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

Classification of culture media by composition:

1. Simple environments(MPB, MPA, gelatin, peptone water). Meat-peptone broth (BCH) is the protein basis of all media.

There are several ways to prepare BCH:

a) in meat water with the addition of ready-made peptone;

b) on digestions of products of hydrolysis of the feedstock using enzymes.

Meat-peptone agar (MPA) - is obtained by adding agar-agar (1.5-3%) to the MPB. If the MPA is distributed diagonally across the tube or vial, it is an agar slant. If the medium is distributed vertically in a test tube with a height of 5-7 cm, it is agar in a column. MPA, frozen in Petri dishes in the form of a stick - plate agar. If the medium has a vertical layer with a height of 2-3 cm, and the diagonal layer is the same size, it is a semi-lined agar.

2. Complex environments are prepared on the basis of simple ones with certain additives (carbohydrates, blood, bile, eggs, whey, milk, salts, growth factors, etc.)

Classification of culture media by initial components:

1. Natural culture media Is a natural product of animal or vegetable origin.

May be:

Vegetable (starting products - soybeans, peas, potatoes, carrots, etc.).

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

Mixed (MPA, Lowenstein - Jensen environment, etc.).

2. Artificial environments contain processed natural products (meat water, digest), substances derived from these products (peptone, yeast and corn extracts) and various additives. This is the largest and most varied composition of the most commonly used group of media. They are prepared according to certain recipes from various infusions or decoctions of animal or vegetable origin with the addition of inorganic salts, carbohydrates and nitrogenous substances.

3. Synthetic media(of known chemical composition) consist of chemically pure compounds in precisely defined concentrations (with the addition of carbohydrates, salts, amino acids, vitamins, etc.). On the basis of these environments, by adding natural or artificial environments to them, semi-synthetic environments are obtained.

Classification of culture media by consistency: Wednesdays are liquid(media without agar), semi-liquid(with agar up to 1%), dense(agar - 1.5-2.5%). Liquid media are more often used to study the physiological and biochemical characteristics of microorganisms, for the accumulation of biomass and metabolic products. Semi-liquid media are usually used for storing cultures, dense ones - for isolating microorganisms, studying the morphology of colonies, diagnostic purposes, quantitative accounting, determining antagonistic properties, etc.

Classification of culture media by purpose: universal (common) and special.

Universal (basic) environments. These media are used for the cultivation of most relatively unpretentious microorganisms or are used as a basis for the preparation of special media, adding blood, sugar, milk, whey and other ingredients necessary for the reproduction of one or another type of microorganism. This group includes: MPB - meat-peptone broth, MPA - meat-peptone agar, MPG - meat-peptone gelatin, etc.

Special environments. Designed for the isolation and selective cultivation of certain types of microorganisms that do not grow on simple media.

There are the following types of special media: enrichment media, elective, differential diagnostic, conservation and storage media.

Enrichment media. Many microorganisms do not grow on conventional media, so carbohydrates (sugar broth or agar) or proteins (whey agar and broth, blood agar and broth) are added to it to increase the nutritional value of the medium. Blood agar or blood broth is obtained by adding 5-10% warmed sterile defibrinated blood of a ram, rabbit, horse, or human to the nutrient medium. The medium is used to isolate streptococci, pneumococci and other bacteria, as well as to study hemolytic activity. Whey broth or whey agar is prepared by adding 15-20% horse or bovine serum to simple media.

2. Electoral (electoral) environments. These media are designed to selectively isolate and accumulate a specific type of microorganism from a material containing several types of microbes. When sowing material containing a mixture of various microorganisms on them, the growth of the species for which this medium will be elective will appear first. The selectivity of the medium is achieved by creating conditions that are optimal for the cultivation of certain microbes (pH, Eh, salt concentration, composition of nutrients), i.e. positive selection. Or by adding to the environment substances that inhibit other microorganisms (bile, high concentrations of NaCl, antibiotics, etc.), i.e. negative selection. This group includes:

Selenite environment- is the best enrichment medium for Salmonella and dysentery microbes in the Sonne. Sodium selenite contained in the medium stimulates the growth of these bacteria and inhibits the growth of the accompanying flora.

Bismuth sulfite agar - contains bismuth salts, brilliant green. Salmonella grow on this medium as black colonies. Other types of bacteria do not grow on this medium.

Yolk Salt Agar (YSA) - medium for isolation of staphylococci, contains up to 10% sodium chloride, which suppresses most of the bacteria contained in the material. In addition, this environment is also differential diagnostic, since the presence of egg yolk allows the detection of the enzyme lecithinase (lecitovitellase), which form pathogenic staphylococci. Lecithinase breaks down lecithin into phosphorcholines and water-insoluble fatty acids, so the environment around the lecithin-positive colonies becomes cloudy and an opalescent zone appears in the form of a "rainbow corolla".

Bile broth elective for salmonella, the reproduction of which is stimulated by the added 10% bile, at the same time inhibiting the growth of accompanying microorganisms.

Alkaline agar or alkaline peptone water they are elective for cholera vibrios, the alkaline reaction of the medium (pH 9.0) does not prevent the growth of cholera vibrios, but inhibits the growth of other microorganisms. 3-5 days F

3. Differential diagnostic environments. Differential diagnostic media are used to differentiate one type of microorganism from another by the nature of their enzymatic activity. The composition of these media is selected in such a way as to clearly identify the most characteristic properties of a certain type of microorganism, based on the characteristics of its metabolism.

Media for detecting the proteolytic and hemolytic ability of microbes, containing protein substances: blood, milk, gelatin, etc. The most common media are meat-peptone gelatin (MPG) curdled horse serum, milk, and blood agar (CA).

Media for studying glycolytic properties include three main components: a nutrient base (broth, agar), a substrate (mono- and disacchars, polyhydric alcohols) and an indicator for detecting the corresponding enzymes. Enzymatic degradation of substrates leads to a shift in pH and a change in the color of the medium. The most common colored media with various carbohydrates (for example, bromothymol blue, BP indicator). Giss media are also widespread, on which differences in the ability to ferment various carbohydrates to form an acid or acid and gas are taken into account.

For the differentiation of enterobacteria, peptone water is used with a set of various carbohydrates, Andrede's indicator and floats that facilitate the detection of gas formation and help to visually determine the change in pH characteristic of various microorganisms. In particular, the shift towards the acidic side causes reddening of the medium with Andrede's reagent or yellowing when using the medium with bromothymol blue, whereas when alkalized, Andrede's indicator and bromothymol blue do not change the color of the medium. For example, for the isolation of pathogenic bacteria from the intestine, media are used that make it possible to differentiate pathogenic microorganisms from the permanent inhabitants of the intestine - microorganisms that decompose lactose.

This environment is the Endo environment. The main components of Endo's medium are MPA, lactose and basic fuchsin, discolored with sodium sulfite. The original culture medium is colored light pink. When lactose is fermented, acetaldehyde is formed, which reacts with sulfite and, released during this process, fuchsin stains the colonies in a bright red color. Therefore, Escherichia coli, which ferments lactose, when growing on this medium, forms red colonies with a metallic sheen, and Salmonella and Shigella are colorless, since they do not ferment lactose.

4. Environment of accumulation, on which there is fast growth certain types of microorganisms.

5. Conservative (transport) media. Designed to preserve microorganisms during transportation to the research site. These media contain additives that prevent the multiplication and death of microbes, which contributes to the preservation of their viability. The most widely used are glycerin mixture (Teague medium), phosphate-buffered mixture and Kari-Blair and Amies media (with and without activated carbon activated carbon), Stuart, etc.

Sterilization of culture media.

All culture media, regardless of their purpose, are poured into clean dishes and sterilized. Most media are sterilized by autoclaving, but under different conditions, depending on their composition.

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

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

3. Media containing protein substances (blood serum, ascitic fluid) are provided by tyndalization or filtration.

4. For sterilization of culture media containing native proteins, filtration through Seitz membrane filters is used.

To control the sterility of the medium after sterilization, it is placed in a thermostat at 37 ° C for 3-5 days. Liquid media should remain transparent, and signs of growth should not appear on the surface and in the thickness of solid nutrient media. In addition to sterility control, chemical control of ready-made media is carried out, which consists in the fact that in several samples of each series the pH, the amount of total and amine nitrogen and chlorides are determined.

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

Nutrient media are classified depending on the source components, consistency, intended use, chemical composition.

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

Mediums of undefined chemical composition. They are subdivided into: 1) environments of animal origin (initial products - meat, fish, eggs, milk, etc.); 2) environment of plant origin (initial products - soybeans, peas, potatoes, carrots, etc.).

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

Media of known chemical composition(synthetic). They include 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 freed as much as possible from ballast organic compounds that are part of conventional media, for example, when receiving diagnostic allergens or when studying the metabolic needs of a microorganism in a particular chemical compound.

By consistency, nutrient media are differentiated into dense, semi-liquid and liquid.

Liquid culture media. Prepared using extracts, hydrolysates, solutions of starting products.

Semi-liquid and solid culture media... The necessary consistency is given to the medium by adding various seals.

Agar-agar (Malay jelly) is a polysaccharide, a processed product of some seaweed. It melts at 80 ... 86 ºС, solidifies 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.). The gelatinous 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 degrade gelatin. Usually 10 ... 20% gelatin is added to nutrient media.

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

Common (basic) environments... They are used for the cultivation of relatively unpretentious microorganisms.

Most often, meat water, Hottinger's digestion, and vegetable hydrolysates are used as initial components for the preparation of basic media.

Meat water: beef is freed from bones, fat, tendons, 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 added with tap water to the original volume, poured into containers, closed with cotton-gauze plugs and sterilized at 120 ° C for 20 minutes.

Hottinger's digestion 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, alkalinized with sodium carbonate solution to pH 7.8 ... 8.0, shaken and chloroform added (10 ml / l), tightly closed, kept in a warm place for 10 days, a hydrolysis product (digest) is obtained.

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

Rice. 32. Preparation of agar slant

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

Meat peptone agar (MPA): add 2 ... 3% of washed finely chopped agar agar to the 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 min. To obtain a beveled 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).

The 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, plates of smaller and larger diameters are produced, as well as disposable plastic ones. V

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

Semi-liquid meat-peptone agar (PFA) is prepared as MPA, but 0.25% agar is added. Boil with stirring until the agar is completely melted, set the pH to 7.2 ... 7.6, filtered hot, sterilized in an autoclave.

Meat-peptone gelatin (MPG): add 10 ... 20% of crushed gelatin to the MPB, heat until the sealant melts, set the pH to 7.2 ... 7.4, boil, filter through a cotton-gauze filter, pour into test tubes and sterilized 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 main digest is diluted with tap water in a ratio of 1: 5 (1: 8) to an amine nitrogen content of 120 mg%, 0.5% sodium chloride, 0.1 g of potassium hydrogen phosphate are added, the pH is set to 7.4 ... 7.6, boiled for 15 ... 20 minutes, filtered through a cotton-gauze or paper filter, poured into containers and sterilized at 120 ° C for 20 ... 30 minutes.

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

The biological industry produces 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 min, filtered through a paper filter, poured into containers and sterilized in an autoclave at 120 ° C for 20 min (pH 7.3).

Nutrient agar contains (g / l): enzymatic hydrolyzate of fodder 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, sterilized at 120 ° C for 20 minutes (pH 7.3).

Enriched environments. Many types of pathogenic bacteria grow poorly on commonly used nutrient media, therefore blood, blood serum, carbohydrates, etc. are added to the main media. Such media are called enriched media.

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

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

Whey broth and blood broth are prepared in a similar manner.

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 of the environment, 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 Wednesday: clean chicken eggs treated with alcohol, quickly passed through the flame of the burner. Opened sterile, the yolks are separated from the proteins. To 60 parts of the yolks add 40 parts of saline (pH 7.0 ... 7.2). The components are mixed, poured into tubes of 4 ... 5 ml and placed in an inclined position in a serum coagulation apparatus. Sterilized 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 for 2 days in a thermostat at 37 ... 38 ° C.

Wednesday Terskikh consists of a phosphate mixture of Zerensen and rabbit serum. Zerensen's 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 bring the volume to 1000 ml with distilled water. The solution is poured into 5 ml test tubes, sterilized at 1.5 atm for 20 minutes. Six to eight drops of sterile rabbit serum inactivated at 56 ° C are added to each tube.

Elective environments(Latin electus - the chosen one). It is nutritious
media for the selective isolation and accumulation of microorganisms of a certain type from materials containing several types of microbes. Electoral environments are extremely diverse in their composition. They include components that ensure the predominant growth of the desired microorganism and
(or) suppressing to one degree or another the growth of concomitant
microflora. By the consistency of this type of environment can be
dense and liquid. Liquid elective media are called enrichment or storage media and are used when placing

the goal is to increase the number of the desired microorganism in the mixed population.

Milk-salt agar is intended for selective cultivation of staphylococci. To molten MPA with a pH of 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. Represents MPA (pH 7.4) with the addition of 10% to the volume of the medium of a 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's indicator are added to the BCH. Sterilized with flowing steam.

Müller'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 (sodium thiosulfate - 50 g, distilled water - 100 ml) are added sterilely , sterilized in a Koch apparatus for 30 minutes.

Kaufman'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 flowing steam for 30 minutes.

Casein-charcoal agar (AMC) with penicillin is used to cultivate 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. Dissolve the components, adjust the pH to 7.2, sterilize at 0.5 ATM for 30 minutes. Before use, 3% yeast extract and 0.2% dry activated carbon and 0.5 U / ml of penicillin are added to molten agar (50 ° C). The components are mixed and poured into Petri dishes.

Differential diagnostic environments. Designed to detect enzymes in microorganisms. In consistency, they can be liquid, semi-liquid, dense. The composition of these media includes a basic nutrient medium that ensures the growth of the microorganism under study, a substrate for the detection of an enzyme, and an indicator, by the color change of which one can judge about a shift in the pH of the medium as a result of the decomposition of the substrate.

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

Giss media are 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, the pH is adjusted to 7.0 ... 7.4, one of the carbohydrate substrates (lactose, glucose, etc.), agar-agar (0.3 ... 0.4 %), and then 1 ml of Andrede's indicator or 0.1 ml of a 1.6% solution of bromothymol blue. The prepared medium is poured into 3 ml test tubes, sterilized by flowing steam for three days in a row for 30 minutes or at 112 ° C for 20 minutes.

Produce dry Giss media with BP indicator - a mixture of water-blue with rosolic acid (ready-made media - semi-liquid consistency).

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

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) with a temperature of 70 ° C add 1 g of lactose, previously dissolved in a small amount of distilled boiled water. Prepare in separate test tubes: 2 ... 3 ml of an alcohol solution of basic fuchsin; 10 ml of 10% aqueous sodium sulfate solution.

1 ml of fuchsin solution is added to a sterile test tube and sodium sulfite solution is added until the fuchsin is discolored. The prepared mixture is poured into molten agar, mixed and poured into Petri dishes. The finished medium is colorless, with the growth of microorganisms that break down lactose on it, the medium is acidified, the discolored fuchsin is restored, and the colony of the microorganism, for example, Escherichia, acquires a red color with a metallic tint. Wednesday is prepared one day before its use. Dry Endo medium is also produced. 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 yellow eosin, 2 g of lactose, 0.2 g of potassium dihydrogen phosphate. Dye solutions are prepared in distilled water, sterilized with flowing steam for 60 minutes. Lactose and potassium dihydrogen phosphate are preliminarily 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 inhibit the growth of accompanying microflora. The medium is produced in the form of a powder; in addition to the nutritious agar base, it includes: bile salts, sodium citrate, sodium thiosulfate, sodium phosphate, brilliant green, soda ash, iodine, sodium chloride, lactose, neutral red. A weighed portion of the powder is dissolved in water, boiled and poured into Petri dishes. The prepared medium is clear or pinkish. Colonies of Salmonella are colorless, Escherichia is lingonberry.

Microorganism cultivation methods. As well as general principles cultivation of microorganisms of various physiological groups has some peculiarities. Cultivation of aerobic and facultative anaerobic bacteria. Dense, liquid or semi-liquid nutrient media, inoculated with pure cultures of microorganisms or the test material, are placed in thermostats (Fig. 34), maintaining the optimum temperature for the given microorganism. At temperatures exceeding the upper limit of the norm, bacteria not only slow down growth, but also quickly die. At temperatures below the optimum, the growth rate of the microorganism gradually slows down.

In mesophiles, the temperature optimum is in the range of 30 ... 37 ºС, in psychrophiles - 10 ... 15 ºС, in 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 parameters (pH, Eh, etc.). Such cultivation of microorganisms is called periodic. If at the same time the liquid nutrient medium is not stirred during the incubation of crops, then this method of cultivation is defined as stationary. In diagnostic bacteriological studies, it is this 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, bottles on a shutter apparatus with a vibration frequency of 150 ... 250 min-1, which facilitates the transfer of oxygen and nutrient components to bacteria.

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

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

The most effective cultivation of bacteria in liquid nutrient media with the maximum yield of bioproduct 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 stirrers with the simultaneous supply of the required amount of sterile air. Fermenters are designed as self-contained systems with automatic regulation of temperature and pH of the medium. Fermenters also carry out continuous (flow) cultivation, in which, unlike a batch culture, fresh nutrients are automatically fed into the medium at a rate equal to the removal of a similar volume of the grown bacteria 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 energetic and constructive metabolism occurs without molecular oxygen 02. In such microorganisms, in the process of respiration, the final acceptors

electrons are carbon monoxide (IV), sulfate ions, fumarate, etc. In addition, molecular oxygen acts destructively on many anaerobes. For example, strict anaerobes die at low oxygen concentrations (bacteroids, fusobacteria), moderate anaerobes are less sensitive ( C. perfringens), aerotolerant anaerobes can grow in a normal atmosphere (lactic acid bacteria). Most disease-causing anaerobes are classified as severe or moderate anaerobes. For their cultivation, special nutrient media and gas mixtures are used. The latter are filled with anaerostats.

Necessary condition the growth of obligate anaerobes is not so much the absence of molecular oxygen as the low TSP of nutrient media. Sharply reducing conditions are achieved by adding reducing (reducing) substances to the media and at the same time 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, muscles, brain, blood clots, chicken egg white, 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 for anaerobiosis, nutrient media are freed from oxygen as much as possible by boiling, as well as by passing inert gases through liquid media or by layering liquid paraffin on the surface of the nutrient medium to prevent contact with atmospheric oxygen.

Kitta-Tarozzi Meat-Peptone Liver Broth (MPPB) 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 (ratio 1: 1). Liver water is mixed with MPB in a ratio of 1: 2, the mixture is boiled, the required pH is set 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 are 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 after that the inoculation is carried out.

For the cultivation of anaerobes, nutrient media with increased viscosity are also used, since the diffusion of oxygen in them is difficult.

Semi-liquid agar: 0.25 ... 0.75% agar, 1% glucose are added to the BCH, the pH is set to 7.4, poured into test tubes in a high column and fractionally sterilized.

They practice the cultivation of anaerobes in dense media.

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

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

Among solid media for the cultivation of anaerobes, blood glucose agar 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% defibriniro - bath blood of a ram 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 growing on this medium reduce sodium sulfite to sodium sulfate, which reacts with ferric chloride, forming a black precipitate of ferrous sulfite; colonies of bacteria are colored black.

For the cultivation of 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 (anoxic) conditions are created in one way or another.

A typical anaerostat is a metal cylinder that is sealed by a rubber-sealed lid (fig. 36). The cover contains a pressure gauge and taps for evacuating air or filling the anaerostat with an inert gas (nitrogen). The air is evacuated 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 thermoregulatory device that independently maintains the temperature at the required level.

Cultivation of microaerophilic bacteria. Although microaerophilic bacteria are aerobic 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, test tubes with crops are placed in a desiccator together with a bottle containing a cotton swab 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 oxygen burns out, and a decrease in its content is sufficient for the growth of microaerophiles.

The most accessible and effective method for cultivating microaerophiles in a semi-liquid medium with 0.1 ... 0.4% agar. In such an environment, convection flows are unable 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 culture of the microaerophile is inoculated with an injection, and the microorganism grows in a zone with an 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.

Cultivation of mushrooms. The best mushroom growth was noted on media with a carbohydrate content of 1 ... 4%. During initial isolation, antibiotics are often added to culture media to inhibit the growth of various accompanying bacteria.

Agar Sabouraud is used for the cultivation of 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 tubes and sterilized at 0.5 atm for 30 minutes. After sterilization, the pH of the medium is 6.9 ... 7.0.

Czapek agar is used for the cultivation of 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 ... Natural pH of the medium is 5.6 ... 5.9. The medium is sterilized at 0.5 ATM for 30 minutes.

Wort-agar is intended for cultivation of pathogens of dermatomycosis and candidiasis. Uncoated 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 agar is suitable for the cultivation of dermatophytes. Peptone - 10 g, glucose - 10 g, bovine 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 stachybotriotoxicosis, 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 mitz. Then the medium is moistened with sterile Petri dishes with filter paper.

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 medium is 5.9 ... 6.2. The liquid medium can be used to moisten the filter paper in Petri dishes for the subsequent cultivation of the mushrooms.

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

Sabouraud's glucose broth is used to cultivate many types of mushrooms. Glucose - 40 g, Peptone - South, distilled water - 1000 ml. Heated to boiling, poured into test tubes and sterilized at 1 atm for 15 minutes.

Cultivation on hair according to Van Breisegem is used to isolate dermatophytes. Healthy sterile hair is attached with collodion to a glass tube. A mushroom culture is applied to the middle of the hair. The tube is placed in a cylinder, on the bottom of which it is poured for moisture a small amount of water. Cultivated at 25 ° C for five to ten days or more.

To isolate the causative agents of histoplasmosis, epizootic lymphangitis, blood agar is used.

TASKS FOR INDEPENDENT WORK

1. Prepare beveled MPA, serum and blood
MPA.

2. Examine the structure of the anaerostat and fermenter.

Control questions

1.What General requirements present to nutrient media?

2. Into what groups are culture media classified?

How anaerobes and microaerophiles are cultivated

SPECIAL (ELECTIVE) NUTRIENT MEDIA

Yeast Growth Media

Reader's synthetic 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 the reproduction of yeast add 2% sugar, for the study of fermentation - 5-10%. The complete synthetic medium contains crystalline vitamins, μg / ml: inositol 5, biotin 0.0001, pantothenic acid 0.25, thiamine 1.0, pyridoxine 0.25, nicotinic acid 0.5. The medium is sterilized in an autoclave at a pressure of 0.1 M Pa for 20 minutes.

Glucose-ammonium environment

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, add yeast (0.2%) or meat (0.3%) extract and grape juice.

Synthetic medium for the detection of 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 shown. 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 lysine medium for the 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 is added, g: inositol 2, calcium pantothenate 0.4, nicotinamide 0.5, hydratthiamine 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 Yeast

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, poured into 5 ml test tubes and sterilized at a pressure of 0.05 MPa for 30 minutes.

Medium for the detection of extraneous yeast morphologically similar to the main culture

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

Medium for the formation of pseudomycelium

Glucose-peptone agar. To 1 liter of tap water add, g: peptone 10, glucose 20, agar 30-35. Sterilized 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 l with tap water, 20 g of glucose, 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 row")

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

The ability of yeast to assimilate carbohydrates by oxidation is investigated 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, agar slant is prepared. The growth of cultures is assessed after 3-4 days.

Yeast Sugar Agar

0.5% sodium chloride, 1% glucose (or 4 or 10% sucrose) and 2% agar, pH 6.8 (with glucose) and 6-6.5 (with sucrose) are dissolved in yeast water. 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 U / ml), penicillin (20-100 U / ml), levomycin (50 mg / L), neomycin (20 U / ml) and etc. They can be added to the environment both together and separately.

Media for ascospore formation

Wednesday Gorodkovoi. Contains in 1 liter of tap water, g: peptone 10, sodium chloride 5, glucose 1 (or 2.5), agar 20; pH of the medium 7.3. Poured into test tubes and sterilized 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 is dissolved, g: potassium hydrogen phosphate 1, potassium dihydrogen phosphate 0.25, magnesium sulfate 0.25, calcium chloride 0.05, agar 20. Sterilized at a pressure of 0.05 MPa for 15 minutes.

Medium for growing osmophilic yeast

For 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). Sterilized 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 settle for 2 hours. At the same time, coagulated colloids settle and the molasses solution is 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 the agar medium, 1.5-2% agar is added. Sterilized 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.

Growing media for 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, sterilized at a pressure of 0.1 MPa for 30 minutes.

Wednesday Czapek

The 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. Agar is leached and added to the specified ingredients, previously dissolved in 1 liter of distilled water, heated with flowing steam, set the pH to 4.0-5.5 with a 10% solution of citric acid or sodium hydroxide. Filtered, poured into test tubes and sterilized with fractional flowing steam 3 times for 30 minutes with an interval of 1 day.

Chapek-Doxa sugar-nitrate agar

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

Ammonia starch agar

The 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. Sterilized 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. Sterilized for 20 minutes at a pressure of 0.05 MPa or fractionally.

This medium is based on yeast water. To prepare yeast water, 70-100 g of fresh pressed yeast (7-10 g of dry yeast) is boiled for 20-30 minutes in 1 liter of distilled water and defended in a high cylinder in the cold for 12 hours. l of water, boil for 30 minutes, filter, adjust the pH to the required value. The prepared medium is sterilized fractionally for 20 min 2-3 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.

Wednesday can also be prepared in ordinary 1% peptone water.

Culture media for lactic acid bacteria

Hydrolyzed milk (according to Bogdanov)

Regular or skim (reverse) milk (pH 7.6-7.8) is boiled for 5 minutes, the vessel is shaken thoroughly and cooled to 45 ° C and 0.5-1 g of pancreatin is added to 1 liter, after 4-7 min. 5 ml of chloroform are added. Placed in a thermostat for 18-20 hours at a temperature of 40 ° C. Pancreatin powder should first be diluted in a little warm water. During the first hours, the milk is stirred several times with the cap open. The hydrolyzed milk is filtered through a paper filter, diluted 2-3 times with water, adjusted to pH 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 the hydrolyzed milk. The mixture is heated to boiling 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 hot with litmus tincture to an intense lilac color. Sterilize with flowing steam (3 times for 30 minutes with an interval of 1 day) or autoclaving for 10 minutes at a pressure of 0.1 MPa.

Malt wort with grain

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

Sucrose Yeast 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 medium 6-6.5.

Sprout 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 whipped 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 is 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 in 2 times and 2% glucose and 1% peptone are added to the broth, Poured into test tubes and sterilized at a pressure of 0.05 MPa for 15 minutes. To obtain a dense medium, add 2% agar.

Wednesday MRS (Wednesday de Mans)

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, Difco 5 yeast extract, meat extract 10, glucose 20, tween- 80 1 ml, medium pH 6-6.5. The medium is filtered and sterilized fractionally for 30 minutes 3 times with an interval of 1 day or in an autoclave at a pressure of 0.05 MPa for 20 minutes. Applied in liquid, semi-liquid and agar form for childbirth Leuconostoc and Lactobacillus.

MRS media (modified by A.A.Lantsier)

Wednesday МРС-1. 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 ( dissolved separately in a small amount of hot distilled water), yeast autolysate (see Appendix 2) 50 ml, liver extract 100 ml. The volume of the liquid is brought to 500 ml with distilled water and 500 ml of hydrolyzed skimmed milk of Bogdanov, previously not sterilized, pH 6.2-6.8, is added. The medium is filtered and sterilized with fractional flowing steam.

Wednesday MRS-2. 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, which creates more anaerobic conditions than a liquid one.

Wednesday MRS-3. Designed for the "motley row" in the identification of lactic acid bacteria. It is based on the MRS-1 medium, but without glucose, hepatic extract and hydrolyzed milk. Carbohydrates and polyhydric alcohols are added in an amount of 0.5%. The amount of agar applied is 0.15%. pH of the medium 7.0. Chlorophenol red (0.004%) serves as an indicator. 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 in the range of pH 4.8-6.4.

Hepatic extract

Fresh beef liver is finely cut and poured with water (for 1 kg of liver, 1 liter of water). The mixture is boiled for 30 minutes and filtered, after which it is sterilized 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 specified order in the malt wort (for Lactobacillus) or yeast water (for Leuconostoc). In the first case, the pH of the medium 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, 0.75 ml of tween-80 and 37.5 g of glucose are dissolved while heating, 5 ml of yeast autolysate, 600 ml of skim milk (skim milk) and 150 ml of melted 2% mesopatamia agar are added. The pH is adjusted 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% mesopatamia agar is layered on top. In the case of weak gas formation, the plug is separated from the main medium, with strong gas formation, it rises high or flies out of the test tube.

Media for the cultivation of mucus-forming bacteria

Option 1. Meat-concrete 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 bromcresol purple 1.4 ml, pH 6.8-7.0. Sterilize with flowing steam 3 times for 45 minutes with an interval of 1 day.

Media for growing putrefactive asporogenic 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% aqueous agar, pour 4-5 ml into test tubes and sterilize for 30 minutes at a pressure of 0.1 MPa. Agar is melted, sterilely combined with milk and poured into Petri dishes, where the test sample is previously introduced.

Growth media for fat-splitting bacteria

Option I. 5 g of peptone and 3 ml of yeast autolysate are added to 1 l of water. Having adjusted the pH to 7.2-7.4, 1.5% agar is added. The agar is melted, the medium is filtered and 1% of hot milk fat is added or olive oil... The mixture is stirred, poured into test tubes and sterilized at a pressure of 0.1 MPa for 15 minutes.

Option 2. To mesopatamia agar add 2-4% milk fat or olive oil. Pour 10 ml into test tubes and sterilize at a pressure of 0.1 MPa for 20 minutes. Shake the medium thoroughly before plating.

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

Growth media for 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.

Anaerobic culture media

Wednesday Vinogradsky. 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 dipped into a test tube so that they cover the bottom. Pour mesopatamia broth with 1% glucose (pH 7.2-7.4) per "/ 2 of the volume of the test tube and lower the float. Pour a 1 cm layer of vaseline oil on top. Sterilize for 15 minutes at a pressure of 0.1 MPa 2 times with an interval 30 minutes.

Growing medium for thermophilic anaerobes producing 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 inoculating the sugar, a layer of sterile petroleum jelly is poured into the test tube. In the presence of hydrogen sulfide-forming bacteria in the sugar, characteristic black colonies are formed in the agar.

microorganism anaerobic cultivation microbiology

For the cultivation of microorganisms, nutrient media of different composition are used, which must contain all the substances necessary for growth. The needs of microorganisms for nutrients are extremely diverse and are determined by the characteristics of their metabolism. Therefore, there are no universal media equally suitable for the growth of all microorganisms.

In the broadest sense of the word, the culture medium must meet the following requirements:

1) include cell accessible energy source. For some organisms (phototrophs), such a source is light, for others - an organic (chemoorganotrophs) or inorganic (chemolithotrophs) substrate.

2) contain all the necessary components for the implementation of design processes in a cage. Moreover, the synthetic abilities of microorganisms can vary from the use of carbon dioxide as the only source of carbon (autotrophs) to the need for more reduced carbon compounds - acids, alcohols, carbohydrates, etc. (heterotrophs).

In the narrow sense of the word any artificial culture medium must meet the following requirements : contain all nutrients necessary for growth in an easily digestible form; have optimal moisture content, optimal viscosity, optimal pH (optimal for a specific microorganism), be isotonic, balanced with a high buffer capacity and, if possible, transparent.

A nutrient medium in microbiology, media are called media containing various compounds of complex or simple composition, which are used for the reproduction of microorganisms, their cultivation and preservation in laboratory or industrial conditions.

The choice of the composition of the nutrient medium depends largely on the goals of the experiment or the industrial process.

There is the following classification of culture media:

· By composition nutrient media are divided into natural, synthetic and semi-synthetic... They can also be classified as environments a certain and undefined composition. Natural are called environments that consist of products of plant or animal origin, having uncertain chemical composition ... Examples of nutrient media of this type are media that are a mixture of degradation products of proteins (casein, mammalian muscles) formed during their hydrolysis. Nutrient media of undetermined composition can also include media obtained on the basis of plant raw materials: potato agar, tomato agar, decoctions of cereals, yeast, beer wort, hay and straw infusions, etc. The main purpose of such nutrient media is isolation, cultivation, biomass production, etc. maintaining cultures of microorganisms.

Among the media undefined composition include environments semi-synthetic... Known compounds are introduced into such an environment as clearly necessary; and also add a small amount of yeast or corn extract (or any other natural product) to meet unknown growth needs. Such media are often used in the case of industrial cultivation of biological objects for the production of metabolic products, for example, for the production of amino acids, antibiotics, vitamins, etc.

Synthetic media are environments certain composition, represented by pure chemical compounds, taken in precisely specified concentrations and ratios of individual elements. Essential components of such media are inorganic compounds (salts) and carbon- and nitrogen-containing substances (typical representatives are glucose and (NH 4) 2 SO 4. Buffer solutions and chelating compounds are often added to such media. Main purpose of such nutrient media - the study of the physiology and metabolism of microorganisms, the isolation of genetic recombinants, etc.

· By appointment environments are divided into the main, elective (selective) and differential diagnostic... TO the main includes media used to grow many bacteria. It is a nutrient broth and nutrient agar. Such media serve as the basis for the preparation of more complex nutrient media. Elective environments provide the preferential development of one or a whole physiological group of microorganisms. For example, to isolate staphylococci, sodium chloride at a concentration of 7.5% can be added to the medium. At this concentration, the growth of other bacteria is inhibited. Elective media are used at the first stage of isolating a pure culture of bacteria, i.e. upon receipt of a cumulative culture.

Differential diagnostic environments are used for the rapid identification of closely related species of microorganisms, to determine the species, in clinical bacteriology, etc. The composition of the differential diagnostic medium includes: a) the main nutrient medium that ensures the reproduction of bacteria; b) a certain chemical substrate, the attitude to which is a diagnostic feature for a given microorganism; c) a colored indicator, the change in color of which indicates a biochemical reaction and the presence of this enzyme system in the microorganism under study. For example, Endo medium can distinguish clones that ferment lactose from clones that do not have this property.

· By consistency environments can be liquid, semi-liquid, solid, free-flowing. Liquid culture media obtained by dissolving in water a certain required set of nutrients, macro- and microelements. In composition, they can be both natural and synthetic.

· Solid state media in the form of dense gels have been used in bacteriology since the time of R. Koch. The most important advantage of using solid media is that microorganisms can be grown on them in the form of colonies formed from individual cells of the population. The preparation of solid nutrient media is achieved by adding certain seals to the liquid media, which can be agar, gelatin, silica gel, carrageenan.

Semi-liquid media contain a gelling agent in low (0.3 - 0.7%) concentration and have a soft jelly-like consistency. Such media are suitable for the study of cell motility and chemotaxis, cultivation microaerophiles.

Loose media are a mass of crushed and moistened raw materials (usually vegetable) to one degree or another. Their main purpose is to use in the food industry (obtaining soy sauce or rice vodka), agriculture(fodder ensiling), etc.

In bacteriological practice, dry nutrient media are most often used, which are obtained on an industrial scale - tryptic hydrolysates of cheap non-food products (fish waste, meat and bone meal, technical casein) with the addition of agar. Dry media are a fairly cheap raw material, can be stored for a long time, are convenient for transportation, have a relatively standard composition, and can be used to quickly and easily prepare culture media on their basis.

The need for the cultivation of microbes is associated not only with the purpose of isolating the causative agent of the disease from the test material and determining its type, but also for the accumulation of microbial mass in the manufacture of biological preparations: vaccines, antigens and allergens. For the cultivation of microorganisms in the laboratory, various artificial nutrient media are used.

Nutrient media are: by consistency - liquid, dense, semi-liquid; by origin - animal, vegetable and synthetic media of constant composition; by appointment - ordinary, or simple, for the cultivation of most microorganisms; special - for the cultivation of microbes that do not grow or grow poorly on conventional nutrient media; differential diagnostic - used to determine the generic or species characteristics of the studied bacterial cultures (hemolytic, saccharolytic, proteolytic, reducing and other properties); selective - for the isolation of microbes of the same genus or species from the material containing the mixture different types microorganisms on which some species grow well, while others do not; enrichment environments (accumulative).

Meat water is the basis of many nutrient media of animal origin. It is prepared from fresh, low-fat beef meat freed from bones, fascia, tendons. Chopped meat (small pieces or minced meat) is poured with distilled water in a ratio of 1: 2 (for 1 kg of meat, 2 liters of water). It is extracted for 12-24 hours, boiled for 1.5-2 hours, filtered, distilled water is added to the original volume, poured into bottles, flasks, closed with cotton-gauze plugs and sterilized in an autoclave.

Regular (simple) environments.

Meat Peptone Broth (BCH)- a liquid nutrient medium. For its preparation, 1% of peptone, 0.5% of chemically pure sodium chloride are added to 1 liter of meat water and boiled. Meat water is weakly acidic, so the BCH is made alkaline - add a small amount of 10-15% KOH or NaOH solution, boil for 2-3 minutes, check the pH using a Michaelns comparator (Fig. 30) or an electropotentiometer. The meat-peptone broth is filtered through a paper filter, poured into test tubes, and autoclaved.

Meat Peptone Agar (MPA)- dense nutrient medium. 2% agar-agar (nitrogen-free organic matter obtained from seaweed) is added to the MPB and boiled until it melts, the pH is set hot, boiled for 5-10 minutes, filtered hot through a cotton-gauze filter, poured into test tubes or cones, autoclavable. After sterilization, hot agar tubes are laid obliquely at an angle of 5-6 °. When solidified, a beveled dense surface is formed.

Meat-peptone semi-liquid agar prepare in the same way as MPA; the difference is that less agar is added - 0.15-0.20-0.25%.

Special nutrient media.

Bouillon Martin. Pork stomachs (or cattle abomasums) are cleaned of fat, fascia, minced in a meat grinder, filled with water 1: 4 and 1% (by volume of liquid) hydrochloric acid is added. The mixture is kept at 50 ° for 24 h, neutralized with a 20% NaOH solution until alkaline by litmus, and autoclaved at 120 ° for 15 min. To prepare the broth, mix equal amounts of meat water and the resulting mixture, boil for 10 minutes, alkalinize with 20% NaOH solution to pH 7.9, boil for 30 minutes, filter, pour into test tubes, autoclave at 0.5 atm (110 °) 30 min.

The addition of 2% agar provides a dense medium - Martin's agar.

Hottinger's broth prepared from tryptic hydrolyzate (digestion) of meat waste - fascia, fat, tendons. .1 kg of meat scraps are poured into 2 liters of boiling water, boiled for 5 minutes, cooled to 45 ° and pancreatin 5.0-10.0 is added, alkalinized to pH 7.8-8.0 with sodium carbonate, shaken and chloroform is added (10 ml 1 l), tightly closed, kept in a warm place for 10 days, shaking daily. The resulting digest is acidified with hydrochloric acid to pH 5.5. Store in a dark place. To prepare a nutrient medium, 100-200 ml of the resulting digestion is added to 1 liter of water, boiled for 1-2 minutes, filtered, alkalinized, sterilized. Hottinger's Agar is prepared in the same way as regular MPA.

Sugar (glucose) broth (or agar) are made as usual media, but 1-2% glucose is added to them and sterilized with flowing steam or autoclaved at 0.5 atm.

Meat-peptone gelatin. 10-20% gelatin (a product obtained from adhesive animal tissues) is added to the MPB, melted and hot set the desired reaction of the medium, passed through a cotton-gauze filter, poured into test tubes, sterilized with flowing steam fractionally.

Meat-Peptone Liver Broth Kitt - Tarozzi- a liquid medium for the cultivation of anaerobes. The liver of cattle is cut into small pieces, poured with water 1: 1, boiled, filtered and liver water is added to the MPB 2: 1 (for 2 liters of MPB 1 liter of liver broth), boiled, set the pH, poured into tubes in a high column, into which pre-put pieces of boiled liver. 1-2 ml of vaseline oil is poured into test tubes on top of the medium and sterilized in an autoclave at 0.5 atm for 30 minutes. ^

Whey broth and serum-precise meat-peptone agar is prepared by aseptic addition to BCH or molten n cooled to 45-50 ° MPA 5-10% sterile horse blood serum (or ram, rabbit), then the whey broth is poured into sterile tubes (flasks).

Serum MPA poured or into test tubes (column). or in Petri dishes.

Differential diagnostic environments. Blood MPA is used to reveal the hemolytic properties of bacteria. Beforehand, blood is aseptically taken into a sterile flask with glass beads (from a ram, horse or rabbit), shaken for 15-20 minutes to separate fibrin from the rest of the blood. Fibrin clots are deposited on beads and defibrinated blood (5-10%) is sterilely added to the melted and cooled MPA, the cones are evenly mixed by gentle rotation and poured into sterile Petri dishes.

Wednesday of Giss. In peptone water (consisting of distilled water, 0.5% NaCl and 1% peptone) add 0.5% carbohydrate (sugar or polyhydric alcohol) and 0.5% Andrede's indicator. Usually, a set of media is prepared with different carbohydrates, each separately. The indicator used is 0.5 g of acidic fuchsin, 16 ml of 4% NaOH solution, 100 ml of distilled water. Media with carbohydrates are poured into test tubes with "gas tubes" (floats) dipped in test tubes upside down. Sterilize media with carbohydrates with fluid steam fractionally. ... Giss media can be liquid and semi-liquid - (without gas stoves), containing 0.25% agar. When a particular carbohydrate is fermented by a microbe growing in a given environment, an acid is formed, under the influence of which the color of the indicator dye is restored. The medium turns red. The gaseous products formed during the fermentation of the carbohydrate accumulate in the floats.

Wednesday Endo. In molten MPA (pH 7.4-7.6), 0.5-1% lactose and 0.5% saturated alcoholic solution of basic fuchsin are added, discolored by adding dropwise 10% sodium sulfate. The medium is boiled and poured into Petri dishes. Lactose fermenting bacteria grow on this medium in the form of red colonies.

Wednesday Levin. Prepare 2% agar on Hottinger's broth, pH 7.2-7.4, sterilize in an autoclave. To 100 ml of ready-made molten agar add: 2 ml of a 0.5% solution of methylene blue, 1.5 ml of 2% eosin (alkaline bacteriological), 2 g of lactose and 0.2 g of dibasic potassium phosphate (NaHPO4). Dye solutions are prepared in distilled water and sterilized in a Koch apparatus. A solution of methylene blue before use is heated in a water bath and added to the agar in a warm form. After adding these components, the medium is thoroughly mixed and poured into Petri dishes. The medium is purple.

Bismuth sulfite agar(Wednesday Wilson-Blair). MPA (pH 7.5) with an indicator containing citric acid bismuth, sodium sulfate, Mohr's salt (ammonium sulfate of iron), dibasic sodium phosphate, glucose and brilliant green. Currently, bismuth-sulfite agar (like Endo agar, Ploskirev's medium) is produced by the industry in dry powder form. 6 g of dry powder is dissolved in 100 ml of distilled water, heated with continuous stirring, poured into sterile test tubes and left in an inclined position. They also use media with chemicals that change color as a result of redox (reducing) processes caused by bacterial enzymes. ... For this, milk is prepared with methylene blue. Fresh skimmed cow's milk is made alkaline with sodium bicarbonate to a slightly alkaline reaction according to litmus paper, a 1% aqueous solution of methylene blue and blue color is added. Sterilized with flowing steam fractionally.

If there is an assumption that the test material contains a small amount of bacteria, then it is recommended to use an accumulation (enrichment) medium. With Shustova: 10% 50% aqueous solution of hyposulfite and 2% Lugol's solution are added to MPA (pH 7.4). Used for the accumulation of paratyphoid bacteria. Wednesday Rappoport: add 1% glucose, 10% bile and 1% Andrade's indicator to the BCH. Sterilized with flowing steam.

Synthetic media used to study the metabolism of bacteria and other biological features. They are composed of chemically pure water-soluble substances in strictly defined quantities: ammonium phosphate, potassium phosphate, sodium chloride, magnesium sulfate, glucose or other carbohydrate, nicotinamide, etc. Dense synthetic media are prepared as needed by adding agar. Sterilized in an autoclave. (Synthetic media Model, Sotton

For the cultivation of yeast and molds, the following media are used

Van Iterson Synthetic Medium: ammonium nitrate (NaH4NO3) 0.5, potassium phosphate monobasic (KH2PO4) 0.5, tap water 1 l. Autoclave at 1 ATM for 20 minutes.

Sabouraud Glucose Agar: glucose 4.0, peptone 1.0, agar 1.8, water 100 ml. Sterilized by autoclaving.

Litman agar with bovine bile: peptone 10.0, glucose 10.0, dehydrated bovine bile 15 ml, crystal violet 0.01, water 1 l, agar 20.0. Sterilized in an autoclave at 1 ATM for 15 minutes. Pour into * cups in a thick layer to prevent dehydration of the medium.