Why are elective nutrient media used? Elective culture media

Nutrient media in microbiology are substrates on which microorganisms and tissue cultures are grown. They are used for diagnostic purposes, isolation and study of pure cultures of microorganisms, production of vaccines and drugs, and for other biological, pharmaceutical and medical purposes.

Classification of microbiological culture media

In microbiology, nutrient media are divided into:
- environments of definite and indefinite composition;
- natural, semi-synthetic and synthetic;
- basic, diagnostic, elective;
- dense, semi-liquid, liquid, dry, granular.

Natural nutrient media are those that are obtained from natural materials: blood, meat, proteins, animal organs, plant extracts and plant materials. Examples of such media include meat broth, whey, beer wort, hay infusions, agar-agar, blood, and bile. Natural environments refer to environments with an uncertain composition, which at different times may have different amounts of certain components.

Semi-synthetic media are also considered media of uncertain composition. They are prepared on the basis of natural nutrient media, but substances are added to them that guarantee active reproduction of the crops. Crops are grown on semi-synthetic media to produce vitamins, amino acids, and antibiotics for industrial pharmaceuticals.

Synthetic media are prepared from ingredients of known composition, in known concentrations and ratios, therefore these media belong to media of a certain composition. With their help, they study the metabolism of microorganisms, their biological and physiological properties, and the possibility of obtaining substances that suppress or, conversely, stimulate their development.

Basic, elective and diagnostic culture media

Basic media are used for growing various microbial cultures, as well as as a basis for obtaining elective and diagnostic media. Basic media, for example, include meat broth, meat agar, wort, and Hottinger broth. For different crops, some components are added to the basic media to stimulate growth - these can be vitamins, amino acids, and natural extracts. Thus, the causative agent of whooping cough is grown on a medium with the addition of blood.

Elective media - media for selective (selective) cultivation of biological crops. The composition of the medium is selected so as to be optimal for one species or group of closely related bacteria and suppress the development of bacteria of other species. For example, adding sodium chloride to the medium in a certain concentration inhibits the growth of all bacteria except staphylococci. With the help of elective crops, pure cultures are obtained for further propagation and accumulation.

Diagnostic media are used to identify microorganisms. Based on changes in the medium and its chemical composition (changes in the color of the medium, the appearance of gas bubbles, etc.), the type of bacteria is determined. Chemical indicator dyes such as crystal violet, malachite green, methylene blue, fusin and others are often added to such media. They help separate close cultures. For example, in the pink Endo medium, tinted with fusin, E. coli forms red colonies, and typhoid and dysentery colonies of bacteria are colorless.

Nutrient media are the basis of bacteriological research. They serve to isolate pure cultures of microbes from the material under study and to study their properties. Nutrient media creates optimal conditions for the proliferation of microorganisms. The media must contain substances necessary for the construction of all components of the cytoplasm, i.e. all sources of growth of a living organism. These primarily include sources of nitrogen, carbon, hydrogen and oxygen.

The source of hydrogen and oxygen in nutrient media is water. The source of nitrogen is organic compounds that are obtained from meat, fish, placenta, milk, eggs, and blood. As a result of hydrolysis with pancreatin or trypsin, these products produce the so-called. hydrolysates containing a large amount of amino acids and peptones, which are well absorbed by most microorganisms. Native protein is digested only by some microorganisms that have exoproteases. Hydrolysates are the basis for preparing media for many microorganisms.

The source of carbon for pathogenic microbes is mainly various carbohydrates: mono- and disaccharides, polyhydric alcohols, organic acids and their salts.

In addition to organogens, bacteria require inorganic compounds containing phosphorus, potassium, sulfur, sodium, magnesium, iron, as well as microelements: cobalt, iodine, manganese, boron, zinc, molybdenum, copper, etc.

The need of microorganisms for inorganic compounds is satisfied by adding salts KH2PO4 K2HPO4 and others to the nutrient medium. Microelements that act as catalysts for chemical processes are needed in negligible quantities and enter the nutrient medium with peptone, inorganic salts and water. Along with the listed organic elements, many microorganisms need growth factors, i.e. in substances that they themselves cannot synthesize. Growth factors must be added to nutrient media in ready-made form. Growth factors include various vitamins, the source of which in nutrient media are products of plant and animal origin added to the nutrient medium, containing nicotinic, pantothenic, parabenzoic acids, vitamins A, B, C, etc.

Nutrients can be absorbed by microbes only under a certain environmental reaction, because the permeability of microbial cell membranes changes depending on the pH of the environment.

Requirements for nutrient media.

1. Culture media must contain the nutrients necessary to feed microbes.

2. Have a pH reaction that is optimal for the type of microbe being grown. -

3. Nutrient media must have sufficient moisture and viscosity, because microbes feed according to the laws of diffusion and osmosis.

4. Be isotonic and have a certain redox potential (rH2).

5. Culture media must be sterile, thereby ensuring the possibility of growing pure cultures.

The need for nutrients and physical conditions for different types of microbes is not the same, and this excludes the possibility of creating a universal nutrient medium.

Based on consistency, there are solid and liquid nutrient media. Dense ones are prepared on the basis of liquid ones by adding adhesive substances to them: agar-agar or gelatin! Agar-agar (jelly in Malay) is a product of plant origin, extracted from seaweed. Agar-agar dissolves in water at a temperature of 80-86°C, hardens at 36-40, and therefore is used to compact nutrient media for growing different groups of microorganisms at their optimal temperature.

Nutrient media are classified according to their composition and purpose.

1.Based on the composition, nutrient media are divided into simple and complex

There is a group of general-purpose environments - simple. This group includes meat-peptone broth (simple nutrient broth), meat-peptone agar (simple nutrient agar), nutritious gelatin. These media are used to grow many pathogenic microbes. General purpose media, or simple nutrient media, are usually prepared from hydrolysates with the addition of peptone and sodium chloride. They are also used as a basis for preparing complex media.

2. The second group includes elective, special and differential diagnostic environments.

Elective environments (selective, selective, accumulation, enrichment). The principle of creating selective nutrient media is based on satisfying the basic biochemical and energy needs of the type of microbe for which they are intended for cultivation, or on the addition of inhibitors that suppress the growth of accompanying microflora. A certain composition and concentration of nutrients, microelements, growth factors at a strictly defined pH value or the addition of inhibitors provide optimal conditions for the cultivation of one or several types of microorganisms. When sowing material containing a mixture of various microbes on them, the growth of the species for which the environment will be selective will be the first to wilt. Examples of elective media are yolk broth, selenite broth, Ploskirev's medium - for growing microbes of the intestinal family, alkaline peptone water - for Vibrio cholerae.

Yolk broth. 10-20% ox bile is added to MPB. Bile inhibits the growth of cocci and aerial flora, but is favorable for the proliferation of salmonella.

Selenite broth. It consists of phosphate broth with the addition of sodium salt of selenite, which is an inhibitor of the growth of coccal flora and Escherichia coli, but does not inhibit the growth of salmonella.

Wednesday Ploskireva. A dense medium containing inhibitors of E. coli, coli, but favorable for the growth of Shigella and Salmonella, the reproduction of which is not inhibited by brilliant green and bile salts.

Peptone water. Contains 1% peptone and 0.5% sodium chloride. The environment is selective for chlorine vibrios, because they multiply better than other bacteria in “hungry environments,” especially with an alkaline reaction, because they themselves secrete acidic waste products.

Special environments. Necessary for cultivating bacteria that do not grow on simple nutrient media. For some organisms, it is necessary to add carbohydrates, blood, and other additional nutrients to simple nutrient media. Examples of simple nutrient media are sugar broth and sugar agar for streptococcus (prepared from MPB and MPA, respectively, to which 0.5-2% glucose is added).

For pneumococci and meningococci, the special medium is whey broth and whey agar (to prepare whey broth, 1 part MPB is mixed with 2 parts fresh serum; to obtain whey agar, 10-25% sterile horse or bovine serum is added to the molten MPA).

Differential diagnostic media are used to determine the species of the microbe under study, based on the characteristics of its metabolism.” According to their purpose, differential diagnostic environments are divided as follows:

1. Media for identifying the proteolytic ability of microbes, containing milk, gelatin, blood, etc.

2. Media with carbohydrates and polyhydric alcohols for

detection of various saccharolytic enzymes.

Indicators are added to the composition of differential diagnostic media designed to identify saccharolytic properties and redox enzymes: neutral red, acid fuchsin, bromothymol blue, aqueous blue with pink acid (BP). By changing its color at different pH values, the indicator indicates the presence of an enzyme and the breakdown of the ingredient introduced into the medium.

Examples of differential diagnostic environments:

Endo environment. Consists of MPA with the addition of 1% lactose and basic fuchsin (indicator) decolorized with sodium sulfite. Endo medium has a slightly pink color. Used in the diagnosis of intestinal infections to differentiate bacteria that decompose lactose to form acidic products from bacteria that do not have this ability. Colonies of lactose-positive microbes (Escherichia coli) are red due to the reduction of fuchsin. Colonies of lactose-negative microorganisms - salmonella, shigella, etc. - are colorless.

Differential diagnostic environments include a short and an extended motley series. It consists of media with carbohydrates (Hiss media), MPB, milk, and meat-peptone gelatin.

Hiss media is prepared on the basis of peptone water, to which chemically pure mono-, di- or polysaccharides (glucose, lactose, starch, etc.) are added.

To detect pH shifts as a result of the formation of acids and the decomposition of carbohydrates, an indicator is added to the media. With a deeper breakdown of carbohydrates, gaseous products (CO2, CH4, etc.) are formed, which are captured using floats - small test tubes lowered upside down into the medium. Media with carbohydrates can also be prepared as dense media with the addition of 0.5-1% agar-agar. Then gas formation is detected by the formation of bubbles (breaks) in the column of the medium.

On the MPB, which is part of the motley series, products formed during the breakdown of amino acids and peptones (indole, hydrogen sulfide) are found. Hydrogen sulfide is detected by placing a strip of filter paper soaked in a solution of lead acetate into the MPB after sowing the culture. When amino acids containing sulfur are broken down, hydrogen sulfide is released, and the paper turns black due to the formation of lead sulfide. A complex indicator can be used to determine indole. Indole is formed by the breakdown of tryptophan and can be detected when this indicator is added to a culture grown on MPB. In the presence of indole, MPB turns green or blue.

Dry environments.

Nutrient agar, as well as the main differential diagnostic media, are currently produced in the form of dry preparations containing all the necessary components. To such powders you only need to add water and boil them, and then, after pouring, sterilize them.

Preservative culture media prevent the death of pathogens and suppress the growth of saprophytes. The most widely used are a glycerin mixture (Tyga's medium), a hypertonic solution, a glycerin preservative with LiCl2, a solution of sodium citrate and sodium deoxycholate (Bengsang-Elliott's medium).

Enrichment media for bacteria

Enrichment media(For example, Kitta-Tarozzi environment, selenite broth, thioglycollate medium) are used to accumulate a certain group of bacteria by creating conditions that are optimal for some species and unfavorable for others. Most often, various dyes and chemicals are used as such agents - bile salts, Na+ tetrathionate, K tellurite, antibiotics, fuchsin, hendian violet, brilliant green, etc.

Elective and selective nutrient media for growing bacteria

Elective and selective environments(For example, environments Wilson-Blair, Endo, Ploskirev, McConkey) are intended for primary sowing of material or for reseeding from preservative or enrichment media in order to obtain a pure culture. Media are prepared taking into account the biochemical and energy needs of microorganisms. Accordingly, blood and serum media are isolated (for example, Leffler, Bordet-Gengou), egg media (for example, Lowenstein-Jensen), etc.

Media for growing anaerobic microbes.Meat-peptone liver broth of China - Tarozzi (MPPB). Fresh or frozen liver (preferably from cattle) is cut into small pieces, poured with an equal amount of tap water, boiled for an hour, filtered through cotton wool and added to 1 part of the resulting extract 3 parts of meat-peptone broth. The mixture is heated to a boil, chemically pure table salt is added (1.25 g per 1 liter of medium) and the pH is adjusted to 7.6-7.8, then boiled for 15 minutes and filtered through a paper or moistened cotton filter. Finely chopped pieces (1.5-2 g) of liver are added to the filtered broth, at the rate of 100 g of liver per 1 liter of broth (the liver is first cleared of films and washed with water). Several such pieces are placed in a test tube, 7-10 ml of broth is poured into a high column, and Vaseline or paraffin oil is layered on its surface.

The broth with pieces of liver is sterilized under an excess pressure of 0.1 MPa V for 30 minutes. To remove oxygen from the test tube before inoculation, the medium is boiled for 10 minutes and quickly cooled with water.

Semi-solid agar for anaerobes. 0.25-0.75% agar-agar and 1% glucose are added to the MPB; pH of the environment is 7.4. The medium is poured into test tubes in high columns. Sterilize with fractionally flowing steam for 15-20 minutes for 3 days.

Media for growing lactic acid bacteria.Milk (whole). Heat to a boil. Pour into a tube bottle and place in a cold place for 10-20 hours to allow the cream to settle. After this time, the skimmed part of the milk is poured through the tube tap into test tubes and closed with cotton stoppers. Sterilize fractionally at 100°C for three days for 20 minutes or at 112°C once for 30 minutes.

Skimmed milk. To obtain skim milk, whole milk is separated and then proceeded in the same way as when using whole milk.

Hydrolyzed milk (according to Bogdanov). Take 1 liter of boiled And of skim milk cooled to 45° C, set the pH to 7.6-7.8, add 0.5 g of pancreatin powder (pre-diluted in a small amount of warm water) or 2-3 g of crushed pancreas and after a few minutes 5 ml of chloroform. After this, the bottle is thoroughly shaken, tightly closed with a cork stopper and placed for 3 days in a thermostat at a temperature of 40 ° C with daily shaking of the liquid. After the specified period, in order to remove chloroform, the bottle is opened, the liquid is filtered and diluted 2-3 times with tap water. Adjust the pH of the medium to 7.0-7.2 and sterilize.

Hydrolyzed milk agar. 1.5-2% agar is added to hydrolyzed milk, melted, poured into test tubes and sterilized under an excess pressure of 0.1 MPa V for 15 minutes. Lactic acid bacilli grow well on this medium.

Whey agar. For 100 ml of tap water, take 7.5 g of agar, boil until completely dissolved, add water to the original volume (i.e. in a volume equal to the volume of evaporated water), add 400 ml of pre-prepared whey, expose to flowing steam for 30 min, filter through a layer of cotton wool, pour into test tubes And sterilize under pressure of 0.05 MPa for 30 minutes.

Cabbage Wednesday. 200 g of chopped cabbage (or alfalfa) are poured into 100 ml of water and boiled in a saucepan for 10 minutes, squeezed through a double layer of gauze. The resulting liquid is filtered and diluted 2 times with tap water. Add 2% glucose and 1% peptone, pour into test tubes and sterilize at three excess pressures of 0.05 MPa for 15 minutes.

Osmophilic yeast growth medium. Add 200 g of preheated honey, 1 g of potassium diphosphate, 0.5 g of magnesium sulfate, 0.5 g of ammonium tartrate, 0.1 g of sodium chloride and 0.1 g of potassium chloride to approximately 1 liter of distilled water. All components are mixed and sterilized under an excess pressure of 0.1 MPa for 20 minutes.

Halophile growing medium. Use ordinary meat-peptone media with the addition of 10-15 to 20-30% table salt. In addition, when producing solid nutrient media, the percentage of agar is increased. Sterilization is carried out under an excess pressure of 0.1 MPa for 20 minutes.

Enrichment media. Muller's environment. To 4.5 g of chemically pure chalk, previously sterilized with dry heat, add 90 ml of MPB and sterilize under an excess pressure of 0.1 MPa for 20 minutes. Prepare: a) hyposulfite solution (50 g of pure crystalline hyposulfite is poured to 100 ml with distilled water, sterilized with flowing steam for 30 minutes); b) iodine solution (20 g of metallic iodine and 25 g of potassium iodide are poured into 100 ml of distilled water). Before sowing, 10 ml of hyposulfite solution and 2 ml of iodine solution are sterilely added to the broth with chalk. Shake the mixture as each ingredient is added. Pour into sterile test tubes or flasks.

Wednesday Killian. To 100 ml of regular MPB, 1 ml of an aqueous solution (1:1000) of brilliant green is sterilely added before use.