What are elective culture media? Classification of nutrient media and methods of their preparation Elective nutrient media purpose of application examples.

Heating radiators - 12/11/2023

The development of nutrient media, and especially dense media, for different types of microorganisms has made it possible to study their cultural, biochemical, antigenic, and virulent properties.

Pasteur used liquid infusions to cultivate microbes. The first developers of solid nutrient media were Koch and his students. They were the first to use potatoes, coagulated whey, gelatin, and meat-extract agar.

Solid media made it possible to obtain pure cultures of microbes and study their properties.

It has become possible to determine the etiological factors of many infectious diseases and to develop preventive and therapeutic drugs.

Cultivation of microbes on solid nutrient media in laboratory conditions made it possible, by obtaining pure cultures, not only to work on the creation of vaccines and diagnostics, but also to study the spectrum of action of chemotherapeutic drugs and antibiotics used for therapeutic purposes, as well as to study the effect of chemical drugs used for preventive purposes. , current and final disinfection.

Making a laboratory diagnosis is associated with clarifying the systematic position of microorganisms obtained in pure culture (pure culture is a collection of microbes of the same species on a nutrient medium).

Obtaining a pure culture is often a necessary condition when studying microorganisms isolated from a patient.

Determining the species of microorganisms includes determining a number of characteristics of microorganisms: cell morphology, the nature of culture growth on various nutrient media, the ability to use certain chemical compounds, relationship to temperature, pH of the environment, oxygen, etc. In addition, to determine the species of microorganisms, they often it is necessary to know metabolic products, antigenic properties, nucleotide composition of cells, biochemical activity associated with a set of enzymes, and much more.

Determination of all these characteristics allows the identification of microorganisms.

Identification of microorganisms is very important in diagnosing infections, establishing the sources and routes of transmission of the pathogen.

In order to be able to identify any microorganism, it is necessary to accumulate individuals in a pure culture and in the required quantity.

To isolate, cultivate, accumulate and preserve microorganisms, they use nutrient media containing all the nutrients necessary for microbes and simulating the habitat of microorganisms in natural conditions.

All culture media must meet the following requirements:
1) the presence of nutrients and growth factors in an easily digestible form;
2) sterility – absence of viable microorganisms and spores;
3) isotonicity - the same content of mineral salts inside and outside the cell. For pathogenic microorganisms that have adapted to a long stay inside the human body, a 0.85% sodium chloride solution is considered isotonic. For microorganisms living, for example, in the ocean (salt water), isotonicity will be created by a different salt concentration, but the requirement for isotonicity of the nutrient media remains;
4) optimal pH of the environment. The redox potential of the nutrient medium is created by hydrogen ions, which contributes to the normal functioning of microbial enzymes;
5) transparency of the environment. Since most bacteria, one of the main signs of growth on nutrient media is turbidity (on liquid media - turbidity, sediment, film or mixed; on solid media - the formation of colonies).

The most important feature of the growth of some microorganisms on nutrient media: for leptospira - the absence of turbidity of the nutrient medium (observation of the growth and reproduction of these microorganisms is carried out using a phase contrast microscope), for mycoplasmas and the causative agent of tuberculosis - slow growth (turbidity or the appearance of colonies no earlier than after 21 days, or even later).

Nutrient media are the basis of microbiological work and their quality often determines the results of the study. Therefore, when selecting media, one should take into account both the requirements of microbes in relation to the substances necessary to maintain their vital functions, and their ability to carry out the exchange of substances between the cell and the environment under given conditions.

Nutrient media must create optimal (best) conditions for the life of microbes.

In order to carry out biosynthesis, growth and reproduction, the cell must receive from the outside in the required quantities all the elements it contains and must be provided with a source of energy. In accordance with exactly what elements are delivered to the cell, the substances of the nutrient medium are called sources of carbon, nitrogen, phosphorus, sulfur, etc.

The compounds in the form of which elements necessary for constructive purposes must be introduced into the media are determined by the synthetic abilities of microorganisms.

The form of the energy source is determined by the method of its production.

The synthetic capabilities of microorganisms and the ways they obtain energy are extremely diverse, and therefore their needs for food sources are different. This fact must be taken into account when preparing nutrient media, clearly realizing that universal media that are equally suitable for the growth of all microorganisms without exception do not exist.

Thus, the composition of nutrient media is determined primarily by the characteristics and diversity of metabolism of microorganisms.

The necessary nutritional elements for microorganisms, like other creatures, are carbon, nitrogen, sulfur, phosphorus, ash elements, and for many microorganisms, various additional nutrients such as vitamins, growth factors, etc.

Many microorganisms, like higher animals, in addition to the normal sources of carbon, nitrogen, mineral salts and other elements that serve them as a source of energy and material for synthesis, also require very significant growth factors. A feature of growth factors is their activity in extremely small quantities.

Growth factor was first discovered by Wilde in 1901 while cultivating yeast. He noticed that when a synthetic medium was inoculated with a small amount of yeast, no growth was observed. However, if, simultaneously with sowing the yeast, yeast killed by boiling is added to the nutrient medium, growth appears. Wilde explained this phenomenon by the presence of a certain growth factor in the yeast cell, which he called “BIOS”.

A study of the nature of BIOS showed that it is a mixture of several components. BIOS was split into two fractions “BIOS”-1 and “BIOS” -2. Both fractions, taken separately, are inactive. Their ability to stimulate microbial growth occurs only when they act together. "BIOS"-1 is an inositol, "BIOS"-2 contains a heterocyclic ring with a free carboxyl group.

Substances similar to BIOS turned out to be necessary as growth factors for many microorganisms. Some microorganisms require the addition of vitamin B to the nutrient medium.

Based on their need for vitamin B, bacteria are divided into four groups:
a) bacteria growing in broth devoid of vitamin B. These bacteria do not grow in synthetic media (typhoid and dysentery bacilli, pyogenic staphylococcus);
b) bacteria that do not require exogenous supply of vitamin B. These bacteria grow in vitamin-free broth and synthetic media (Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, anthrax, etc.). Microbes of this group are able to synthesize vitamin B themselves;
c) bacteria that grow poorly on vitamin-free media (meningococcus, the causative agent of diphtheria);
d) bacteria that do not grow on vitamin-free media (hemolytic streptococcus, pneumococcus).

It has been established that B vitamins have the ability to stimulate growth and acid formation in propionic acid and lactic acid bacteria.

The influenza bacillus, the causative agents of whooping cough and chancroid, require a growth factor consisting of X and Y factors. Both of these factors are found in the blood, as well as in potatoes and other plant extracts.

X - factor is thermostable, it is hematin and can be replaced by some inorganic iron compounds that have oxide or catalase activity. Hematin and other iron compounds are necessary for the synthesis of cytochromes involved in respiration processes.

Y - factor has a vitamin nature, is destroyed during autoclaving, and is produced by bacteria, yeast, animal and plant cells.

Anaerobic microbe Bac. sporogenes uses unsaturated fatty acid as a growth factor. Its presence is also necessary for the growth of Cl.botulinum and Cl. Perfringens. This substance is formed by many aerobic bacteria, for example, typhoid, tuberculosis bacilli, and mold fungi. Obviously, this substance is necessary for the life of all microorganisms, but anaerobic clostridia lack the ability to synthesize it themselves.

A very active growth factor with universal biological distribution is pantothenic acid. Nicotinic acid amide serves as a growth factor for staphylococci. Without it, staphylococci do not grow on synthetic media containing hydrolyzed gelatin, tryptophan, tyrosine, cystine and glucose. Nicotinamide is synthesized by intestinal and typhoid bacilli, as well as Vibrio cholerae.

Growth factors include some amino acids (necessary for protein synthesis), purine and pyrimidine bases (used for the construction of nucleic acids), etc. Many growth factors are part of various enzymes and play the role of catalysts in biological processes. The question of bacterial growth factors is very important. On the one hand, it helps to understand the physiological role of meat water, which is used to prepare laboratory nutrient media for the cultivation of many microorganisms. On the other hand, resolving the issue of growth factors makes it possible to more widely use synthetic media for cultivating microbes.

Nutrient media for the same microorganism may be different depending on the objectives of the study. For example, media suitable for long-term maintenance of the vital activity of microbial cultures may differ greatly from media intended for the production of certain metabolic products, when it is necessary to stimulate certain aspects of the vital activity of microbes. Special environments are needed for the formation of spores and other life cycle forms.

If possible, nutrient media should be standardized, i.e., contain constant amounts of individual ingredients. For ease of monitoring the growth of crops and monitoring environmental contamination by foreign microorganisms, nutrient media must be transparent.

Based on their composition, nutrient media are divided into natural, synthetic and semi-synthetic.

Natural media are usually called media consisting of products of animal or plant origin and having a complex, uncertain chemical composition. The basis of such media are various parts of green plants, animal tissue, malt, yeast, fruits, vegetables, manure, soil, sea water, lakes and mineral springs. Most of them are used in the form of extracts or infusions.

Many microorganisms develop well in natural media, since such media, as a rule, contain all the components necessary for the growth and development of microbes. However, media with an uncertain composition are of little use for studying the physiology of the metabolism of microorganisms, since they do not allow one to take into account the consumption of a number of components of the medium and to find out what substances are formed during the development of microorganisms.

Natural media of uncertain composition are used mainly for maintaining cultures of microorganisms, accumulating their biomass and for diagnostic purposes.

Media of uncertain composition also include the so-called semi-synthetic media. Their composition, along with compounds of a known chemical nature, includes substances of uncertain composition. Such media are especially widely used in industrial microbiology for the production of amino acids, vitamins, antibiotics and other important products of microbial activity.

An example of such media is meat peptone broth (MPB), which, along with meat extract and peptone, which have a complex composition, includes sodium chloride, potassium phosphate, and sometimes glucose or sucrose. Semi-synthetic media also include potato media with glucose and peptone.

Synthetic media are media that contain only certain, chemically pure compounds, taken in exactly the specified concentrations.

Synthetic media are most convenient for studying the metabolism of microorganisms. Knowing the exact composition and quantity of components included in the environment, it is possible to study their consumption and transformation into the corresponding metabolic products.

Nutrient media are selective, differential diagnostic and preservative.

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.).

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.

Preservative media are intended for primary seeding and transportation of the test material. They prevent the death of pathogenic microorganisms and suppress the development of saprophytes. An example is the glycerin mixture used to collect stool in tests carried out to detect certain types of bacteria.

Based on their physical state, media are divided into liquid, dense, semi-liquid and granular. To clarify the physiological and biochemical characteristics of microorganisms, as well as to accumulate their biomass or metabolic products, it is most convenient to use liquid media. Solid media are used to isolate pure cultures, obtain isolated colonies, store cultures and quantify microorganisms, determine their antagonistic properties, and in a number of other cases. Semi-solid culture media are typically used for longer-term storage of microbial cultures. To compact media, agar is used - agar, gelatin and silicic acid gel.

In industrial microbiology, so-called bulk nutrient media are used. Such media include, for example, boiled millet, bran soaked in a nutrient solution, etc.

Nutrient media can be simple or complex. Simple liquid nutrient media include peptone water, meat - peptone broth (MPB). Dense simple nutrient media include meat - peptone agar (MPA) and meat - peptone gelatin.

Simple nutrient media, especially MPB and MPA, serve as the basis for making more complex media from them by adding various substances to them that increase the nutritional value of the substrate. For example, adding glucose produces sugar broth or sugar agar; ascites - agar and ascites - broth are obtained by adding ascitic fluid; Whole blood is a component of blood agar and blood broth, and the addition of blood serum produces serum media (agar or broth).

Media of a more complex composition are usually intended for the cultivation of microbes that are demanding on nutrient substrates and do not reproduce on simple media. Such microorganisms include pathogens of gonorrhea, diphtheria, brucellosis, tularemia, syphilis, relapsing fever, rhinoscleroma, tuberculosis, etc.

Nutrient media are classified depending on the initial components, consistency, intended purpose, and chemical composition.

Depending on the chemical composition and initial components, the following types of nutrient media are distinguished.

Environments of uncertain chemical composition. They are divided into: 1) media of animal origin (initial products - meat, fish, eggs, milk, etc.); 2) media of plant origin (initial products - soybeans, peas, potatoes, carrots, etc.).

Some products are used in their natural form (potatoes, carrots, milk, etc.), but more often animal and plant tissues are subjected to various processing (extraction, enzymatic or acid hydrolysis).

Media of known chemical composition(synthetic). They contain known chemical compounds (salts, carbohydrates, amino acids, vitamins, etc.) in an optimal quantitative ratio. Synthetic nutrient media are used when the grown cell mass needs to be maximally freed from ballast organic compounds included in conventional media, for example, when obtaining diagnostic allergens or when studying the metabolic needs of a microorganism for a particular chemical compound.

Based on consistency, nutrient media are differentiated into solid, semi-liquid and liquid.

Liquid nutrient media. Prepared using extracts, hydrolysates, solutions of initial products.

Semi-liquid and solid nutrient media. The required consistency is given to the medium by adding various sealants.

Agar-agar (Malayan jelly) is a polysaccharide, a product of the processing of certain seaweeds. Melts at 80...86 ºС, hardens at 40 ºС. To obtain dense media, it is added in an amount of 1.5...2%, less often 3%; semi-liquid - 0.3...0.7%.

Gelatin is an extract from tissues containing a lot of collagen (bones, cartilage, tendons, etc.). Gelatin gel melts at 25 °C, which makes it inconvenient for growing microorganisms with a temperature optimum of 37...38 °C. In addition, a number of bacteria secrete proteolytic enzymes that decompose gelatin. Typically, 10...20% gelatin is added to the culture media.

According to their intended purpose, they distinguish between commonly used (basic), enriched, special, selective (selective) and differential diagnostic nutrient media.

Common (basic) environments. They are used for cultivating relatively unpretentious microorganisms.

The starting components for the preparation of basic media are most often meat water, Hottinger's digest, and plant hydrolysates.

Meat water: beef is freed from bones, fat, tendons and passed through a meat grinder. Minced meat is poured with tap water in a ratio of 1: 2, boiled for 1 hour. After boiling, the meat water is cooled, filtered through a cotton-gauze filter, then topped up with tap water to the original volume, poured into containers, closed with cotton-gauze stoppers and sterilize at 120 °C for 20 minutes.

Hottinger's digest is prepared from meat waste by tryptic hydrolysis. Fat, fascia, tendons are finely chopped, poured with boiling water in a ratio of 1:2, boiled, cooled to 45 ° C and pancreatin is added, alkalized with sodium carbonate solution to pH 7.8...8.0, shaken and added chloroform (10 ml/l), tightly closed, kept in a warm place for 10 days, a hydrolysis product (digest) is obtained.

Meat-peptone broth (MPB). To 1 liter of meat water add 1% peptone and 0.5% sodium chloride, setting

Rice. 32. Preparation of agar slants

The required pH is achieved by fractional addition of a 10% solution of sodium hydroxide or potassium hydroxide. Filter through a paper filter, pour into flasks, test tubes and sterilize at 120 °C for 15...20 minutes.

Meat-peptone agar (MPA): add 2...3% of washed, finely chopped agar-agar to MPB, heat until the agar melts, bring to a boil, check the pH while hot, then, if necessary, bring it to the desired value ( 7.2...7.6), filtered through a cotton-gauze filter. Filtered hot agar is poured into test tubes and flasks, sterilized by autoclaving at 1 atm for 20...30 minutes. To obtain a slanted agar surface convenient for inoculation, after sterilization, the tubes with molten MPA are left at room temperature until compacted in an inclined position (the end with the stopper is raised) (Fig. 32).

Cultivation of microorganisms on solid nutrient media in Petri dishes is widely used. The diameter of a standard Petri dish (Fig. 33) is about 10 cm; dishes of smaller and larger diameters, as well as disposable plastic ones, are produced. IN

standard sterile Petri dishes are poured over a burner flame with about 20 ml of melted and cooled to 45...50 "C nutrient agar, the dishes are placed on a horizontal surface until the agar hardens.

Semi-liquid meat-peptone agar (MPA) is prepared like MPA, but 0.25% agar is added. Boil with stirring until the agar completely melts, set the pH to 7.2...7.6, filter while hot, sterilize in an autoclave.

Meat-peptone gelatin (MPG): 10...20% of crushed gelatin is added to MPB, heated until the sealant melts, the pH is set to 7.2...7.4, boiled, filtered through a cotton-gauze filter, poured into test tubes and sterilize fractionally in a Koch apparatus for three days for 20 minutes or once in an autoclave at 112°C for 15 minutes.

Hottinger's broth: Hottinger's basic digest is diluted with tap water in a ratio of 1:5 (1:8) to an amine nitrogen content of 120 mg%, add 0.5% sodium chloride, 0.1 g of potassium hydrogen phosphate, set the pH to 7.4... 7.6, boil for 15...20 minutes, filter through a cotton-gauze or paper filter, pour into containers and sterilize at 120 °C for 20...30 minutes.

Hottinger's agar is prepared by adding 2% agar agar to Hottinger's broth.

Biological industry enterprises produce ready-made nutrient broth and agar in the form of dry powder.

Nutrient broth contains (g/l): tryptic hydrolyzate of sprat - 10.05, sodium chloride - 4.95. A 15 g sample of powder is dissolved in 1 liter of distilled water, boiled for 2 minutes, filtered through a paper filter, poured into containers and sterilized in an autoclave at 120 ºC for 20 minutes (pH 7.3).

Nutrient agar contains (g/l): enzymatic hydrolyzate of feed yeast - 12.0; agar - 12.5; sodium chloride - 5.5. A sample of powder weighing 36 g is dissolved in 1 liter of distilled water, boiled for 3 minutes, filtered through a cotton filter, and sterilized at 120 °C for 20 minutes (pH 7.3).

Enriched environments. Many types of pathogenic bacteria do not grow well on commonly used nutrient media, so blood, blood serum, carbohydrates, etc. are added to the basic media. Such nutrient media are called enriched.

Serum and blood agars: 5...10% of sterile defibrinated sheep (rabbit) blood or blood serum (horse, cattle, rabbit) is added to melted and cooled to 45...50°C sterile nutrient agar. To obtain defibrinated blood from a sheep, blood is taken aseptically from the jugular vein with a sterile needle into a sterile bottle (or flask) with glass (porcelain) beads or balls, shaken with rotational movements for 15...20 minutes to prevent blood clotting. Fibrin remains on the beads.

The components are mixed, poured into Petri dishes, test tubes and left until the nutrient medium hardens.

Whey and blood broths are prepared in a similar way.

Solutions of carbohydrates (glucose, etc.) are sterilized by flowing steam or filtration and added in an amount of 0.5...1% to the nutrient medium.

Special environments. This is the name given to media developed taking into account the specific growth needs of a number of bacteria. For example, McCoy's yolk medium for the causative agent of tularemia, Terskikh's medium for the cultivation of Leptospira, etc.

McCoy's medium: clean chicken eggs are treated with alcohol and quickly passed through a burner flame. Open sterilely, yolks are separated from whites. To 60 parts of yolks add 40 parts of physiological solution (pH 7.0...7.2). The components are mixed, poured into 4...5 ml test tubes and placed in an inclined position in a serum clotting apparatus. Sterilize on the first day at 75 °C for 1 hour, on the second day at 85 °C for 30 minutes. To control sterility, the prepared media are kept in a thermostat for 2 days at 37...38 °C.

Terskikh's medium consists of a phosphate mixture of Zerensen and rabbit serum. Zerensen mixture: solution A: sodium hydrogen phosphate - 11.876 g, distilled water - 1000 ml; solution B: potassium dihydrogen phosphate - 9.078 g, distilled water - 1000 ml. To 90 ml of solution A add 10 ml of solution B and adjust the volume with distilled water to 1000 ml. The solution is poured into 5 ml test tubes and sterilized at 1.5 atm for 20 minutes. Add six to eight drops of sterile rabbit serum inactivated at 56 °C to each tube.

Elective environments(Latin electus - chosen one). It's nutritious
environments for the selective isolation and accumulation of microorganisms of a certain type from materials containing several types of microbes. Elective environments are extremely diverse in their composition. They include components that ensure the preferential growth of the desired microorganism and
(or) suppressing to one degree or another the growth of accompanying
microflora. According to the consistency of this type of medium, it can be
dense and liquid. Liquid elective media are called enrichment or accumulation media; they are used when placing

The goal is to increase the amount of the desired microorganism in a mixed population.

Milk-salt agar is intended for selective cultivation of staphylococci. To molten MPA with pH 7.2...7.4, containing 5...7.5% sodium chloride, add 10% sterile skim milk, mix and pour into Petri dishes.

Shustova's medium is intended for the isolation of Salmonella. It is an MPA (pH 7.4) with the addition of 10% to the volume of the medium 50% aqueous solution of sodium thiosulfate and 2% Lugol's solution.

Rappoport medium is intended for the cultivation of Salmonella. 1% glucose, 10% bile, 1% Andrede indicator are added to the MPB. Sterilize with flowing steam.

Muller's medium is intended for the cultivation of Salmonella. 90 ml of MPB is poured into a flask with 4.5 g of sterile chalk, sterilized in an autoclave at 120 °C for 30 minutes. Then 2 ml of Lugol’s solution and 10 ml of sodium thiosulfate solution are sterilely added (sodium thiosulfate - 50 g, distilled water - 100 ml) , sterilized in a Koch apparatus for 30 minutes.

Kauffman's medium is an enrichment medium for Salmonella. To 100 ml of Müller's medium add 1 ml of an aqueous solution of brilliant green, diluted 1: 1000, and 5 ml of sterile bovine bile. The mixture is sterilized with running steam for 30 minutes.

Casein charcoal agar (CCA) with penicillin is used for the cultivation of Bordetella. To 1000 ml of distilled water add casein hydrolyzate - 20 ml, sodium chloride - 5 g, potassium chloride - 0.2 g, calcium chloride - 0.002 g, sodium carbonate - 0.4 g, magnesium chloride - 0.025 g, potassium hydrogen phosphate - 0 .24 g, soluble starch - 1 g, cystine - 0.01 g, agar -20 g. The components are dissolved, the pH is set to 7.2, sterilized at 0.5 atm for 30 minutes. Before use, add 3% yeast extract and 0.2% dry activated carbon and 0.5 U/ml penicillin to the molten agar (50 °C). The components are mixed and poured into Petri dishes.

Differential diagnostic environments. Designed to detect enzymes in microorganisms. The consistency can be liquid, semi-liquid, dense. The composition of these media includes the main nutrient medium that ensures the growth of the microorganism under study, a substrate for detecting the enzyme, and an indicator, the color change of which indicates a shift in the pH of the medium as a result of the breakdown of the substrate.

Nutrient media of this type include Gissa, Endo, Ploskirev, Levin, etc.

Hiss media is used to study the enzymatic properties of isolated cultures of microorganisms. To 100 ml of distilled water add 1% peptone, 0.5 g of sodium chloride. The components are dissolved, filtered through a paper filter, set the pH to 7.0...7.4, add one of the carbohydrate substrates (lactose, glucose, etc.), agar-agar (0.3...0.4 %), and then 1 ml of Andrede indicator or 0.1 ml of 1.6% bromothymol blue solution. The prepared medium is poured into 3 ml test tubes and sterilized with flowing steam for three days in a row for 30 minutes or at 112 °C for 20 minutes.

They produce dry Hiss media with the BP indicator - a mixture of aqueous blue with rosolic acid (ready-made media have a semi-liquid consistency).

Dense differential diagnostic media are used for the primary isolation of pathogens from the material. In addition to the known substrate, their composition often includes substances that impart selective properties to the nutrient medium.

Endo medium contains lactose as a substrate and is designed to differentiate bacteria that differ in their ability to break down lactose.

To 1000 ml of molten MPA (pH 7.4) at a temperature of 70 ° C, add 1 g of lactose, previously dissolved in a small amount of distilled boiled water. In separate test tubes prepare: 2...3 ml of an alcohol solution of basic fuchsin; 10 ml of 10% aqueous sodium sulfate solution.

Add 1 ml of fuchsin solution to a sterile test tube and add sodium sulfite solution until the fuchsin becomes discolored. The prepared mixture is poured into molten agar, mixed and poured into Petri dishes. The prepared medium is colorless; when microorganisms that break down lactose grow on it, the medium becomes acidified, the discolored fuchsin is restored, and the colony of the microorganism, for example Escherichia, acquires a red color with a metallic tint. The medium is prepared one day before its use. They also produce Endo dry medium. Before use, a certain amount of powder is added to distilled water, boiled and poured into Petri dishes.

Levin's medium is similar in purpose to Endo's medium, but contains a different indicator (eosin with methylene blue). To 100 ml of molten MPA (pH 7.2...7.4) add 2 ml of a 0.5% aqueous solution of methylene blue, 1.5 ml of a 2% solution of eosin yellow, 2 g of lactose, 0.2 g potassium dihydrogen phosphate. Dye solutions are prepared in distilled water and sterilized with running steam for 60 minutes. Lactose and potassium dihydrophosphate are pre-diluted in a small amount of sterile distilled water and boiled. Colonies of lactose-positive bacteria on this medium are purple-black.

Ploskirev's agar is intended for the isolation of salmonella, contains lactose as a substrate and components that suppress the growth of accompanying microflora. The medium is produced in the form of a powder; in addition to the nutrient agar base, it contains: bile salts, sodium citrate, sodium thiosulfate, sodium phosphate, brilliant green, soda ash, iodine, sodium chloride, lactose, neutral red. A sample of the powder is dissolved in water, boiled and poured into Petri dishes. The prepared medium is clear or pinkish. Salmonella colonies are colorless, Escherichia colonies are lingonberry-colored.

Methods for cultivating microorganisms. Along with the general principles, the cultivation of microorganisms of various physiological groups has some features: Cultivation of aerobic and facultative anaerobic bacteria. Solid, liquid or semi-liquid nutrient media seeded with pure cultures of microorganisms or the material being studied are placed in thermostats (Fig. 34) that maintain the optimal temperature for a given microorganism. At temperatures above the upper limit of normal, bacteria not only slow down their growth, but also quickly die. At temperatures below the optimum, the growth rate of the microorganism gradually slows down.

For mesophiles, the temperature optimum is in the range of 30...37 ºС, for psychrophiles - 10... 15 ºС, for thermophiles - 50...60 ºС.

Microorganisms in the process of cultivation on nutrient media, provided that no additional substances are added to the media, gradually slow down and then stop their growth due to depletion of the nutrient substrate, changes in the optimal values of biophysical indicators (pH, Eh, etc.). Such cultivation of microorganisms is called periodic. If the liquid nutrient medium is not stirred during the incubation of crops, then this method of cultivation is defined as stationary. For diagnostic bacteriological studies, this is the cultivation method that is usually used. In the biological industry, in the production of vaccines and other biological products, when it is necessary to achieve the maximum yield of bacterial mass or exotoxins, periodic cultivation in liquid media with intensive mixing is used.

For such tasks, aerobic bacteria are cultivated in flasks and bottles on Schüttel devices with an oscillation frequency of 150... 250 min-1, which facilitates the transfer of oxygen and nutritional components to the bacteria.

Rice. 35. Scheme of a fermenter for deep cultivation of aerobic microorganisms.

1 – air inlet; 2 – air outlet; 3 – bumpers; 4 – stirrer; 5 - bubbler

The most effective cultivation of bacteria in liquid nutrient media with maximum yield of bioproducts is achieved in fermenters. Fermenters (reactors) are metal or glass culture vessels with a capacity of 500 ml to 1000 l (Fig. 35). When cultivating bacteria in fermenters, the medium is stirred special mixers with the simultaneous supply of the required amount of sterile air. Fermenters are designed as autonomous systems with automatic regulation of temperature and pH of the environment. Fermenters also carry out continuous (flow) cultivation, in which, unlike batch culture, fresh nutritional components are automatically supplied to the medium at a rate equal to the removal of a similar volume of grown bacterial culture. Such continuous cultivation in a well-regulated system can in principle be continued indefinitely.

Cultivation of anaerobic bacteria. Obligate anaerobes are bacteria in which energy and constructive metabolism occurs without molecular oxygen 02. In such microorganisms, in the process of respiration, final acceptors

electrons are carbon monoxide (IV), sulfate ions, fumarate, etc. In addition, molecular oxygen has a detrimental effect on many anaerobes. For example, strict anaerobes die at low oxygen concentrations (bacteroides, fusobacteria), moderate anaerobes are less sensitive ( C. perfringens), aerotolerant anaerobes can grow in normal atmosphere conditions (lactic acid bacteria). Most pathogenic anaerobes are classified as strict or moderate anaerobes. For their cultivation, special nutrient media and gas mixtures are used. Anaerostats are the last to be filled.

A necessary condition for the growth of obligate anaerobes is not so much the absence of molecular oxygen as the low TRC of nutrient media. Sharply reducing conditions are achieved by adding reducing (reducing) substances to the media and simultaneously removing molecular oxygen from them. The chemical compounds listed in Table 1 are added to the nutrient media as reducing substances.

1. Reducing substances for the cultivation of anaerobes

For the same purpose, boiled pieces of liver, muscle, brain, blood clots, chicken egg whites, and rye grains are added to the nutrient media. It is believed that substances rich in SH groups have a strong reducing effect in these tissues.

To create conditions of anaerobiosis, nutrient media are freed as much as possible from oxygen by boiling, as well as by passing inert gases through liquid media or by layering petroleum jelly on the surface of the nutrient medium to prevent contact with atmospheric oxygen.

Meat-peptone liver broth (MPLP) Kitta-Tarozzi is a traditional medium for the cultivation of anaerobes. It is based on liver water, which is prepared by boiling small pieces of beef liver in water (1:1 ratio). Liver water is mixed with MPB in a ratio of 1:2, the mixture is boiled, the required pH is adjusted and poured into 10 ml test tubes. Pieces of boiled liver (reducing agent) are added to the test tubes and then 2 ml of vaseline oil is added. Autoclave at 0.5 atm for 20 minutes. Before use, the test tubes with the medium are boiled in a water bath, then cooled and only then inoculated.

To grow anaerobes, nutrient media with high viscosity are also used, since the diffusion of oxygen in them is difficult.

Semi-liquid agar: add 0.25...0.75% agar and 1% glucose to the MPB, set the pH to 7.4, pour into test tubes in a high column and fractionally sterilize.

They practice the cultivation of anaerobes in dense media.

Sugar agar in Veyon tubes: add 1% glucose to 2% MPA, set the pH to 7.4. The seed material is added to the melted and cooled to 48...50 "C medium, mixed and poured into sterile narrow glass tubes - Veyon tubes (length 20...25 cm, diameter 1...1.5 cm). The ends of the tubes closed with sterile rubber stoppers.Colonies of anaerobes grow in the thickness of the nutrient medium.

Cultivation of anaerobes on the surface of solid nutrient media in Petri dishes is widely used.

Among the solid media for cultivating anaerobes, blood agar with glucose and iron sulfite agar are often used.

Glucose-blood agar: to 3% MPA (pH 7.2...7.4), melted and cooled to 50°C, add 1...2% sterile glucose solution, 15...20% defibrin -a bath of sheep's blood and poured into Petri dishes.

Iron sulfite agar (Wilson-Blair medium): to 100 ml of 3% MPA (pH 7.4) with 1% glucose at a temperature of 60 °C add 10 ml of 20% sodium sulfite solution and 1 ml of 8% ferric chloride solution, then the medium is poured into Petri dishes. Anaerobes, when growing on this medium, reduce sodium sulfite to sodium sulfate, which reacts with ferric chloride to form a black precipitate of ferrous sulfite; bacterial colonies are colored black.

To cultivate anaerobes on the surface of solid nutrient media, it is not enough to add reducing agents to them. Culture vessels with crops are placed in sealed chambers (anaerostats), in which anaerobic (oxygen-free) conditions are created in one way or another.

A conventional anaerostat is a metal cylinder, which is hermetically sealed with a lid with a rubber gasket (Fig. 36). The lid contains a pressure gauge and taps for pumping out air or filling the anaerostat with inert gas (nitrogen). The air is pumped out using a vacuum pump, the valve is tightened and the anaerostat with test tubes or cups is placed in a thermostat. The usual residual pressure in the anaerostat is about 10 mm Hg. Art. Anaerostats have been created, the removal of oxygen from which occurs due to its reaction with hydrogen in

presence of a catalyst (platinum, palladium). Hydrogen is pumped into the chamber from a cylinder through a reducer. Some anaerobic chambers are equipped with heating elements with a thermostatic device that independently maintains the temperature at the required level.

Cultivation of microaerophilic bacteria. Although microaerophilic bacteria are aerobes by type of respiration, they do not grow in a normal atmosphere (21% oxygen), but with a reduced oxygen content. For example, Campylobacter fetus grows in an atmosphere containing no more than 6% oxygen. Such an atmosphere can be created in sealed thermostats, anaerostats, replacing part of the air with compressed carbon monoxide (IV) from a cylinder, or in a conventional desiccator. In the latter case, the test tubes with the crops are placed in a desiccator along with a bottle containing cotton wool moistened with alcohol or a candle. The cotton wool (candle) is lit and the lid of the desiccator is closed. The flame dies out as the oxygen burns out, and the decrease in its content is sufficient for the growth of microaerophiles.

The most accessible and effective method for cultivating microaerophiles is in a semi-liquid medium with 0.1...0.4% agar. In such an environment, convection currents are not able to mix the upper, oxygen-rich layers of the medium with the lower ones, which creates an oxygen concentration gradient in the medium filling the test tube. The microaerophile culture is inoculated with a prick, and the microorganism grows in a zone with optimal oxygen content, usually in the form of thin disks at a distance of several millimeters to several tens of millimeters from the surface of the medium.

Mushroom cultivation. The best growth of mushrooms was observed on media with a carbohydrate content of 1...4%. During primary isolation, antibiotics are often added to the culture media to suppress the growth of various associated bacteria.

Sabouraud agar is used for cultivating pathogens of dermatomycosis and candidiasis. Glucose - 4 g, peptone - 1 g, agar - 1.8 g, distilled water - 100 ml. After dissolving the agar, the medium is filtered, poured into test tubes and sterilized at 0.5 atm for 30 minutes. After sterilization, the pH of the medium is 6.9...7.0.

Capek agar is used for cultivating many types of mushrooms. Glucose - 30 g, sodium nitrate - 2 g, potassium dihydrogen phosphate - 1, magnesium sulfate - 0.5 g, potassium chloride - 0.5 g, ferrous sulfate -0.0012 g, agar - 20 g, distilled water - 1000 ml . The natural pH of the environment is 5.6...5.9. The medium is sterilized at 0.5 atm for 30 minutes.

Wort agar is intended for the cultivation of pathogens of dermatomycosis and candidiasis. Unhopped malt wort is diluted with tap water in a ratio of 1: 2 (to a sugar content of 7%), pH is set to 6.5...6.7, 2% agar is added, boiled, filtered, sterilized at 0.5 atm for 30 minutes. .

Litman's agar is suitable for culturing dermatophytes. Peptone - 10 g, glucose - 10 g, ox bile - 15 g, crystal violet - 0.01 g, agar - 20 g, distilled water - 1000 ml. The medium is sterilized at 1 atm for 15 minutes.

Van Iterson's medium is intended for the isolation of toxic fungi from feed that cause stachybotryotoxicosis, dendrodochiotoxicosis, etc. Ammonium nitrate - 0.5 g, potassium dihydrogen phosphate - 0.5 g, tap water - 1000 ml. The medium is sterilized at 1 atm 30 mc. Then sterile Petri dishes with filter paper are moistened with the medium.

Czapek's liquid medium. Glucose - 30 g, sodium nitrate - 2 g, potassium dihydrogen phosphate - 1 g, magnesium sulfate - 0.5 g, potassium chloride - 0.5 g, ferrous sulfate - 0.001 g, distilled water - 1000 ml. The medium is sterilized at 0.5 atm for 30 minutes. After sterilization, the pH of the environment is 5.9...6.2. The liquid medium can be used to moisten filter paper in Petri dishes for subsequent cultivation of fungi.

Bilay medium is intended for obtaining macroconidia of fungi. Potassium nitrate - 2 g, potassium dihydrogen phosphate - 1 g, magnesium sulfate -0.5 g, potassium chloride -0.5 g, iron sulfate - traces, soluble starch - 0.1 g, sucrose - 0.1 g, glucose - 0.1 g, distilled water - 1000 ml. The medium is poured into 5 ml test tubes and a strip of filter paper is inserted into each test tube so that most of it is above the solution. The medium is sterilized at 1 atm for 20 minutes.

Sabouraud glucose broth is used to cultivate many types of mushrooms. Glucose - 40 g, peptone - South, distilled water - 1000 ml. Heat to a boil, pour into test tubes and sterilize at 1 atm for 15 minutes.

Cultivation on hair according to Van Breusegem is used for isolating dermatophytes. Healthy sterile hair is attached by collodion to a glass tube. A fungal culture is applied to the middle of the hair. The tube is placed in a cylinder, at the bottom of which a small amount of water is poured for humidity. Cultivate at 25°C for five to ten days or more.

Blood agar is used to isolate pathogens of histoplasmosis and epizootic lymphangitis.

TASKS FOR INDEPENDENT WORK

1. Prepare MPA slant, serum and blood
MPA.

2. Study the structure of the anaerostat and fermenter.

Control questions

1.What are the general requirements for nutrient media?

2.What groups are nutrient media classified into?

How are anaerobes and microaerophiles cultivated?

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 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 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.

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 medium

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.

What do you understand by differential nutrient media?

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.

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.

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