Sugar broth and sugar agar prepared by adding one or another carbohydrate in an amount of 0.5 to 1% to ordinary broth or molten plain agar. The carbohydrate is dissolved in a small amount of water and then added to the nutrient medium. Sterilization of sugar broth and agar is carried out in a Koch apparatus for 3 days in a row for 1 hour each.
Blood, serum or ascitic fluid agar prepared under strict asepsis. 20-25% of sterile serum or ascitic fluid or 5-25% of sterile defibrinated blood are added to the finished slightly alkaline agar, melted and again cooled to 45°, poured into test tubes or vials. After thorough mixing (avoid the formation of bubbles and foam), the agar in the test tubes is mowed down, and the contents of the vials are poured into Heidenreich dishes. Broth with serum, ascitic fluid or blood prepared in the same way as agar, but without heating. To control sterility, the broth is placed in a thermostat for 48 hours at 37°.

Differential diagnostic nutrient media. Composition and purpose.

Endo environment. Prepared immediately before use. To 100 ml of meat-peptone agar or Hottinger broth agar (pH 7.6), sterilely add 1 g of chemically pure lactose dissolved in 5 ml of sterile water, cool to 70° and add a mixture of 0.5 ml of a saturated solution of basic fuchsin and 1 .25 ml of freshly prepared 10% sodium sulfide solution, shaken and poured into cups. To dry, open cups are placed in a thermostat at 37° for 30 minutes.

On Endo medium, E. coli produces red colonies with a metallic tint, and bacteria of the typhoid-paratyphoid and dysentery group produce colorless colonies.

Variegated series of carbohydrates (Hiss medium). To 100 ml of water add 1 g of peptone and 0.5 g of table salt. Peptone and salt are dissolved in hot water for a few minutes and filtered through a paper filter (the solution should be completely clear). Set pH to 7.0. 1% of one of the carbohydrates used is dissolved in the specified medium, floats are lowered to the bottom of the test tubes to catch gas (indicating the deep breakdown of sugars) and an indicator is added. The following is used as an indicator:

a) litmus tincture - 5 ml per 100 ml of medium. In an acidic environment, litmus tincture shows redness, in an alkaline environment it turns blue;

b) azolitmin, which is a purified litmus preparation, its potassium salt (0.25 g of azolitmin is added per 1 liter of medium);

c) bromothymol blau - per 1 liter of medium, 1 ml of 1.6% alcohol solution of the indicator (the medium becomes green, turns blue when an alkali forms, turns yellow when an acid forms);

d) Andrede indicator, which consists of 1 g of acid fuchsin, 400 ml of distilled water and 64 ml of normal sodium hydroxide solution. 1% indicator is added to the nutrient medium. The medium in the test tube should acquire a straw-yellow color without a pink tint. In an acidic environment, the color of the medium changes to red.

The indicator is added in order to determine changes in the reaction of the nutrient medium occurring under the influence of carbohydrate fermentation, i.e., to identify the biochemical activity of microbes. The latter is of great importance for the microbiological diagnosis of a number of infectious diseases (typhoid fever, dysentery, cholera, etc.). Media with carbohydrates and an indicator are poured into sterile tubes and sterilized fractionally at 100° for 3 days.

Short motley row. Changes during growth of E. coli, S. typhi.

Methods for isolating pure cultures of aerobes.

Drigalski method

Isolation of a pure culture of anaerobes.

See practical skills

Virological methods. Purpose, principle.

Virological method

Main stages:

1. Collection of the test material.

2. Selection based on the principle of cytotropism and obtaining a sensitive test system, determining its viability.

3. Infection of the selected system.

4.Indication of the virus based on the detection of its nucleic acid, antigens, hemagglutinin, CPD, inclusions.

5. Identification and titration of the virus is carried out on the basis of:

a) determination of virus antigens using immunological reactions (RIF, ELISA, RPGA, RSK, RN, VIEF, etc.); b) pathohistological examination of organs and tissues; c) CPP; d) clinical symptoms, biological tests (keratoconjunctival, etc.).

Virus detection methods

When cell cultures are infected with viruses, various visible manifestations of the virus’ action can be obtained:

1. Cytopathic effect of the virus on cell culture (CPE)– the occurrence of visible morphological degenerative changes in it;

2. Acquisition of the ability of an infected cell culture to hemadsorption– to the adsorption of erythrocytes on the surface of the cell layer;

3. Formation in an infected cell culture under a dense layer of a special agar coating of characteristic plaques, which are “negative colonies” of viruses;

4. Intracellular inclusions

Classification of culture media:

Natural– consist of products of animal or plant origin and have an uncertain chemical composition. For example: vegetable and fruit juices, animal tissues, blood, milk, eggs, etc. (IPA, MPB).

Semi-synthetic– the composition includes compounds of known chemical nature and substances of unknown composition. For example: MPB with glucose, Endo medium, Sabouraud medium.

Synthetic– contain only chemically pure compounds in precise concentrations. Used in laboratory experiments. For example: environment of Chapek, Omelyansky, Ushinsky, etc.

Purpose of culture media

Universal(general purpose) - suitable for growing many types of microorganisms and used as a basis for special nutrient media. Examples: MPB, MPA, Hottinger's medium, GRM, thioglycollate medium.

Special used in cases where microorganisms do not grow on simple media. These include blood, serum agar, whey broth, ascitic broth, ascites agar and others.

1. Elective environments- some microorganisms grow faster and more intensively on them than other types of bacteria. For example, 1% alkaline peptone water is an elective medium for vibrios cholera, Roux and Leffler's medium for diphtheria pathogens.

2. Selective - thanks to selective additives (bile, paints, antibiotics, etc.) they are able to suppress the development of some types of microorganisms, but do not affect other types. Examples: Müller's medium is selective for typhoid-paratyphoid bacteria, furazolidone-tween agar is selective for corynebacteria and micrococci. The addition of antibiotics to the media makes them selective for fungi (eg Sabouraud's medium, etc.).

3. Differential diagnostic- a group of media that make it possible to determine the biochemical properties of microorganisms and differentiate them. They are divided into media for determining proteolytic, peptolytic, saccharolytic, hemolytic, lipolytic, and reducing properties (Endo, Levin, Ploskirev, Gissa media).

4. Preservative (transport) -

designed to preserve the viability of microorganisms from the moment of collection

biomaterial before culture for diagnostics

Liquid(broths) – study of physiological and biochemical characteristics and accumulation of microorganism biomass

Semi-liquid(1% agar) – storage of cultures and cultivation of anaerobes

Dense(3-5% agar) – isolation of pure cultures, accumulation, quantitative recording, study of cultural properties, antagonistic relationships

Bulk– storage of seed in industry (millet, bran)

Dry– produced by industry for the preparation of nutrient media

Transport system with Stuart environment

Stewart's medium is a semi-solid, nutrient-poor substrate for the preservation and transport of a wide range of pathogenic microorganisms, such as Neisseria gonorrhoeae, Haemophilus influenzae, Corynebacterium diphteriae, Trichomonas vaginalis, Streptococcus sp., Salmonella sp., Shigella sp. etc. The most demanding microorganisms survive in this environment for more than a day, others – up to several days.

The presence of thioglycolate in the medium suppresses the enzymatic activity of bacteria, and the absence of nitrogen prevents their reproduction.

Transport system with environment Keri Blair

Keri Blair's transport medium is a modification of Stewart's basic transport medium designed specifically for fecal specimens.

Glycerophosphate, which is a metabolite of some enterobacteria ( Escherichia coli, Klebsiella pneumoniae, etc.), replaced by inorganic phosphate,

Methylene blue was removed and the pH of the medium was increased to 8.4.

Keri Blair's medium allows the preservation of most pathogens, including fastidious microorganisms such as Neisseria sp., Haemophilus sp., Streptococcus sp..

This medium is standard for transporting anaerobes.

Transport system with Ames environment

The Ames transport medium is another modification of the basic Stewart transport medium, in which glycerophosphate is replaced by inorganic phosphate, since glycerophosphate is a metabolite of some enterobacteria ( Escherichia coli, Klebsiella pneumoniae, ets.) and may support the growth of some gram-negative microorganisms.

Methylene blue has been replaced with pharmaceutical grade activated carbon.

Calcium and magnesium were added to the medium to maintain bacterial cell permeability.

This environment is capable of supporting microorganisms such as Neisseria sp., Haemophilus sp., Corynebacteria, Streptococci, Enterobacteriaceae etc., however, the best results are obtained by cultivation within the first 24 hours.

Universal enrichment media: Meat peptone agar (MPA) and Meat peptone broth (MPB)

They are the main media for inoculating microorganisms to check the purity of cultures before biochemical and serotyping.

They are used for cultivating and counting unpretentious microorganisms. In semi-liquid form, the medium can be used to store control (reference) microorganisms.

Universal storage environments Hottinger environment

Designed for the cultivation of various microorganisms, such as enterobacteria, Pseudomonas aeruginosa, staphylococci, and some types of streptococci. If necessary, it can be enriched with carbohydrates and salts.

Contains Hottinger's hydrolyzate, which is obtained by enzymatic hydrolysis of minced meat (beef) with pancreatin, followed by filtration and the addition of chloroform as a preservative.

Universal storage environments:Mueller-Hinton environment

This medium is used for cultivation Neisseria sp. and to determine the sensitivity of microorganisms to antimicrobial agents.

Wednesday McConkey

MacConkey media are recommended as differential media for the selective isolation of enterobacteria and related gram-negative bacilli.

Lactose-positive strains grow with pink or red colonies that may be surrounded by a zone of bile salt precipitation.

The red color appears as a result of acidification of the medium by lactose decomposition products (when the pH drops below 6.8) and the adsorption of neutral red.

Strains that do not ferment lactose (Shigella, Salmonella) usually form transparent, colorless colonies and do not change the environment.

Differential diagnostic environments:Endo environment

This medium was developed by Endo as a culture medium for the differentiation of lactose fermenting and non-fermenting microorganisms. It is used for microbiological examination of water, wastewater, dairy and other food products.

Sodium sulfite and basic fuchsin have an inhibitory effect on gram-positive microorganisms. Lactose is decomposed by microorganisms to aldehyde and acid. The aldehyde, in turn, releases fuchsin from the fuchsin-sulfite complex, enhancing the red color of the colonies. In E. coli, this reaction is very pronounced and is accompanied by crystallization of fuchsin, which is manifested by a greenish metallic sheen (muchsin gloss) of the colonies.

Differential diagnostic environments:Yolk salt agar

This medium is used as a selective medium for isolating clinically significant staphylococcal cultures.

Mannitol is a fermentable and differentiating substrate as well as a carbon source.

Addition (up to 5% v/v) of egg yolk emulsion makes it possible to determine the lipase activity of microorganisms. The emulsion in a saline environment becomes transparent, therefore, in the presence of lipase activity, a yellow opaque zone is formed around the colonies.

Differential diagnostic environments:Wilson-Blair or Bismuth Sulfite Agar

Selective medium for the isolation of Salmonella.

Peptic digest of animal tissue and meat extract serve as a source of nitrogenous nutrients, carbon, sulfur, B vitamins and trace elements necessary for the growth of these bacteria.

Brilliant green inhibits the growth of all gram-positive bacteria. Glucose is a fermentable carbohydrate. Ferrous sulfate can detect hydrogen sulfide production.

Bismuth is a heavy metal that inhibits the growth of most gram-negative intestinal bacteria except Salmonella.

Salmonella reduce ferrous sulfate in the presence of glucose and bismuth sulfite to ferrous sulfide, which turns their colonies black.

Special elective environments:Loeffler's environment

This medium with the addition of horse serum is used for cultivation Corynebacterium diphtheriae from clinical material and subcultures of pure cultures of these microorganisms.

A high serum concentration helps determine the proteolytic activity of microorganisms, as well as pigment formation. Peptone and meat extract provide microorganisms with essential nutrients. Glucose is a fermentable substrate and source of energy.

Special selective media:Kampilobakagar

Selective medium for Campylobacter which consisted of blood agar base with sheep blood or horse blood and antibiotics.

Antimicrobial components significantly inhibit the growth of normal microflora, promoting growth and excretion from feces Campylobacter fetus ssp. jejuni.

The presence of amphotericin B in the supplement significantly or completely suppresses the growth of fungi; cephalothin introduced later enhances the suppression of normal intestinal microflora.

Colonies Campylobacter fetus ssp. jejuni have a mucous character, flat gray with irregular outlines or raised, round, without hemolysis.

Some strains may produce yellow-brown or pinkish colonies.

Merging growth or swarming may occur on a moist surface of the medium.

Elective media were introduced into microbiological practice by S.N. Vinogradsky and M. Beyerinck. These are nutrient media in which, by adding one or more chemical compounds, optimal conditions are created for the growth and reproduction of one type of microorganism (or group of related microorganisms) and unfavorable conditions for all others. Such media are used mainly for isolating a pure culture of microorganisms from their natural habitats and for accumulating a mass of cultures (chemical method for isolating a pure culture). For example, a nutrient medium, which is coagulated horse serum, is a selective medium for diphtheria bacteria, alkaline peptone water for Vibrio cholera, bile broth for the causative agent of typhoid fever, liver broth for Brucella, etc.

The accumulation of microbes in selective nutrient media in many cases serves as an important preliminary step in the isolation of pure cultures from the original test materials (for example, Vibrio cholerae or typhoid bacteria from the feces of patients or carriers, etc.).

What do you understand by differential nutrient media?

Differential diagnostic media are those that contain, in addition to substances that ensure the growth and development of microorganisms, substances used as a substrate for certain enzymes. Based on the qualitative change in the substrate, the presence of a particular enzyme is determined (assessed using an indicator that reacts to the presence of substrate decomposition products in the nutrient medium).

Each type of microorganism is characterized by a fairly stable set of enzymes. Determination of a set of enzymes using differential diagnostic media makes it possible to differentiate the types of microorganisms. For example, blood agar allows you to detect the enzyme hemolysin, His media - saccharolytic enzymes (carbohydrases), gelatin is used to take into account the proteolytic properties of microbes, etc.

Blood agar. The presence of the hemolysin enzyme is judged by the destruction of red blood cells and the formation of a light zone around microbes grown on blood agar.

His media. The presence of enzymes - carbohydrases, which break down carbohydrates into acid, is indicated by a change in the pH of the medium towards the acidic side and a change in the color of the nutrient medium. The difference in the set of enzymes can be used to check the purity of the isolated culture, as well as to quickly differentiate one species from others during the initial study of inoculations of infectious material.

Chemical reagents

1. What are chemical reagents and what are they used for?

Chemical reagents- substances used in laboratory practice to carry out various chemical reactions.

In most cases, chemical reagents are individual substances, but quite often they have a complex composition. There is no generally accepted classification of chemical reagents; most often they are divided into analytical chemical reagents and everything else.

2. In veterinary medicine, for what purposes are they used?

In veterinary medicine, chemical reagents are used for analytical and diagnostic purposes in clinical, veterinary-sanitary, hygienic, expert, biochemical and other laboratory studies. Research methods used and developed in biological and clinical practice require a wide range of chemical reagents that must satisfy a wide variety of requirements. For example, clinical and biochemical studies require highly purified substrates for enzymes, the enzymes themselves, reagents for specific groups (SH, NH3, COOH groups, etc.), etc. For carrying out inorganic and organic syntheses, as well as for qualitative and quantitative analyses, incl. during veterinary and sanitary control in various industries, analysis) of medicines, when conducting veterinary, sanitary and hygienic analyzes of food products, air, water, etc., a large number of a wide variety of highly purified chemical reagents are used.

1.By 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.

Also, according to their composition, they distinguish protein, protein-free and mineral media.

2. By origin environments are divided into artificial and natural (natural).

Natural nutrient media may contain components of animal (for example, blood, serum, bile) or plant (for example, pieces of vegetables and fruits) origin.

3.According to purpose allocate preservative media(for primary sowing and transportation), enrichment environment(for the accumulation of a certain group of bacteria), culture media(universal simple, complex special and for toxin formation), media for isolation and accumulation (preservative, enrichment and selective) and identification environment(differential and elective-differential).

Preservative culture media prevent the death of pathogens and suppress the growth of saprophytes. The most widely used are a glycerin mixture, a hypertonic solution, a glycerin preservative with LiCl 2, a solution of sodium citrate and sodium deoxycholate.

Enrichment media for bacteria

Enrichment media(for example, Kitta-Tarozzi medium, 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, gentian violet, brilliant green, etc.

Also by appointment distinguish between environments elective, special and differential diagnostic.

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 onto them, the growth of the species for which the environment will be selective will appear first. Examples of elective media are bile broth, selenite broth, Ploskirev's medium - for growing microbes of the intestinal family, alkaline peptone water - for Vibrio cholerae.

Bile broth. 10-20% ox bile is added to MPB. Bile suppresses 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 Vibrio cholerae, 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).

Differential diagnostic environments 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 detection proteolytic ability microbes containing milk, gelatin, blood, etc.

2. Media with carbohydrates and polyhydric alcohols for the detection of various saccharolytic enzymes.

The following 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 rosolic 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 short and extended motley row. It consists of media with carbohydrates (Hiss media), MPB, milk, and meat-peptone gelatin.

Hiss media are 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 (CO 2, CH 4, 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. For determining indole you can use a complex indicator. 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.

In practical bacteriological laboratories they are widely used micro and express methods for an indicative study of the biochemical properties of microorganisms. There are many test systems for this purpose. The most commonly used system of indicator papers (SIB). SIBs are disks of filter paper impregnated with solutions of sugars or other substrates in combination with indicators. Such disks are lowered into a test tube with a culture grown in a liquid nutrient medium. The change in color of the disk with the substrate is used to judge the functioning of the enzyme. Micro-test systems for studying the identification of enterobacteria are represented by disposable plastic containers with media containing various substrates with the addition of indicators. Sowing a pure culture of microorganisms into such test systems allows you to quickly identify the ability of bacteria to utilize citrates, glucose, sucrose, release ammonia, indole, decompose urea, lysine, phenylalanine, etc.

SPECIAL (ELECTIVE) NUTRITION MEDIA

Yeast Growth Media

Synthetic Reader Medium

The composition of the medium includes, g/l: ammonium sulfate 3, magnesium sulfate 0.7, calcium nitrate 0.04, sodium chloride 0.5, potassium dihydrogen phosphate 1.0, potassium hydrogen phosphate 0.1. Initial pH 6.6. Calcium nitrate, which is not used by yeast, can be omitted from the medium. To study yeast reproduction, add 2% sugar, to study fermentation - 5-10%. The complete synthetic medium contains crystalline vitamins, mcg/ml: inositol 5, biotin 0.0001, pantothenic acid 0.25, thiamine 1.0, pyridoxine 0.25, nicotinic acid 0.5. Sterilize the medium in an autoclave at a pressure of 0.1 M Pa for 20 minutes.

Glucose-ammonium medium

Contains in 1 liter of tap water the following substances, g: ammonium sulfate 5, potassium dihydrogen phosphate 0.85, potassium hydrogen phosphate 0.15, magnesium sulfate 0.5, sodium chloride 0.1, calcium chloride 0.1, glucose 20, agar 20 For enrichment with growth factors, yeast (0.2%) or meat (0.3%) extract and grape juice are added.

Synthetic medium for identifying imperfect yeasts

Contains in 1 liter of tap water the following substances, g/l: glucose 50, lysine 3, potassium dihydrogen phosphate 1, magnesium sulfate 1, iron sulfate - traces. Each component is dissolved in water separately and added in the order indicated. Agar (1.5%) is added to the medium, melted, poured into test tubes and sterilized for 20 minutes at a pressure of 0.05 MPa.

Complete medium with lysine for detection of imperfect yeasts

Contains in 1 liter of tap water the following substances, g/l: glucose 50, magnesium sulfate 1, potassium dihydrogen phosphate 2, potassium lactate 12 ml (50% solution), /, (+) lysine monohydrate 1, vitamin solution (per 100 ml of sterile distilled water add, g: inositol 2, calcium pantothenate 0.4, nicotinamide 0.5, hydrattiamine 0.1, agar 20; pH of the medium - 5-5.2. The medium is poured into 15 ml test tubes and sterilized for 15 minutes at a pressure of 0.1 MPa.

Acetate medium for detection of imperfect yeasts

For 1 liter of tap water, take 10 g of sodium acetate, 10 g of ammonium chloride, 5 g of glucose, 3 ml of yeast autolysate, pour into 5 ml test tubes and sterilize at a pressure of 0.05 MPa for 30 minutes.

Medium for identifying foreign yeasts morphologically similar to the main culture

10 g of peptone, 2 g of potassium hydrogen phosphate are dissolved in 500 ml of distilled water and filtered. 15 g of agar are melted in the filtrate, 10 g of glucose, 0.4 g of eosin and 0.065 ml of methylene blue (90% alcohol solution) are added, adjusted to 1000 ml with hot distilled water, poured into test tubes and sterilized for 15 minutes at a pressure of 0. 1 MPa. The color disappears during sterilization and reappears when cooled. The medium is stored for no more than 2 months.

Medium for pseudomycelium formation

Glucose peptone agar. To 1 liter of tap water add, g: peptone 10, glucose 20, agar 30-35. Sterilize for 30 minutes at a pressure of 0.05 MPa. If necessary, you can add yeast or meat extract (0.5%) or cook in liquid form.

Potato agar. 100 g of peeled, washed, thinly sliced ​​potatoes are infused in a cool place for several hours with 300 ml of tap water. The extract is filtered, 230 ml of the extract is brought to 1 liter with tap water, 20 g of glucose and 30-35 g of agar are added, melted and sterilized for 1 hour at a pressure of 0.075 MPa.

Yeast water with carbohydrates (“color series”)

The ability of yeast to cause fermentation of carbohydrates is determined using yeast water with 2% of the test sugar (glucose, maltose, sucrose, lactose, raffinose, etc.). The medium is poured into test tubes with floats, Dunbar tubes and sterilized with fractionally flowing steam. The results after sowing are recorded after 2 days, if necessary after 7 days of cultivation at a temperature of 30 °C.

The ability of yeast to metabolize carbohydrates by oxidation is studied on a medium of the following composition, r/l: ammonium sulfate 5, potassium dihydrogen phosphate 1, magnesium sulfate 0.5, autolitate 1, test sugar 10, agar 20. The medium is poured into test tubes, sterilized for 30 minutes at pressure 0.05 MPa, prepare agar slant. Culture growth is assessed after 3-4 days.

Yeast agar with sugar

0.5% sodium chloride, 1% glucose (or 4 or 10% sucrose) and 2% agar are dissolved in yeast water, pH 6.8 (with glucose) and 6-6.5 (with sucrose). The medium is poured into test tubes or flasks and sterilized at a pressure of 0.05 MPa for 30 minutes.

Antibiotic media

For the preferential development of yeast and suppression of accompanying bacteria, broad-spectrum antibiotics are introduced into the media: streptomycin (100 units/ml), penicillin (20-100 units/ml), levomycin (50 mg/l), neomycin (20 units/ml) and etc. They can be added to the environment either together or separately.

Media for ascospore formation

Wednesday Gorodkova. Contains in 1 liter of tap water, g: peptone 10, sodium chloride 5, glucose 1 (or 2.5), agar 20; The pH of the environment is 7.3. Pour into test tubes and sterilize for 15 minutes at a pressure of 0.1 MPa.

McClary acetate agar. To 1 liter of distilled water add, g: sodium acetate 8.2, potassium chloride 1.8, glucose 1, yeast extract 2.5, agar 15. Autoclave for 15 minutes at a pressure of 0.1 MPa.

Wednesday Starkey. In 1 liter of tap water dissolve, g: potassium hydrogen phosphate 1, potassium dihydrogen phosphate 0.25, magnesium sulfate 0.25, calcium chloride 0.05, agar 20. Sterilize at a pressure of 0.05 MPa for 15 minutes.

Osmophilic yeast growth medium

To 1 liter of glucose syrup (50-60% DM) add 5 g of peptone and 20 g of agar. Peptone can be replaced with yeast water (50 ml). Sterilize at a pressure of 0.05 MPa.

Molasses wort

200-300 g of thick molasses are mixed with water in a ratio of 1: 3, heated to a temperature of 95 ° C and allowed to stand for 2 hours. In this case, the coagulated colloids settle and the molasses solution becomes clarified. 3% diammonium phosphate is added to the solution, diluted with water to 5-8% DM and poured into test tubes or flasks. To prepare an agar medium, add 1.5-2% agar. Sterilize at a pressure of 0.05 MPa for 30 minutes in an autoclave or fractionally for 1 hour with an interval of 20-24 hours 3 times.

Media for growing filamentous fungi

Beet agar

Well-washed sugar beets are cut into slices, filled with tap water (20 g of beets per 1 liter of water) and boiled for 30 minutes. The filtrate is brought to the original volume with water, 2% agar is added and sterilized at a pressure of 0.1 MPa for 30 minutes.

Beet pulp

Clean beets are ground on a grater, placed in Petri dishes and, without turning over, sterilized at a pressure of 0.1 MPa for 30 minutes.

Wednesday Capek

Composition of the medium, g/l: sucrose or glucose 30, potassium dihydrogen phosphate 1.0, sodium nitrate 2.0, magnesium sulfate 0.5, potassium chloride 0.05, ferrous sulfate 0.1, agar 20. A sample of agar is leached and added These ingredients, previously dissolved in 1 liter of distilled water, are heated with flowing steam, and the pH is adjusted to 4.0-5.5 with a 10% solution of citric acid or sodium hydroxide. Filter, pour into test tubes and sterilize with fractionally flowing steam 3 times for 30 minutes at an interval of 1 day.

Czapek-Dox sugar nitrate agar

Option 1. For 1 liter of distilled water take, g: sucrose 20, potassium hydrogen phosphate 0.5, magnesium sulfate 0.5, sodium chloride 0.5, potassium nitrate 1, traces of iron sulfate, calcium carbonate 2-5, agar 20.

Option 2. For 1 liter of distilled water take, g/l: sucrose 30, ammonium nitrate 2.5, potassium dihydrogen phosphate 1, magnesium sulfate 1, ferrous sulfate 0.01, agar 20.

Glucose-starch medium

The same salt components as in Czapek's sucrose nitrate agar, but instead of sucrose, 25 g of soluble starch and 5 g of glucose are taken.

Starch ammonium agar

Composition of the medium, g/l: soluble starch 10, calcium carbonate 3, potassium hydrogen phosphate 1, magnesium sulfate 1, sodium chloride 1, ammonium sulfate 1, agar 20. Sterilize for 30 minutes at a pressure of 0.05 MPa.

Wednesday Saburo

To 100 ml of sterile yeast water add, g: peptone 5, glucose 4, agar 1.8-2. Sterilize for 20 minutes at a pressure of 0.05 MPa or fractionally.

The basis of this medium is yeast water. To prepare yeast water, 70-100 g of fresh pressed yeast (7-10 g of dry yeast) are boiled for 20-30 minutes in 1 liter of distilled water and left in a high cylinder in the cold for 12 hours. The settled liquid is decanted, another 1 is added. l of water, boil for 30 minutes, filter, adjust the pH to the required value. The prepared medium is sterilized in 2-3 minute increments of 20 minutes with an interval of 1 day. To 100 ml of sterile yeast water add 1% peptone, 2% agar, after dissolving the agar, add 4% glucose or maltose, filter, pour into test tubes and sterilize at a pressure of 0.05 MPa for 20 minutes.

The medium can also be prepared using regular 1% peptone water.

Media for growing lactic acid bacteria

Hydrolyzed milk (according to Bogdanov)

Regular or skim milk (pH 7.6-7.8) is boiled for 5 minutes, the vessel is thoroughly shaken and cooled to a temperature of 45 ° C and 0.5-1 g of pancreatin is added per 1 liter, after 4-7 min add 5 ml of chloroform. Place in a thermostat for 18-20 hours at a temperature of 40 °C. Pancreatin powder should be pre-diluted in a small amount of warm water. During the first hours, the milk is stirred several times with the stopper open. Hydrolyzed milk is filtered through a paper filter, diluted 2-3 times with water, pH is set to 7.0-7.2 and sterilized for 15 minutes at a pressure of 0.1 MPa or for 20 minutes at a pressure of 0.05 MPa.

Hydrolyzed milk agar

1.5-2.0% agar is added to hydrolyzed milk. The mixture is heated to a boil and kept until the agar is completely dissolved. The hot medium is filtered through a cotton filter, poured into test tubes or flasks and sterilized at a pressure of 0.1 MPa for 10-15 minutes.

Skim milk with indicator

Fresh skim milk heated to a boil is tinted while hot with litmus tincture to an intense lilac color. Sterilize with flowing steam (3 times for 30 minutes at an interval of 1 day) or by autoclaving for 10 minutes at a pressure of 0.1 MPa.

Malt wort with spent grain

Malt wort is prepared, but without separating the spent grain (12-15% DM). Pour into test tubes, add sterile chalk (2-4%) and sterilize at a pressure of 0.05 MPa for 30 minutes.

Yeast sucrose agar

To identify Lactobacillus And Leuconostoc use a medium prepared on the basis of yeast water with the addition of 0.5% sodium chloride, 10% sucrose and 2% agar; pH of the environment is 6-6.5.

Sprouting medium

25 g of malt (barley) sprouts are boiled for 10 minutes with 500 ml of water and, after cooling to a temperature of 45-50 ° C, filtered through a linen bag, clarified with beaten chicken protein, boiled again and filtered through a paper filter to remove coagulated protein. 1.5% peptone, 2% sugar, 2% agar are added to the solution and sterilized for 30 minutes at a pressure of 0.05 MPa.

Cabbage Wednesday

200 g of chopped white cabbage are poured into 1 liter of water, boiled for 10 minutes, squeezed through two layers of gauze. The resulting liquid is filtered through a folded filter, diluted 2 times and 2% glucose and 1% peptone are added to the decoction. Pour into test tubes and sterilize at a pressure of 0.05 MPa for 15 minutes. To obtain a solid medium, add 2% agar.

MRS medium (de Man's medium)

The composition of the medium includes, g/l: manganese sulfate 0.05, magnesium sulfate 0.2, potassium hydrogen phosphate 2, ammonium nitrate 2, sodium acetate 5, peptone 10, Difko yeast extract 5, meat extract 10, glucose 20, tween- 80 1 ml, medium pH 6-6.5. The medium is filtered and sterilized in fractional steps of 30 minutes 3 times with an interval of 1 day or in an autoclave at a pressure of 0.05 MPa for 20 minutes. Used in liquid, semi-liquid and agar form for childbirth Leuconostoc And Lactobacillus.

MRS media (modified by A.A. Lanzier)

MRS-1 environment. Dissolve in 200 ml of distilled water, g: manganese sulfate 0.05, magnesium sulfate 0.2, cysteine ​​0.2, potassium hydrogen phosphate 2, ammonium citrate 2, sodium acetate 5, glucose 20, peptone 10, Tween-80 1 ml ( dissolve separately in a small amount of hot distilled water), yeast autolysate (see Appendix 2) 50 ml, liver extract 100 ml. The volume of liquid is adjusted to 500 ml with distilled water and 500 ml of Bogdanov’s hydrolyzed skim milk, not previously sterilized, pH 6.2-6.8, is added. The medium is filtered and sterilized with fractionally flowing steam.

MRS-2 environment. Designed for museum storage of strains Lactobacillus. Prepared on the basis of MRS-1 medium with the addition of 0.15% agar. The result is a semi-liquid medium, creating more anaerobic conditions compared to a liquid one.

MRS-3 environment. Designed for the “variegated series” when identifying lactic acid bacteria. It is based on the MPC-1 medium, but without glucose, liver extract and hydrolyzed milk. Carbohydrates and polyhydric alcohols are added in an amount of 0.5%. The amount of agar added is 0.15%. pH of the environment is 7.0. The indicator is chlorophenol red (0.004%). The indicator is dissolved in 1-2 ml of ethyl alcohol and added to the medium before sterilization. Chlorophenol red gives a color transition from red-violet to yellow within the pH range of 4.8-6.4.

Liver extract

Fresh beef liver is finely cut and filled with water (1 kg of liver: 1 liter of water). Boil for 30 minutes and filter, then sterilize at a pressure of 0.05 MPa for 20 minutes.

Wednesday 10

For 1 liter of unhopped beer wort (8% DM) or 1 liter of yeast water add, g: manganese sulfate 0.05, magnesium sulfate 0.2, cystine or cysteine ​​0.2, potassium hydrogen phosphate 2, ammonium citrate 0.2, acetate sodium 2.5, sucrose 20, peptone 10, yeast autolysate 50 ml. Each component is dissolved in the indicated order in malt wort (for Lactobacillus) or yeast water (for Leuconostoc). In the first case, the pH of the environment is 5.5, in the second - 6.0. Add 1.5% agar and sterilize with flowing steam. Sterile chalk can be added to Petri dishes.

In 150 ml of filtered tomato juice, dissolve 0.75 ml of Tween-80 and 37.5 g of glucose while heating, add 5 ml of yeast autolysate, 600 ml of skim milk (skimmed milk) and 150 ml of melted 2% meat-peptone agar. The pH is set to 7.0. The medium is poured into test tubes of 6-7 ml, sterilized at a pressure of 0.05 MPa for 20 minutes, cooled, inoculated with the studied lactic acid bacteria and 1-2 ml of melted 2% meat-peptone agar is layered on top. In the case of weak gas formation, the plug is separated from the main medium; in case of strong gas formation, it rises high or flies out of the test tube.

Media for growing mucus-forming bacteria

Option 1. Meat meat agar with 10% sucrose.

Option 2. Composition, g/l: raw sugar 40, sodium hydrogen phosphate 2, sodium chloride 0.5, magnesium sulfate 0.1, ferrous sulfate 0.01, calcium carbonate 10, agar 20.

Wednesday Wittenbury

The composition of the medium includes, g/l: meat extract 5, peptone 5, yeast autolysate 50 (or yeast extract 50), 1.6% solution of bromocresol purple 1.4 ml, pH 6.8-7.0. Sterilize with flowing steam 3 times for 45 minutes at an interval of 1 day.

Media for growing putrefactive asporogenous bacteria

Milk agar

Skim milk is poured into 5 ml test tubes and sterilized with flowing steam or in an autoclave for 20 minutes at a pressure of 0.05 MPa. Separately, prepare 3% water agar, pour 4-5 ml into test tubes and sterilize for 30 minutes at a pressure of 0.1 MPa. The agar is melted, sterilely combined with milk and poured into Petri dishes, where the test sample has previously been added.

Media for growing fat-digesting bacteria

Option I. To 1 liter of water add 5 g of peptone and 3 ml of yeast autolysate. Having established a pH of 7.2-7.4, add 1.5% agar. The agar is melted, the medium is filtered and 1% hot milk fat or olive oil is added. Mix, pour into test tubes and sterilize at a pressure of 0.1 MPa for 15 minutes.

Option 2. 2-4% milk fat or olive oil is added to meat peptone agar. Pour 10 ml into test tubes and sterilize at a pressure of 0.1 MPa for 20 minutes. Shake the medium thoroughly before adding it to the cups.

An example of such a medium is gelatin with hydrolyzed milk. 10% gelatin is added to hydrolyzed milk (you can use hydrolyzed casein or meat-extract broth), allow it to swell and heat with stirring to a temperature of 50 °C. pH of the environment is 7.0-7.2. The medium is filtered and sterilized in test tubes at a pressure of 0.075 MPa for 20 minutes.

Media for growing acetic acid bacteria

Add 4 vol. to malt wort or cabbage medium. % ethyl alcohol and 20 units/ml of the antibiotic monomycin, which inhibits the growth of lactic acid bacteria.

Media for growing anaerobes

Winogradsky Wednesday. In 1 liter of distilled water dissolve, g: potassium hydrogen phosphate 1, magnesium sulfate 0.5, manganese sulfate 20, glucose 20, sodium chloride and ferric chloride - traces.

Wednesday Kita-Tarozzi. Pieces of liver or meat, boiled and washed with water, are lowered into a test tube so that they cover the bottom. Pour meat-peptone broth with 1% glucose (pH 7.2-7.4) to "/ 2 volumes of the test tube and lower the float. Pour a layer of vaseline oil 1 cm high on top. Sterilize for 15 minutes at a pressure of 0.1 MPa 2 times at intervals 30 min.

Medium for growing thermophilic anaerobes that produce hydrogen sulfide

The composition of the medium includes, g/l: peptone 10, ferrous sulfate 1, agar 20. Before filling, a clean iron nail is placed in each test tube. After seeding the sugar, a layer of sterile petroleum jelly is poured into the test tube. If sugar contains hydrogen sulfide-producing bacteria, characteristic black colonies form in agar.