Growth and reproduction of bacteria. Reproduction phases

Reproduction of bacteria by fission is the most common method of increasing the size of a 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 most of their species have a form of asexual reproduction by fission. Bacteria rarely reproduce by budding. Sexual reproduction of bacteria is present in a primitive form.

Rice. 1. The photo shows a bacterial cell at the stage of division.

Genetic apparatus of bacteria

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

Nucleoid

A nucleoid is an analogue of a nucleus. It is located in the center of the cell. It contains DNA, the carrier of hereditary information in a folded form. Unwound DNA reaches a length of 1 mm. The nuclear substance of a bacterial cell does not have a membrane, a nucleolus or a set of chromosomes, and does not divide by mitosis. Before dividing, the nucleotide is doubled. During division, the number of nucleotides increases to 4.

Rice. 2. The photo shows a bacterial cell in a section. A nucleotide is visible in the central part.

Plasmids

Plasmids are autonomous molecules folded into a ring of double-stranded DNA. Their mass is significantly 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 the bacterial cell.

Rice. 3. The photo shows a bacterial plasmid.

Stages of division

After reaching a certain size characteristic of an adult cell, division mechanisms are launched.

DNA replication

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

DNA replication is carried out with the help of enzymes DNA polymerases. During replication, the hydrogen bonds in double-stranded DNA are broken, resulting in two single-stranded daughter DNAs being formed from one DNA. Subsequently, when the daughter DNAs have taken their place in the separated daughter cells, they are restored.

As soon as DNA replication is complete, 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 sections

In Bacillus subtilis, the process of DNA replication ends with the exchange of two DNA sections.

After cell division, a bridge is formed through which the DNA of one cell passes into another. Next, both DNAs are intertwined. Some sections of both DNA stick together. At the sites of adhesion, DNA segments are exchanged. One of the DNA goes along the jumper back into the first cell.

Rice. 5. Variant of DNA exchange in Bacillus subtilis.

Types of bacterial cell divisions

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

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

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

Methods for separating bacteria

Division by breaking

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

Sliding division

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

Split split

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

Rice. 6. The photo shows rod-shaped bacteria forming chains (anthrax bacilli).

Rice. 7. The photo shows a sliding method for separating E. coli.

Rice. 8. The splitting method of separating corynebacteria.

Type of bacterial clusters after division

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

Globular bacteria arranged one by one, two by two (diplococci), in packets, in chains, or like bunches of grapes. Rod-shaped bacteria - in chains.

Spiral shaped bacteria- chaotic.

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

Rice. 10. In the photo there are diplococcus bacteria - Streptococcus pneumoniae.

Rice. 11. The photo shows Sarcina bacteria. Coccoid bacteria cluster together in packets.

Rice. 12. The photo shows streptococcus bacteria (from the Greek “streptos” - chain). Arranged in chains. They are causative agents of a number of diseases.

Rice. 13. In the photo, the bacteria are “golden” staphylococci. Arranged like “bunches of grapes”. The clusters are golden in color. They are causative agents of a number of diseases.

Rice. 14. In the photo, the coiled Leptospira bacteria are the causative agents of many diseases.

Rice. 15. The photo shows rod-shaped bacteria of the genus Vibrio.

Bacterial division rate

The rate of bacterial division is extremely high. On average, one bacterial cell divides every 20 minutes. Within just one day, one cell forms 72 generations of offspring. Mycobacterium tuberculosis divides slowly. The entire division process 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 reproductive cells) are not formed, but some cells exchange genetic material ( genetic recombination).

Gene transfer occurs as a result conjugation- unidirectional transfer of part of the genetic information in the form plasmids upon contact of bacterial cells.

Plasmids are small DNA molecules. They are not associated with the chromosome genome and are capable of doubling autonomously. Plasmids contain genes that increase the resistance of bacterial cells to unfavorable environmental conditions. Bacteria often pass these genes on to each other. Transfer of genetic 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 characteristics. This is how the ability of bacteria to exhibit drug resistance is transmitted. The transfer of antibiotic resistance between disease-causing populations is particularly dangerous for humanity.

Rice. 17. The photo shows the moment of conjugation of two E. coli.

Phases of bacterial population development

When inoculated 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.

Breeding delay phase

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

Logarithmic phase

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

Negative acceleration phase

During this phase, there is a decline in the reproduction rate, 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

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

Death Acceleration Phase

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

Logarithmic death phase

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

Decrease rate phase

During this phase, the remaining living bacterial cells enter a dormant state.

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

Rice. 19. In the photo, a colony of Pseudomonas aeruginosa is blue-green, a colony of micrococci is yellow, a colony of Bacterium prodigiosum is blood-red, and a colony of Bacteroides niger is black.

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. The 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 lines magnetic field Earth and find sulfur, oxygen and other substances it needs so much. 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.

Bacterial growth

When a bacterial cell begins to divide, two DNA molecules move to opposite ends of the cell. Next, the cell is divided into two equal parts, which are separated from each other and increase to their original size. The division speed of many bacteria averages 20 - 30 minutes. Within just one day, one cell forms 72 generations of offspring.

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

Rice. 22. The photo shows the moment of cell division.

Growth factors

For the growth of bacteria, certain substances (growth factors) are necessary, some of which are synthesized by the cell itself, some of which come from the environment. The need for growth factors is different for all bacteria.

The need for growth factors is a constant feature, which makes it possible to use it for identifying bacteria, preparing nutrient media, and using it 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 to minimum quantities and unchanged. Row chemical substances This group is part of cellular enzymes.

Rice. 23. The photo shows 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 a component of coenzyme A.
• Vitamin B6 (pyridoxine). Takes part in amino acid metabolism.
• 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(para-aminobenzoic acid). It is a growth factor for many bacteria, including those inhabiting the human intestines. Folic acid is synthesized from para-aminobenzoic acid.
• Gemin. It is a component of some enzymes that take part in oxidation reactions.
• Kholin. Takes part in the reactions of cell wall lipid synthesis. It is a supplier of 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 nucleic acids.
• Amino acids. These substances are components of cell proteins.

Requirement for growth factors of certain bacteria

Auxotrophs To ensure life, they require the supply of chemicals from the outside. For example, clostridia are not able to synthesize lecithin and tyrosine. Staphylococci require the supply of lecithin and arginine. Streptococci require the supply of fatty acids - components of phospholipids. Corynebacteria and Shigella require nicotinic acid. Staphylococcus aureus, pneumococci and Brucella require vitamin B1. Streptococci and tetanus bacilli - in pantothenic acid.

Prototrophs independently synthesize the necessary substances.

Rice. 24. Different environmental conditions have different effects on the growth of bacterial colonies. On the left is steady growth in the form of a slowly expanding circle. On the right is rapid growth in the form of “shoots”.

Studying the need of bacteria for growth factors allows scientists to obtain a large microbial mass, so necessary in the manufacture of antimicrobial drugs, serums and vaccines.

Read more about bacteria in the articles:

Bacterial proliferation is a mechanism for increasing the number of microbial populations. Bacterial division is the main method of reproduction. After dividing, the bacteria must reach adult size. Bacteria grow by quickly absorbing nutrients from their environment. Growth requires certain substances (growth factors), some of which are synthesized by the bacterial cell itself, and some of which 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.

Table of contents of the topic "Respiration (aerobic, anaerobic). Catabolism in bacteria. Constructive metabolism (plastic metabolism). Growth of bacteria in culture.":
1. Propionic acid fermentation. Butyric acid and acetone butyl fermentation. Homoacetate fermentation. Energy production by oxidative phosphorylation. Breath.
2. Catabolism of carbohydrates in bacteria. Glycolysis. Glycolytic oxidation pathway. The Embden-Meyerhof-Parnassus path. Pentose phosphate oxidation pathway. Warburg-Dickens-Horecker-Racker scheme.
3. Entner-Doudoroff pathway in bacteria. Krebs cycle. The tricarboxylic acid cycle in bacteria.
4. Catabolism of nitrogen-containing organic compounds by bacteria. Amino acids. Decarboxylation and deamination of amino acids by bacteria. Stickland mechanism.
5. Catabolism of fats and fatty acids by bacteria. Endogenous energy metabolism of bacteria.
6. Constructive metabolism (plastic metabolism). Carbon compounds for biosynthetic reactions of bacteria. Biosynthesis of amino acids and proteins by bacteria.
7. Biosynthesis of nucleotides and nucleic acids by bacteria.
8. Biosynthesis of oligosaccharides and polysaccharides by bacteria. Biosynthesis of lipids (fats) by bacteria.
9. Regulation of microbial metabolism. Allosteric proteins.

Actually under bacterial growth usually involve the coordinated replication of all bacterial components. Since the division of a bacterial cell leads to the formation of two individuals, their number grows exponentially: 2 0 -2 1 -2 2 -2 3 -..2 n. The quality of the nutrient medium and growing conditions have a regulatory effect on the growth of bacteria.

Cell Population Growth in a limited living space (periodic culture) can be divided into at least four phases(Figure 4-12).

Rice. 4-12. Bacterial culture growth.

After being introduced into the environment, bacteria adapt to its conditions and multiply relatively slowly ( lag phase). Then comes exponential growth phase (exponential phase). Further, the environment is depleted, toxic metabolic products accumulate in it, which is manifested by a decrease in the rate of reproduction and a cessation of the increase in the number of cells ( stationary phase).

Thus, growth in periodic culture obeys laws that are valid not only for unicellular, but also for multicellular organisms. Subsequently, the bacterial culture may die or be significantly reduced (die-off phase). Spore-forming species enter the sporulation stage; in non-spore-forming species, the formation of anabiotic forms is possible (see below). In some cases, a growth acceleration phase (the beginning of the exponential phase) and a growth deceleration phase (the transition to the stationary phase) are additionally distinguished.

Lag phase of bacterial growth corresponds to a period of physiological adaptation, including enzyme induction, synthesis and assembly of ribosomes. The duration of the phase depends mainly on the age of the inoculum of bacteria and the previous cultivation conditions. If the inoculum is taken from an old culture (in the stationary growth phase), then the bacteria need time to adapt to new conditions. If the sources of energy and carbon in the new environment differ from those available in the previous culture, then adaptation to new conditions may require the synthesis of new enzymes that were not previously needed.

Exponential phase of bacterial growth (logarithmic) is characterized by the maximum rate of cell division. For a particular species of bacteria under specific growth conditions, the generation time (that is, the time required for the number of bacteria to double) is constant throughout the logarithmic phase, but varies among various types and strains, and also depends on the composition of the medium and cultivation conditions. The generation time on the optimal medium can be short (for E. coli 20 minutes) or long (for Mycobacterium tuberculosais 6 hours). In this phase, the maximum accumulation of bacterial metabolites (for example, toxins, bacteriocins) occurs in the medium.

Stationary phase of bacterial growth. During this period, the availability of essential nutrients becomes a limiting factor. A balance is established between cell growth and division and the process of cell death. Spore-forming bacteria (for example, the genera Bacillus and Clostridium) are able to enter the sporulation phase, which is activated when the bacteria are in conditions of limited nutrition. At a certain point, the ratio of dying, newly formed and resting cells becomes stable; such a state is known as the maximum stationary phase. The biomass of bacteria in the stationary phase is referred to as the “yield” or “biomass yield” (the difference between the maximum and initial biomass); or “economic coefficient”, if the increase in biomass is related to a unit of growth-limiting substrate.

Dieback phase (recession, lysis) includes a period of logarithmic death, which turns into a period of decreasing rate of bacterial death. Reasons for the death of bacteria in normal nutrient media are not completely clear. It is understandable that acids accumulate in the medium (during the growth of Escherichia, Lactobacillus). Sometimes bacteria are destroyed by their own enzymes (autolysis). The rate of death varies widely depending on the living conditions and characteristics of the microorganism (for example, enterobacteriaceae die off slowly, and bacilli die off quickly).

The intense processes of anabolism and catabolism in the cell lead to rapid cell growth.

Bacterial growth is an orderly increase in the number and size of all cell components, provided that all necessary chemical elements, which leads to an increase in its mass. Nutrient substrates must contain these elements in a metabolically accessible form. Cell growth is not unlimited. After reaching a critical size, the cell undergoes division or reproduction.

Most bacteria divide by transverse binary fission or cytokinesis. In most gram-positive bacteria, division occurs through the synthesis of a transverse septum running from the periphery to the center. The cells of most gram-negative bacteria divide by constriction. The division process is repeated at approximately equal intervals of time (from several minutes to several days), which is an individual genetic characteristic of the microbial species. As a result of reproduction, the number of cells in the population sharply increases.

Reproduction or reproduction in bacteria is the division of supercoiled nucleoid DNA into two daughter strands, each of which is further completed by a complementary strand and the formation of two daughter cells simultaneously occurs (semi-conservative method).

Reproduction is characterized generation time(the time interval during which the number of cells doubles) and such a concept as bacteria concentration(number of cells in 1 ml).

When bacteria are introduced into a nutrient medium, they grow and multiply until the content of any of the necessary components of the medium reaches a minimum, after which growth and reproduction stop. If throughout this entire time we do not add nutrients and do not remove the final products of metabolism, then we get static bacterial culture. A static (batch) culture of bacteria behaves like a multicellular organism, with genetic growth limitation. If we construct a graph with time on the abscissa axis and the number of cells on the ordinate axis, we obtain a curve describing the dependence of the number of cells formed on the time of reproduction, which is called growth curve.

Growth curve of bacteria in a nutrient medium. On this curve, several phases can be distinguished, replacing each other in a certain sequence (Fig. 11):

1. Initial - lag phase(English) lag- to lag behind). Covers the period of time between the inoculation of bacteria and the start of reproduction. Its duration is on average 2-5 hours and depends on the composition of the nutrient medium and the age of the crop being sown. During the lag phase, bacterial cells adapt to new cultivation conditions and inducible enzymes are synthesized.

2. Exponential (logarithmic) phase. Characterized by a constant maximum rate of cell division, a phase of geometric growth with a sharp increase in the population of microorganisms (2 in degree n). The rate of reproduction depends on the type of bacteria and the nutrient medium. The cell doubling time is called generation time, which varies depending on the type of bacterial culture: in bacteria of the genus Pseudomonas it is equal to 14 minutes, and Mycobacterium 18 - 24 hours. The size of the cells and the protein content in them remain constant during the exponential phase. The bacterial culture in this phase consists of standard cells.

Rice. 11. Phases of bacterial reproduction

3. Stationary phase(phase of equilibrium of reproduction and death of microbial cells). Occurs when the number of cells stops increasing. Since the growth rate depends on the concentration of nutrients, when the content of nutrients in the nutrient medium decreases, the growth rate also decreases. A decrease in growth rate also occurs due to the high density of bacterial cells, a decrease in the partial pressure of oxygen, and the accumulation of toxic metabolic products. The duration of the stationary phase is several hours and depends on the type of bacteria and the characteristics of their cultivation.

4. Dieback phase or death - a decrease in population size due to a decrease and lack of conditions for the reproduction of microorganisms. Occurs due to the accumulation of acidic metabolic products or as a result of autolysis under the influence of its own enzymes. The duration of this phase ranges from ten hours to several weeks.

This dynamics is typical for periodic crops with gradual depletion of nutrients and accumulation of metabolites. The constant presence of the bacterial population in the logarithmic growth phase is observed in a continuous culture, which is achieved by gradual dosing of nutrients, control of the density of the bacterial suspension and removal of metabolites. This process of growing microorganisms is called flow cultivation (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.

Bacterial growth – This is an increase in bacterial cell size without increasing the number of individuals in the population. Cell growth is not unlimited. After reaching a critical size, the cell undergoes division.

Reproduction of bacteria - a process that ensures an increase in the number of individuals in a population. Bacteria are characterized by a high reproduction rate.

Growth always precedes reproduction. Bacteria reproduce by transverse binary fission, in which two identical daughter cells are formed from one mother cell. In most gram-positive bacteria, division occurs through the synthesis of a transverse septum running from the periphery to the center. The cells of most gram-negative bacteria divide by constriction.

The process of bacterial cell division begins with the replication of chromosomal DNA. Replication begins in one selected region, called origin, which has a specific nucleotide sequence. One or two replication forks may occur here. More than 20 enzymes are involved in the replication process. Since bacterial DNA is double-stranded, it must be divided before replication. This process involves the enzymes helicase, which unwinds the double helix, and topoisomerase, which prevents the formation of secondary curls. SSB protein binds to single-stranded DNA, preventing it from refolding into a double helix. As a result, a replication fork is formed. The synthesis of new DNA chains is carried out by the enzyme DNA polymyrase. To carry out the polymerization reaction of nucleotides on the template of the parent chain, polymerase requires a primer. The primer is a short RNA nucleotide chain, complementary to the template strand, with a free 3/- end. After the DNA strand has begun to be synthesized, the RNA primer is removed. Since the DNA strands in a duplex are antiparallel, the direction of unwinding of the double strand coincides only with the direction of DNA synthesis on one template, which is called the leading one. On the complementary strand, DNA is synthesized in short fragments, which are subsequently stitched into one strand by DNA ligases. The bacterial DNA replication process continues until all the DNA is doubled.

When bacteria are added to the nutrient medium, they grow and multiply until the content of one of the necessary components of the medium reaches a minimum, after which growth and reproduction stop. If we do not add nutrients and do not remove end products of metabolism, we get a statistical bacterial culture.

Bacterial reproduction phases:

1. Initial(lag phase) covers the period of time from the moment the bacteria are inoculated until the start of their growth. Its duration averages 2-5 hours and depends on the composition of the nutrient medium.

2. Exponential(logarithmic) phase. Characterized by a constant maximum rate of cell division. This speed depends on the type of bacteria and the nutrient medium. The time it takes for cells to double is called generation time. This time varies from several minutes to several hours.

3. Stationary phase occurs when the number of cells stops increasing. With a decrease in the concentration of nutrients in the nutrient medium, a decrease in the partial pressure of oxygen, and the accumulation of toxic metabolic products, the growth rate of bacteria decreases. The duration of the stationary phase is several hours and depends on the type of bacteria.

4. Dieback phase occurs due to the accumulation of acidic metabolic products or as a result of autolysis under the influence of its own enzymes. The duration of this phase ranges from ten hours to several weeks.

3.2. Nutrient media, principles of their classification, requirements for nutrient media, conditions for cultivating microorganisms.

The basis of bacteriological work is nutrient media, often determining the results of the study by their quality.

Basic requirements for nutrient media:

1. Culture media must contain all the nutrients necessary to feed the microbe, i.e. have nutritional value.

2. Have sufficient humidity

3. Have an optimal pH (7.2-7.6) acidity of the environment.

4. Be isotonic (NaCl concentration 0.87%), for halophilic bacteria the salt concentration is 1% or higher.

5. Have an optimal electronic potential, indicating the content of dissolved oxygen in the medium. It should be high for aerobes and low for anaerobes.

6. Be transparent so that bacterial growth is visible, especially in liquid media.

7. Be sterile (so that there are no other bacteria).

To prepare nutrient media, products of animal origin (meat, fish, blood, eggs, milk) and products of plant origin (potatoes) are used. Synthetic nutrient media composed of chemical compounds are also used.

The source of nitrogen for bacteria is simple ammonium compounds, amino acids or peptones; carbon source – sugar, polyhydric alcohols, organic acids. The need of bacteria for inorganic elements is satisfied by salts added to the nutrient medium: NaCl, KN 2 PO 4, K 2 HPO 4.

Depending on the consistency, nutrient media can be: liquid, semi-liquid and dense. The density of the medium is achieved by adding agar. Agar is a polysaccharide obtained from algae. It melts at a temperature of 100 oC, cools at a temperature of 45-50 oC. For semi-liquid media, agar is added at a concentration of 0.5%, for dense media - 1.5-2%. Liquid media do not contain agar-agar.

The composition of nutrient media can be simple and complex. Simple media include peptone water, meat peptone broth, meat peptone agar, Hottinger agar. Complex ones are simple ones with the addition of an additional nutritional component (sugar, whey, bile broths, blood, whey, yolk-salt agars, Keith-Tarozzi, Wilson-Blair medium).

Depending on the purpose of the environment, they are divided into:

1. General purpose – for cultivating most bacteria (meat peptone agar, blood agar).

2. Special purpose:

a) elective environments– these are the media on which a specific microorganism grows. For example, alkaline agar with a pH of 9 is used to isolate Vibrio cholerae.

b) With enrichment steps– these are environments that stimulate the growth of a particular microorganism, inhibiting the growth of others. For example, magnesium and selenite media stimulate the growth of bacteria of the genus Salmonella, inhibiting the growth of E. coli.

c) differential diagnostic environments serve to study the enzymatic activity of bacteria (Hiss medium).

d) combined nutrient media combine an selective medium that suppresses the growth of accompanying flora and a differential diagnostic medium (Ploskirev’s medium for isolating Shigella, bismuth-sulfite agar for Salmonella). Both of these media inhibit the growth of E. coli.

To differentiate prototrophic and auxotrophic bacteria, selective media are used. Prototrophs grow on a minimal medium containing only salts and carbohydrates, since they themselves are able to synthesize the metabolites they need for development. Auxotrophs need media containing certain amino acids, vitamins, i.e. growth factors.

Preparation of nutrient media is one of the most important and difficult areas of bacteriological work.

Currently, the medical industry has organized the production of canned media. Dry nutrient media are contained in plastic jars with tight-fitting lids to ensure an airtight seal.

Bacteria cultivation conditions:

1. Availability of a complete nutrient medium.

2. A certain cultivation temperature (optimal temperature 37 0 C).

3. A certain cultivation atmosphere. Strict aerobes require oxygen, so they grow well on the surface of agar Petri dishes or in a thin top layer of liquid medium. For the growth of aerobes in the deep layer of a liquid medium, it is necessary to continuously mix or shake the nutrient media so that oxygen is distributed throughout the entire volume of the medium. For facultative anaerobes, the same methods are used. Microaerophiles multiply at reduced partial pressure of oxygen. The CO 2 concentration should be 1-5%. To do this, special CO 2 incubators are used or the crops are placed in desiccators in which a hot candle is installed. For the growth of obligate anaerobes, it is necessary to exclude access to oxygen. To do this, add oxygen-reducing substances (thioglycolic acid) to the nutrient media, regenerate liquid nutrient media from air oxygen by boiling them, use oxygen absorbers by placing them in hermetically sealed “gaspack” containers, and use anaerostats.

4. Cultivation time (18-48 hours). For the cultivation of Mycobacterium tuberculosis (3-4 weeks).

5. Lighting. Light is required to grow phototrophic bacteria.

In industrial conditions, to obtain biomass of bacteria or fungi in order to obtain antibiotics, vaccines, and diagnostic drugs, cultivation is carried out in apparatus (fermenters) with strict adherence to the optimal parameters for growth and propagation of crops.

Nutrition of bacteria.

Nutrition refers to the processes of entry and exit of nutrients into and out of cells. Nutrition primarily ensures cell reproduction and metabolism.

Essential nutrients include: carbon, oxygen, hydrogen, nitrogen, phosphorus, potassium, magnesium, calcium. In addition to organogens, microelements are necessary. They provide enzyme activity. These are zinc, manganese, molybdenum, cobalt, copper, nickel, tungsten, sodium, chlorine.

Bacteria have a variety of sources of nutrients.

Depending on the source of carbon, bacteria are divided into: 1) autotrophs (use inorganic substances - CO 2); 2) heterotrophs (use organic C-hexoses, polyhydric alcohols, amino acids);

Nutrition processes must provide the energy needs of the bacterial cell. Based on energy sources, microorganisms are divided into: 1) phototrophs - the source is solar energy; 2) chemotrophs - obtain energy through redox reactions; 3) chemolithotrophs - use inorganic compounds; 4) chemoorganotrophs - use organic substances.

Medical microbiology studies bacteria that are heterochemoorganotrophs.

Bacterial growth factors are vitamins, amino acids, purine and pyrimidine bases, the presence of which accelerates growth. Among bacteria there are: 1) prototrophs (able to synthesize the necessary substances themselves); 2) auxotrophs (need growth factors).

Microorganisms assimilate nutrients in the form of small molecules, so proteins, polysaccharides and other biopolymers can serve as sources of nutrition only after they are broken down by exoenzymes into simpler compounds.

Paths for the entry of metabolites and ions into the microbial cell: I. Passive transport (without energy costs): simple diffusion; 2) facilitated diffusion (along a concentration gradient, with the help of carrier proteins). II. Active transport (with energy consumption, against a concentration gradient; in this case, the substrate interacts with a carrier protein on the surface of the cytoplasmic membrane).

There are modified versions of active transport - the transfer of chemical groups. Phosphorylated enzymes act as carrier proteins, so the substrate is transported in a phosphorylated form. This transfer of a chemical group is called translocation.

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

Batch 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

3rd phase - stationary - reproduction - equal to mortality

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

External factors may have

• Bacteriostatic effect- suppress the reproduction and growth of bacteria
• Bactericidal effect- cause the death of 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 reaction catalyzed - oxyreductases, lyases, transferases, hydrolases, etc.
2. By localization, endoenzymes catalyze reactions inside the cell. Exoenzymes - released from the bacterial cell, catalyze the breakdown
3. Genetic control of formation - constitutive (during the entire life cycle, not affected by 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.

The importance of enzymes.

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

2. Bacterial enzymes determine their pathogenicity

3. Enzymatic properties are used in microbiological industry

Determination of bacterial enzymes

Proteases break down proteins into amino acids, urea, indole, hydrogen sulfide, and ammonia. On media with protein, proteases are detected by the release of these products. Gelatin is used to liquefy the medium. On coagulated whey by its liquefaction and on milk by its clearing. Casein will break down and the protein will coagulate. At the MPB for the release of indole and hydrogen sulfide gases, 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 MPB or MPA, add an acid formation indicator + carbohydrate + float for gas formation. According to this principle, the Gies and Pestrel environments are created. If the light of the environment changes, gas is released, which means that carbohydrates are being broken down. Monosaccharides are used. Based on this principle, panels, tablets, paper indicator systems, NIB - systems of indicator papers, energy tubes and instruments for measuring 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 the yolk, which contains a lot of lipids and the destruction of lipids is accompanied by clearing of the medium

Cultivation of microorganisms.

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

1. Study of microbiological properties

2. To diagnose infections

3. To obtain a biological product - from bacteria or obtained with the help of bacteria.

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

1. Availability of a complete nutritional environment.
2. Optimal 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 only grow 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 medium.
3. Continuous cultivation - using flow-through 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 the bottom, near the wall, and can form a film on the surface of the medium. Colonies form on solid 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

SR colonies 0 intermediate - slightly uneven edges and surface.

Features of the 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. the use of special devices - anaerostats and desiccators. In them, oxygen is absorbed either by chemical absorbers or pumped out from the device.
3. Adding reducing substances to the medium - substances that oxidize easily and quickly - carbohydrates, cysteine, pieces of parenchymal organs, ascorbic acid. Based on this principle, an environment for anaerobes has been created - Kit-Tarozzi - an environment for anaerobes. It contains MPB, carbohydrate and pieces of liver, which contain cysteine.
4. Special sowing methods. Sowing under oil, sowing in a Veillon-Vignan tube, sowing according to Fortner. Aerobes and anaerobes populate the cup - Aerobes absorb oxygen and an anaerobic environment is obtained.

Isolation of pure cultures.

Pure culture- a population of microorganisms of one type, isolated on a liquid or solid nutrient medium in large quantities.

Purposes of allocation.

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

Stages of isolating a pure culture of aerobes.

1. Study of the mixture - smear staining according to Gram.
2. Separation of the mixture and obtaining colonies. Separation is carried out 1) According to Drigalsky - with strokes on the surface of the agar. The material is taken with a loop and inoculated onto agar. Sowing a Spatula into several Cups. 2) Serial dilution method. 3) Kokha - method of serial dilutions in molten agar.
3. Colony frequency check, smear, gram stain
4. Subculture of material from colonies onto agar slants to accumulate a pure culture. The isolated pure culture is identified by its properties - morphological, tinctorial, cultural, enzymatic, and others.

Isolation of a pure culture of anaerobes

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

2. Obtaining colonies according to Zeisler, a colony of anaerobes is obtained on the surface of an agar in Anaerostat, according to Weinberg, colonies are obtained in Veillon-Vignal tubes.

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

4. Reseeding of the Colonies on Kittarotsi medium, accumulation of anaerobic, pure culture.

5. Identification, determination of the type of anaerobes.

Other methods for isolating pure cultures.

1. Optimal temperatures can be used
2. Release of spores when the mixture is heated for 10 minutes at 80 degrees
3. Using the swarming phenomenon - spreading beyond the crop area.
4. Shukevich's method is the isolation of a pure culture of microorganisms with creeping growth.
5. Bacterial filterability is the ability to pass through filters with a certain amount of spores. Treatment of the mixture with ultraviolet rays, ultrasound, antisera, obtaining a pure culture of microorganisms resistant to these factors.
6. During electrophoresis of the mixture. Organisms with a certain charge will accumulate at the anode or cathode.
7. Use a micromanipulator. Take a cell under a microscope and get a pure culture - a clone - the offspring of one microbial cell. Use of selective growth media.
8. Bile, thiurite salts, sodium chloride, antibiotics are added to the nutrient media, and the pure culture resistant microorganisms.
9. Differential diagnostic environments can be used.
10. You can use a biological method. White mice are infected intraperitoneally with a mixture of bacteria and due to tropism, the bacteria accumulate in a certain organ.

Bacterial pigments.

These are dyes secreted by the bacterial cell; their synthesis is genetically determined. By 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 - color only colonies
2. Soluble pigments - can be soluble in alcohols, water

Pigments are usually formed from bacteria that are found in the air microflora, from antibiotic-resistant microorganisms, because they are secondary metabolites and pigments are often formed in light.

Function of pigments

1. Pigments are involved in metabolism
2. Increases antibiotic resistance
3. Increases resistance to UV rays by protecting areas sensitive to photo-oxidation

L-forms of bacteria.

Opened in 1935 These are microorganisms that lack a cell wall, but retain the ability to grow and reproduce. 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 when environmental conditions change.
2. Morphologically similar in certain species of bacteria. They are polymorphic - spherical, gram-negative. They form colonies of type A - small colonies on the surface of the medium and colonies of type B - dark center, and raised edges, the colonies grow into the nutrient medium.
3. Anaerobes or microaerophiles
4. L-forms have many methods of reproduction - binary fission, budding, fragmentation, combined.
5. They have reduced virulence, lack adhesion, and have altered antigenic properties.
6. They are able to reverse - return to their original bacterial form

And cause infections that are difficult to treat.

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, and compliment.

Unculturable forms of NFB bacteria

These are bacteria that have metabolic activity, but do not grow on nutrient media; the transition to an uncultivable 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 meaning of uncultivated forms. In this form they are stored in o external environment between epidemics and when they enter a macroorganism, they can be recultivated - revived - this explains the presence of naturally focal diseases.

Revealing -

1. Direct cell count

2. Detection of DNA activity

3. Genetic research methods.