doses, it inhibits phagocytosis, causes toxicosis, dysfunction of the cardiovascular system, thrombosis, endotoxic shock. LPS of some bacteria is part of immunostimulants (prodigiosan,

pyrogenal). Bacterial cell wall peptidoglycans have a strong adjuvant effect on SI cells.

H-AG is part of bacterial flagella, its basis is the flagellin protein. Thermolabile.

K-AG is a heterogeneous group of superficial, capsular AG bacteria.

They are in a capsule. They contain mainly acidic polysaccharides, which include galacturonic, glucuronic and iduronic acids. There are variations in the structure of these antigens, on the basis of which, for example, 75 types (serotypes) of pneumococci, 80 types of Klebsiella, etc. are distinguished. Capsular antigens are used to prepare meningococcal, pneumococcal, and Klebsiella vaccines. However, administration of high doses of polysaccharide antigens can induce tolerance.

Antigens of bacteria are also their toxins, ribosomes and enzymes.

Some microorganisms contain cross-reactive - antigenic determinants found in microorganisms and humans / animals.

In microbes of various species and in humans, there are common, similar in structure, AG. These phenomena are called antigenic mimicry. Often, cross-reactive antigens reflect the phylogenetic commonality of these representatives, sometimes they are the result of a random similarity in conformation and charges - antigen molecules.

For example, Forsman's AG is found in barach erythrocytes, salmonella, and in guinea pigs.

Group A hemolytic streptococci contain cross-reacting antigens (in particular, M-protein) that are common with antigens of the endocardium and glomeruli of human kidneys. Such bacterial antigens cause the formation of antibodies that cross-react with human cells, which leads to the development of rheumatism and post-streptococcal glomerulonephritis.

The causative agent of syphilis has phospholipids similar in structure to those found in the heart of animals and humans. Therefore, the cardiolipin antigen of the heart of animals is used to detect antibodies to spirochete in sick people (Wassermann reaction).

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 a property of bacteria as variability, the ability to carefully select and inherit genetic information aimed at the preservation and development of 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 unicellular organisms, the first stage of identification relies on the general properties of bacteria inherent in all prokaryotes (non-nuclear cells):

microscopic dimensions (not visible to the naked eye);
huge metabolic rate and, as a result, growth and reproduction;
rapid adaptation to the changed conditions of existence;
the ability to change in a short time with the transfer of heredity;

Another feature common to all unicellular organisms is their 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 negative temperatures of the stratosphere. True, curious researchers have found a place on earth where it is not so easy to find bacteria - separate parts of the Atacama Desert (South America). This land has not seen rain for decades, and possibly hundreds of years. Even bacteria gave up - water is necessary for any form of protein life.

Identification of bacteria by species

Scientists separate bacteria by species, or rather, they are trying to do it. Presumably (well, science does not know for sure!) There are millions of species of bacterial cells. But science can "recognize by sight" only a few tens of thousands, whose characteristics are well studied. For example, bifidobacteria and lactobacilli are necessary for digestion, the properties of lactic acid bacteria and yeast fungi are used in industry, pathogenic microorganisms carry diseases or cause food poisoning, forming dangerous toxins, etc.

For species identification of bacteria, you need to know the following properties:

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

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

The analysis of all these properties together helps to determine the type of bacterial cell. Such identification makes it possible to distinguish "good" bacteria that bring benefits from harmful pathogenic microbes with negative properties. Strictly speaking, this division is rather conditional. 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 as soon as the population of these bacteria grows above the boundary parameters, there is a danger of poisoning with toxins that are dangerous to health.

What do bacteria look like

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

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

micrococci (single cell);
diplococci (two cells interconnected);
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 bunch of grapes).

2. Sticks distinguish:

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:

vibrio - slightly curved;
spirilla - one or more turns (up to four);
more than four whorls have borelli (from 4 to 12) and (Dr. Bykov's favorite curse, syphilis pathogens) treponema (from 14 to 17 small coils);
leptospira is similar to 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 representatives of the species may be shaped like a very short rod (almost a ball), while others are elongated, approaching filamentous cells. The length in this case depends on the composition of the medium, the presence and percentage of oxygen, the method of cultivation (artificial cultivation) of microorganisms.

With the size of unicellular a little easier:

the smallest (brucella);
medium (bacteroid, E. coli);
large (bacilli, clostridia).

The structure of microorganisms

Common to all prokaryotes 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, referred to by analogy as organelles. For different types of bacteria, this set is not the same, but there is a certain mandatory minimum that is present in each bacterium:

nucleoid (analogous to the nucleus);
cell wall (outer layer of various thicknesses);
cytoplasmic membrane (thin film between the internal semi-liquid 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 a bacterium 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 staining microorganisms using aniline dyes. It turned out that, depending on the structure of the outer shell, the 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 (rinsing is performed in both cases, but only in one case it washes out the paint). Bacteria are divided into two large groups based on the thickness of their cell walls:

gram-positive (thick wall can be stained);
gram-negative (thin wall does not hold the dye).

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

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

Capsule - superficial (above the cell membrane) mucous layer, formed as a reaction to the environment. That is, in comfortable conditions, the bacterium may well do without a capsule, but at the slightest threat it protects itself with a soft shell, which gives additional security.
Flagella are long (longer than the body of the bacterium) filamentous organs of movement. They work as a kind of engine, allowing the cell to move freely.
Pili - very small villi on the surface of the bacterium (thinner and shorter than flagella). Pili do not move the cage, but help it securely anchor in the chosen place.
Spores are solid inclusions that form inside bacteria as a reaction to the threat of death (lack of water, aggressive environment). They allow the cell to survive hard times (sometimes a bacterium can "sleep" for years and 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. So, chlorosomes are responsible for the production of oxygen from the energy of sunlight (photosynthesis); gas vacuoles give the cell buoyancy; lipids and volutin store food and energy reserves, etc.

Growth and reproduction

Accurate identification and industrial production require pure cultures of bacteria - a population grown from a single cell in the laboratory. And for this you need to know their biological properties - in what conditions and how microorganisms grow and multiply. Growth is an increase in the 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, that is, the cell divides in two in the middle, forming two identical organisms. The budding method differs from binary fission only in form - a protrusion is formed on the cell surface, where a half of the divided nucleus substitute (nucleoid) moves, then the protrusion grows and separates from the mother cell.

A more sophisticated method is genetic recombination, which resembles sexual reproduction. The essence of the method is that part of the DNA enters the cell from the outside (when bacteria come into contact with each other, with the help of bacteriophages, or as a result of the absorption of the genetic material of dead cells). As a result, this method gives two genetically modified cells that carry information from both "parents". The properties of the altered cell may differ significantly from its predecessors. This method of reproduction allows bacteria to adapt to changing conditions, perhaps it was he who served as the basis for the emergence of intelligent life on the planet.

In addition, the recombinant breeding 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 respiration and nutrition of the cell

Depending on the relationship to oxygen, bacteria differ in:

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

Identification by the method of respiration and nutrition is important for creating comfortable conditions for growing bacterial cultures on artificial media and in biotechnologies.

Due 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 suitable for further use to nature.

Enzymes and toxins of bacteria (biochemical activity)

Microorganisms produce protein substances - enzymes (Latin "sourdough") or enzymes (Greek "sourdough"), 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 involved in cell metabolism.
exoenzymes are extracellular (released into the environment), they carry out digestion outside the bacterial cell.

The properties of microorganisms to secrete certain enzymes are used to identify the type of unicellular, since this is a constant and unchanging feature that is unique to this type of cell. Distinguish:

Saccharolytic properties of the cell - 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.
The proteolytic properties of microorganisms are the 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 secrete proteolytic enzymes into the external environment, which break down proteins to intermediate products (peptones, amino acids) and / or to final degradation products (hydrogen sulfide, ammonia). Protein digestion and blood clotting depend on proteolytic enzymes.

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

The 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. Distinguish:

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

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

Some bacteria have hemolytic properties, that is, they secrete toxins that destroy red blood cells (hemolysins). In the natural process of erythrocyte renewal, the hemolytic properties of cells are necessary, but they can become dangerous in the pathological development of the process.

Bacteria are ubiquitous and diverse. There are “good”, beneficial microorganisms, but there are also harmful, pathogenic microbes that provoke 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.

Introduction.Identification- determination (establishment) of the microbe's species affiliation. At present, the generally accepted identification method is based on the study of a certain set of the most important phenotypic features of the microorganism under study. The criterion for identification is the presence in a microbe of a set of basic features characteristic of a given species (taxonometric characters). The species is established according to the international taxonomy of bacteria (Bergey's Manual of Systematic Bacteriology).

To main species features bacteria include:

Morphology of the microbial cell;

Tinctorial properties - staining features using simple and complex staining methods;

Cultural characteristics - features of microbial growth on nutrient media;

in biochemical signs - the presence in bacteria of enzymes necessary for the synthesis or splitting (fermentation) of various chemical compounds.

In bacteriological practice, saccharolytic and proteolytic enzymes are most often studied.

To additional features, used for identification include:

Presence of species-specific antigens (see Chapter 10);

Susceptibility to species-specific bacteriophages (see Chapter 5);

Species resistance to certain antimicrobials (see Chapter 8);

For pathogenic bacteria, the production of certain virulence factors (see Chapter 9).

Fine intraspecific identification up to biovar (serova-ra, fagovar, fermentovar, etc.) - titration - based on the detection of the appropriate marker: antigen (serotyping, see Chapter 10), sensitivity to a typical bacteriophage (phage typing, see Chapter 5), etc.

In recent years, modern biochemical and molecular biological methods of identification have been developed and started to be applied: chemoidentification, analysis of nucleic acids: restriction analysis, hybridization, polymerase chain reaction (PCR), ribotyping, etc.

▲ Lesson plan

▲ Program

1. Identification of bacteria.

2. Study of the biochemical properties of aerobic and anaerobic bacteria.

▲ Demo

1. Unsown "variegated row".

2. Options for changing the "variegated row".

3. "Motley row" for anaerobic bacteria.

4. Micromethod for studying the biochemical properties of bacteria.

5. Growth of pigment-producing bacteria.

▲ Assignment to students

1. Draw options for changing the "variegated row".

2. Assess the results of screening out pure culture: note the presence or absence of growth of the inoculated culture, as well as the presence of foreign bacteria.

3. Make sure that the isolated culture is pure, for this, prepare a smear and stain it according to the Gram method.

4. Put a catalase sample on the glass and evaluate its result.

5. Take into account the results of determining the biochemical activity of isolated pure cultures.

6. Using the identification table, on the basis of the studied morphological, tinctorial, cultural and enzymatic properties, identify the isolated microbes.

▲ Guidelines

Biochemical identification. To assess the biochemical activity of bacteria, the following are used: reactions:

1) fermentation - incomplete breakdown of the substrate to

Intermediate products, such as the fermentation of carbohydrates with the formation of organic acids;

2) oxidation - complete breakdown of the organic substrate to CO 2 and H2O;

3) assimilation (utilization) - the use of a substrate for growth as a source of carbon or nitrogen;

4) dissimilation (degradation) of the substrate;

5) substrate hydrolysis.

The classical (traditional) method of identifying microbes by biochemical characteristics is to inoculate a pure culture on differential diagnostic media containing certain substrates in order to assess the ability of a microorganism to assimilate this substrate or determine the end products of its metabolism. The study takes at least 1 day. An example is the assessment of the saccharolytic activity of bacteria (the ability to ferment carbohydrates) by seeding on Hiss media - a short and long "motley row".

Identification of bacteria by biochemical characteristics using "variegated series" media. A short "variegated series" includes liquid Hiss media with mono- and disaccharides: glucose, lactose, sucrose, maltose and with 6-hydric alcohol - mannitol. In a long "motley row" along with the listed carbohydrates, media with a variety of monosaccharides (arabinose, xylose, rhamnose, galactose, etc.) and alcohols (glycerol, dulcitol, inositol, etc.) are introduced. To assess the ability of bacteria to ferment carbohydrate, an indicator (Andrede's reagent or others) is added to the media, which makes it possible to detect the formation of acidic cleavage products (organic acids), and a "float" to detect the release

from 2 .

A pure culture of the studied microorganism is seeded with a loop in the media of the "variegated row". The cultures are incubated at 37°C for 18-24 hours or more. If the bacteria ferment the carbohydrate to form acidic products, a change in the color of the medium is observed; when the carbohydrate decomposes to acid and gaseous products, along with the color change, a gas bubble appears in the float If media with semi-liquid agar is used, then the formation of gas is recorded by the break of the column.In the absence of fermentation, the color of the medium does not change.Since bacteria do not ferment all, but only certain carbohydrates, which are part of Hiss media, certain for each type, a rather motley picture is observed, therefore a set of media with carbohydrates and a color indicator is called the "variegated row" (Fig. 3.2.1; on the insert).

For determination of proteolytic enzymes produce a culture of bacteria with an injection into a column of 10-20% gelatin,

peptone water. Cultures in gelatin are incubated at 20-22°C for several days. In the presence of proteolytic enzymes, bacteria liquefy gelatin, forming a figure resembling a funnel or herringbone.

In crops in peptone water*, the cleavage products of amino acids are determined after incubation for 2-3 days at 37 °C by setting reactions to ammonia, indole, hydrogen sulfide and etc.

reaction to ammonia. A narrow strip of litmus paper is fixed under the cork so that it does not come into contact with the nutrient medium. Blue paper indicates the formation of ammonia.

Reaction to indole. Ehrlich's method: 2-3 ml of ether is added to a test tube with a culture of bacteria, the contents are vigorously mixed and a few drops of Ehrlich's reagent (an alcoholic solution of paradimethylamidobenzaldehyde with hydrochloric acid) are added. In the presence of indole, a pink coloration is observed, with careful layering a pink ring is formed (see Fig. 3.2.1).

reaction to hydrogen sulfide. A narrow strip of filter paper moistened with iron sulfate is placed in a test tube with peptone water and fixed under the stopper so that it does not come into contact with the nutrient medium. When hydrogen sulfide is released, insoluble iron sulfide (FeS) is formed, turning the paper black (see Fig. 3.2.1). H 2 S production can also be determined by inoculating a bacterial culture by injection into a column with a nutrient medium containing reagents for detecting H 2 S (a mixture of salts: iron sulfate, sodium thiosulfate, sodium sulfite). A positive result - the medium turns black due to the formation of FeS.

detection of catalase. A drop of 1-3% hydrogen peroxide solution is applied to a glass slide and a loop with a bacterial culture is introduced into it. Catalase decomposes hydrogen peroxide into oxygen and water. The release of gas bubbles indicates the presence of catalase in this type of bacteria.

In bacteriological practice, they sometimes confine themselves to studying the saccharolytic and proteolytic features of the studied bacteria, if this is sufficient for their identification. If necessary, investigate other signs, for example, the ability to restore nitrates, carboxylation of amino acids, the formation of oxidase, plasmacoagulase, fibrinolysin and other enzymes.

The results of work on the identification of the isolated culture are recorded (Table 3.2.1).

Biochemical tests of the 2nd generation, based on the use of concentrated substrates and more sensitive methods for detecting end products of the reaction,

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.

The culture conditions also depend on the properties of the respective microorganisms. Most pathogenic microbes are grown on nutrient media at 37°C for 12 days. However, some of them require longer periods. For example, pertussis 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 of their cultivation, the method of deep cultivation is used, which consists in continuous aeration and mixing of the nutrient medium. The depth 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 - anaerostats. Anaerobes are grown on nutrient media containing reducing substances (glucose, sodium formic acid, etc.), which reduce the redox potential.

In diagnostic practice, pure cultures of bacteria are of particular importance, which are isolated from the test material taken from a patient or environmental objects. For this purpose, artificial nutrient media are used, which are divided into basic, differential diagnostic and elective of the most diverse composition. The choice of a 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 on the surface of the medium with a loop and rubbed with a spatula to obtain isolated colonies that have grown from one cell. Subculture of an isolated colony onto a slant agar medium in a test tube results in a pure culture.

For identification, i.e. determining the generic and species affiliation of a selected culture, most often they study phenotypic characteristics:

a) the 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 chromography and other methods are used.

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

Pure Culture Isolation Methods

In order to isolate a pure culture of microorganisms, it is necessary to separate the numerous bacteria that are in the material from one another. This can be achieved through methods that are based on two principles − mechanical and biological dissociation 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 in carrying out serial serial dilutions of material that contains microbes in a sterile liquid nutrient medium. This technique is rather painstaking and imperfect in operation, since it does not allow controlling the number of microbial cells that enter the test tubes during dilutions.

This disadvantage does not Koch method (plate dilution method ). R. Koch used dense 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 content of which was later poured onto sterile glass plates. After gelation of the medium, it was cultivated at the optimum temperature. Isolated colonies of microorganisms were formed in its thickness, which can be easily transferred to a fresh nutrient medium using a platinum loop to obtain a pure culture of bacteria.

Drygalski method is a more advanced method that is widely used in everyday microbiological practice. First, the test material is applied to the surface of the medium in a Petri dish with a pipette or a loop. Using a metal or glass spatula, carefully rub it into the medium. The cup is kept open during inoculation and rotated gently to distribute the material evenly. Without sterilizing the spatula, they spend it on the material in another Petri dish, if necessary - in the third. Only after that, the spatula is dipped in a disinfectant solution or fried in a burner flame. On the surface of the medium in the first dish, as a rule, we observe a continuous growth of bacteria, in the second - a dense growth, and in the third - growth in the form of isolated colonies.

Colonies according to the Drygalski method

Stroke method today is used in microbiological laboratories most often. 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 cup. The excess material is removed and occupied in parallel strokes from edge to edge of the cup. After a day of incubation of crops at the optimum temperature, isolated colonies of microbes grow on the surface of the dish.

Stroke method

To obtain isolated colonies, you can use a swab, which was used to collect the test material. The Petri dish with a nutrient medium is slightly opened, a tampon is introduced into it, and the material is carefully rubbed into the surface of the dish, gradually returning the tampon and the dish.

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

Methods for isolating pure cultures based on the biological principle

The biological principle of separation of bacteria provides for a purposeful 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 sholeraeetc) and anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringensand etc.). If the material from which anaerobic pathogens should be isolated is preheated and then cultivated under anaerobic conditions, then these bacteria will grow.

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

3. Resistance of microbes to the action of 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 for tuberculosis, is pre-treated with an equal volume of 10% sulfuric acid solution, and then sown on nutrient media. Foreign flora dies, and mycobacteria, as a result of their resistance to acids, grow.

Vibrio cholerae (Vibrio sholerae) , 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). Already after 4-6 hours, characteristic signs of growth appear on the surface of the medium in the form of a delicate bluish film.

4. Mobility of bacteria. Some microbes (Proteus vulgaris) tend to creeping growth and are able to quickly spread over the surface of something moist environment. To isolate such pathogens, they are seeded in a drop of condensation liquid, which is formed when the column of slant agar is cooled. After 16-18 years, they spread to the entire surface of the environment. 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 peculiarities of the metabolism 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, for the isolation of these pathogens, elective nutrient media (yolk-salt agar, beckoning-salt agar) are used, which contain precisely this substance. Other bacteria practically do not grow at this concentration of sodium chloride.

6. Introduction of some antibiotics (nystatin) is used to inhibit the growth of fungi in material that is heavily contaminated with them. Conversely, the addition of the antibiotic penicillin to the medium promotes the growth of the 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 aggression enzymes, they are able to penetrate intact skin. To do this, the hair on the body of a laboratory animal is shaved and the test material, which contains the 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 show high sensitivity to different 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 concepts such as strain and pure culture microorganisms. Under the strain understand microbes of the same species, which are isolated from different sources, or from the same source, but at different times. Pure culture of bacteria are microorganisms of the same species, descendants of one microbial cell, which have grown on (in) a nutrient medium.

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

First day (1 stage research) pathological material is taken into a sterile container (test tube, flask, vial). 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 sown. Then it is carried out with a bacteriological loop (used most often), with a spatula - by the Drygalsky method, with a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimum temperature (37 ° C) for 18-48 hours. The purpose of the stage is to obtain isolated colonies of microorganisms.

However, sometimes in order to pile up the material, it is sown on liquid nutrient media.

On the second day (Phase 2 of the study) on the surface of a dense nutrient medium, microorganisms form a 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), so their composition is quite homogeneous. Features of bacterial growth on nutrient media are a manifestation of their cultural properties.

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

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

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

First, attention is paid to the characteristics of the growth of microorganisms on the medium and a smear is made, 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 dye) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, already in appearance and features of their growth, it is possible to draw a conclusion about the type of isolated pathogens. Determining the species of bacteria by their morphological features is called morphological identification. Determining the type of pathogens by their cultural characteristics is called cultural identification.

However, these studies are not enough to make a final conclusion about the type of isolated microbes. Therefore, they study the biochemical properties of bacteria. They are quite varied.

Identification of bacteria.

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

In order to establish the species affiliation of bacteria, their antigenic structure is often studied, that is, they are identified by antigenic properties. Each microorganism has in its composition different antigenic substances. In particular, representatives of the Enterobacteriaceae family (Yescherichia, Salmonella, Shigels) contain envelope O-antigen, flagella 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 definition of a bacterial species is called serological identification.

Sometimes bacteria are identified by infecting laboratory animals with a pure culture and observing the changes that pathogens cause in the body (tuberculosis, botulism, tetanus, salmonellosis, and the like). Such a method is called identification by biological properties. As objects, guinea pigs, white mice and rats are most often used.

APPS

(tables and charts)

Physiology of bacteria

Scheme 1. Physiology of bacteria.

reproduction

cultivation on nutrient media

Table 1. General table of bacterial physiology.

		Characteristic
		The process of acquiring energy and substances.
		A set of biochemical processes, as a result of which the energy necessary for the vital activity of microbial cells is released.
		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.	Under laboratory conditions, microorganisms are grown on nutrient media that must be sterile, transparent, moist, contain certain nutrients (proteins, carbohydrates, vitamins, trace elements, etc.), have a certain buffering capacity, have an appropriate pH, 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 the cell in a hypertonic solution - the exfoliation of protoplasm from the cell membrane occurs. Removal of water from the cell, drying suspend the processes of metabolism. Most microorganisms tolerate drying well. With 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. Determine the most important biological properties of bacteria and usually consist of combinations of 20 amino acids. Bacteria contain diaminopimelic acid (DAP), which is absent in human and animal cells. Bacteria contain more than 2000 different proteins that are in structural components and are 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 the nucleic acids of eukaryotic cells: a 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 found 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 bacterial cell wall, 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 greatest amount of lipids (up to 40%).
Minerals	Found in the ashes after cells are burned. Phosphorus, potassium, sodium, sulfur, iron, calcium, magnesium, as well as trace elements (zinc, copper, cobalt, barium, manganese, etc.) are detected in large quantities. They are involved in the regulation of osmotic pressure, pH, redox potential , activate enzymes, are part of enzymes, vitamins and structural components of microbial cells.

Table 1.2. Nitrogen bases.

Table 1.2.1 Enzymes

	Characteristic
Definition	Specific and effective protein catalysts present in all living cells.
	Enzymes reduce the activation energy, ensuring the flow of such chemical reactions that without them could only take place at high temperature, overpressure, and under other non-physiological conditions that are unacceptable for a living cell.
	Enzymes increase the reaction rate 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. For binding to the substrate, a certain part of the enzymatic protein molecule is responsible - its catalytic center. In this case, an intermediate enzyme-substrate complex is formed, which then decomposes with the formation of a reaction product and a free enzyme.
Varieties	Regulatory (allosteric) enzymes perceive various metabolic signals and, in accordance with them, change their catalytic activity.	Effector enzymes - enzymes that catalyze certain reactions (for details, see 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 medium and its pH. For many pathogenic microorganisms, the optimum temperature is 37°C and pH 7.2-7.4.

ENZYME CLASSES:

microorganisms synthesize various enzymes belonging to all six known classes.

Table 1.2.2. Classes of effector enzymes

	Enzyme class	Catalyzes:
	Oxidoreductase	Electron transfer
	Transferases	Transfer of various chemical groups
	Hydrolases	Transfer of functional groups to a water molecule
		Attachment of double bond groups and reverse reactions
	Isomerases	Transfer of groups within a molecule to form isomeric forms
		The 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 by formation in a bacterial cell

	Characteristic	Notes
Iiducible (adaptive) enzymes "substrate induction"	Enzymes, the concentration of which in the cell increases sharply in response to the appearance of an inductor substrate in the environment. Synthesized by a bacterial cell only in the presence of this enzyme in the substrate medium
Repressible Enzymes	The synthesis of these enzymes is suppressed as a result of excessive accumulation of the product of the reaction 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	Enzymes of glycolysis
Multienzyme complexes	Intracellular enzymes combined structurally and functionally	Respiratory chain enzymes located on the cytoplasmic membrane.

Table 1.2.4. Specific Enzymes

	Enzymes	Identification of bacteria
	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, is catalase-negative.
	Peroxidase	Lactic acid bacteria accumulate peroxidase, an enzyme that catalyzes the oxidation of organic compounds under the action of H2O2 (it is reduced to water).
	Arginine dihydrolase	A diagnostic feature that distinguishes 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. The use of bacterial enzymes in industrial microbiology.

	Enzymes	Application
	Amylase, cellulase, protease, lipase	To improve digestion, ready-made preparations of enzymes are used, which facilitate the hydrolysis of starch, cellulose, protein and lipids, respectively.
	Yeast invertase	In the manufacture of sweets to prevent the crystallization of sucrose
	pectinase	Used to clarify fruit juices
	Clostridial collagenase and Streptococcal streptokinase	Hydrolyze proteins, promote healing of wounds and burns
	Lytic enzymes of bacteria	Secreted into the environment, act on the cell walls of pathogenic microorganisms and serve as an effective tool in the fight against the latter, even if they have multiple resistance to antibiotics
	Ribonucleases, deoxyribonucleases, polymerases, DNA ligases and other enzymes that modify nucleic acids in a targeted manner	Used as a toolkit 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 the reactions of biosynthesis and energy metabolism.
Exoenzymes	Released into the environment.	They are released by the cell into the environment and catalyze the reactions of hydrolysis of complex organic compounds into simpler ones, 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 (enzymes of aggression)

Enzymes
Lecitovitellase Lecithinase	Breaks down cell membranes	Inoculation of the test material on the nutrient medium JSA Result: cloudy area around colonies on LSA.
Hemolysin	Destroys red blood cells	Inoculation of the test material on a blood agar nutrient medium. Result: complete area of hemolysis around colonies on blood agar.
Coagulase-positive cultures	Causes blood plasma to clot	Inoculation of the test material on sterile citrated blood plasma. Result: plasma clotting
Coagulase-negative cultures	Mannitol production	Sowing on a nutrient medium mannitol under anaerobic conditions. Result: The appearance of colored colonies (in the color of the indicator)
Enzymes		The formation of some enzymes in the laboratory
Hyaluronidase	Hydrolyzes hyaluronic acid - the main component of connective tissue	Sowing the test material on a nutrient medium containing hyaluronic acid. Result: in test tubes containing hyaluronidase, no clot formation occurs.
Neuraminidase	It cleaves sialic (neuraminic) acid from various glycoproteins, glycolipids, polysaccharides, increasing the permeability of various tissues.	Detection: reaction for the determination of 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 environments such as Hiss environment, Olkenitsky environment, Endo environment, Levin environment, Ploskirev 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 character of gelatin liquefaction is noted. Proteolytic activity is also determined by the formation of protein decomposition products: indole, hydrogen sulfide, ammonia. For their determination, microorganisms are inoculated into meat-peptone broth.
Enzymes identified by end products	Alkali formation Acid formation Hydrogen sulfide formation Formation of ammonia, etc.	To distinguish some types of bacteria from others on the basis of their enzymatic activity, differential diagnostic environments

Scheme 1.2.8. Enzyme composition.

ENZYME COMPOSITION OF ANY MICROORGANISM:

Determined by its genome

Is a stable feature

Widely used for their identification

Determination of saccharolytic, proteolytic and other properties.

Table 1.3. Pigments

	Pigments	Synthesis by a microorganism
	Fat-soluble carotenoid pigments in red, orange or yellow	They form sarcins, mycobacterium tuberculosis, 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, prodigiosin	Formed by some serations
	The water-soluble phenosine pigment is pyocyanin.	Produced by Pseudomonas aeruginosa bacteria (Pseudomonas aeruginosa). In this case, the nutrient medium with a neutral or alkaline pH turns blue-green.

Table 1.4. Luminous and aroma-producing microorganisms

		Condition and characteristic
	Glow (luminescence)	Bacteria cause the glow of those substrates, such as fish scales, higher fungi, decaying trees, food products, on the surface of which they multiply. Most luminous bacteria are halophilic species that can multiply at elevated salt concentrations. They live in the seas and oceans and rarely in fresh water. All luminous bacteria are aerobes. The glow mechanism is associated with the release of energy in the process of biological oxidation of the substrate.
	aroma formation	Some microorganisms produce volatile aromatic substances, such as acetic-ethyl and acetic-amyl esters, which impart flavor to wine, beer, lactic acid and other food products, as a result of which they are used in their production.

Table 2.1.1. Metabolism

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

Synthesis (cell components)

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

Biosynthesis of monomers:

amino acids nucleotides fatty acid monosaccharides

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 mounted - 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 provides a 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 reactions of biological synthesis.	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 cleavage reactions.	When high-molecular compounds are cleaved, the energy necessary for the biosynthesis reaction is released, that is, dissimilation occurs. During the breakdown of glucose, 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, which is dissipated in the form of heat.
Anoxic or incomplete or anaerobic or fermentation or dissimilation.	The substances formed at this stage with the participation of enzymes undergo further cleavage. 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, 40% of the energy is stored in the ATP molecule, the rest is dissipated in the form of heat. In all cases, the breakdown of one glucose molecule produces two ATP molecules.
The stage of aerobic respiration or oxygen splitting.	When oxygen enters the cell, the substances formed during the previous stage are oxidized (broken down) to final products CO 2 andH 2 O.	The total equation of aerobic respiration:

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 for pyruvic acid recovery.

Pathways for the formation of pyruvic acid from carbohydrates

Scheme 2.2.5. pyruvic acid.

Glycolytic pathway (Embden-Meyerhof-Parnassus pathway)

Entner-Doudoroff path

Pentose phosphate pathway

Table 2.2.5. Fermentation.

Type of fermentation	Representatives	Final product	Notes
lactic acid		Form lactic acid from pyruvate	In some cases (homoenzymatic fermentation) only lactic acid is formed, in others also by-products.
Formic acid	Enterobacteriaceae	Formic acid is one of the end products. (along with it - side)	Some types of enterobacteria break down formic acid to H 2 and CO 2 /
Butyric		Butyric acid and by-products	Some species 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 synthesizing system, ion exchange.

	Element name	Characteristic
	Ribosomal subunits 30S and 50S	In the case of bacterial ribosomes, the 70S subunit contains the 50S rRNA (~3000 nucleotides in length) and the 30S subunit contains the 16S rRNA (~1500 nucleotides in length); a large ribosomal subunit, in addition to a “long” rRNA, also contains one or two “short” rRNAs (5S rRNA of bacterial ribosomal subunits 50S or 5S and 5.8S rRNA of eukaryotic large ribosomal subunits). (for details see Fig. 2.3.1.)
	Messenger RNA (mRNA)
	A complete set of twenty aminoacyl-tRNAs that require the appropriate amino acids, aminoacyl-tRNA synthetases, tRNAs, and ATP to form	It is an amino acid charged with energy and associated with tRNA, ready for delivery to the ribosome and incorporation into 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 are involved in the organization of the active complex (708-complex) of the 30S and 50S subunits, 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 elongation (elongation) of the synthesized polypeptide chain (peptidyl). Protein termination or release factors (eng. - release factors - RF) provide codon-specific separation of the polypeptide from the ribosome and the end of protein synthesis.
	Element name	Characteristic
	Protein termination factors	(for prokaryotes - RF-1, RF-2, RF-3)
	Some other protein factors (associations, dissociations of subunits, releases, etc.).	Protein translation factors necessary for the functioning of the system
	Guanosine triphosphate (GTP)	For the implementation of translation, the participation of GTP is necessary. The need 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 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, potassium ions are used to bind tRNA to ribosomes. Ions of iron, magnesium 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, a high osmotic pressure is maintained.
iron intake	For a number of pathogenic and conditionally pathogenic bacteria (Escherichia, Shigella, etc.), the consumption of iron in the host organism 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 medium to synthesize compounds containing these elements (sulfur-containing amino acids, phospholipids, etc.).
	For the growth and reproduction of bacteria, mineral compounds are needed - 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 They are 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.

In order to synthesize, bacteria need...

Nutrients

Table 2.4.1. Energy metabolism (biological oxidation).

Process

Necessary:

Synthesis of structural components of a microbial cell and maintenance of vital processes

Sufficient amount of energy.

This need is met by biological oxidation, which results in the synthesis of ATP molecules.

Energy (ATP)

Iron bacteria receive the energy released during the direct oxidation of iron (Fe2+ to Fe3+), which is used to fix CO2, sulfur metabolizing bacteria provide themselves with energy due to the oxidation of sulfur-containing compounds. However, the vast majority of prokaryotes obtain energy through dehydrogenation.

Energy is also received in the process of breathing (for a detailed table, see the corresponding section).

Scheme 2.4. Biological oxidation in prokaryotes.

Breakdown of polymers into monomers

Carbohydrates

glycerin and fatty acids

amino acids

monosaccharides

Cleavage under anoxic 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 energy to cells necessary for the manifestation of life.
		The ATP molecule is synthesized as a result of the transfer of an electron from its primary donor to the final acceptor.
		Respiration is biological oxidation (splitting). Depending on what is the final electron acceptor, there are breath: Aerobic - during aerobic respiration, molecular oxygen O 2 serves as the final electron acceptor. Anaerobic - inorganic compounds serve as the final electron acceptor: NO 3 -, SO 3 -, SO 4 2-
	Energy mobilization	Energy is mobilized in oxidation and reduction reactions.
	Reaction Oxidation	The ability of a substance to donate electrons (to be oxidized)
	Recovery Reaction	The ability of a substance to accept 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

Biosynthesis	Of what	Notes
biosynthesis of carbohydrates	Autotrophs synthesize glucose from CO2. 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 in the host organism.
Lipid biosynthesis	Lipids are synthesized from simpler compounds - products of protein and carbohydrate metabolism.	Acetyl-carrying proteins play an important role. Auxotrophic microorganisms - consume ready-made in the host organism 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 with the help of DNA - dependent RNA - polymerase.

The transfer of information about the structure of the protein to the ribosomes occurs with the help of mRNA.

Broadcast (transmission)

The process of protein biosynthesis.

The process of deciphering the genetic code in mRNA and its implementation in the form of a polypeptide chain.

Since each codon contains three nucleotides, the same genetic text can be read in three different ways (starting from 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. Chains of electron transfer from the primary hydrogen donor (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 types of nutrition.

Organogen element	Food types	Characteristic
Carbon (C)	Autotrophs	They themselves synthesize all the carbon-containing components of the cell from CO 2.
	Heterotrophs	They cannot satisfy their needs at the expense of CO 2, they use ready-made organic compounds.
	Saprophytes	Food source - dead organic substrates.
		The source of nutrition is the living tissues of animals and plants.
	Prototrophs	Satisfy their needs with atmospheric and mineral nitrogen
	Auxotrophs	They need ready-made organic nitrogenous compounds.
Hydrogen (H)	The main source is H 2 O
Oxygen (O)	The main source is H 2 O

Table 3.1.2. Energy transformation

Table 3.1.3. Ways to carbon feed

Energy source	Electron donor	Way of carbon feeding
Sunlight energy	inorganic compounds	Photolithoheterotrophs
Sunlight energy	organic compounds	Photoorganoheterotrophs
Redox reactions	inorganic compounds	Chemolithoheterotrophs
Redox reactions	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.	Along the concentration gradient
Facilitated diffusion	Permease proteins are involved.	Along the concentration gradient
active transport	Permease proteins are involved.
Translocation of chemical groups	In the process of transfer, chemical modification of nutrients occurs.	Against the concentration gradient

Table 3.3. transport of nutrients from the bacterial cell.

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

Table 4. Height.

	concept	Concept definition.
		Irreversible increase in the amount of living matter, most often due to cell division. If in multicellular organisms an increase in body size is usually observed, then in multicellular organisms the number of cells increases. But even in bacteria, an increase in the number of cells and an increase in cell mass should be distinguished.
	Factors affecting the growth of bacteria in vitro.	Culture media: Mycobacterium leprae are not capable of in vitro Temperature (grow in range): Mesophilic bacteria (20-40 o C) Thermophilic bacteria (50-60 o C) Psychrophilic (0-10 o C)
	Assessment of bacterial growth	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 volume unit.

growth factors

Amino acids

vitamins

Nitrogenous bases

Table 4.1. growth factors

	growth factors	Characteristic	Function
Amino acids			Many microorganisms, especially bacteria, require certain amino acids (one or more) because they cannot synthesize them on their own. Such microorganisms are called auxotrophic for those amino acids or other compounds that they are unable 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	They are part of membrane lipids
		Phospholipids	They are part of membrane lipids
		Fatty acid	They are components of phospholipids
		Glycolipids	Mycoplasmas form part of the cytoplasmic membrane
			Mycoplasmas form part of the cytoplasmic membrane
vitamins (mainly 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	Hemophilus bacteria, Mycobacterium tuberculosis

Table 5. Breathing.

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

Table 5.1. Classification by type of breathing.

bacteria	Characteristic	Notes
Strict anaerobes	Energy exchange occurs without the participation of free oxygen. The synthesis of ATP during the consumption of glucose under anaerobic conditions (glycolysis) occurs due to the 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, therefore, accumulating in the presence of oxygen has a bactericidal effect on them; Strict anaerobes lack a system for regulating the redox potential (redox potential).
Strict aerobes	Able to receive energy only by breathing and therefore necessarily need molecular oxygen. Organisms that obtain energy and form ATP with the help of only oxidative phosphorylation of the substrate, where only molecular oxygen can act as an oxidizing agent. The growth of most aerobic bacteria stops at an oxygen concentration of 40-50% or more.	Strict aerobes include, for example, representatives of the genus Pseudomonas
bacteria	Characteristic	Notes
Facultative anaerobes	Grows in the presence or 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 0 2 to fermentation in the absence of 0 2 .
microaerophiles	A microorganism that, unlike strict anaerobes, requires the presence of oxygen in the atmosphere or nutrient medium for its growth, but in lower concentrations compared to the oxygen content in ordinary air or in normal tissues of the host organism (unlike aerobes, which require normal oxygen for growth). oxygen content in the atmosphere or nutrient medium). Many microaerophiles are also capnophiles, meaning they require an increased concentration of carbon dioxide.	In the laboratory, such organisms are easily cultivated in a "candle jar". A "candle jar" is a container into which a burning candle is brought in before being sealed with an airtight lid. The flame of the candle will burn until it is extinguished from lack of oxygen, resulting in an atmosphere saturated with carbon dioxide and reduced in oxygen in the jar.

Table 6. Characteristics of reproduction.

Scheme 6. Dependence of the generation duration on various factors.

Generation duration

Type of bacteria

population

Temperature

The composition of the nutrient medium

Table 6.1. phases of bacterial reproduction.

	Phase	Characteristic
	Initial stationary phase	It lasts 1-2 hours. During this phase, the number of bacterial cells does not increase.
	Lag phase (reproduction delay phase)	It is characterized by the beginning of intensive cell growth, but the rate of their division remains low.
	Log phase (logarithmic)	Differs in the maximum rate of cell reproduction and an exponential increase in the number of bacterial populations
	Phase of negative acceleration	It is characterized by less activity of bacterial cells and lengthening of the generation period. This occurs as a result of the depletion of the nutrient medium, the 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.
	Doom phase	It occurs at a constant rate and is replaced by UP-VSH phases of a decrease in the rate of cell death.

Scheme 7. Requirements for nutrient media.

Requirements

Viscosity

Humidity

Sterility

Nutrition

Transparency

isotonicity

Table 7. Reproduction of bacteria on nutrient media.

Nutrient medium	Characteristic
Dense culture media	On dense 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 shape with jagged edges, rough, indented.
Liquid culture media	Bottom growth (sediment) Surface growth (film) Diffuse growth (uniform turbidity)

Table 7.1. Classification of nutrient media.

Classification	Kinds	Examples
Composition		MPA - meat-peptone agar MPB - meat-peptone broth PV - peptone water
Composition		blood agar JSA - yolk-salt agar Hiss media
By appointment	Main
	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-liquid agar
Origin	natural
	Semi-synthetic
	Synthetic	Simmonson

Table 7.2. Principles of pure cell culture isolation.

Mechanical principle

biological principle

1. Fractional dilutions by L. Pasteur

2. Plate dilutions by 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;

d - spore formation;

e - temperature optimum;

e - selective sensitivity of laboratory animals to bacteria

Table 7.2.1. Stages of pure cell culture isolation.

	Stage	Characteristic
	1 stage research	Take pathological material. It is studied - appearance, consistency, color, smell and other signs, a smear is prepared, painted and examined under a microscope.
	Stage 2 research	On the surface of a dense nutrient medium, microorganisms form a 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), so their composition is quite homogeneous. Features of bacterial growth on nutrient media are a manifestation of their cultural properties.
	3 stage research	The nature of the growth of a pure culture of microorganisms is studied and its identification is carried out.

Table 7.3. Identification of bacteria.

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

Which of the processes 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?

oxidoreductases

All classes

Transferases

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

Iuducible

constitutional

Repressible

Multienzyme complexes

Pathogenicity enzyme secreted by Staphylococcus aureus?

Neuraminidase

Hyaluronidase

Lecithinase

fibrinolysin

What is the function of proteolytic enzymes?

Protein breakdown

Fat breakdown

Breakdown of carbohydrates

Alkali formation

Fermentation of enterobacteria?

lactic acid

Formic acid

propionic acid

Butyric

What mineral compounds are used to bind tRNA to ribosomes?

Biological oxidation is...?

reproduction
cell death

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

Prototrophs

Heterotrophs

Autotrophs

Saprophytes

Nutrient media differ:

Composition

By consistency

By appointment

For all of the above

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

Phase of negative acceleration
Stationary phase

The duration of generation depends on?

age

Populations

All of the above

In order to establish the species affiliation of bacteria, their antigenic structure is often studied, that is, they are identified, which one?

biological

Morphological

Serological

Biochemical

Drygalski's surface seeding method is referred to as...?

Mechanical principles of pure culture isolation

Biological principles for isolating a pure culture

Bibliography

1. Borisov L. B. Medical microbiology, virology, immunology: a textbook for honey. universities. - M .: LLC "Medical Information Agency", 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 honey. universities. - St. Petersburg: SpecLit, 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 exercises in medical microbiology, virology and immunology / ed. V. V. Tets. – M.: Medicine, 2002.

Introduction 6

The composition of bacteria in terms of their physiology. 7

Metabolism 14

Nutrition (nutrient transport) 25

Breath 31

Reproduction 34

Microbial communities 37

APPENDICES 49

References 105

Biochemical properties mostly typical of the genus Salmonella. Distinctive features are: the absence of gas formation during the 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. only cause disease in humans. The source of infection is a sick or bacteriocarrier, which release the pathogen into the external environment with feces, urine, saliva. The causative agents of these infections, like other salmonella, are stable in the external environment, persist in soil and water. S. Typhi can become non-cultivable. Food products (milk, sour cream, cottage cheese, minced meat, jelly) are a favorable environment for their reproduction. The transmission of the pathogen is carried out by water, which currently plays a significant role, as well as by alimentary and contact household routes. The infectious dose is approximately 1000 cells. The natural susceptibility of humans to these infections is high.

Pathogenesis and clinical picture. Once in the small intestine, the causative agents of typhoid and paratyphoid invade the mucous membrane during

effector proteins TTSS-1, forming the primary focus of infection in Peyer's patches. It should be noted that the osmotic pressure in the submucosa is lower than in the intestinal lumen. This contributes to the intensive synthesis of the Vi-antigen, which increases the antiphagocytic activity of the pathogen and suppresses the release of pro-inflammatory tissue mediators by the cells of the submucosa. The consequence of this is the absence of the development of inflammatory diarrhea at the initial stages of infection and the intensive multiplication of microbes in macrophages, leading to inflammation of the 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, the causative agents of typhoid and paratyphoid are carried throughout the body with blood flow, settling in the reticuloendothelial elements of parenchymal organs: the liver, spleen, lungs, and also in the bone marrow, where they multiply in macrophages. From the Kupffer cells of the liver, Salmonella through the bile ducts, into which they diffuse, enter the gallbladder, where they also multiply. Accumulating in the gallbladder, salmonella cause inflammation and reinfection of the small intestine with bile flow. The re-introduction of Salmonella into Peyer's patches leads to the development of hyperergic inflammation in them according to the type of 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 with functional insufficiency of the latter leads to the formation of a bacteriocarrier. Salmonella can also persist in the gallbladder for a long time, excreted in the faeces 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 with urine, sweat, and mother's milk. Diarrhea begins at the end of the 2nd or the beginning of the 3rd week of the disease, from that time the pathogens are sown from the feces.