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Growth and reproduction

The term "growth" means an increase in the cytoplasmic mass of an individual cell or group of bacteria as a result of the synthesis of cellular material (eg, protein, RNA, DNA). Having reached a certain size, the cell stops growing and begins to multiply.

The reproduction of microbes means their ability to self-reproduce, to increase the number of individuals per unit volume. Otherwise, we can say: reproduction is an increase in the number of individuals of a microbial population.

Bacteria reproduce mainly by simple transverse division (vegetative reproduction), which occurs in different planes, with the formation of diverse combinations of cells (grape bunch - staphylococci, chains - streptococci, pairs - diplococci, bales, packages - sarcins, etc.). The division process consists of a number of successive stages. The first stage begins with the formation of a transverse septum in the middle part of the cell (Fig. 6), which initially consists of a cytoplasmic membrane that divides the cytoplasm of the mother cell into two daughter cells. In parallel with this, a cell wall is synthesized, which forms a full-fledged partition between two daughter cells. In the process of bacterial division, an important condition is the replication (doubling) of DNA, which is carried out by DNA polymerase enzymes. When DNA is duplicated, hydrogen bonds are broken and two strands of DNA are formed, each of which is located in daughter cells. Further, daughter single-stranded DNA restore hydrogen bonds and again form double-stranded DNA.

DNA replication and cell division occurs at a certain rate inherent in each type of microbe, which depends on the age of the culture and the nature of the nutrient medium. For example, the growth rate of Escherichia coli ranges from 16 to 20 minutes; in mycobacterium tuberculosis, division occurs only after 18-20 hours; a mammalian tissue culture cell takes 24 hours. Consequently, bacteria of most species reproduce almost 100 times faster than tissue culture cells.

The process of reproduction of microbial culture on a non-replaceable medium proceeds unevenly. It defines four main phases.

1. The initial phase (lag phase), or resting phase. At this time, the culture adapts to the nutrient medium. In the microbial cell, the content of RNA increases, and with its help, the synthesis of the necessary enzymes occurs.

2. Exponential (logarithmic) phase characterized by a maximum increase in cells in culture, it goes exponentially (1, 2.4, 8, 16, 256, etc.). At this time, the majority of young and biologically active cells are in the medium. At the end of the phase, when the medium is depleted, the substances necessary for a given microbe disappear, the amount of oxygen decreases, an increase in metabolic products occurs - culture growth slows down. The curve gradually assumes a horizontal direction.

3. stationary phase, or period of maturity, graphically represents a line running parallel to the x-axis. There comes a balance between the number of newly formed and dead cells. The amount of medium decreases, the density of cells in the population increases, the toxic effect of metabolic products increases - all this causes cell death.

4. Dying phase. In this phase, not only a decrease, but also a change in cells is observed. Degraded forms appear, as well as spores. After a few weeks or months, the culture dies. This happens because toxic waste products not only inhibit, but also kill microbial cells.

Thus, thanks to the processes of metabolism, the vital activity of the microbial cell is maintained. Aerobes need oxygen to breathe, while anaerobes use nitrate and sulfate respiration and fermentation. Microorganisms assimilate organic and inorganic substances from the external environment, oxidizing which they receive the necessary energy and plastic elements. The result is cell growth. Having reached the necessary stage of maturity, the cell reproduces by simple division. In the course of their life activity, microorganisms gradually consume nutrients, releasing their metabolites into the environment, thereby changing the composition of the environment and making it unsuitable for life.

Reproduction of bacteria by fission is the most common method of increasing the size of the microbial population. After division, bacteria grow to their original size, which requires certain substances (growth factors).

The methods of reproduction of bacteria are different, but for most of their species, a form of asexual reproduction is inherent in the division method. Bacteria rarely reproduce by budding. Sexual reproduction of bacteria is present in a primitive form.

Rice. 1. In the photo, a bacterial cell is in the division stage.

The genetic apparatus of bacteria

The genetic apparatus of bacteria is represented by a single DNA - the chromosome. DNA is closed in a ring. The chromosome is located in a nucleotide that does not have a membrane. The bacterial cell contains plasmids.

Nucleoid

The nucleoid is analogous to the nucleus. It is located in the center of the cell. DNA is localized in it - the carrier of hereditary information in a folded form. The untwisted DNA reaches a length of 1 mm. The nuclear substance of a bacterial cell does not have a membrane, a nucleolus and a set of chromosomes, and is not divided by mitosis. Before division, the nucleotide is doubled. During division, the number of nucleotides increases to 4.

Rice. 2. In the photo, a bacterial cell on a cut. A nucleotide is visible in the central part.

Plasmids

Plasmids are autonomous molecules folded into a ring of double-stranded DNA. Their mass is much less than the mass of a nucleotide. Despite the fact that hereditary information is encoded in the DNA of plasmids, they are not vital and necessary for a bacterial cell.

Rice. 3. The photo shows a bacterial plasmid.

Division stages

After reaching a certain size inherent in an adult cell, division mechanisms are launched.

DNA replication

DNA replication precedes cell division. Mesosomes (folds of the cytoplasmic membrane) hold DNA until the process of division (replication) is completed.

DNA replication is carried out with the help of DNA polymerase enzymes. During replication, hydrogen bonds in 2-stranded DNA are broken, as a result of which two daughter single-stranded ones are formed from one DNA. Subsequently, when the daughter DNA has taken its place in the separated daughter cells, they are restored.

As soon as DNA replication is completed, a constriction appears as a result of synthesis, dividing the cell in half. First, the nucleotide undergoes division, then the cytoplasm. Cell wall synthesis completes division.

Rice. 4. Scheme of bacterial cell division.

Exchange of DNA segments

In hay bacillus, the DNA replication process is completed by the exchange of 2 DNA segments.

After cell division, a bridge is formed, along which the DNA of one cell passes into another. The two DNAs then intertwine. Some stretches of both DNA stick together. At the sites of adhesion, DNA segments are exchanged. One of the DNA goes back to the first cell along the jumper.

Rice. 5. Variant of DNA exchange in hay bacillus.

Types of bacterial cell divisions

If cell division is ahead of the division process, then multicellular rods and cocci are formed.

With synchronous cell division, two full-fledged daughter cells are formed.

If a nucleotide divides faster than the cell itself, then multinucleotide bacteria are formed.

Ways to separate bacteria

Division by breaking

Division by breaking is characteristic of anthrax bacilli. As a result of this division, the cells break at the joints, breaking the cytoplasmic bridges. Then they repel each other, forming chains.

sliding separation

With sliding separation after division, the cell separates and, as it were, slides over the surface of another cell. This separation method is typical for some forms of Escherichia.

split split

With a split division, one of the divided cells describes an arc of a circle with its free end, the center of which is the point of its contact with another cell, forming a Roman five or cuneiform (corynebacterium diphtheria, listeria).

Rice. 6. In the photo, rod-shaped bacteria forming chains (anthrax rods).

Rice. 7. In the photo, a sliding method for separating Escherichia coli.

Rice. 8. Splitting method for separating corynebacteria.

View of bacterial clusters after division

Accumulations of dividing cells have a variety of shapes, which depend on the direction of the division plane.

globular bacteria arranged one at a time, two at a time (diplococci), in bags, in chains, or like bunches of grapes. Rod-shaped bacteria - in chains.

Spiral bacteria- chaotic.

Rice. 9. The photo shows micrococci. They are round, smooth, white, yellow and red in color. Micrococci are ubiquitous in nature. They live in different cavities of the human body.

Rice. 10. In the photo, diplococcus bacteria - Streptococcus pneumoniae.

Rice. 11. Sarcina bacteria in the photo. Coccoid bacteria are combined into packets.

Rice. 12. In the photo, streptococcus bacteria (from the Greek "streptos" - a chain). Arranged in chains. They are the causative agents of a number of diseases.

Rice. 13. In the photo, the bacteria are "golden" staphylococci. Arranged like "bunch of grapes". The clusters have a golden color. They are the causative agents of a number of diseases.

Rice. 14. In the photo, the convoluted bacteria of leptospira are the causative agents of many diseases.

Rice. 15. In the photo, rod-shaped bacteria of the genus Vibrio.

bacterial division rate

The rate of division of bacteria is extremely high. On average, one bacterial cell divides every 20 minutes. Within only one day, one cell forms 72 generations of offspring. Mycobacterium tuberculosis divides slowly. The whole process of division takes them about 14 hours.

Rice. 16. The photo shows the process of streptococcus cell division.

Sexual reproduction of bacteria

In 1946, scientists discovered sexual reproduction in a primitive form. In this case, gametes (male and female germ cells) are not formed, however, some cells exchange genetic material ( genetic recombination).

Gene transfer occurs as a result of conjugations— unidirectional transfer of a part of genetic information in the form plasmid upon contact between bacterial cells.

Plasmids are small DNA molecules. They are not associated with the chromosome genome and are able to duplicate autonomously. Plasmids contain genes that increase the resistance of bacterial cells to adverse environmental conditions. Bacteria often pass these genes on to each other. The transfer of gene information to bacteria of another species is also noted.

In the absence of a true sexual process, it is conjugation that plays a huge role in the exchange of useful traits. This transfers the ability of bacteria to exhibit drug resistance. For humanity, the transmission of antibiotic resistance between disease-causing populations is especially dangerous.

Rice. 17. In the photo, the moment of conjugation of two Escherichia coli.

Phases of development of a bacterial population

When sowing on a nutrient medium, the development of the bacterial population goes through several phases.

Initial phase

The initial phase is the period from the moment of sowing to their growth. On average, the initial phase lasts 1-2 hours.

Reproductive delay phase

This is the phase of intensive growth of bacteria. Its duration is about 2 hours. It depends on the age of the culture, the period of adaptation, the quality of the nutrient medium, etc.

logarithmic phase

In this phase, the peak of the rate of reproduction and increase in the bacterial population is noted. Its duration is 5 - 6 hours.

Phase of negative acceleration

In this phase, a decline in the rate of reproduction is noted, the number of dividing bacteria decreases and the number of dead bacteria increases. The reason for the negative acceleration is the depletion of the nutrient medium. Its duration is about 2 hours.

Stationary maximum phase

In the stationary phase, an equal number of dead and newly formed individuals is noted. Its duration is about 2 hours.

Accelerated Death Phase

In this phase, the number of dead cells progressively increases. Its duration is about 3 hours.

Phase of logarithmic death

In this phase, bacterial cells die off at a constant rate. Its duration is about 5 hours.

Decreasing phase

In this phase, the remaining living bacterial cells go into a dormant state.

Rice. 18. The figure shows the growth curve of a bacterial population.

Rice. 19. The photo shows blue-green colonies of Pseudomonas aeruginosa, yellow colonies of micrococci, blood-red colonies of Bacterium prodigiosum and black colonies of Bacteroides niger.

Rice. 20. The photo shows a colony of bacteria. Each colony is the offspring of a single cell. In a colony, the number of cells is in the millions. a colony grows in 1-3 days.

Division of magnetically sensitive bacteria

In the 1970s, bacteria living in the seas were discovered that had a sense of magnetism. Magnetism allows these amazing creatures to navigate along the lines of the Earth's magnetic field and find sulfur, oxygen and other substances that are so necessary for it. Their "compass" is represented by magnetosomes, which consist of a magnet. When dividing, magnetically sensitive bacteria divide their compass. In this case, the constriction during division becomes clearly insufficient, so the bacterial cell bends and makes a sharp fracture.

Rice. 21. The photo shows the moment of division of a magnetically sensitive bacterium.

Bacteria growth

At the beginning of bacterial cell division, two DNA molecules diverge to different ends of the cell. Next, the cell is divided into two equal parts, which are separated from each other and increase to the original size. The rate of division of many bacteria is on average 20-30 minutes. Within only one day, one cell forms 72 generations of offspring.

The mass of cells in the process of growth and development quickly absorbs nutrients from the environment. This is facilitated by favorable environmental factors - temperature, a sufficient amount of nutrients, the necessary pH of the environment. Aerobic cells require oxygen. For anaerobes, it is dangerous. However, the unlimited reproduction of bacteria in nature does not occur. Sunlight, dry air, lack of food, high ambient temperature and other factors have a detrimental effect on the bacterial cell.

Rice. 22. In the photo, the moment of cell division.

growth factors

Bacterial growth requires certain substances (growth factors), some of which are synthesized by the cell itself, and some come from the environment. All bacteria have different requirements for growth factors.

The need for growth factors is a constant feature, which makes it possible to use it for the identification of bacteria, the preparation of nutrient media and use in biotechnology.

Bacterial growth factors (bacterial vitamins) are chemical elements, most of which are water-soluble B vitamins. This group also includes hemin, choline, purine and pyrimidine bases, and other amino acids. In the absence of growth factors, bacteriostasis occurs.

Bacteria use growth factors in minimal amounts and unchanged. A number of chemicals in this group are part of cellular enzymes.

Rice. 23. In the photo, the moment of division of a rod-shaped bacterium.

The most important bacterial growth factors

• Vitamin B1 (thiamine). Takes part in carbohydrate metabolism.
• Vitamin B2 (riboflavin). Takes part in redox reactions.
• Pantothenic acid is an integral part of coenzyme A.
• Vitamin B6 (pyridoxine). Takes part in the metabolism of amino acids.
• Vitamins B12(cobalamins are substances containing cobalt). They take an active part in the synthesis of nucleotides.
• Folic acid. Some of its derivatives are part of enzymes that catalyze the synthesis of purine and pyrimidine bases, as well as some amino acids.
• Biotin. Participates in nitrogen metabolism, and also catalyzes the synthesis of unsaturated fatty acids.
• Vitamin PP(a nicotinic acid). Participates in redox reactions, the formation of enzymes and the metabolism of lipids and carbohydrates.
• Vitamin H(paraaminobenzoic acid). It is a growth factor for many bacteria, including those inhabiting the human intestine. Folic acid is synthesized from para-aminobenzoic acid.
• Gemin. It is an integral part of some enzymes that take part in oxidation reactions.
• Choline. Takes part in the reactions of lipid synthesis of the cell wall. It is a supplier of the methyl group in the synthesis of amino acids.
• Purine and pyrimidine bases(adenine, guanine, xanthine, hypoxanthine, cytosine, thymine and uracil). Substances are needed mainly as components of nucleic acids.
• Amino acids. These substances are the constituents of cell proteins.

Need for growth factors of some bacteria

Auxotrophs to ensure life, they need the supply of chemicals from outside. For example, clostridia are unable to synthesize lecithin and tyrosine. Staphylococci need the intake of lecithin and arginine. Streptococci need the intake of fatty acids - components of phospholipids. Corynebacteria and Shigella need nicotinic acid intake. Staphylococcus aureus, pneumococcus and brucella need vitamin B1 intake. Streptococci and tetanus bacilli - in pantothenic acid.

Prototrophs independently synthesize the necessary substances.

Rice. 24. Different environmental conditions affect the growth of bacterial colonies in different ways. On the left - stable growth in the form of a slowly expanding circle. On the right - rapid growth in the form of "shoots".

Studying the need of bacteria for growth factors allows scientists to obtain a large microbial mass, which is so necessary in the manufacture of antimicrobials, sera and vaccines.

Read more about bacteria in the articles:

Reproduction of bacteria is a mechanism for increasing the number of microbial populations. Bacterial division is the main mode of reproduction. After division, the bacteria should reach the size of adults. Bacteria grow by rapidly absorbing nutrients from their environment. Growth requires certain substances (growth factors), some of which are synthesized by the bacterial cell itself, and some come from the environment.

By studying the growth and reproduction of bacteria, scientists are constantly discovering the beneficial properties of microorganisms, the use of which in everyday life and in production is limited only by their properties.

Bacterial activity is characterized by growth- the formation of structural and functional components of the cell and the increase in the bacterial cell itself, as well as reproduction- self-reproduction, leading to an increase in the number of bacterial cells in the population.

bacteria multiply by binary fission in half, less often by budding. Actinomycetes, like fungi, can reproduce by spores. Actinomycetes, being branching bacteria, reproduce by fragmentation of filamentous cells. Gram-positive bacteria divide by ingrowth of the synthesized division partitions into the cell, and gram-negative bacteria divide by constriction, as a result of the formation of dumbbell-shaped figures, from which two identical cells are formed.

Cell division preceded replication of the bacterial chromosome according to a semi-conservative type (the double-stranded DNA chain opens and each strand is completed by a complementary strand), leading to the doubling of the DNA molecules of the bacterial nucleus - the nucleoid.

DNA replication occurs in three stages: initiation, elongation, or chain growth, and termination.

Reproduction of bacteria in a liquid nutrient medium. Bacteria seeded in a certain, unchanging volume of the nutrient medium, multiplying, consume nutrients, which subsequently leads to the depletion of the nutrient medium and the cessation of bacterial growth. The cultivation of bacteria in such a system is called periodic cultivation, and the culture is called periodic. If the cultivation conditions are maintained by continuous supply of fresh nutrient medium and the outflow of the same volume of culture fluid, then such cultivation is called continuous, and the culture is called continuous.

When growing bacteria on a liquid nutrient medium, near-bottom, diffuse, or surface (in the form of a film) culture growth is observed. The growth of a periodic culture of bacteria grown on a liquid nutrient medium is divided into several phases, or periods:

1. lag phase;

2. phase of logarithmic growth;

3. stationary growth phase, or maximum concentration

bacteria;

4. phase of bacterial death.

These phases can be depicted graphically as segments of the bacterial reproduction curve, which reflects the dependence of the logarithm of the number of living cells on the time of their cultivation.

Lag phase- the period between sowing bacteria and the beginning of reproduction. The duration of the lag phase is on average 4-5 hours. At the same time, bacteria increase in size and prepare for division; the amount of nucleic acids, protein and other components increases.

Logarithmic (exponential) growth phase is a period of intensive division of bacteria. Its duration is about 5-6 hours. Under optimal growth conditions, bacteria can divide every 20-40 minutes. During this phase, bacteria are the most vulnerable, which is explained by the high sensitivity of the metabolic components of a rapidly growing cell to inhibitors of protein synthesis, nucleic acids, etc.

Then comes the stationary growth phase., at which the number of viable cells remains unchanged, constituting the maximum level (M-concentration). Its duration is expressed in hours and varies depending on the type of bacteria, their characteristics and cultivation.

The death phase completes the process of bacterial growth, characterized by the death of bacteria in conditions of depletion of the sources of the nutrient medium and the accumulation of metabolic products of bacteria in it. Its duration varies from 10 hours to several weeks. The intensity of growth and reproduction of bacteria depends on many factors, including the optimal composition of the nutrient medium, redox potential, pH, temperature, etc.

Reproduction of bacteria on a dense nutrient medium. Bacteria growing on dense nutrient media form isolated rounded colonies with even or uneven edges (S- and R-forms), of different consistency and color, depending on the bacterial pigment.

Water-soluble pigments diffuse into the nutrient medium and color it. Another group of pigments is insoluble in water but soluble in organic solvents. And, finally, there are pigments that are insoluble neither in water nor in organic compounds.

The most common pigments among microorganisms are carotenes, xanthophylls, and melanins. Melanins are insoluble black, brown or red pigments synthesized from phenolic compounds. Melanins, along with catalase, superoxide cismutase, and peroxidases, protect microorganisms from the effects of toxic oxygen peroxide radicals. Many pigments have antimicrobial, antibiotic-like effects.

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 formation - constitutive (during the entire life cycle, the presence of a substrate does not affect), 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. You can use the 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 unculturable form can be observed in many microorganisms when exposed to unfavorable 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 the external environment between epidemics and, if 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 research methods.

Concepts of growth and reproduction of bacteria

For microbiological diagnostics, the study of microorganisms, and for biotechnological purposes, microorganisms are cultivated on artificial nutrient media.

Under bacteria growth understand the increase in the mass of cells without changing their number in the population as a result of the coordinated reproduction of all cellular components and structures. An increase in the number of cells in a population of microorganisms is denoted by the term "reproduction". It is characterized by the generation time (the time interval during which the number of cells doubles) and such a concept as the concentration of bacteria (the number of cells in 1 ml).

In contrast to the mitotic cycle of division in eukaryotes, the reproduction of most prokaryotes (bacteria) proceeds by binary fission, and actinomycetes by budding. Moreover, all prokaryotes exist in the haploid state, since the DNA molecule is represented in the cell in the singular.

Bacterial population

When studying the process of bacterial reproduction, it must be taken into account that bacteria always exist in the form of more or less numerous populations, and the development bacterial population in a liquid nutrient medium in batch culture can be considered as a closed system. There are 4 phases in this process:

• 1st - initial, or lag phase, or retardation phase,- characterized by the beginning of intensive cell growth, but the rate of their division remains low;
• 2nd - logarithmic, or log phase, or exponential phase,- characterized by a constant maximum rate of cell division and a significant increase in the number of cells in the population;
• 3rd - stationary phase- occurs when the number of cells in the population ceases to increase. This is due to the fact that there is a balance between the number of newly formed and dying cells. The number of living bacterial cells in the population per unit volume of the nutrient medium in the stationary phase is referred to as the M-concentration. This indicator is a characteristic feature for each type of bacteria;
• 4th - dying phase (logarithmic death)- characterized by a predominance of the number of dead cells in the population and a progressive decrease in the number of viable cells in the population. The cessation of growth in the number (reproduction) of the population of microorganisms occurs due to the depletion of the nutrient medium and / or the accumulation of metabolic products of microbial cells in it. Therefore, by removing metabolic products and/or replacing the nutrient medium, regulating the transition of the microbial population from the stationary phase to the dying phase, it is possible to create an open biological system that seeks to eliminate the dynamic balance at a certain level of population development.

This process of growing microorganisms is called flow culture (continuous culture). Growth in continuous culture makes it possible to obtain large masses of bacteria during flow cultivation in special devices (chemostats and turbidistats) and is used in the production of vaccines, as well as in biotechnology to obtain various biologically active substances produced by microorganisms.

To study metabolic processes throughout the cell division cycle, it is also possible to use synchronous crops- such cultures of bacteria, all members of the population of which are in the same phase of the cycle. This is achieved through special cultivation techniques.

However, after several simultaneous divisions, the synchronized cell suspension gradually switches back to asynchronous division, so that the number of cells increases further, no longer stepwise, but continuously.

Colonies

When cultivated on dense nutrient media, bacteria form colonies- visible to the naked eye, the accumulation of bacteria of the same species, which is most often the offspring of one cell.

Colonies of bacteria of different species are different:

• form;
• size;
• transparency;
• color;
• height;
• the nature of the surface and edges;
• consistency.

The nature of the colonies is one of the taxonomic features of bacteria.