Principles of cultivation of microorganisms - microbiology with the technique of microbiological research. Open Library

Microorganisms in the nature around us are everywhere: in soils, water bodies, on the surfaces of various objects, people and animals are inhabited by them. All this can serve as sources of microbial contamination of food, drugs, and production lines. The cultivation of bacteria is necessary to study their properties, needs, and characteristics. This, in turn, is an important step in the development of various drugs, laboratory diagnosis of diseases, the calculation of industrial reactors, and much more.

General concepts

The cultivation of bacteria in microbiology is understood as the cultivation of microorganisms carried out in the laboratory. In turn, microbes that have grown on a selected nutrient medium are called a culture. Cultures can be mixed if they are formed by different types of microorganisms, and pure if they are represented by only one type of bacteria.

If only one cell is placed in, and a group of individuals is obtained as a result of its reproduction, then this set of microorganisms is called a clone. When a clone develops to the point where it becomes visible to the naked eye, this collection of bacteria is called a colony.

Usually the cultivation of bacteria isolated from different sources is carried out separately from each other. Each such separately grown group of microbes is called a strain. So, if one type of staphylococcus is isolated from three sources (or different portions of the same product, different people), speak of three strains of this type of staphylococcus aureus.

Bacterial growth factors

These include various amino acids, lipids, purine bases and other compounds necessary for the development of microorganisms. Some microbes can independently produce the substances they need, while others need to receive them in finished form. According to the needs of microorganisms in certain growth factors, identification and differentiation of bacteria are carried out. Also, this parameter is important for the correct production of a nutrient medium for laboratory and biotechnological work:

Amino acids. Bacteria may require one particular amino acid or group of acids. So, clostridia need leucine and tyrosine, streptococci need leucine and arginine. Microorganisms that need to obtain amino acids from outside for growth are called auxotrophic.
Purine and pyrimidine bases, as well as their derivatives (adenine, guanine and others). They are an important factor in the growth of many types of streptococci.
Vitamins. They are part of the coenzymes required by bacteria. So, nicotinic acid, as well as its amide, which are part of NAD and NADP, is needed by Shigella. Thiamine, as an integral part of pyrophosphate, is required by Staphylococcus aureus, pneumococcus, brucella. Pantothenic acid, which is part of the CoA coenzyme, is required by tetanus bacilli and certain types of streptococcus. Cytochromes, and hence the folic acid, hemes and biotin that form them, are necessary for Mycobacterium tuberculosis and Haemophilus influenzae.

Environment requirements

Conditions for nutrient media for the cultivation of bacteria:

Nutrition. They must contain substances, moreover, in an easily digestible form, necessary for microorganisms to feed and replenish energy. These include organogens and minerals. Some microorganisms additionally require vitamins and amino acids that they cannot synthesize.
Optimal pH level. It affects the permeability of the cell membrane and, accordingly, the ability to absorb nutrients by the bacterium. Most often, the pH value should be at the level of 7.2-7.4. Many microorganisms in the course of their life produce products with acidic or alkaline reactions, and in order for the pH of the nutrient medium not to change, it must have buffering properties.
Isotonicity. The osmotic pressure in the nutrient medium for cultivating bacteria should have the same values as inside the microbial cells. Usually it corresponds to a 0.5% NaCl solution.
Sterility. This is due to the fact that the appearance of foreign bacteria will distort the results of studying the analyzed strain.
Humidity level. This indicator, along with the consistency of the medium, should have optimal characteristics for a particular type of bacteria.
Redox potential (RH2). It shows the ratio of substances that donate and accept electrons, as well as the level of oxygen saturation of the nutrient medium. For aerobes and anaerobes, the conditions for cultivating bacteria differ somewhat in this indicator. Anaerobic microorganisms reproduce best at RH2 values not exceeding 5, and aerobic microorganisms at least 10.
Uniformity. It is important that the culture medium contains constant amounts of its individual ingredients. In addition, clear solutions are preferred, in which it is easier to monitor the growth of the culture or notice its contamination.

Types of culture media

The choice of one or another medium for growing microorganisms is influenced by many factors, among which are the characteristics of their nutrition and the purpose of the study. The main features underlying the classification of nutrient media are:

1. Components. According to the initial substances used to create the substrate, there are:

natural, which are prepared from animal or vegetable products (for example, meat, milk, fruits) and are suitable for growing mixed crops;
semi-synthetic, in which expensive natural food products are replaced with non-food products (for example, bone meal, blood clots), and which are optimal for the cultivation of bacteria certain types or excretion of products of their vital activity from the environment;
synthetic ones, which are prepared from precise amounts of chemical compounds, have a known constant composition and are easily reproduced.

2. Consistency (density). There are environments:

liquid;
dense;
semi-liquid.

The last two are prepared from special solutions or liquid substances with the addition of agar-agar or gelatin to create the required density. In addition, a dense environment for the growth of bacteria is clotted blood serum, potatoes, silica gel media, carrageenan.

3. Composition. On this basis, the environments are:

simple ones, the list of which is short, are meat-peptone broth (MPB), Hottinger broth and agar, meat-peptone agar (MPA), nutrient gelatin and peptone water.
complex, prepared from simple ones with the addition of blood, whey, carbohydrates and other substances.

4. Appointment. The following nutrient media are distinguished:

the main ones serve to grow many pathogenic microbes (usually of a simple composition);
special ones are used for the isolation and cultivation of bacteria that do not grow on simple substrates;
elective (they are also selective) are suitable for isolating a specific type of bacteria and inhibit the growth of associated microbes (selectivity is created by adding certain substances to the media, such as antibiotics or salts, or by adjusting the pH);
differential diagnostics make it possible to distinguish one type of bacteria from another by assessing the enzymatic activity, for example, the environment;
preservatives are needed for initial inoculation with subsequent transportation of samples, as they prevent the death of microorganisms, as well as inhibit the growth of other bacteria.

Media preparation

The most important step in the cultivation of anaerobic bacteria is the preparation of a suitable nutrient medium. After the optimal parameters are selected, proceed to the following stages:

weighing, by selecting a sample of components on an analytical balance;
dissolution carried out in distilled water heated to 70 ° C, and phosphates, micro- and macrosalts are separately dissolved;
boiling in a water bath for two minutes;
pH determination performed with indicator paper or potentiometer;
filtration through wet cloth or paper filters for liquid as well as molten dense media, and through a cotton-gauze filter for agar media;
bottling performed on 3/4 capacity;
sterilization depending on the composition of the medium;
control for sterility is carried out by settling for two days in a thermostat, followed by viewing;
chemical control to establish the pH and the content of the necessary elements;
biological control by trial inoculation.

Sterilization of glassware and media

One of the basic principles of bacterial cultivation is sterility. The growth and development of foreign microorganisms can affect the characteristics of the nutrient medium by changing its chemical composition and pH. Sterilization is the main condition for growing pure cultures. In practice, this term refers to methods for the destruction of absolutely all life forms on the surface and in the volume of sterilized objects. Vessels, instruments used, media, and other items used in the course of the study are subjected to sterilization.

Some types of sterilization:

Calcination. Sterilization of loops and needles for sowing, glass slides, some tools can be performed using a burner or spirit lamp.
Boiling. Suitable for handling syringes, needles, food products but does not kill bacterial spores.
Dry heat sterilization. It is carried out in a special drying cabinet and is suitable for processing flasks, test tubes and other laboratory glassware.
Steam sterilization. Carried out in an autoclave, this method is highly effective. But it is not suitable for nutrient media containing proteins or any other compounds that break down at high temperatures. More sparing can be called tyndalization. It is carried out in a Koch boiler and combines the germination of spores with their destruction.
Pasteurization. It is used for media that change their properties when boiled (for example, milk, wine, beer), it is able to rid them of non-spore-bearing microorganisms. The processing temperature is only 50-60 ° C for fifteen to thirty minutes. In some cases, cold sterilization is used, carried out using filters or UV rays.

Conditions for cultivating bacteria

The growth and development of bacteria is possible only under certain factors and the values of each of them:

1. Temperature. There are three groups of bacteria that differ in temperature preferences:

thermophiles, or heat-loving microbes, grow at 45-90 ° C, which means they do not multiply in human and animal organisms;
psychrophiles, or cold-loving microorganisms, prefer temperatures in the range of 5-15 ° C and are grown in refrigerators;
mesophiles, develop at a temperature of 25-37 ° C, they include the bulk of bacteria.

2. Light. It is a feature of the cultivation of phototrophic bacteria, since they carry out the photosynthetic process. But for most microbes, lighting is not a prerequisite. And even vice versa, solar ultraviolet can suppress their development.

3. Water. All microorganisms need water in an accessible (liquid) form. That is why bacteria practically do not develop in frozen foods.

4. This principle of bacterial cultivation has already been discussed in detail above.

5. Aeration. oxygen like chemical element, is integral part water and a considerable number of compounds used for growing microorganisms. Gaseous oxygen can also be contained in water and other liquids in dissolved form. A significant part of bacteria needs a constant supply of oxygen molecules. But for a number of microorganisms, it is unnecessary, or, worse, gaseous oxygen is toxic to them, since they do not have catalase and peroxidase, which destroy toxic respiratory products. Therefore, the most important step in the cultivation of anaerobic bacteria is the removal of O 2 molecules from the nutrient medium.

6. Cultivation of microorganisms. The cultivation of aerobic and anaerobic bacteria is carried out in different layers of the environment and in different modes.

Cultivation of aerobic microorganisms

The cultivation of aerobic bacteria requires molecular oxygen. To obtain pure cultures of aerobes that can be successfully used in medicine and Food Industry, the following methods are used:

surface cultivation on dense media or in liquid media (their thin layer), when oxygen comes directly from the air;
deep cultivation in liquid media, when an increase in the amount of oxygen dissolved in them is achieved by constant aeration.

Cultivation of anaerobic microorganisms

The basic principle of cultivating bacteria of this type is their minimal contact with atmospheric oxygen. Providing conditions for their growth is much more difficult than for aerobes. The following methods are used to isolate anaerobes from molecular O2:

Physical. This method of cultivation of anaerobic bacteria is reduced to their cultivation in a special vacuum apparatus - a microanaerostat. The air in it is replaced by a special gas mixture of nitrogen with the addition of 10% hydrogen and 5% carbon dioxide.
Chemical. These include: the use of absorbing agents (eg Fe, Na 2 S 2 O 4 , CuCl) or reducing agents (eg ascorbic acid).
Biological. It comes down to the co-cultivation of aerobes and anaerobes in a closed system. This method of cultivating bacteria involves seeding one half of a Petri dish with some of the aerobic species of bacteria, and the other half with the studied anaerobe. Its development will begin at the moment when all the oxygen is used up.

The following seeding methods are suitable for cultivating anaerobic bacteria:

in the surface layer;
in the surface layer filled with sterile paraffin;
in the thickness of a dense nutrient medium;
in deep layers of viscous media.

Obtaining pure culture

Microbiologists in their work usually deal with samples inhabited by many different types of microbes. However, to determine the systematic position of microorganisms (family, genus, species), as well as to study their characteristics, it is necessary to isolate them and grow a pure culture. They are essential in many food industries, such as cheese, bread, kvass, wine, etc. The cultivation of lactic acid bacteria makes it possible to obtain an essential component for the production of fermented milk products, dough, cocoa, silage and even plastic.

The isolation method in a dense medium is based on the mechanical separation of microbial cells followed by their isolated cultivation. The sample is transferred into a sterile volume of water or saline (10-100 ml volume) and then shaken for two minutes. In order to extract microorganisms located in the thickness of the material under study (for example, sausages or cheese), first rubbing the sample pieces with sterile instruments with sand is performed. The material that has undergone preliminary preparation, weighing 1 g or a volume of 1 ml, is diluted with sterile water by 10, 100, 1000, etc. times. Choose the degree of dilution that gives the concentration of cells corresponding to the capabilities of the method.

The subsequent cultivation of microorganisms consists in the preparation of a nutrient medium. Usually a dense medium (MPA) is chosen. It is first melted and cooled to 45-50 °C, and only then it is poured into several Petri dishes (three to five pieces), on the bottom of which swabs from the test substance of various concentrations are placed. Next, mixing of the still not frozen nutrient medium and the material introduced into it is carried out. This is how cells are fixed at various points in the volume of the substrate.

Next, the Petri dishes are placed in a thermostat for 2 days at 22°C. During this time, the cells multiply to such an extent that the colony formed by each of the cells becomes visible to the naked eye. Each of them is a pure culture of the type of bacteria from whose cells it has grown.

After that, from Petri dishes, microorganisms are subcultured into separate test tubes filled with a nutrient medium. In this way, pure cultures are isolated from a mixed sample. This method bears the name of its developer - R. Koch. It is also commonly called the cup method, or depleting sowing. After obtaining pure cultures of various types of bacteria, their form is established, spores, and families are detected.

All work must be carried out according to the principles of asepsis. To avoid premature development of microorganisms, the study should be carried out immediately after sampling. Tap water is analyzed after draining the first portions, since they may contain microbes accumulated in pipes and taps. The microflora of fruits, berries and vegetables is mainly located on the surface (peel), therefore, washings are performed from it. To do this, place the fetus in a sterile container and pour it necessary quantity water. Then they are shaken quite vigorously and the water is poured into another container. Crops from cloth products are also obtained by swabs, but beforehand, pieces of a given size are cut out of them.

To isolate a pure culture of microorganisms, to study their biological properties for the purpose of identification, as well as to obtain biomass, it is necessary to propagate microorganisms in a laboratory. Cultivation, or cultivation, of microbes is possible only when certain conditions are created for their vital activity. Most bacteria, yeasts, molds are cultivated on artificial nutrient media. Viruses and rickettsia reproduce only in living cells, tissue culture, chick embryos or in the body of an animal.

Artificial media used for the cultivation of microorganisms must meet certain requirements: be easily digestible, with the necessary composition of nitrogenous and carbohydrate substances, vitamins, the required concentration of salts, with a certain pH (pH of the medium); have buffer properties; have an optimal redox potential .

The culture media must also contain a sufficient amount of water and must be sterile, i.e. free of microorganisms before inoculation. The source of nitrogen in the media can be various organic, rarely inorganic compounds. Often, peptone, which is a product of incomplete protein hydrolysis, is added to protein-free media. Proteolytic microorganisms can use gelatin (“animal jelly”) as a nitrogenous substance. Carbohydrates, alcohols, and some organic acids are more often the source of carbon in nutrient media.

Various natural products can be used to prepare artificial nutrient media: milk, blood, whey, meat, egg yolk, potatoes and others. organic matter and mineral salts.

Artificial nutrient media according to their purpose are divided into four main groups: universal, special, selective (elective) and differential diagnostic.

Universal media include meat-peptone broth and meat-peptone agar, on which many types of pathogenic and non-pathogenic bacteria grow.

Special media are used to grow bacteria that do not multiply on universal media. Special foods include food with milk, blood serum, with the addition of animal blood, t-chukose, etc. They grow lactic acid bacteria, pathogenic and other microorganisms.

In selective (elective) environments, only certain types of bacteria develop well. Such media include enrichment media in which the species of interest grows faster than the accompanying bacteria. For example, Kessler's medium, which contains gentian violet and bovine bile, is selective for gram-negative Escherichia coli resistant to these substances and at the same time selective for sensitive gram-positive

bacteria.

Differential diagnostic media are used to differentiate certain types of bacteria according to their cultural and biochemical properties. These include:

media for determining proteolytic activity (meat peptone gelatin - NRM, milk agar, etc.);

media for determining the fermentation of carbohydrates (Giess, Eido, Ploskirev, etc.);

media for determining hemolytic capacity (blood agar and other media supplemented with animal blood);

environments for determining the reducing (reducing) ability of microorganisms (Wilson-Blair medium);

selective media used to differentiate between prototrophic and auxotrophic bacteria.

According to the consistency, nutrient media can be dense, semi-liquid and liquid. To obtain media of dense consistency, 2–2.5% agar or 10–20% gelatin are added to liquid media. Semi-liquid media are obtained by adding 0.5-1.0% agar. Agar (in Malay “jelly”) is a dense fibrous substance obtained from red algae and forming a dense gel (jelly) in aqueous solutions. It consists mainly of polysaccharides (70-75%). The main components of agar are high-molecular substances agarose and agaropeptin, which are not broken down and absorbed by microorganisms. In this regard, agar is not a nutrient substrate, it is added to the media solely to obtain a dense consistency. Agar melts in water at 100°C and solidifies at 40-43°C. It is produced in the form of yellowish plates or a grayish-white powder.

The osmotic conditions necessary for the vital activity of microbes are created in the nutrient medium by the addition of sodium chloride or a certain combination of salts of sodium phosphate and potassium phosphate OH) ions. It is the logarithm of the absolute concentration of hydrogen ions.

The pH of the neutral reaction corresponds to 7.0. In this case, the number of hydrogen ions is equal to the number of hydroxyl ions. A reading below 7.0 indicates an acidic reaction, while a reading above 7.0 indicates an alkaline reaction. Microorganisms have adapted to develop in conditions with an extremely wide range of pH - from 2.0 to 8.5. Most saprophytic and pathogenic microorganisms are cultivated in a slightly alkaline environment with a pH of 7.2-7.4. For the cultivation of lactic acid bacteria, yeasts and molds, an acid reaction of the medium, pH 5.0-6.5, is necessary.

Currently, many nutrient media are produced in the form of ready-made dry semi-finished products containing all the ingredients necessary for the vital activity of microorganisms. To prepare the nutrient medium, the powder is diluted with water, the resulting mixture is boiled, the required pH value is set and sterilized.

Great importance for the growth and reproduction of microorganisms on artificial nutrient media have temperature conditions. In relation to the temperature regime, all microorganisms are divided into three groups: psychrophilic (cold-loving), mesophilic (medium), thermophilic (heat-loving). The temperature limits of reproduction in psychrophiles range from 0 to 20 °C, in mesophiles - from 20 to 45 °C, in thermophiles - from 45 to 70 °C.

When growing aerobes, crops are cultivated in thermostats with access to atmospheric oxygen, that is, under normal conditions. For the cultivation of anaerobes, anoxic conditions are created that can be achieved by physical, chemical and biological methods. Anaerobic thermostats are also used.

Physical Methods based on the creation of a vacuum in special devices - anaerostats or vacuum desiccators, in which crops are first placed, and then a vacuum is created in the devices.

Sometimes the air in anaerostats is replaced carbon dioxide, nitrogen or other inert gas. The access of oxygen to the nutrient medium can be hindered if anaerobes are cultivated in the depth of a nutrient agar column or inside sealed glass tubes. Anaerobic conditions can also be created in simpler ways: using a layer of agar poured over crops on a dense nutrient medium, or using vaseline oil, which covers a liquid nutrient medium (Kitta-Tarozzi medium). Chemical methods are that in a desiccator With chemical substances, such as pyrogallol and alkali, are placed in crops, the reaction between which occurs with the absorption of oxygen.

biological method is based on the simultaneous cultivation of aerobes and anaerobes on dense nutrient media in hermetically sealed Petri dishes. In this case, oxygen is absorbed by growing aerobes sown on one half of the medium, after which the growth of anaerobes begins, which are sown on the other half.

8.1. Methods for reproduction of bacteria

The terms 'bacteria' and 'growth of bacteria' are often used interchangeably, although the terms are strictly speaking different things. Bacterial growth is understood as an increase in the size of a bacterial cell, and bacterial reproduction is an increase in the number of bacterial cells. However, when referring to the bacterial population as a whole, in this case, the term "growth" refers to an increase in the number of individuals in the population. In the latter case, it is more correct to use the term "growth of a bacterial culture".

A. The main mode of reproduction for most bacteria is binary division.

1. In gram-positive bacteria, binary fission occurs by forming partitions from opposite ends of the cell wall to the center, where both parts of the septum merge, thereby forming two independent cells.

2. In gram-negative bacteria, binary fission occurs through the formation constriction: the cell seems to become thinner in the middle until it breaks into two independent cells.

B. A number of bacteria can divide by budding(e.g. Francisella, mycoplasmas).

B. Those bacteria that form filamentous forms can divide by their fragmentation(e.g. actinomycetes, mycoplasmas).

D. Streptomycetes have a way of reproduction exospores.

D. Chlamydia has special cycle of development(see below).

8.2. Chlamydia development cycle

BUT. elementary body performs an infectious function - penetrates into the host cell by invagination of the site of adsorption.

B. Reticular(or initial) corpuscle multiplies by binary fission in the resulting cytoplasmic vesicle and forms a microcolony, which is visible under microscopy as a cytoplasmic inclusion.

B. The transitional form from the reticular body to the elementary body is intermediate corpuscle. After many new elementary bodies have been formed in the microcolony, it merges with the cell membrane and “poured” outwards as a “harvest” of multiplying chlamydia, whose elementary bodies go in search of new host cells.

8.3. Cultivation of microorganisms and classification of artificial nutrient media

There are two basic principles for the cultivation of microorganisms - in vivo and in vitro.

1. According to them consistency artificial culture media are classified into solid, semi-liquid and liquid (Figure 8-3).

a. Dense nutrient media can be agarized and folded.

1 . agarized Nutrient media are called so because agar, a polysaccharide extracted from seaweed of certain species and used to compact nutrient media in bacteriology, is introduced as a sealant into their composition according to the same algorithm as starch or gelatin in everyday life. To obtain a dense nutrient medium, an agar concentration of 1.5–2% is sufficient. In this case, either Petri dishes (“plate agar”) or test tubes (if there is a bevel - “beveled agar” or “jamb”, in its absence - “agar column”) can be used.

2 . Vintage nutrient media are solid media containing blood serum or enriched with another protein (egg, for example), which are compacted (as a result of protein denaturation) when they are heated during the sterilization process.

b. semi-liquid culture media contain a small amount of agar (about 0.5%).

in. Liquid culture media do not contain sealants. In principle, any liquid medium can be turned into a solid medium either by adding agar to it, or by folding the protein contained in it (observing in both cases the necessary concentration of the sealant).

2. In your own way composition artificial nutrient media are classified into natural and synthetic.

a. natural artificial nutrient media are prepared on the basis of decoctions or extracts of meat, fish, vegetables, and other natural products. Natural nutrient media, in turn, are classified into simple and complex. It is predominantly natural nutrient media that are used in the work of bacteriological laboratories of the so-called "practical health care".

1 . Simple natural nutrient media, in fact, are such decoctions or extracts. Simple natural nutrient media include:

- meat-peptone agar (MPA) and meat-peptone broth (MPB), belonging to the same group, since agar is not digested by the vast majority of bacteria, and, therefore, it does not change the composition of the medium - only its concentration;

- gelatin;

- milk;

- pieces of vegetables.

2 . Complex natural nutrient media are obtained by adding any substance (dye, sugar, antibiotic, blood, etc.) to simple natural media.

b. Synthetic artificial nutrient media are obtained by mixing pure chemicals (usually salts) in a solution. Unlike natural nutrient media, these are so-called media of known composition, since the amount of substances contained in them is precisely specified by the recipe for their preparation. Synthetic artificial nutrient media are mainly used in bacteriological laboratories of scientific institutions.

3. In your own way appointment artificial nutrient media are classified into basic, elective (selective), differential diagnostic and preserving.

a. Main artificial nutrient media are so named because they are used to carry out the main work of a bacteriologist - the accumulation of a pure culture, its "revival" after long-term storage, etc. The main nutrient media, in turn, are divided into universal and special.

1 . Same universal the basic nutrient medium can be used for the cultivation of many types of bacteria. In their composition, these are simple natural nutrient media. Bacteria that can be cultured on such media are called bacteria with simple nutritional requirements.

2 . Special the main nutrient medium is used for the cultivation of a particular species or group of bacteria. Bacteria that need special nutrient media for their cultivation are called bacteria with complex nutrient requirements.

b. Electoral (selective, selective, enriched) artificial culture media are media containing substances that are used by certain types of bacteria and do not favor or even prevent the growth of other bacteria. Such media serve to isolate a specific type of bacteria from pathological material. It is rather difficult to isolate those types of bacteria for which such media have not been developed.

in. Differential diagnostic artificial nutrient media are media that allow one species of bacteria to be distinguished from others by their enzymatic activity (most often by the color of the colonies they form) or cultural properties.

G. Preservative artificial nutrient media are media used, for example, in the delivery of pathological material to a bacteriological laboratory; since the metabolic activity of bacteria on them is reduced to almost zero, the bacteria persist, but do not multiply.

8.4. Requirements for the conditions for cultivating bacteria

In order to successfully cultivate bacteria on artificial nutrient media, it is necessary to take into account not only their nutritional needs (simple or complex, as mentioned above), but also the cultivation temperature, the reaction of the medium (pH), as well as the aeration conditions necessary for a particular species. .

A. By optimal temperature cultured bacteria are classified into three groups: thermophiles, mesophiles and psychrophiles.

1. Optimum cultivation temperature thermophiles is 50 - 60°С. By understandable reasons thermophiles are not the subject of medical microbiology.

2. The vast majority of bacteria of medical importance grow best at human body temperature, i.e. 37°C. Such bacteria are called mesophiles.

3. A number of human pathogenic bacteria grow best at lower temperatures (6 to 20°C) and are called psychrophiles.

B. Depending on the required culture medium reactions Bacteria are divided into three main groups, two of which have specific terms.

1. Those that grow best on acidic nutrient media are called acidophiles.

2. Bacteria, for the cultivation of which it is necessary to use alkaline media, are called alkaliphiles.

3. The vast majority of human pathogenic bacteria grow on media with neutral pH. No special terms are used to refer to this group of bacteria.

B. According to the requirements for aeration conditions during cultivation, bacteria can be divided into four main groups.

1. obligate aerobes require constant access of air to the surface of the nutrient medium during cultivation.

2. Anaerobes, on the contrary, are cultivated in an airless environment.

3. They require a special gas composition for their cultivation microphiles(reduced oxygen content) and capnophiles(high carbon dioxide content).

4. Facultative anaerobes grow under any aeration conditions.

8.5. The nature of bacterial growth on artificial nutrient media

The nature of bacterial growth depends, first of all, on what nutrient medium - liquid or solid - is used for cultivation.

BUT. For liquid nutrient media (for example, meat-peptone broth) for the whole variety of bacteria, four forms of growth can be distinguished.

1. Most bacteria form diffuse haze(Figure 8-4).

2. The so-called "Koch's bacteria", so named because their main pathogenic representatives were discovered by Koch - bacilli, mycobacteria and vibrios - form on the surface of the broth film.

3. Streptococci are characterized by the so-called benthic or parietal growth- sediment or small flakes at the wall of the test tube with a clear broth.

4. The causative agent of the plague - Yersiniapestis - grows in the form films on the surface of the broth from which weights descend, similar to stalactites, sediment can also form.

B. On dense In nutrient media (for example, meat-peptone agar), the growth of bacteria depends on the method of inoculation.

1. If the inoculation density is high, then the bacteria form a continuous coating on the surface of the agar - the so-called "growth lawn' or 'confluent growth'.

2. If the inoculation is carried out in such a way that each bacterial cell lies on the surface of the agar at a great distance from the others, then, after repeated divisions, it forms an isolated colony(they also talk about isolated growth"). And since a colony is the result of the reproduction of a single cell, it is, with some assumptions, considered as a clonal culture. It is from the material of a separate, isolated colony that the so-called "pure culture" is obtained in the process of the cultural method of research - a culture containing cells of only one type. All the huge variety, in their appearance, of colonies (Fig. 8-5) can be reduced to two main types.

a. S-shape colonies ("smooth") - homogeneous, with smooth edges, domed, moist, transparent or translucent. All S-shaped colonies are similar to each other, differing in different types of bacteria or their variants in size, and in the case of pigmentation or growth on differential diagnostic media, in color. S-shaped colonies form:

- Gram-negative rods, except for the causative agent of plague (Yersiniapestis).

b. R-shape colonies ("rough") - not homogeneous, with uneven edges, with a wide variety of options for location relative to the surface of the nutrient medium (from towering above it to immersed in it, i.e. located below the surface of the nutrient medium), opaque. The R-shapes of colonies of various bacteria can differ sharply from each other, sometimes comparative turns are used to describe them (for example, they talk about colonies that look like a daisy flower in the causative agent of diphtheria, similar to cauliflower or a wart - in pathogens of tuberculosis, etc. .). R-shaped colonies form:

- Gram-positive rods

Plague causative agent (Yersinia pestis).

8.6. Growth stages of a batch bacterial culture

When growing bacteria in a liquid nutrient medium, it is possible to constantly select the grown bacterial mass, remove the metabolic products of bacteria, and add a new complete nutrient medium. Those. constantly maintain optimal cultivation conditions. In this case, the culture will constantly grow at maximum speed. Such a culture is called chemostatic, because special devices are used to obtain it - chemostats - which make it possible to perform the above manipulations. Such cultures are used in industrial microbiology to obtain useful substances - products of microbial metabolism (antibiotics, amino acids, etc.). If the bacterial culture is grown in a test tube (this is the method used in medical microbiology), then over time, the products of bacterial metabolism accumulate in the nutrient medium, and the nutrient medium, on the contrary, is depleted. As a result, in order for the culture not to die, it must be periodically transferred to a fresh nutrient medium. Such a culture is called periodic. In its growth, it goes through nine stages (phases) of development.

A. After adding the inoculum (inoculation dose) to the nutrient medium, lag phase(Figure 8-6). Cell division does not occur at this stage - bacteria, as it were, adapt to a new habitat; while some of them may die.

B. Then comes the phase positive acceleration(Fig. 8-7). Bacterial cells begin to divide and the rate of their division is constantly increasing.

C. Finally, the rate of cell division will reach a maximum value and remain so for some time (Fig. 8-8). it exponential phase (phase logarithmic growth).

D. However, over time, the amount of nutrients in the medium decreases, and the concentration of metabolic products of bacterial cells increases. As a result, the conditions for the reproduction and growth of bacteria worsen, and the rate of division of bacterial cells decreases - the phase negative acceleration(Fig. 8-9). However, at this stage, the number of living bacterial cells in culture is still increasing, albeit at a constantly decreasing rate.

E. The stage during which at any given moment in time the number of newly appeared living bacterial cells is equal to the number of dead ones is called stationary phase maximum(Figure 8-10). A bacterial culture that has reached the maximum concentration of living bacterial cells possible under the given conditions of cultivation in this growth phase is called stopped.

E. Then the number of living bacterial cells begins to decrease at an increasing rate. This stage of bacterial culture growth is called the phase accelerated death(Figure 8-11).

G. The stage during which this rate of decrease in living bacterial cells becomes maximum is called the phase log death(Figure 8-12).

3. Over time, however, this speed begins to decrease - a phase begins decrease in death rate(Figure 8-13).

I. In the end, the culture will for some time consist of a minimum number of living bacterial cells. This stage of bacterial growth is called stationary phase minimum(Figure 8-14). The time that a bacterial culture can hold out at this minimum until it dies depends both on the type of microorganisms and on the cultivation conditions.

8.7. Methods for creating anaerobic conditions for the cultivation of bacteria

Depending on the method of creating an airless environment, all methods of creating anaerobic conditions for cultivating bacteria are divided into physical, chemical and biological. The Kitta-Tarozzi method, which combines physical, chemical and biological methods of creating an airless habitat for microorganisms, stands apart.

A. Air, more precisely oxygen in the air, can be removed from the culture medium of bacteria physical methods.

1. Can be used anaerostat- a vessel with a hermetic lid, from which air is sucked off by a pump. Petri dishes with crops are placed in such a vessel and, after pumping out the air, they put it (if it allows this in size - such small anaerostats are called microanaerostats) in a thermostat, where bacteria are cultivated.

2. There are, in fact, varieties of anaerostats in which the removed air is replaced by some inert gas, for example - in Kip's apparatus- hydrogen.

3. One of the most common methods for creating anaerobic conditions in the cultivation of bacteria is used in the so-called Vignal-Villon pipes. This method is easy to implement and does not require any special equipment. In Viñal-Villon tubes, deep cultivation bacteria. To do this, the bacterial culture is diluted in a molten and cooled nutrient medium (in test tubes or pipettes - hence the name “tubes”) in such a way that bacterial cells were at a considerable distance from one another. When the nutrient medium solidifies, the bacterial cells turn out to be “immured” in its thickness, and during cultivation, each of them forms a separate colony (naturally, without access to atmospheric oxygen).

4. Anaerobic conditions for the cultivation of bacteria are also created in the case of their inoculation by injection into high column semi-liquid agar. The injection site is immediately tightened and the bacteria grow in the thickness of the nutrient medium.

5. The air dissolved in the liquid nutrient medium can be removed by boiling. When a liquid is heated, the air dissolved in it escapes into the atmosphere, which we observe in the form of "seething". Such a process is called regeneration nutrient medium. So that subsequently, during slow cooling, the air does not dissolve again in the nutrient broth, it is cooled very quickly (for example, under a stream of cold water).

6. To minimize diffusion atmospheric air into the nutrient medium, its surface is covered with a layer of liquid oil (for example, vaseline). In this case, one speaks of cultivation under oil».

7. In deep culture, it can be difficult to access the desired colony and extract it from the depth of the nutrient medium. Facilitates this task Peretz method: a melted and cooled nutrient agar medium mixed with a bacterial culture is poured into a Petri dish, on the surface of which a glass slide is carefully placed, which is slightly pressed into the nutrient agar. Those colonies that grow directly under this glass, after removing the latter, become easily accessible.

B. Chemical Methods for creating anaerobic conditions for the cultivation of bacteria are divided into two groups.

1. To bind oxygen in the air, it can be carried out in a closed volume (for example, in a desiccator with a ground-in lid) chemical reaction, which flows with the absorption of air.

a. AT Aristovsky's method for this purpose, bulk ingredients are used. In the development of this method, the modern microbiological industry produces special kits with which you can create a gas mixture, both with the complete absence of oxygen, and with the presence of it, as well as carbon dioxide and nitrogen, in certain concentrations necessary for the cultivation of bacteria with "non-standard" aeration requirements.

b. AT Omelyansky method liquid ingredients (pyrogallol and caustic potash) are used for this purpose.

2. You can add oxygen-binding substances to the liquid nutrient medium. Such substances are called reducing. These include, for example, glucose, thioglycolic acid and a number of others.

B. As biological method of creating anaerobic conditions for the cultivation of bacteria is the most common (in various modifications) Fortner method. Its principle is that in a closed volume (for example, in a waxed Petri dish), anaerobes and the so-called "greedy aerobe" are simultaneously cultivated - a type of bacteria that intensively absorbs oxygen during its growth. The most commonly used enterobacterium is Serracia marcescens. The aerobe destroys all the oxygen in the closed volume, thereby creating conditions for the growth of the anaerobe.

G. Kitt-Tarozzi method consists in using the nutrient medium of the same name for the cultivation of anaerobes. The Kitta-Tarozzi medium consists of a meat-peptone broth containing glucose (as a reducing substance), regenerated and filled with a layer of oil, pieces of a parenchymal organ (more often a liver) are placed at the bottom of the tube to adsorb the air dissolved in the meat-peptone broth.

To isolate a pure culture of microorganisms, to study their biological properties for the purpose of identification, as well as to obtain biomass, it is extremely important to propagate microorganisms in a laboratory. Cultivation, or cultivation, of microbes is possible only when certain conditions are created for their vital activity. Most bacteria, yeasts, molds are cultivated on artificial nutrient media. Viruses and rickettsia reproduce only in living cells, tissue culture, chick embryos or in the body of an animal.

Artificial media used for the cultivation of microorganisms must meet certain requirements: be easily digestible, with the necessary composition of nitrogenous and carbohydrate substances, vitamins, an extremely important concentration of salts, with a certain pH value (pH of the medium); have buffer properties have an optimal redox potential.

Nutrient media must also contain a sufficient amount of water and must be sterile, that is, do not contain microorganisms before sowing. The source of nitrogen in the environments are various organic, rarely inorganic compounds. Often, peptone, which is a product of incomplete protein hydrolysis, is added to protein-free media. Proteolytic microorganisms can use gelatin (“animal jelly”) as a nitrogenous substance. Carbohydrates, alcohols, and some organic acids are more often the source of carbon in nutrient media.

Various natural products can be used to prepare artificial nutrient media: milk, blood, whey, meat, egg yolk, potatoes and other organic substances and mineral salts.

Artificial nutrient media according to their purpose are divided into four main groups: universal, special, selective (elective) and differential diagnostic.

Universal media include meat-peptone broth and meat-peptone agar, on which many types of pathogenic and non-pathogenic bacteria grow.

In selective (elective) environments, only bacteria of certain species develop well. Such media include enrichment media in which the species of interest grows faster than the accompanying bacteria. For example, Kessler's medium, which contains gentian violet and bovine bile, is selective for gram-negative Escherichia coli resistant to these substances and at the same time selective for sensitive gram-positive

bacteria.

Differential diagnostic media are used to differentiate certain types of bacteria according to their cultural and biochemical properties. These include:

media for determining proteolytic activity (meat-peptone gelatin - NRM, milk agar, etc.);

media for determining the fermentation of carbohydrates (Giess, Eido, Ploskirev media, etc.);

media for determining hemolytic ability (blood agar and other media with the addition of animal blood);

environments for determining the reducing (reducing) ability of microorganisms (Wilson-Blair medium);

selective media used to differentiate prototrophic and auxotrophic bacteria.

According to the consistency, nutrient media are dense, semi-liquid and liquid. To obtain media of dense consistency, 2–2.5% agar or 10–20% gelatin are added to liquid media. Semi-liquid media are obtained by adding 0.5-1.0% agar. Agar (in Malay “jelly”) is a dense fibrous substance obtained from red algae and forming a dense gel (jelly) in aqueous solutions. It consists mainly of polysaccharides (70-75%). The main components of agar are high-molecular substances agarose and agaropeptin, which are not broken down and absorbed by microorganisms. In this regard, agar is not a nutrient substrate, it is added to the media solely to obtain a dense consistency. Agar melts in water at 100°C and solidifies at 40-43°C. It is produced in the form of yellowish plates or a grayish-white powder.

The osmotic conditions necessary for the life of microbes are created in a nutrient medium by the addition of sodium chloride or a certain combination of salts of sodium phosphate and potassium phosphate hydroxide (OH) ions. It is the logarithm of the absolute concentration of hydrogen ions.

Today, many nutrient media are produced in the form of ready-made dry semi-finished products containing all the ingredients necessary for the life of microorganisms. To prepare a nutrient medium, the powder is diluted with water, the resulting mixture is boiled, set extremely importance pH and sterilized.

Temperature conditions are of great importance for the growth and reproduction of microorganisms on artificial nutrient media. In relation to the temperature regime, all microorganisms are divided into three groups: psychrophilic (cold-loving), mesophilic (medium), thermophilic (heat-loving). The temperature limits of reproduction in psychrophiles range from 0 to 20 °C, in mesophiles - from 20 to 45 °C, in thermophiles - from 45 to 70 °C.

Physical Methods are based on creating a vacuum in special anaerostat devices or in vacuum desiccators, in which crops are first placed, and then a vacuum is created in the devices.

Sometimes the air in anaerostats is replaced with carbon dioxide, nitrogen, or another inert gas. The access of oxygen to the nutrient medium can be hindered if anaerobes are cultivated in the depth of a column of nutrient agar or inside sealed glass tubes. Anaerobic conditions can also be created in simpler ways: using a layer of agar, poured over crops on a dense nutrient medium, or using vaseline oil, which covers a liquid nutrient medium (Kitt-Tarozzi medium). Chemical methods are that in a desiccator With crops are placed with chemicals, for example pyrogallol and alkali, the reaction between which occurs with the absorption of oxygen.

biological method is based on the simultaneous cultivation of aerobes and anaerobes on dense nutrient media in hermetically sealed Petri dishes. At the same time, oxygen is absorbed by growing aerobes sown on one half of the medium, after which the growth of anaerobes begins, which are sown on the other half.

Universal Tool for the production of crops is a bacterial loop. In addition to it, a special bacterial needle is used for inoculation with an injection, and metal or glass spatulas are used for inoculation on Petri dishes. For inoculation of liquid materials, Pasteur and graduated pipettes are used along with the loop. The former are pre-made from sterile fusible glass tubes, which are pulled out on a flame in the form of capillaries. The end of the capillary is immediately sealed to maintain sterility. For Pasteur and graduated pipettes, the wide end is covered with cotton wool, after which they are placed in special cases or wrapped in paper and sterilized.

When reseeding the bacterial culture take the test tube in the left hand, and with the right hand, grasping the cotton plug IV and V with fingers, take it out, passing it over the flame of the burner. Holding the loop with the other fingers of the same hand, they collect the inoculum with it, and then close the test tube with a stopper. Then, a loop with inoculum is introduced into the test tube with slant agar, lowering it to the condensate in the lower part of the medium, and the material is distributed in a zigzag motion over the slant surface of the agar. After removing the loop, burn the edge of the test tube and close it with a cork. The loop is sterilized in the flame of a burner and placed in a tripod. The test tubes with inoculations are written over r, indicating the date of inoculation and the nature of the inoculum (study number or culture name).

Crops "lawn" produced with a spatula on nutrient agar in a Petri dish. To do this, having slightly opened the lid with the left hand, the inoculum is applied to the surface of the nutrient agar with a loop or pipette. Then the spatula is passed through the flame of the burner, it is cooled on the inside of the lid and the material is rubbed over the entire surface of the medium. After the incubation of the inoculation, a uniform continuous growth of bacteria appears.

Nutrient medium in microbiology, media containing various compounds of complex or simple composition are called, which are used for the propagation of bacteria or other microorganisms in laboratory or industrial conditions.

Culture media are prepared from animal or vegetable products. Of great importance is the presence in the nutrient medium of growth factors that catalyze the metabolic processes of the microbial cell (group B vitamins, nicotinic acid, etc.).

Built environments prepared according to certain recipes from various infusions or decoctions of animal or vegetable origin with the addition of inorganic salts, carbohydrates and nitrogenous substances.

In bacteriological practice most often dry nutrient media are used, which are obtained on the basis of the achievements of modern biotechnology. For their preparation, economically profitable non-food raw materials are used: blood substitutes that have expired (hydrolysin-acid hydrolyzate of animal blood, aminopeptide - enzymatic blood hydrolyzate; biotechnology products (fodder yeast, fodder lysine, grape flour, proteincolysin). Dry nutrient media can be stored for a long time, are convenient for transportation and have a relatively standard composition.

By consistency nutrient media can be liquid, semi-liquid, dense. Dense media are prepared by adding 1.5-2% agar to a liquid medium, semi-liquid - 0.3-0.7% agar. Agar is a product of the processing of a special type of seaweed, it melts at a temperature of 80-86 ° C, solidifies at a temperature of about 40 ° C and, when solidified, imparts density to the medium. In some cases, gelatin (10-15%) is used to obtain dense nutrient media. A number of natural nutrient media (clotted blood serum, folded egg white) are dense in themselves.

For the intended purpose environments are divided into basic, elective and differential diagnostic.

To the main include the media used to grow many bacteria. These are tryptic hydrolysates of meat, fish products, animal blood or casein, from which a liquid medium is prepared - nutrient broth and dense - nutrient agar. Such media serve as the basis for the preparation of complex nutrient media - sugar, blood, etc., that satisfy the nutritional needs of pathogenic bacteria.

elective nutrient media are intended for selective isolation and accumulation of microorganisms of a certain type (or a certain group) from materials containing a variety of foreign microflora. When creating elective nutrient media, they proceed from the biological features that distinguish these microorganisms from most others. For example, the selective growth of staphylococci is observed at an increased concentration of sodium chloride, vibrio cholerae - in an alkaline environment, etc.

Differential diagnostic nutrient media are used to distinguish between individual species (or groups) of microorganisms. The principle of constructing these environments is based on the fact that different types bacteria differ from each other in biochemical activity due to an unequal set of enzymes.

special group make up synthetic and semi-synthetic nutrient media. The composition of synthetic media includes chemically pure substances: amino acids, mineral salts, carbohydrates, vitamins. Semi-synthetic media additionally include peptone, yeast extract and other nutrients. These media are most often used in research work and in the microbiological industry in the production of antibiotics, vaccines, and other drugs.

In recent years, in order to save nutrient media and accelerate the identification of certain microorganisms (enterobacteria, staphylococci, streptococci, etc.), the so-called microtest systems (MTS) have been used. They are polystyrene plates with wells containing sterile differential diagnostic media. Sterilization of MTS is carried out by UV irradiation. Microtest systems are especially convenient for mass bacteriological studies in practical laboratories.