Cultivation, differentiation and isolation of certain types of microorganisms became possible only with the use of nutrient media. These are special substances that create favorable conditions for the reproduction and growth of a certain type of microbes and fungi, that is, pure cultures, which opens up the possibility of studying their properties and effects on the body.
Today, nutrient media are used both in medicine and in other areas, for example, in the food industry, where different kinds microorganisms to improve food quality, increase shelf life, taste and aromatic properties. Thus, pure cultures isolated through the use of nutrient media are used in bakery production, in the wine and vodka industry, in the creation of cheeses and dairy products, in the production of organic acids, in the fermentation and preservation of vegetables and fruits, and in pharmacology.
However, the majority of microbiological research is in the medical sector. That is why clinics and laboratories are the main buyers of substrates.
BioVitrum is a leading manufacturer and supplier of medical, diagnostic and laboratory equipment, as well as consumables. One of the activities is the production of nutrient media for the cultivation and cultivation of microorganisms. The company's products are made from high quality raw materials, which contain components of horse and sheep blood. The advantage of source material supplied from the UK is the special conditions in which the animals are raised. Their food does not contain antibiotics, which guarantees the sterility of culture media.
BioVitrum offers to buy nutrient media of all types that meet European standards.
Environment Requirements
Substrates produced today, used for the cultivation, differentiation and isolation of microorganisms, must meet certain criteria:
- Nutrition . The environment is vital for the nutrition and energy needs of the crops grown. Namely, it contains vitamins, mineral and organic (natural) substances, trace elements included in cellular composition and enzyme-activating and non-naturally produced amino acids.
- The presence of hydrogen ions. Since microorganisms are able to feed only if the cell membrane is permeable, an optimal pH balance is required to ensure it.
For pathogenic microbes, a weakly alkaline nutrient medium is needed; for pathogens of tuberculosis, a weakly acidic reaction is suitable.
- Buffer environment. This property ensures the stability of the pH balance, neutralizing the decay products.
- Isotonicity is an indicator of pressure inside the cell and in the environment, which should be at the same level for most microorganisms.
- Sterility of the environment. It is important to exclude the presence of foreign microbes that can affect the growth of the crop of interest.
Media must have humidity, so dense and dry media require pre-treatment. The media must have redox characteristics, transparency. Such an indicator as uniformity provides the possibility of growing various microorganisms.
Classification
Each culture needs certain conditions for abundant cell reproduction, their intensive growth and optimal development, so it is difficult to create a universal environment. In addition, depending on the objectives of the research, the parameters of the nutrient medium change.
Today, several types of media have been created that differ in properties:
- According to the composition of the initial components, they are classified into synthetic and natural. The latter are made from vegetable or animal raw materials. To reduce costs, non-food products are used, such as bone meal or coagulated blood clots. Synthetic is obtained from organic, that is, natural and mineral components.
- According to the consistency, the media are divided into liquid, dense, semi-liquid. The increase in density is carried out by adding gelatin or agar-agar. The last ingredient is not a nutrient for microbes, its task is to create optimal density. At the same time, the melting point of agar reaches 80-100 degrees, which makes it possible to grow microorganisms that need to create greenhouse conditions on media containing agar. Gelatin is an animal protein, so substrates containing it can be used at room temperature. Dense substances include coagulated blood serum, egg white, potatoes. Some are produced in the form of powders and require dissolution and bringing to the desired consistency before use.
- The composition of nutrient media is simple and complex. The former are meat-and-peptoid broths, nutritious gelatin, and peptoid waters. The second type, in addition to the original components, includes substances that promote the growth of certain bacteria. There are also special media used where bacteria cannot grow on a normal substance.
- Elective nutrient media. They are used to isolate a specific type of bacteria by inhibiting the growth of others. Liquid media of this category are called accumulative.
- Differential diagnostic media are used to isolate one culture by its enzymatic activity. Preservative substrates are used when transporting materials to the study site.
Media preparation
The accuracy of the results obtained depends on the quality of the nutrient medium, therefore, in addition to following the recipe, it is necessary to adhere to the following requirements:
- Sterile dishes. In industrial conditions, where the production is carried out on a large scale, the cooking of media is carried out in special boilers. In laboratories, when it is necessary to obtain a small amount of culture media, enamel or glassware should be used that do not emit acids and alkalis. All tanks are pre-washed, rinsed and dried.
- Previously unused glassware is sterilized in hydrochloric acid, in which it is left overnight, after which it is rinsed in accordance with the established regimen.
The process of preparing nutrient media itself consists of the following steps:
- Cooking, which is carried out either on fire and a water bath (for laboratory conditions), or in boilers and autoclaves with steam supply (on an industrial scale).
- Setting the optimal pH ratio requires the use of paper indicators, potentiometers, glass electrodes.
- Clarification is carried out by introducing into the substrate, beaten with water, egg white or blood serum, which, during the cooking process, entrain suspended particles into the sediment.
- Liquid media are subjected to filtration, and based on molten gelatin. To do this, use fabric and paper moistened filters. Purification of agar substrates is significantly more difficult, since the main component solidifies quickly. Therefore, this process is often replaced by settling.
- Sterilization is carried out for each substrate in a certain period of time and at the required temperature. These parameters are specified in the cooking recipe.
The final stage is the packaging of nutrient media in dishes - these are vials, test tubes, Petri dishes. The vessel is only 2/3 filled because during sterilization the medium expands in volume and can reach the plug, which will affect the purity and properties of the substrate.
The product is poured using funnels, syringes, pipettes or other devices.
Each vessel is labelled. The name of the product is applied to the vessels, the quantity and date of production are indicated.
The finished environment goes through several stages of control. The first test for sterility is carried out by placing the product in a thermostat.
The nutrient medium is sent to laboratories for chemical testing, the purpose of which is to establish an accurate pH level.
Biological control is carried out, which consists in determining the nutritional qualities of the medium.
Types of culture media
According to their purpose, the nutrient compositions of the medium produced today can be divided into the following categories:
- Universal environments. They are suitable for propagation of various types of crops.
- Selective or selective environments. Used to isolate one type of microorganism.
- Differential diagnostic media, which open up the possibility of distinguishing bacteria by their enzymatic properties, that is, waste products.
- Special environments. They are used to grow those crops that are unable to reproduce on universal substrates.
- Differential selective media are used for rapid identification of bacteria.
- Semi-synthetic nutrient compositions of the medium, which include components of natural origin.
BioVitrum offers a wide range of nutrient media, which are supplied both in vials of various sizes and ready-made in Petri dishes. The latter are sealed with a specially designed cellulose film, which ensures the sterility of the medium and a shelf life of up to 60 days.
The advantage of BioVitrum is prompt delivery, competitive prices, compliance with GOST standards. All nutrient compositions of the medium are manufactured at our own facilities, equipped with high-tech equipment from Oxoid LTD raw materials from a well-known UK manufacturer.
In BioVitrum you can purchase 18 products listed in the catalog, which are subject to multi-stage control. Products are supplied with a complete set of documentation and markings in Russian.
100 r first order bonus
Select the type of work Course work Abstract Master's thesis Report on practice Article Report Review Test Monograph Problem solving Business plan Answers to questions creative work Essay Drawing Essays Translation Presentations Typing Other Increasing the uniqueness of the text PhD thesis Laboratory work Help online
Ask for a price
In microbiology, a nutrient medium is a medium containing various compounds of complex or simple composition, which are used for the reproduction of microorganisms in laboratory or
industrial conditions. Any culture medium must comply with the following
requirements: contain all the nutrients necessary for growth in an easily digestible form; have optimal moisture, viscosity, pH, be isotonic, balanced with a high buffer
capacity and, if possible, transparent. Substances that cells are unable to synthesize on their own - called growth factors. Organisms that need their addition n-Xia auxotrophic for the respective connections. A group capable of growth on simple environments, prototrophic. Oligotrophic(on the poor), the opposite group for them is bacteria copiotrophic– able to grow on rich food substrates.
Composition nutrient media are divided into natural and synthetic. Natural media are media that consist of products of plant or animal origin that have an indefinite chemical composition. Examples - a mixture of degradation products of proteins formed during their hydrolysis. The action of enzymes such as trypsin, pancreatin, papain leads only to partial (incomplete) hydrolysis of proteins, resulting in the formation of peptones. The main purpose of such nutrient media is isolation, cultivation, biomass production and culture maintenance.
The media of indeterminate composition also include media 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 industrial cultivation of biological objects to obtain metabolic products.
Synthetic media- these are media of a certain composition, represented by pure chemical compounds taken in precisely specified concentrations and ratios of individual elements.
The main purpose of such nutrient media is to study the features of physiology and metabolism.
microorganisms, isolation of genetic recombinants, etc.
By appointment: Elective environments provide development of one or the whole physiological group of microorganism. Differential diagnostic environments are used for rapid identification of closely related microorganism species, for species identification, in clinical bacteriology. The principle of constructing differential-diagnostic media is based on the fact that different types of bacteria differ from each other in biochemical activity and have an unequal set of enzymes that break down the substrates that make up the nutrient medium.
The composition of d.-d. include: a) the main nutrient medium that ensures the reproduction of bacteria; b) a specific chemical substrate, the relationship to which is a diagnostic feature for a given microorganism; c) a color indicator, the color change of which indicates a biochemical reaction and the presence of this enzyme system in the microorganism under study.
By consistency environments can be liquid, semi-liquid,solid, loose. Liquid products are obtained by dissolving a certain essential set of nutrients in water. in-in, macro- and microelements. The growth of microorganisms in a liquid medium can occur in periodic (closed) system, in this case, after the inoculation of the medium, neither the addition nor the removal of any components, except for the gas phase, occurs. At flowing (continuous) cultivation is characterized by a constant supply of fresh nutrients at a rate equal to the rate of removal of the medium ( open system). The preparation of solid media is achieved by adding certain sealants to liquid media (agar, gelatin, silica gel, carrageenan). Loose media are used in industrial microbiology for the cultivation of certain producers of physiologically active compounds. Such media include, for example, boiled millet, bran.
Part Hiss environments includes a basic background (peptone and K2HPO4), an indicator (bromothymol blue, bromocresol purple, Andrede) and one of the carbohydrates or alcohols studied. Distinguish between small and
a large motley row of Hiss. The small Hiss series includes the following carbohydrates and alcohols: glucose, sucrose, lactose, maltose, and mannitol. The large motley series of Giss includes, in addition to those that form a small series, other carbohydrates and alcohols, such as arabinose, rhamnose, sorbitol, dulcite, etc. Giss media can be used in a liquid or semi-liquid state (0 is added to the liquid medium, 5% agar)
The release of metabolic products is recorded by changing the pH of the medium. For example, if the Hiss medium contains the bromthymol blue indicator, then its color will change depending on the pH as follows: pH = 7.0 - green; pH > 7.0 - blue; pH< 7,0 – желтый.
microorganism anaerobic cultivation microbiology
For the cultivation of microorganisms, nutrient media of various 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 a broad sense, the nutrient medium must meet the following requirements:
1) include cell accessible energy source. For some organisms (phototrophs) this source is light, for others it is an organic (chemoorganotrophs) or inorganic (chemolithotrophs) substrate.
2) contain all necessary components for the implementation of constructive 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 growth medium must meet the following requirements : contain all the nutrients necessary for growth in an easily digestible form; have optimal moisture content, optimal viscosity, optimal pH (optimal for a particular microorganism), be isotonic, balanced with high buffering capacity and, if possible, transparent.
Nutrient medium in microbiology, media containing various compounds of complex or simple composition are called, 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 to a large extent on the objectives of the experiment or industrial process.
There is the following classification of nutrient media:
· Composition nutrient media are divided into natural, synthetic and semi-synthetic. They can also be classified as media certain and indefinite composition. natural are called media that consist of products of plant or animal origin that have undetermined 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 indeterminate composition can also include media obtained on the basis of plant materials: potato agar, tomato agar, decoctions of cereals, yeast, beer wort, infusions of hay and straw, etc. The main purpose of such nutrient media is isolation, cultivation, obtaining biomass and maintenance of cultures of microorganisms.
Among the environments indeterminate 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 industrial cultivation of biological objects to obtain metabolic products, for example, to obtain 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. Mandatory components of such environments are inorganic compounds(salts) and carbon- and nitrogen-containing substances (typical representatives are glucose and (NH 4) 2 SO 4. Often, such media are added buffer solutions and chelating compounds. Main purpose such nutrient media - the study of the characteristics of the physiology and metabolism of microorganisms, the isolation of genetic recombinants, etc.
· By appointment environments are divided into main, elective (selective) and differential diagnostic. To main include the media used to grow many bacteria. This is nutrient broth and nutrient agar. Such media serve as the basis for the preparation of more complex culture media. Elective environments provide preferential development of one or the 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. when receiving an enrichment culture.
Differential diagnostic environments are used for rapid identification of closely related species of microorganisms, for determining 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 specific chemical substrate, the relationship to which is a diagnostic feature for a given microorganism; c) a color indicator, the color change of which indicates a biochemical reaction and the presence of this enzyme system in the microorganism under study. For example, Endo's medium makes it possible to distinguish clones that ferment lactose from clones that do not.
· By consistency environments can be liquid, semi-liquid, solid, friable. Liquid culture media obtained by dissolving in water a certain essential set of nutrients, macro- and microelements. In composition, they can be both natural and synthetic.
· Media in the solid state 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 in a population. The preparation of solid nutrient media is achieved by adding certain sealants to liquid media, which can be agar, gelatin, silica gel, carrageenan.
Semi-liquid media contain a gel-forming substance in a low (0.3 - 0.7%) concentration and have a soft jelly-like consistency. Such media are suitable for studying cell motility and chemotaxis, cultivating microaerophiles.
Bulk environments are a mass of more or less crushed and moistened raw materials (usually vegetable). Their main purpose is the use in the food industry (obtaining soy sauce or rice vodka), agriculture(ensiling fodder), 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 fairly cheap raw materials, can be stored for a long time, are convenient for transportation, have a relatively standard composition, and culture media can be quickly and easily prepared on their basis.
Media for growing anaerobic microbes.Meat peptone liver broth Keith - Tarozzi (MPPB). Fresh or frozen liver (preferably cattle) is cut into small pieces, poured with an equal mass of tap water, boiled for an hour, filtered through cotton wool and added to 1 part of the obtained extract 3 parts of meat-peptone broth. The mixture is heated to a boil, chemically pure common salt is added (1.25 g per 1 liter of medium) and the pH is adjusted to 7.6-7.8, after which it is boiled for 15 minutes and filtered through a paper or moistened cotton filter. Finely chopped pieces (1.5-2 g) of liver are added to the filtered broth, based on 1 liter of broth 100 g of liver (the liver is preliminarily cleaned of films and washed with water). Several such pieces are placed in a test tube, 7-10 ml of broth is poured in a high column, vaseline or paraffin oil is layered on its surface.
The broth with liver pieces is sterilized under an overpressure of 0.1 MPa in within 30 min. In order to remove oxygen from the test tube, the medium is boiled for 10 minutes before inoculation and quickly cooled with water.
Semi-fluid agar for anaerobes. To the MPB add 0.25-0.75% agar-agar and 1% glucose; medium pH 7.4. The medium is poured in high columns into test tubes. Sterilized with fractional flowing steam for 15-20 minutes for 3 days.
Media for growing lactic acid bacteria.Milk (whole). Heat up to a boil. Pour into a tube flask and put for 10-20 hours in a cold place to settling the cream. After this time, the skimmed part of the milk is poured through the tap of the tube into test tubes, closed with cotton plugs. Sterilize fractionally at 100 ° C for three days for 20 minutes or at 112 ° C once for 30 minutes.
Skimmed milk. To obtain skimmed milk, whole milk is separated and then proceed in the same way as when using whole milk.
Hydrolyzed milk (according to Bogdanov). Take 1 liter boiled and cooled to 45 ° C skim milk, set the pH to 7.6-7.8, add 0.5 g of pancreatin powder (previously diluted in a small amount warm water) or 2-3 g of crushed pancreas and after a few minutes 5 ml of chloroform. After that, the bottle is thoroughly shaken, tightly closed with a cork stopper and placed for 3 days in a thermostat at a temperature of 40 ° C with daily shaking of the liquid. After the specified period, in order to remove chloroform, the bottle is opened, the liquid is filtered and diluted 2-3 times with tap water. Bring the pH of the medium to 7.0-7.2 and sterilize.
Agar from hydrolyzed milk. 1.5-2% agar is added to hydrolyzed milk, melted, poured into test tubes and sterilized under an overpressure of 0.1 MPa in within 15 min. Lactic acid sticks grow well on this medium.
Serum agar. 7.5 g of agar are taken per 100 ml of tap water, boiled until completely dissolved, water is added to the original volume (i.e., in a volume equal to the volume of evaporated water), 400 ml of pre-prepared whey is added, exposed to flowing steam for 30 min, filtered through a layer of cotton wool, poured into test tubes and sterilized under a pressure of 0.05 MPa for 30 minutes.
Cabbage Wednesday. 200 g of chopped cabbage (or alfalfa) is poured into 100 ml of water and boiled in a saucepan for 10 minutes, squeezed through a double layer of gauze. The resulting liquid is filtered and diluted 2 times with tap water. 2% glucose and 1% peptone are added, poured into test tubes and sterilized three times with an overpressure of 0.05 MPa for 15 minutes.
Medium for growing osmophilic yeast. Approximately 1 liter of distilled water is added with 200 g of preheated honey, 1 g of potassium diphosphate, 0.5 g of magnesium sulfate, 0.5 g of ammonium tartrate, 0.1 g of sodium chloride and 0.1 g of potassium chloride. All components are mixed and sterilized under an overpressure of 0.1 MPa for 20 minutes.
Growth medium for halophiles. Usual meat-peptone media are used with the addition of 10-15 to 20-30% salt. In addition, in the manufacture of dense nutrient media, the percentage of agar is also increased. Sterilization is carried out under an overpressure of 0.1 MPa for 20 minutes.
enrichment media. Mueller Wednesday. To 4.5 g of chemically pure chalk, previously sterilized by dry heat, add 90 ml of MPB and sterilize under an overpressure of 0.1 MPa for 20 minutes. Prepare: a) hyposulfite solution (50 g of pure crystalline hyposulfite is poured up to 100 ml with distilled water, sterilized with flowing steam for 30 minutes); b) iodine solution (20 g of metallic iodine and 25 g of potassium iodide are poured up to 100 ml with distilled water). Before sowing, 10 ml of hyposulfite solution and 2 ml of iodine solution are sterilely added to the broth with chalk. Shake the mixture as you add each ingredient. Pour into sterile test tubes or flasks.
Wednesday Killian. Sterile addition of 1 ml to 100 ml of regular BCH before use aqueous solution(1:1000) brilliant green.
Bacteria research requires meticulous work with numerous equipment and instruments. In order for microorganisms to multiply as quickly as possible in laboratory conditions and to be able to maintain normal vital activity, special nutrient media are used. Their composition and biophysical conditions are suitable for the active growth of a bacterial culture.
nutrient media. Microbiology and other applications
Colonies of bacteria in the laboratory are grown on Petri dishes, which are filled with jelly-like or semi-liquid contents. These are nutrient media, the composition and properties of which are as close as possible to natural ones for the qualitative growth of culture.
For example, such an elective environment is suitable only for the reproduction of Escherichia coli. Then, from the sowing of many bacteria on a Petri dish, we will see only colonies of the same Escherichia coli and no more. Before starting work, it is necessary to have a good knowledge of the metabolism of the studied bacterium in order to successfully select it from a mixture of other species.
Solid, semi-solid and liquid culture media
Bacteria can be grown not only on solid substrates. Nutrient media differ from each other in terms of state of aggregation, which depends on the composition during manufacture. Initially, they all have a liquid consistency, and when gelatin or agar is added in a certain percentage, the mixture solidifies.
Liquid culture media are usually found in test tubes. If it becomes necessary to grow bacteria under such conditions, add a solution with a culture sample and wait 2-3 days. The result may be different: a precipitate appears, a film appears, small flakes float, or a cloudy solution forms.
Solid growth media is often used in microbiological research to study the properties of bacterial colonies. Such media are always transparent or translucent, so that it is possible to correctly determine the color and shape of the culture of microorganisms.
Media preparation
It is very easy to prepare such substrates as meat-peptone mixtures based on broth, gelatin or agar. If you need to make a solid or semi-liquid substrate, add 2-3% or 0.2-0.3% gelatin or agar, respectively, to the liquid. They play a major role in the hardening of the mixture, but they are not a source of nutrients. Thus, nutrient media are obtained that are suitable for the growth of a bacterial culture.