Height- this is a coordinated increase in all components of the cell, the result of growth is reproduction.

Reproduction of bacteria– increase in the number of cells in the population.

In the process of growth, the bacterial cell increases by 2-3 times, it is intensely stained and RNA accumulates. Under favorable conditions, growth ends with reproduction. In bacteria, reproduction occurs by dividing in half - binary fission is the main method of reproduction.

growth curve characterizes the growth and reproduction of bacteria in certain environmental conditions. The growth curve is obtained from a batch culture study.

periodical culture- this is a population of m / o that develops in a limited volume of the environment without the supply of nutrients.

Phase 1 - initial - bacteria grow, but do not multiply

Phase 2 - phase of logarithmic growth - bacteria multiply intensively, their number increases in a logarithmic progression.

3 phase - stationary - reproduction - equal to mortality

Phase 4 - death - metabolic products accumulate, nutrients are depleted and bacteria die.

External factors can have:

    bacteriostatic action - inhibit the reproduction and growth of bacteria

    bactericidal action - cause the death of bacteria

Reproduction of bacteria.

It begins with the replication (doubling) of the genome, and then division occurs.

Bacteria have vegetative replication - information is transferred from the parent cell to the daughter cell.

In bacteria, replication is self-regulating - the genome contains genes responsible for replication.

Replication is semi-conservative in nature - daughter cells receive an evenly distributed genetic material (one strand of DNA is maternal, the second is newly synthesized).

Replication starts from a certain point, from which the DNA unwinds, a replication fork is formed, and the SSB protein is synthesized, which prevents the re-twisting of the strands. The process is carried out by DNA polymerase, which is able to attach complementary nucleotides to the free 3" end.

The synthesis of complementary regions is triggered by primer loading. This is a section of RNA that is complementary to template DNA and the primer has a free 3 "end. Filling with a primer starts DNA synthesis, Okazaki fragments are built on the matrix, which are sewn into a single thread by DNA ligases. In a bacterial cell, 2 identical DNA strands are formed, which are pulled apart along the poles cells and after replication, bacterial division starts.

The division begins with the elongation of the cytoplasmic membrane, an intercellular septum is formed along the equator, along which the bacterium divides binary and 2 identical daughter cells are formed.

Indicators of growth and reproduction of bacteria:

    Increase in cell size

    The concentration of bacteria - the number of cells in 1 ml

    Density of bacteria - the mass of bacteria in mg per ml

    Generation time is the time it takes for the number of cells to double.

22. Cultivation and methods for isolating a pure culture of aerobes and anaerobes.

Cultivation m/o- This is the production of a large number of bacteria on a nutrient medium.

Purpose of cultivation: Micro study biological properties, For the diagnosis of infections and To obtain a biological product from bacteria or obtained using bacteria.

Bacteria cultivation conditions:

    The presence of a complete growth medium.

    Optimum t (≈37 0 C)

    Culture atmosphere (with or without O2).

    Culture time - visible growth in 18-48 hours, or for some 3-4 weeks (tbc)

    Illumination of the nutrient medium for photosynthetic (grow only in the presence of light).


Growth and reproduction of microorganisms

Growth is an increase in the number chemical components microbial cell. To characterize the growth of microorganisms, the concept of bacterial mass is used, which is expressed by the density of bacteria (dry weight per 1 ml). The reproduction of microbes is described by the number of bacteria, reflecting the concentration of cells in 1 ml. There is no strict proportionality between the increase in the number of bacteria and the bacterial mass. This is explained by the fact that not all cells in the bacterial population are viable - some of them are dead, some are at different stages of destruction. Participating in the creation of the bacterial mass, such cells do not participate in the further reproduction of bacteria. Bacteria reproduce by direct division. In this case, a constriction is formed or the cytoplasmic membrane begins to grow inward, perpendicular to the longitudinal axis of the cell with the formation of a disk-cell plate.

This plate can sometimes be incomplete and has a hole in the center of which connects both sister cells. Subsequently, a side wall grows into the cell plate, which forms a transverse partition dividing the cell plate into two parts, each of which goes to one of the formed cells. The central opening, not separated by a transverse septum or plate, is called plasmodesmos. Plasmodesmos plays a role in connecting the cells of some bacteria into long chains or groups. In addition to the above, the process of division of bacterial cells can occur by ligation. The number of bacterial cells in the process of reproduction increases exponentially. For most bacteria, the generation time is 20 - 30 minutes.

Growth and reproduction of bacteria manifest themselves differently depending on the cultivation conditions. On dense nutrient media, a manifestation of the growth and reproduction of bacteria is the appearance of colonies, which are visually distinguishable clusters of bacterial cells. Colonies are characterized by a set of specific features, on the basis of which pure cultures of bacteria can be identified. These signs include: sizes (large, medium, small, microscopic); shape (round, flattened, etc.); color, depending on the formation of pigments by bacteria; surface (convex, flat, matte, shiny, etc.); the nature of the edges (smooth, rough, etc.); consistency (homogeneous, pasty, mucous, etc.); transparency (transparent, cloudy).

The growth process begins with a growth retardation phase, or lag phase. During this period, intense metabolic activity of bacteria occurs, the result of which is the preparation of the cell for rapid reproduction. The phase begins with the introduction of bacteria into the medium. Its duration depends on the age of the sown culture (it is longer when the old culture is introduced), the composition of the medium, temperature and other factors.

Growth of a bacterial cell. The increase in the biomass of cellular protoplasm resulting from the synthesis of plastic material in the process of nutrition is called growth. Microbes grow rapidly, reaching within a short time the limit of their physiological maturity.

Fig.1. Development cycle of hay bacillus

1 - young hay stick; 2 - hay stick, shed flagella; 3 - cell chain division; 4 - formation of flagella in chains; 5 - spore formation

Cell division. A cell that has reached a certain middle age, begins to divide, and in the nutrient medium, an increase in the bacterial population - culture is simultaneously observed. Cell division is preceded by the formation of a cytoplasmic membrane, which usually forms in the middle of a bacterial cell. During cell division, DNA replication occurs. In this case, hydrogen bonds are broken and two chains (helices) of DNA are formed, each of which is present in daughter cells. Then single-stranded DNAs are joined by hydrogen bonds and double-stranded DNAs reappear, possessing genetic information. Cell division is considered complete when the newly formed cells are separated by a cytoplasmic septum.


Fig.2. Electron diffraction patterns of ultrathin sections of dividing bacteria

a - staphylococcus, dividing by the formation of a division septum (indicated by arrows), × 32,000; b - Escherichia coli - as a result of the formation of division constriction (indicated by arrows); 1 - cell wall, 2 - cytoplasmic membrane, 3 - nucleoid; × 22,000.

Reproduction - binary fission of bacteria, rickettsiae, protozoa, etc. In this case, two new individuals are formed, endowed with the genetic information of the mother cell. This method of reproduction is called integral, and viruses reproduce in a disjunctive way, i.e., by separate synthesis of their components - nucleic acid and protein in the host cell.

Bacterial cells divide faster initial stages population growth. In the later stages, division is slower, some of the mother cells die, and in certain types bacteria appear various inclusions.

In favorable conditions, the rate of reproduction of bacteria is very high. Every 15–20 minutes, two individuals are obtained from one individual. According to the calculations of some researchers, if a microbe produces two individuals in just one hour, then in a day the number of microbial cells reaches 16.5 million. which can fill the pools of all seas and oceans.

With such reproduction, microbial cells could cover the entire surface of not only the seas and oceans, but also the continents. Consequently, the life of people on our planet would be impossible. However, microbial reproduction does not exist absolute law geometric progression. Their growth and reproduction are negatively affected by the antagonistic relationships of microorganisms, depletion of the nutrient medium, oxygen deficiency, accumulation poisonous products the vital activity of microbes. These factors prevent continuous cell division.

Bayle's long-term studies and observations have established that in a liquid nutrient medium in a certain volume, the maximum development of cells with a limiting number occurs. Within 24 hours under the same conditions, the concentration of cells in 1 ml of the liquid medium is established: for E. coli and paratyphoid B bacteria - 1.5 billion, for Grigoriev's dysentery bacteria - Shiga and staphylococci - 300 billion, for typhoid fever bacilli - 800 billion. numeric expression It is customary to call the M-concentration (M is the maximum) of microbes. Under normal growing conditions, the M-concentration of cells is the limit of microbial accumulation. It is interesting to note that if such a quantity of microbes is inoculated into a fresh nutrient medium that is equal to the M-concentration, then the number of cells does not increase, and if the number of cultivated microbes exceeds the M-concentration, the excess ones die.

Reproduction of bacteria in a population

To understand the patterns of reproduction of microbes in a population, pure cultures are studied. However, microbes in natural and artificial conditions are found in associations. bacterial population a collection of bacteria that multiply in a certain volume of a liquid medium in a test tube, flask, etc. is called. With the continuous growth of bacteria on the surface of a dense nutrient medium in a test tube, the totality of all cells in it is considered to be a single population. In the case of the growth of isolated colonies, each of them can be considered a separate population, since they do not communicate with each other.

When microbes are grown on dense nutrient media, certain features of their growth are revealed, i.e., colonies appear, representing the offspring of one or more cells. Appearance colonies, their shape, color, transparency, size and other properties are hallmarks for each type of bacteria. A number of bacterial species that have flagella on agar give a continuous growth covering the entire surface of the dish (Proteus vulgaris). Sporogenic species differ in the nature of the colonies, forming opaque colonies with a matte surface.

The growth of bacterial cells in liquid nutrient media is characterized by uniformity, which is not the case on dense nutrient media. However, even with this method, some features of bacterial growth can be observed. Species that form dry colonies on dense media give a variety of sediments in a clear broth. Species that form soft and moist colonies generally give a homogeneous growth, evenly disturbing the nutrient medium.

For a culture growing in a liquid medium, aeration has great importance. It is known that in test tubes or flasks, only the upper layers of the liquid come into contact with atmospheric air, and because of this, some obligate aerobes, for example, Mycobacterium tuberculosis, Vibrio cholerae, etc., accumulate on the surface, forming a delicate film.

Growth phases of a bacterial population

In 1918 Buchanan, studying the characteristics of bacterial reproduction, proposed a curve indicating the number of cells in each period of time. The dynamics of bacterial reproduction is characterized by the following phases, indicated by Roman numerals.

In the initial phase (segment I), bacteria adapt to new living conditions from the moment they are sown on a nutrient medium. In this phase, bacteria do not multiply. The duration of the initial phase is 1–2 hours.

The initial phase of reproduction (section II) is characterized by increased metabolic processes, growth rate and cell division. However, bacterial growth is slow. The duration of this phase is 2 hours.

In the logarithmic phase (segment III), there are accelerated growth and cell division. In this phase of maximum reproduction, morphological, cultural, biochemical, antigenic, and virulent properties typical of each bacterial species are formed. The duration of the phase is 5–6 hours.

The deceleration phase (segment IV) occurs after the active growth and reproduction of bacterial cells. By this time, the conditions in the environment are changing; accumulation of toxic products of metabolism nutrients decreases, the pH of the medium does not meet the individual needs of individual microbes, hydrogen acceptors are consumed, the release of energy and the rate of cell division slow down, the generation time decreases, the number of dying cells increases. The duration of the phase is 2 hours.

The stationary phase (segment V) is characterized by a constant concentration of living cells in the nutrient medium. Moderate multiplication of cells does not lead to an increase in microbial mass. In this phase, a balance is established between the number of dying and emerging cells. The duration of the phase is 2 hours.

The death acceleration phase (segment VI) is characterized by an imbalance between reproduction and accelerated cell death. This phase lasts 4–5 hours.

In the phase of logarithmic death (segment VII), massive cell death occurs at a constant rate. The duration of the phase is about 5 hours.

The phase of decreasing the rate of death (segment VIII) is characterized by the fact that the surviving bacteria go into a dormant state.

The phases of bacterial reproduction in time depend on the type of bacteria, the quality of the nutrient medium, its concentration, temperature and aeration. Therefore, the duration of each phase is indicated approximately. Under optimal conditions, cell division in a number of individuals occurs in different time, for example, Escherichia coli divide after 15-20 minutes, typhoid bacteria - 20-25 minutes, streptococci - 25-30 minutes, Mycobacterium tuberculosis - 18-20 hours.

The duration of the cell death phase is related to the species characteristics of bacteria. The death period of pneumococcus lasts 2-

3 days, and E. coli - months. In the dying stage, the cells stain weakly, and some of them do not perceive dyes. In addition, the forms of bacteria, their biochemical activity and antigenic properties change.



In order to study microorganisms, determine the etiological factors infectious diseases, deal with the prevention and treatment of infectious diseases and solve many other issues related to microorganisms, it is necessary to have them in sufficient quantities, which means creating all conditions for normal growth and reproduction of microorganisms.

The term "reproduction" of microbes means their ability to self-reproduce, to increase the number of individuals.

Reproduction of microorganisms occurs by transverse division, budding, spore formation, reproduction.

The growth of microorganisms means an increase in the mass of microbes as a result of the synthesis of cellular material and the reproduction of all cellular components and structures.

Bacteria, spirochetes, actinomycetes, fungi, rickettsiae, mycoplasmas, protozoa, chlamydia are said to reproduce, while viruses and phages (microbial viruses) reproduce.

The reproduction of microorganisms corresponds to certain patterns. The rate of division of microorganisms is different, it depends on the type of microbe, the age of the culture, the characteristics of the natural and artificial nutrient medium, temperature, carbon dioxide concentration and many other factors.

In the process of reproduction, microorganisms at various stages undergo morphological and physiological changes (in shape, size, staining, biochemical activity, sensitivity to physical and chemical factors etc.).

Microorganisms have age-related variability, i.e. individuals change at different stages of growth, maturation and aging. These changes are observed in the normal cycle individual development microorganism, which depends on the nature of the organism, on the complexity of its structure and sequence in development.

Bacteria have the simplest cycle of development among microorganisms. They reproduce by simple transverse division in different planes. Depending on this, cells can be arranged randomly, in clusters, chains, packages, in pairs, in fours, etc.

A characteristic feature of bacteria, which distinguishes them from numerous animals and plants, is their extraordinary rate of reproduction.

Each bacterial cell, on average, undergoes division within half an hour, which is due to increased metabolism, the speed with which the nutrient material enters the cell.

The factor inhibiting the reproduction of bacteria is the depletion of the nutrient substrate and poisoning. environment decay products.

There are eight main phases of reproduction in bacteria.

1. The initial stationary phase, which is a period of time of one to two hours from the moment of sowing bacteria on a nutrient medium. No reproduction occurs during this phase.

2. The phase of reproduction delay (lag - phase), during which the reproduction of bacteria occurs very slowly, and their growth rate increases. The duration of the second phase is about two hours.

3. The phase lasts five to six hours. The third phase is characterized maximum speed division, reduction in cell size.

4. Phase of negative acceleration (lasts about two hours). The rate of reproduction of bacteria decreases, the number of dividing cells decreases.

5. Stationary phase, lasting about two hours. The number of new bacteria is almost equal to the number of dead individuals.

6. Phase of cell death acceleration (lasts about three hours).

7. Phase of logarithmic cell death (lasts about five hours), in which cell death occurs at a constant rate

8. Phase of decrease in the rate of death. The surviving individuals go into a state of rest.

The duration of the breeding phases is not a constant value. It can be different depending on the type of microorganisms and cultivation conditions.

The development cycle of coccoid bacteria is reduced to the growth of the cell and its subsequent division. rod-shaped asporogenic bacteria young age grow, reach a maximum size, then divide into two daughter cells, which repeat the same cycle. In bacilli and clostridia, the process of spore formation is included in the development cycle under certain conditions.

Spirochetes and rickettsia, like bacteria, reproduce by binary fission.

Among mycoplasmas, all elementary bodies of a spherical or ovoid shape have the ability to reproduce. In the process of development, several filamentous outgrowths appear on the elementary body, in which spherical bodies are formed. Gradually, the threads become thinner and chains are formed with clearly defined spherical bodies. Then the threads are divided into fragments and the spherical bodies are released.

Reproduction of some mycoplasmas occurs by budding of daughter cells from larger spherical bodies. Mycoplasmas reproduce by transverse fission if the processes of mycoplasma division proceed synchronously with the replication of the nucleoid DNA. If synchrony is disturbed, filamentous multinucleoid forms are formed, subsequently dividing into coccoid cells.

Actinomycetes and fungi have two various stages development: the stage of vegetative growth, in which the formation of mycelium is characteristic and the stage of formation of spores that form on spore-bearing plants.

An important feature of actinomycetes and fungi is a significant variety of ways of their reproduction. They are characterized by vegetative, asexual and sexual reproduction.

Vegetative propagation is carried out by dividing into fragments of hyphae, followed by the formation of individual rod-shaped and cocci-shaped cells.

Asexual reproduction occurs vegetatively (growth of fragments of hyphae or their individual cells) and with the help of more or less specialized bodies reproduction (spores and conidia). The most frequent, asexual, way of reproduction is manifested in the formation of exogenous and endogenous spores. Exospores or conidia are formed at the ends of fruiting hyphae, but are enclosed within a common sac - sporangia. Hyphae that carry sporangia are called sporangiophores. Sporangiophores can be straight, wavy, spiral.

Sexual reproduction occurs through special bodies- ascospores, basidiospores, the formation of which is preceded by the sexual process. According to the biological purpose, spores of actinomycetes and fungi are dormant, serving to preserve the species for a certain period and serving for rapid reproduction.

Spores of actinomycetes and fungi are produced by each individual in in large numbers, since, unlike bacterial spores, they serve mainly the purposes of reproduction. They are less resistant to environmental factors than bacterial spores.

In protozoa, as well as in actinomycetes and fungi, along with reproduction by division, there is also a sexual process.

Chlamydia, viruses and phages have peculiar cycles of development.

Reproduction of chlamydia begins with the penetration of elementary bodies into a sensitive tissue cell by endocytosis. These bodies in the vacuole of the cell turn into vegetative forms, called initial or reticular bodies, which have the ability to divide. Reticular bodies have a lamellar cell wall, and in the cytoplasm there are loosely located nuclear fibrils and numerous ribosomes. After repeated division, the reticular bodies turn into intermediate forms, from which a new generation of elementary bodies develops. The whole cycle of development of chlamydia lasts 40-48 hours and ends with the formation of a microcolony of chlamydia in the cytoplasm of the host cell.

After the rupture of the vacuole wall and the complete destruction of the host cell, the microcolonies of chlamydia, being outside the whole cell, break up into independent elementary bodies, and the cycle of penetration of chlamydia into the cell with their subsequent reproduction is repeated.

The reproduction of viruses is characterized by a sequence of individual stages.

1. Stage of adsorption. Virions are adsorbed on the surface structures of the cell. In this case, the interaction of complementary structures of the virion and the cell, which are called receptors, occurs.

2. The stage of penetration of the virion into the host cell. The ways of introduction of viruses into cells sensitive to them are not the same. Many virions enter the cell by pinocytosis, when the resulting pinocytic vacuole "pulls" the virion into the cell. Some viruses enter the cell directly through its membrane.

3. The stage of destruction of the outer shell and capsid of the virion with the help of proteolytic enzymes of the host cell. In some virions, the process of destruction of their shell begins at the stage of adsorption, in others - in the pinocytic vacuole, in others - directly in the cytoplasm of the cell with the participation of the same proteolytic enzymes.

4. Stage of viral protein synthesis and nucleic acid replication. After the complete or partial release of the viral nucleic acid, the process of viral protein synthesis and nucleic acid replication begins.

5. Assembly stage or virion morphogenesis. The formation of virions is possible only under the condition of a strictly ordered connection of viral structural polypeptides and their nucleic acid, which is ensured by the self-assembly of protein molecules around the nucleic acid. In some viruses, this process occurs in the cytoplasm, in others, in the nucleus of the host cell. In complexly organized viruses with an outer shell, further assembly occurs in the cytoplasm during their release from the cell.

6. The stage of release of virions from the host cell. A number of complex viruses leave the host cell, while the cells remain viable for some time, and then die. Simple virions leave the cell through the holes formed in its shell, the host cell dies, not maintaining viability for some time.

In some cases, the reproduction of virions in cells can occur over many months and even years. Viruses are shed through the cell wall. When such cells divide, the virions are transferred to daughter cells, which in turn begin to produce viral particles.

There are three types of interaction between a virus and a cell: productive, abortive, and virogenic.

Productive the type of interaction is the formation of new virions.

Abortive the type of interaction can be abruptly interrupted at the stage of viral nucleic acid replication or viral protein synthesis, or virion morphogenesis.

Virogenic the type is characterized by the incorporation (integration) of the viral nucleic acid into the DNA of the cell, which ensures synchronous replication of the viral and cellular DNA.

During phage reproduction, it is also adsorbed on the cell surface (stage 1) as a result of the interaction of amino groups of proteins localized in the peripheral part of the phage tail process and negatively charged carboxyl groups on the surface of the bacterial cell.

There are reversible and irreversible phases of adsorption. The reversible phase is characterized by the fact that fixed phages can be separated from the cell by vigorous agitation or the concentration of ions can be sharply reduced. The released phages retain their viability.

During the second irreversible phase of adsorption, the phage does not separate from the microbial cell body. The adsorption process takes several minutes. Under the influence of an enzyme located in the tail process of the phage, a hole is formed in the body of the microbial cell at the site of attachment of the phage, through which the phage DNA penetrates into the cell. The phage shell remains outside (stage 2).

Some phages introduce their nucleic acid into the cell without prior mechanical damage. cell wall. During the latent period following the penetration of the phage nucleic acid into the cell, the biosynthesis of the phage nucleic acid and phage capsid proteins takes place.

There is a synthesis of enzymes necessary for the replication of phage nucleic acid and structural proteins of the phage (stage 3).

In the fourth stage, the hollow phage particles are filled with the phage nucleoacid and mature phages are formed. Phage morphogenesis is carried out.

At the end of the latent period, the infected microbial cells are lysed and mature phage particles are released (stage 5).

It is believed that the adsorption of the phage lasts 40 minutes, the latent period is 75 minutes. The entire cycle of interaction between a phage and a microbial cell lasts a little more than three hours.

The introduction of a phage into a microbial cell is not always accompanied by its lysis. Often, the interaction of a phage with a microbial cell leads to the formation of lysogenic cultures.

By the nature of the interaction with the microbial cell, temperate and virulent phages are distinguished. The state of lysogeny is caused by temperate phages. Lysogenic microbial cells are resistant to virulent phages. Virulent phages cause the formation of new phages and the lysis of the microbial cell.

The growth curve characterizes the growth and reproduction of bacteria under certain environmental conditions. The growth curve is obtained from a batch culture study.

periodical culture This is a population of microorganisms that develops in a limited volume of the environment without the supply of nutrients.

Phase 1 - initial - bacteria grow but do not multiply

Phase 2 - lg growth phase - bacteria multiply intensively

3 phase - stationary - reproduction - equal to mortality

Phase 4 - death - metabolic products accumulate, the nutrient medium is depleted, bacteria die.

External factors may have

  • Bacteriostatic action- inhibit the reproduction and growth of bacteria
  • Bactericidal action- kill bacteria

bacterial enzymes

- Enzymes specific proteins that catalyze chemical reactions. Enzymes cause a redistribution of electron densities and some deformation of the substrate molecule. This leads to a weakening of intramolecular bonds, the activation energy decreases and the reaction accelerates.

Classification of enzymes -

  1. According to the type of catalyzed reaction - oxidoreductases, lyases, transferases, hydrolases, etc.
  2. By localization - endoenzymes - catalyze reactions inside the cell. Exoenzymes - secreted from the bacterial cell, catalyze the breakdown
  3. Genetic control of education - constitutive (during the whole life cycle, does not affect the presence of a substrate), inducible - they are formed in response to the presence of a substrate
  4. According to the substrate - proteolytic - break down proteins, saccharolytic - break down carbohydrates, lipolytic - break down fats.

Importance of enzymes.

1. The synthesis of enzymes is determined, therefore, the determination of enzymatic properties serves to identify organisms

2. Enzymes of bacteria determine their pathogenicity

3. Enzymatic properties are used in the microbiology industry

Determination of bacterial enzymes

Proteases break down proteins into amino acids, urea, indole, hydrogen sulfide, ammonia. On media with protein, proteases are detected by isolating these products. Use gelatin, liquefaction of the medium. On curdled whey according to its liquefaction and on milk according to its clarification. Casein - will break down, the protein will coagulate. At the BCH for the release of indole gas and hydrogen sulfide, which are detected using indicator papers

Determination of enzymes that break down carbohydrates - saccharolytic. These enzymes break down carbohydrates into aldehydes, acids, carbon dioxide, and H2. To determine them, use the MPB or MPA, add an indicator of acid formation + carbohydrate + float for gas formation. According to this principle, the environments of Gis and Pestrel are created. If the light of the environment changes, gas is released, then carbohydrates are being split. Monosaccharides are used. On this principle, panels, tablets, paper indicator systems, NIB - systems of indicator papers, an energy tube and devices for recording enzymatic activity are created. (Carbonic acid is formed => indicators with Ph are needed)

Lipolytic enzymes - lipases - are detected on JSA - yolk-salt agar, which contains yolk, in which there are many lipids and the destruction of lipids is accompanied by enlightenment of the medium

Cultivation of microorganisms.

This is getting a large number of bacteria on a nutrient medium. Purposes of cultivation. Cultivation is carried out for

1. Study of microbiological properties

2. To diagnose infections

3. To obtain a biological product - from bacteria or obtained using bacteria.

Such drugs can be therapeutic, diagnostic, prophylactic. Conditions for cultivating bacteria

  1. The presence of a complete nutrient medium.
  2. Optimum temperature
  3. The cultivation atmosphere is either oxygen or its absence.
  4. Cultivation time - visible growth after 18-48 hours, but some - tuberculosis for example - 3-4 weeks
  5. Light Some will grow only in the presence of light.

Methods for cultivating aerobes

  1. Stationary - on the surface of the agar
  2. Method of deep cultivation with medium aeration. Aeration is carried out to dissolve oxygen in the environment.
  3. Continuous cultivation - use flowing nutrient media.

Cultural properties of microorganisms. These are features of bacterial growth on nutrient media.

On liquid nutrient media, bacteria cause turbidity of the medium, can form sediment - near-bottom, parietal, and can form a film on the surface of the medium. Colonies form on dense nutrient media.

The colony- an isolated accumulation of microorganisms of the same species on a dense nutrient medium. It has a certain size, surface, edge, shape, consistency, structure, color.

Colony types

S-smooth - round shape, smooth edges, smooth surface.

R-colonies - rough, uneven edges, striated surface

Colony SR 0 intermediate - slightly uneven edges and surface.

Features of cultivation of anaerobes. For the cultivation of anaerobes, oxygen-free conditions are created. This is achieved

  1. Regeneration of the nutrient medium - the nutrient medium is boiled and dissolved oxygen leaves the medium.
  2. use of special devices - anaerostats and desiccators. In them, oxygen is absorbed either by chemical absorbers or is pumped out of the device.
  3. Adding reducing substances to the medium - substances that are easily and quickly oxidized - carbohydrates, cysteine, pieces of parenchymal organs, ascorbic acid. On this principle, an environment for anaerobes was created - Keith-Tarozzi - an environment for anaerobes. It contains BCH, carbohydrate and pieces of liver which contain cysteine.
  4. Special seeding methods. Sowing under oil, sowing in the Veyon-Venyan tube, sowing according to Fortner. Aerobes and anaerobes are populated on a cup - Aerobes absorb oxygen and an anaerobic environment is obtained.

Isolation of pure cultures.

pure culture- a population of microorganisms of the same species, isolated on a liquid or solid nutrient medium in large quantities.

Selection goals.

  1. Diagnosis of infections. The isolation of pure cultures is the basis of the bacteriological method. Based on the isolation of pure culture and its identification. Identification is the definition of a species.
  2. Obtaining biological products
  3. The study of the biological properties of bacteria
  4. Sanitary and hygienic research

Stages of isolating a pure culture of aerobes.

  1. Examining the mixture - smear Gram stain.
  2. Separation of the mixture and obtaining colonies. Dissociation is carried out 1) According to Drygalsky - strokes on the surface of the agar. Loop take the material and inoculate on agar. Sowing Spatula on several Cups. 2) Serial dilution method. 3) Koch - method of serial dilutions in molten agar.
  3. Colony frequency check, smear, gram stain
  4. Subculturing material from colonies onto agar slant to accumulate a pure culture. The selected pure culture is identified by properties - morphological, tinctorial, cultural, enzymatic, and others.

Isolation of pure culture of anaerobes

1. Accumulation of anaerobes. The mixture is inoculated on the Kittarocy medium and heated at a temperature of 80 degrees for 10 minutes. Anaerobes that form spores are preserved, while others - vegetative forms die. Then the nutrient medium is cultivated, the spores germinate, and accumulate

2. Obtaining colonies according to Zeisler, anaerobic colonies are obtained on the surface of the agar in the Anaerostat, according to Weinberg, colonies are obtained in Veyon-Vignal tubes.

3. Checking the frequency of colonies - smear, Gram stain

4. Reseeding Colonies on Kittarocy medium, accumulation by anaerobes, pure culture.

5. Identification, determination of the type of anaerobe.

Other ways to isolate pure cultures.

  1. Optimum temperatures can be used
  2. Isolation of spores when the mixture is heated for 10 minutes at 80 degrees
  3. Using the phenomenon of swarming - spreading beyond the area of ​​​​sowing.
  4. The Shukevich method is the isolation of a pure culture of microorganisms with creeping growth.
  5. Filterability of bacteria - the ability to pass through filters with a certain size of spores. Treatment of the mixture with ultraviolet rays, ultrasound, antisera, obtaining a pure culture of microorganisms resistant to these factors.
  6. By electrophoresis of the mixture. Organisms with a certain charge will accumulate at the anode or cathode.
  7. Use a micromanipulator. Under a microscope, take a cell and get a pure culture - a clone - the offspring of one microbial cell. The use of elective nutrient media.
  8. Bile, thiurite salts, sodium chloride, antibiotics are added to nutrient media, and a pure culture of resistant microorganisms is isolated.
  9. You can use differential diagnostic environments.
  10. Can be used biological method. White mice are infected intraperitoneally with a mixture of bacteria and due to tropism, the bacteria accumulate in a specific organ.

bacteria pigments.

These are dyes secreted by a bacterial cell, their synthesis is genetically determined. According to the chemical structure, pigments can be carotenoids - red-yellow, pyrroles - green, phenosine dyes - blue-green and melanin - black enzymes.

Yellow - golden staphylococcus, blue-green - Pseudomonas aeruginosa

Pigments are divided into

  1. Insoluble pigments - stain only colonies
  2. Soluble pigments - can be soluble in alcohols, water

Pigments are formed, as a rule, in bacteria that are in the air microflora, in antibiotic-resistant microorganisms, because. they are secondary metabolites and pigments are often formed in the presence of light.

Function of pigments

  1. Pigments are involved in metabolism
  2. Increase resistance to antibiotics
  3. Increases UV resistance by protecting areas sensitive to photooxidation

L-forms of bacteria.

Opened in 1935 These are microorganisms that lack a cell wall, but retain the ability to grow and multiply. L forms are formed in most heterotrophs and fungi. Factors inducing L transformation -

1. Antibiotics

2. Amino acids - glycine, methionine, leucine and some others.

3. Enzymes - lysozyme.

4. Factors of macroorganism - macrobodies, compliment

These factors either destroy the cell wall or act on the cell genome and the synthesis of cell wall components does not occur.

PropertiesLforms.

  1. L forms ensure the survival of bacteria under changing environmental conditions.
  2. Morphologically similar in certain types of bacteria. They are polymorphic - spherical, gram-negative. They form type A colonies - small colonies on the surface of the medium and type B colonies - a dark center and raised edges, the colonies grow into the nutrient medium.
  3. Anaerobes or microaerophiles
  4. L-forms have many ways of reproduction - binary fission, budding, fragmentation, combined.
  5. They have reduced virulence, they lack adhesion, and they have altered antigenic properties.
  6. They are able to reverse - return to their original bacterial form

And cause difficult-to-treat infections.

This is due to the fact that L - forms are resistant to antibiotics and they are resistant to the protective factors of the macroorganism, to antibodies, phagocytosis, compliment.

Uncultivated forms of NFB bacteria

These are bacteria that have metabolic activity, but do not grow on nutrient media, the transition to an uncultivated form can be observed in many microorganisms, when exposed to adverse conditions. This transition is genetically controlled. The transition is carried out under the influence of factors

  1. Temperature, especially low
  2. Salt concentration
  3. Aeration of the environment
  4. Amount of Nutrients

The value of uncultivated forms. In this form, they are stored in external environment between epidemics and when they enter the macroorganism, they can be recultivated - revived - this explains the presence of naturally focal diseases.

Identification -

1. Direct cell count

2. Detection of DNA activity

3. genetic methods research.

Reproduction of microorganisms - an increase in the concentration of microorganisms in a unit volume of the medium, aimed at preserving the species.

Microorganisms are characterized by:

    a variety of methods of reproduction;

    switching from one breeding method to another;

    the possibility of using several methods at the same time;

    high reproduction rate.

Methods of reproduction of microorganisms

I. Sexual withbreeding method seen only in eukaryotes.

II. Asexual methods of reproduction.

    Equal area binary transverse fission (simple division, isomorphic division, mitosis) observed in most unicellular microorganisms (bacteria, rickettsia, protozoa, yeast), as a result, two new daughter full-fledged individuals are formed, endowed with the genetic information of the mother cell, symmetrical with respect to the longitudinal and transverse axes, the mother cell itself disappears.

At the same time, in the majority of Gram+ bacteria, division occurs by the synthesis of a transverse septum extending from the periphery to the center (Fig. 63A). The cells of most Gram bacteria divide by cell constriction (the cell becomes thinner in the middle) (Fig. 63B).

    Budding (unequal binary fission) seen in members of the genus Francisella And Mycoplasma and yeast-like fungi. When budding, the mother cell gives rise to a daughter cell: at one of the poles of the mother cell, a small outgrowth (bud) is formed, which increases in the process of growth. Gradually, the kidney reaches the size of the mother cell, after which it separates. The renal CS is completely synthesized anew (Fig. 63B). In the process of budding, symmetry is observed with respect to only the longitudinal axis. There are morphological and physiological differences between mother and daughter cells. The new daughter cell adapts better to changing conditions.

    Fragmentation of filamentous forms characteristic of the genus Actinomyces And Mycoplasma.

    Exospore formation characteristic of Streptomycetes, yeast-like and mold fungi.

    special development cycle tiya seen in Chlamydia. Only vegetative forms of chlamydia (reticular or initial bodies) are capable of dividing in the cells of the macroorganism. Their cycle, consisting of several divisions, ends with the formation of intermediate forms, from which elementary bodies are formed, giving rise to vegetative forms. After destruction of the vacuole wall and host cell, the elementary bodies are released and the cycle repeats. The cycle lasts 40-48 hours.

    Multiple division described for one group of unicellular cyanobacteria. Multiple fission is based on the principle of equal binary fission. The difference lies in the fact that in this case, after binary fission, the resulting daughter cells do not grow, but they again undergo division (Fig. 63D).

Multiple division (schizogony) also described in protozoa (malarial plasmodia): nuclear material divides into many nucleoli, surrounded by sections of the cytoplasm, resulting in the formation of many daughter cells.

Mechanism and phases of simple division

A. Growth to a certain degree of maturity. Cell growth is not unlimited, and after reaching a certain size, the bacterial cell begins to divide. Cell growth slows down during division and starts again after division.

B. Karyokinesis ( DNA replication and d nucleoid division). From the mature cytoplasm, a signal arrives that activates the initiator gene on DNA. Microorganisms under the action of the initiator gene synthesize an initiator protein that acts on the replicator gene - a special section of DNA from which DNA duplication and division into two strands begin.

The division of the DNA molecule (replication) occurs according to a semi-conservative mechanism and normally always precedes cell division. DNA replication begins at the point of attachment of the circular chromosome to the CMP, where the enzymatic apparatus responsible for replication is localized.

The mechanism of DNA replication is expressed in the breaking of hydrogen bonds between its two polynucleotide chains, their unwinding and synthesis with the help of DNA polymerase along each old chain of new chains with a complementary base sequence. After divergence into daughter cells along one old and one new polynucleotide chain, hydrogen bonds are restored between them and a semi-conservative double-stranded DNA is formed.

Normally, there is a certain temporal relationship between chromosome replication and bacterial cell division. Impacts of various chemicals And physical factors, leading to the suppression of DNA replication, stop cell division. However, under certain conditions, the connection between both processes can be broken, and cells are able to divide in the absence of DNA synthesis.

B. Cytokinesis (cell division). In parallel with the replication of DNA molecules, the membrane is synthesized near the mesosome, in the area of ​​DNA contact with the CPM. The formation of a septum leads to cell division. The moment initiating cell division is the end of DNA replication. This leads to the separation of daughter DNA molecules and the formation of separate chromosomes. The newly formed daughter cells separate from each other.

Inhibition of membrane synthesis before the end of replication leads to disruption of the division process: the cell stops dividing and grows in length. In some bacteria, the formation of a septum does not lead to cell division: multi-chambered cells are formed.

D. The divergence of the resulting daughter cells occurs as a result of lysis of the middle layer of the COP. If, after repeated division in the same plane, the cells do not diverge, they form chains of rod-shaped (bacillus) or spherical(Streptococcus) cells or paired cells(Neisseria) . Separation of cells is possible with the isolation of one of the cells by moving along the surface of the other, as a result, the bacteria are located disorderly (Escherichia). If, during separation, one of the daughter cells, without breaking away from the division point, moves along an arc, a V-shaped form (Corynebacterium, Bifidobacterium). After binary fission and divergence of cells in several planes, cell clusters different shapes: bunches (Staphylococcus), packages (Sarcina) (Fig. 65). If nucleoid division precedes cell division, multinucleoid microorganisms. Under the influence of unfavorable external factors(bile salts, UV rays, surfactants, antibiotics) cell division can stop while maintaining its growth. In this case, the formation of elongated filiform cells.

Rice. 65. Division of cocci

Generation period- the time interval during which the number of bacteria doubles. The rate of reproduction of microorganisms and the period of generation depend on the type of microorganism, the size and properties of the inoculum, the composition of the nutrient medium, its pH, aeration, incubation temperature, and other factors. Under favorable conditions, many microorganisms divide in 15–30 minutes. (E. coli, S. Typhi). In whimsical microorganisms, division is carried out after 45–90 minutes (Streptococcus, Corynebacterium) and even after 18 hours (M. tuberculosis).