The complex nutrient medium is. Nutrient microbiological media. Bacteriological research method

In the composition of the nutrient media, it is customary to divide on natural (or natural) and synthetic.

Natural(Natural) are usually called mediums that consist of animal or vegetable products. The basis of such environments are vegetable and fruit juices, milk, animal fabrics (meat, fish, liver, etc.), diluted blood, decoctions or extracts derived from natural substrates, such as meat, malt, potatoes, soil, yeast, etc. Many microorganisms develop on natural environments, since they contain all the components necessary for growth and development. Natural media are mainly used to maintain the cultures of microorganisms and the accumulation of biomass. However, these media have a complex, indefinite chemical composition and are little suitable for studying the metabolism of microorganisms.

The number of natural media widespread in laboratory practice includes meat-pepton broth (MPB), non-chopped beer wort, yeast and potato media, soil extract.

Syntheticcalled the medium, which includes only certain chemically pure compounds taken in exactly these quantities. Synthetic media are most convenient for the study of the metabolism of microorganisms. Knowing the accurate composition and number of components included in the medium, you can explore their consumption and transformation into appropriate exchange products.

Currently, microbiologists have a sufficient number of synthetic media that are not inferior in its qualities complex natural environments.

Semi synthetic mediaalso belong to the environments of an indefinite composition. Their main components are known compounds (carbohydrates, ammonium salts, nitrates, phosphates, etc.), and the component of an indefinite composition (corn extract, yeast autolysate, casein hydrolyzate, etc.) is contained in relatively low concentrations. Such environments are particularly widely used in the biotechnology of amino acids, vitamins, antibiotics, etc.

In purpose, elective and differential diagnostic environments distinguish.

Elective mediadesigned to release pure cultures of microorganisms from the medium of their natural habitat (water, soil, food, etc.).

Differential diagnostic environments- These are such environments with which you can quickly distinguish (differentiate) some types of microorganisms from others. Their composition is selected with such a calculation so that it is possible to clearly identify the most characteristic properties of this species. An example of such a medium to identify intestinal sticks in natural substrates (water, food) can serve as an ENDO environment. Intestinal wand forms crimson colonies with metal glitter on this environment. Differential-diagnostic media is particularly widely used in sanitary and medical microbiology for the rapidly approximate definition of individual groups of microorganisms.

Nutrient media are liquid, dense and bulk. The basis liquid mediais water. These include decoctions and extracts (natural media) or solutions of chemicals and other components (synthetic and semi-synthetic media).

Dense mediumit is obtained by adding to the liquid medium of gelatin or agar-agar (the substance produced from seaweed contains mainly polysaccharides). The lack of gelatin is the low melting point (24-27 ° C), and the agar-agar melts at 100 ° C and freezes at 40-45 ° C. When developing in a liquid medium, the culture of microorganisms form suspensions, a precipitate or film, or development on a dense medium - a colony.

Agricultural nutrients are extremely widely used in microbiological practice. They are used: to explore the nature of the growth and classification of microorganisms; for quantitative accounting of microorganisms; to release pure cultures of microorganisms with microbiological analysis of air, water, soil, etc.; To forward the cultures of microorganisms for the distance, for example, the culture of yeast to the plants will be sent on the Suslovaya agar; For long-term storage of crops. The dense nutrient media possess even the advantage that the contamination of crops from the outside on the liquid medium in most cases is unnoticed,

Microorganisms are cultured on nutritional environments. The nutrient media is divided into groups depending on the properties.

By physical state

Liquid media;

Semi-liquid environments;

Solid (dense) medium;

Liquid medium Present infusions, decoctions, broths prepared on the basis of meat, fish, vegetables (natural media), as well as a composition of certain concentrations of chemical compounds (artificial media). Semi-liquid environments It is prepared by adding 0.5-0.9% agar-agar to liquid media (jelly-forming substance obtained from marine algae). TO dense nutrient media include mediums containing 2-3% agar.

By complexity Nutrient media are divided into:

- simple, or conventional environments (pepton water, meat-pepton broth, meat-pepton agar);

- sophisticated, or special environments (blood agar, ascitic agar and broth, meat-pepton sugar broth, whey agar and broth, rolled serum, blood broth).

By origin Nutrient media are divided into:

Natural environments;

Semi-synthetic media;

Synthetic media.

Natural nutrient media - These are natural organic environments of non-permanent composition, which include animal or vegetable products. These include peptons, blood, decoctions and extracts derived from natural substrates (meat, fish, cereals).

Semi-synthetic media In addition to organic and inorganic substances of a known composition, natural origin products (potato medium with glucose, yeast medium).

Synthetic nutrient media Consist of certain amounts of organic and inorganic chemical compounds of a known composition.

Nutrient set Allocate:

- minimal environmentswhich contain only power supplies sufficient to grow;

- rich environmentswhich includes many additional substances.

In action from the appointment of nutrient media distinguish:

Main environments;

Elective (selective) medium;

Differential diagnostic environments;

Accumulative media (enrichment environment).

TO basic mediathe meat-pepton agar and meat-pepton broth. Most bacteria grow on these environments.

Differential diagnostic environments - These are complex media to study the biochemical properties of bacteria. These media are used to determine the type of bacteria.

Elective (selective) nutrient media contain substances that overwhelming some bacteria, and not affecting the growth of other bacteria. These environments serve to highlight a certain type of bacteria from mixed populations.

Cumulative nutrients (enrichment environment) are environments on which certain types of crops grow faster and intensively associated.

Bacteriological research method

The purpose of bacteriological research It is the allocation of a pure culture of the pathogen, its identification and determination of sensitivity to antibacterial drugs.

Bacteriological research method includes 4 stages:

Sowing the material under study on the nutrient medium;

Selection of a pure culture of the pathogen;

Identification of the pathogen (defining the type of bacteria) and the determination of sensitivity to antibacterial drugs;

Accounting for results and issuance of concluding.

For cultivation of bacteria use the nutrient media to which a number of requirements are presented.

1. Nutrition. Bacteria must contain all the necessary nutrients.

2. Isotonic. Bacteria should contain a set of salts to maintain osmotic pressure, a certain concentration of sodium chloride.

3. Optimal pH (acidity) of the medium. The acidity of the medium ensures the functioning of bacteria enzymes; For most bacteria is 7.2-7.6.

4. Optimal electronic potential indicative of the content in the dissolved oxygen medium. It should be high for aerobones and low for anaerobes.

5. Transparency (so that the growth of bacteria is visible, especially for liquid media).

6. Sterility.

Classification of nutrient media.

1. By origin:

1) Natural (milk, gelatin, potatoes, etc.);

2) artificial - media prepared from specially prepared natural components (peptone, aminoptide, yeast extract, etc.);

3) synthetic - the medium of a known composition, cooked from chemically pure inorganic and organic compounds.

2. In terms of composition:

1) Simple - meat beepton agar, meat beepton broth;

2) Complex are simple with the addition of an additional nutrient component (blood, chocolate agar): sugar broth, bullon, whey agar, yolk-salt agar, Wednesday Kitta Tarozzi.

3. By consistency:

1) solid (contain 3-5% agar-agar);

2) semi-liquid (0.15-0.7% agar-agar);

3) Liquid (do not contain agar-agar).

4. For appointment:

1) general purpose - for the cultivation of most bacteria (meat-beep agar, meat-beep broth, blood agar);

2) Special purpose:

a) elective - environments on which bacteria are growing only of one species (kind), and the genus of others is suppressed (alkaline broth, 1% pepton water, yellow-salt agar, casein-coal agar, etc.);

b) Differential-diagnostic - environments, on which the growth of some types of bacteria differs from the growth of other types of one or another properties, more often than biochemical (Wednesday, Levin, Gis, Plothery, etc.);

c) enrichment environment - mediums in which the reproduction and accumulation of bacteria-causative agents of any kind or type (selenitic broth) occurs.

To obtain pure culture, it is necessary to own methods for the release of pure crops:

1. Mechanical disabilities (Stroke method by firing loop, method of breeding in agar, distribution over the surface of a solid nutrient with a spatula, a DRIGALS method).

2. The use of elective nutrient media.

The colony is visible to the naked eye, an isolated accumulation of bacteria on a solid nutrient medium.

When preparing nutrient media, it is necessary to take into account the need of cultured microorganisms in various cell elements. There are various classifications of nutrient media.

Classification of nutrient media in composition:

1. Simple environments(MPB, MPa, gelatin, pepton water). The meat-pepton broth (MPB) is the protein basis of all environments.

There are several ways to prepare MPB:

a) on meat water with the addition of finished peptone;

b) on the digests of hydrolysis products of the original raw materials using enzymes.

Meat-pepton agar (MPa) is obtained by adding agar-agar (1.5-3%) to the IPT. If the MPa is distributed to the diagonal of the test tube or the bottle is a beveled agar. If the medium is distributed in the tube vertically high 5-7 cm, it is agar with a column. MPa, frozen in Petri dishes in the form of a podcass - a plate agar. If the medium has a vertical layer with a height of 2-3 cm, and the diagonal layer of the same value, it is a semi-sized agar.

2. Complex mediaprepared on the basis of simple with certain additives (carbohydrates, blood, bile, eggs, serum, milk, salt, growth factors, etc.)

Classification of nutrient media on source components:

1. Natural nutrient media- This is a natural product of animal or vegetable origin.

May be:

Vegetable (source products - soy, peas, potatoes, carrots, etc.).

Animals (source products - meat, fish, eggs, milk, animal fabrics, bile, blood serum, etc.).

Mixed (MPa, Wednesday Levsenshtein - Yensen, etc.).

2. Artificial mediacontain recycled natural products (meat water, power), substances obtained from these products (peptone, yeast and corn extracts) and various additives. This is the largest and most diverse in the composition of the most frequently used group of media. They are prepared according to certain recipes from various infusions or decoctions of animal or vegetable origin with the addition of inorganic salts, carbohydrates and nitrogen substances.

3. Synthetic media(known chemical composition) consist of chemically pure compounds in precisely established concentrations (with the addition of carbohydrates, salts, amino acids, vitamins, etc.). Based on these environments, adding natural or artificial media to them, semi-synthetic media receive.

Classification of nutrient consistency media:wednesdays are liquid(environment without agar), semi-winged(with agar up to 1%), dense(Agarey - 1.5-2.5%). Liquid environments are more often used to study the physiological biochemical features of microorganisms, for the accumulation of biomass and exchange products. Semi-liquid media is commonly used for storage of crops, dense - to highlight microorganisms, studying the morphology of colonies, diagnostic purposes, quantitative accounting, identifying antagonistic properties, etc.

Classification of nutrient media for intended purpose:universal (commonly used) and special.

Universal (basic) media.These media are used to cultivate the majority of relatively unpretentious microorganisms or used as a basis for the preparation of special environments, adding blood, sugar, milk, serum and other ingredients necessary for the reproduction of one or another type of microorganisms. This group includes: MPB - meat-pepton broth, MPa - meat-pepton agar, MPJ - meat-pepton gelatin, etc.

Special environments.Designed to highlight and selectively cultivate certain types of microorganisms that do not grow on simple environments.

The following types of special environments distinguish: enrichment media, elective, differential diagnostic, preserving and accumulation media.

Enrichment media.Many microorganisms do not grow on conventional media, so carbohydrates (sugar broth or agar) or proteins (serum agar and broth, blood agar and broth) are added to it. Blood agar or blood broth are obtained by adding 5-10% to the nutrient medium, 5-10% heated sterile defibrous blood blood, rabbit, horses, humans. The medium is used to release streptococci, pneumococci and other bacteria, as well as to study hemolytic activity. Serum broth or whey agar is obtained by adding 15-20% horsepie or bovine serum by adding 15-20%.

2. Elective (selective) media.These media are intended for selective release and accumulation of microorganisms of a certain type of material containing several types of microbes. When crushing on them, a material containing a mixture of various microorganisms is used to appear before the growth of the type for which this medium will be electively. The selectivity of the medium is achieved by creating conditions that are optimal for the cultivation of certain microbes (pH, eh, the concentration of salts, the composition of nutrients), i.e. positive selection. Or by adding substances to the medium that depress other microorganisms (bile, high concentrations of NAS1, antibiotics, etc.), i.e. Negative selection. This group includes:

Selenitic environment- It is the best enrichment medium for salmonella and dysenteric microbes zone. Selenite sodium contained in the medium stimulates the growth of these bacteria and suppresses the growth of the concomitant flora.

Bismuth Sulfit Agar -contains bismuth salts, diamond greens. Salmonella grow on this medium in the form of black colonies. Other types of bacteria on this growth medium do not give.

Salt agar (zva) -the medium for the release of staphylococci, contains up to 10% sodium chloride, which suppresses the majority of bacteria contained in the material. In addition, this medium is both differential-diagnostic, since the presence of egg yolk allows you to identify the enzyme lecithinase (lecitelelate), which forms pathogenic staphylococci. Lecithinase splits lecithin on phosphorcholines and fatty acids insoluble in water, therefore the medium around lecinazo-positive colonies is muttered and an opalescent zone appears in the form of a "rainbow".

Bulconelectives for Salmonella, the reproduction of which stimulates the added 10% bile, while at the same time the growth in the concomitant microorganisms.

Alkaline agaror alkaline pepton waterelective for cholera vibions, alkaline reaction medium (pH 9.0) does not prevent the growth of cholera vibions, but inhibits the growth of other microorganisms. 3-5 days. J.

3. Differential diagnostic environments.Differential diagnostic media is used to differentiate one type of microorganisms from the other by the nature of their enzymatic activity. The composition of these media is selected with such a calculation to clearly identify the most characteristic properties of a certain type of microorganisms, based on the features of its metabolism.

The medium to identify the proteolytic and hemolytic ability of microbes containing protein substances in their composition: blood, milk, gelatin, etc. The most common environments are meat-pepton gelatin (MPH). Rolling in the serum, milk and blood agar (ka).

Environments for the study of glycolithic properties include three main components: nutritional base (broth, agar), substrate (mono- and disahar, polyhydric alcohols) and indicator for identifying relevant enzymes. Enzymatic splitting of substrates leads to a pH shift and a change in the color of the medium. The most common non-ferrous media with various carbohydrates (for example, with bromcto-blue, indicator BP). The GISS medium is also widespread, which take into account differences in the ability to ferment various carbohydrates with the formation of acid or acid and gas.

For differentiation enterobacteria, pepton water with a set of different carbohydrates, an indicator of Andrene and floats, facilitating gas formation detection and help visually determine the change in pH, characteristic of various microorganisms. In particular, the shift in the acidic side causes redness of the environment with Andrene's reagent or yellowing when using the medium with bromismo blue, whereas when pacing the indicator of Andre and brommatome blue does not change the color of the medium. For example, the media that allow differentiate pathogenic microorganisms from the permanent inholes of the intestines to the separation of pathogenic bacteria from the intestine.

Such a medium is an ENDO environment. The main components of the ENDO environment are MPa, lactose and basic fuchsine discovered sodium sulfite. The initial nutrient medium is painted in light pink color. When saving lactose, acetaldehyde is formed, which reacts with sulfite and, released at the same time, Fuchin paints colonies into bright red color. Therefore, an intestinal wand that ferments lactose, with growth on this medium, forms red colonies with a metal glitter, and salmonella and Schigella are colorless, as they do not fermentrate lactose.

4. Wednesdays of accumulation,on which there are rapid growth of certain types of microorganisms.

5. Preservative (transport) media.Designed to preserve microorganisms during transportation to the place of study. These media contain additives, preventing the reproduction and death of microbes, which contributes to the preservation of their viability. The largest application was found a glycerin mixture (a tig medium), phosphate buffer mixture and Kari-Blair medium, amiz (with activated carbon and without activated carbon), stewart, etc.

Sterilization of nutrient media.

All nutrient media regardless of their purpose is poured into clean dishes and sterilize. Most media are sterilized by autoclaving, but at various modes depending on their composition.

1. Synthetic media and all agar media that do not contain in their composition of native protein and carbohydrates are sterilized 15-20 minutes in an autoclave at a temperature of 115-120 ° C and a pressure of 1-1.5 atmosphere.

2. Mediums with carbohydrates and milk (which contains lactose), the nutritious gelatin is sterilized by fluid at a temperature of 100 ° C fractionally or in an autoclave at 112 ° C and at a pressure of up to 1 atmosphere.

3. The mediums that include protein substances (blood serum, ascitic fluid) are associated with tyondalization or filtration.

4. For the sterilization of nutrient media containing native proteins, use filtering through the zater membrane filters.

To control the sterility medium after sterilization is placed in a thermostat at 37 ° C by 3-5 days. Liquid media should remain transparent, and signs of growth should not appear on the surface and in the thicker of dense nutrient media. In addition to controlling sterility, the chemical control of finished media is pro-damaged, which lies in the fact that the pH is determined in several two sides of each series, the amount of general and amine nitrogen and chlorides.

There is also biological media monitoring. In this case, several samples of the medium are seeded with a laboratory culture of that microbe for which the medium is prepared and the nature of its growth is studied. Only after the mediums have withstood control, they can be used for their intended purpose.

In composition, it is customary to allocate natural or natural media of an indefinite composition and synthetic media.

Natural (natural)call media that consist of animal or plant products. Such media includes vegetable or fruit juices, animal fabrics, milk, decoctions or extracts derived from natural substrates, etc. Many microorganisms develop well on natural environments, since such environments contain all the components necessary for growth and development. However, these media have a complex, non-permanent chemical composition and are little suitable for studying the metabolism of microorganisms, as they are difficult to take into account the consumption of a number of components and the formation of metabolic products. Natural environments are mainly used to maintain the cultures of microorganisms, biomass accumulation and for diagnostic purposes. Natural media widespread in laboratory practice includes meat-pepton broth, non-churred beer wort, yeast and potato medium, soil extract.

Synthetic media - These are environments in which only compounds of a certain chemical composition, taken in exactly these quantities. Synthetic media are widely used in the study of metabolism, physiology and biochemistry of microorganisms. To develop the composition of synthetic media providing the growth of microorganisms or enhanced biosynthesis of any product activity, it is necessary to know the features of the metabolism of this organism and the need for power sources. Currently, microbiologists have a sufficient number of synthetic media that are not inferior in their qualities with natural media of an indefinite composition. Synthetic media may have a relatively large set of components, but can be quite simple in composition.

Along with natural and synthetic media, so-called semi-synthetic medium. The main components of the semi-synthetic media are compounds of the known chemical composition - carbohydrates, ammonium salts, phosphates, etc. However, their composition always includes substances of uncertain composition, such as yeast autolysate, soil extract or casin hydrolyzate. These environments are widely used in industrial microbiology to obtain amino acids, vitamins, antibiotics and other important productivity products of microorganisms.

In purpose, elective and differential diagnostic (indicator) media distinguish.

Elective media Designed to highlight microorganisms from their natural habitats. They provide the predominant development of a certain group of microorganisms, which is characterized by the generality of physiological properties.

Differential diagnostic environments (Indicator) make it possible to quickly distinguish some types of microorganisms from others or identify some of their features. An example of an indicator medium for the detection of intestinal sticks in natural substrates is an agarized ENDO environment. Bacteria from the genus Escherichia on this medium form pink and raspberry colonies with metal glitter, and the bacteria of the genus Salmonella is colorless.

Differential-diagnostic media is particularly widely used in sanitary and medical microbiology for the rapid identification of certain groups of microorganisms.

In physical state, liquid, bulk and dense media are distinguished.

Liquid medium We are widely used to clarify the physiological biochemical features of microorganisms, for the accumulation of biomass or exchange products, as well as maintaining and storing many microorganisms that are poorly developing on dense media.

Bulk medium It is used mainly in industrial microbiology for cultivating some producers of physiologically active compounds, as well as in collections for the preservation of microorganisms. Such environments include, for example, a strained millet, bran, etc.

Dense medium Used to highlight pure crops, in diagnostic purposes for describing the colonies, to determine the number of microorganisms, their antibiotic activity, for storing crops in the collection and in a number of other cases. For the purpose of sealing environments, agar or gelatin are used. The tight base can serve as the plates of silica gel, which impregnate the nutrient medium.

Most often, agar, which is a complex polysaccharide, is used to compact the nutrient media, which includes agarose and agurectin. Agar is obtained from algae and produced in the form of plates, stalks or powder. Most microorganisms do not use it as a substrate for growth. In water, it forms a gel that melts about 100 0 s and hardens at a temperature of 40 0 \u200b\u200bC.

Gelatin is an extract obtained from substrates rich in collagen - bones, cartilage, tendons, scales. The gel-produced gel melts at a temperature of 25 0 s, which is lower than the usual incubation temperature of many microorganisms (30-37 0 s). In addition, the gelatin is diluted with proteolytic enzymes that many microorganisms stand out on Wednesday. These properties of gelatin limit its use as a sealing agent. Gelatin is mainly used in diagnostic purposes - to identify the proteolytic activity of microorganisms, as well as to obtain gigantic and deep yeast colonies.

The tight base can serve as a silica gel plate, which is a substance of inorganic nature.