1. Nutrient requirements of microorganisms
Cultivation of microorganisms- This is one of the main techniques in microbiology. For the growth and development of microorganisms in nature and in laboratory conditions, the presence of nutrients for energetic and constructive reactions is necessary. Requirements different groups The relationship of microorganisms to energy sources and chemical elements is determined by their metabolic capabilities. Growing and maintaining microbial cultures in the laboratory is based on modeling the natural living conditions of a given organism in the laboratory, as well as on knowledge of the characteristics of metabolism.
The main biogenic elements are carbon, nitrogen, phosphorus, oxygen, hydrogen, sulfur. These are components of proteins, carbohydrates and fats, as well as nucleic acids. These elements are required in significant quantities (g/l) and are therefore called macroelements. Macroelements also include potassium, magnesium, sodium, calcium and iron ions. They perform various functions in the cell. For example, K + is necessary for the activity of a large number of enzymes and in particular enzymes of protein synthesis. Ca 2+ determines the resistance of bacterial endospores to heat. Mg 2+ stabilizes ribosomes, many enzymes and cell membranes. Fe 2+ and Fe 3+ are part of cytochromes and cofactors of electron transfer proteins.
Trace elements needed in micromolar quantities are metal ions such as chromium, cobalt, copper, molybdenum, manganese, nickel, selenium, tungsten, vanadium, zinc, usually found in enzymes and cofactors. For example, Co 2+ is a component of vitamin B 12, Cu 2+ is part of cytochrome oxidase and cupredoxins, Mn 2+ activates enzymes that catalyze the transfer of phosphate groups, Mo 2+ is part of nitrogenase and nitrate reductase, Ni 2+ is a component of urease, hydrogenase , cofactor F 430, Zn 2+ is part of carbonic anhydrase, DNA and RNA polymerases, etc. The amounts of microelements necessary for microorganisms are contained in ordinary tap water. When working with distilled water, microelements are added specifically in the form of solutions of their mineral salts. Some groups of microorganisms exhibit specific needs. Thus, diatoms, which include in their cell walls significant amounts of silicon compounds require adding them to the medium in high concentration.
Biogenic elements must be present in the nutrient medium in a form accessible to microorganisms. Typically, metal ions, sulfur, phosphorus and trace elements are added to the medium in the form of mineral salts. The mineral basis of the environment (mineral background) is almost the same for most microorganisms.
Sources of carbon and nitrogen in the environment can be both inorganic compounds (CO 2 , N 2 , carbonates, nitrites, nitrates, ammonium salts) and organic substances of varying degrees of complexity and oxidation (sugars, alcohols, organic acids and amino acids, oligosaccharides, peptides etc.). If a microorganism requires a set of carbon or nitrogen sources, then various extracts and hydrolysates of a mixture of proteins and polysaccharides of uncertain composition are used (wort, milk protein hydrolysate, peptone, etc.).
Typically, laboratory environments contain nutrients in higher concentrations than are found in natural habitats. For different microorganisms, the limits of the values of physicochemical factors in which growth can occur differ significantly. Therefore, an important condition for successful cultivation is maintaining optimal values of parameters such as pH, temperature, light, aeration, etc.
2. Types of media and methods of cultivating microorganisms
Varied nutrient media, used in microbiological practice for the cultivation of microorganisms, are divided according to composition, physical condition and purpose.
Based on the composition of the media, they are divided into natural and synthetic. Synthetic media are used to study metabolism in microorganisms. They have a specific chemical composition with an exact indication of the concentration of each compound. Natural media are used to accumulate microbial biomass and are widely used for primary isolation from natural substrates, since their composition allows them to satisfy the nutritional needs of many groups of microorganisms. They contain products of animal or plant origin, rich in various organic substances, having a complex and variable composition. Natural media are often prepared on the basis of meat-peptone broth (MPB) and malt wort. MPB is a boiled extract of minced meat with the addition of peptone and table salt. It is rich in nitrogen-containing organic compounds, but poor in carbohydrates. Malt wort, on the other hand, contains predominantly carbohydrates. It is obtained by infusing ground malt in tap water with gradual heating. Malt is the name given to sprouted and dried barley grains. During the preparation of wort, barley starch is hydrolyzed and sugars are extracted into water. Depending on the batch of grain, the concentration of sugars in the wort may vary. It is expressed in degrees Balling (o B), which approximately corresponds to the percentage of sugars in the solution. Wort with different concentrations of sugars is used to grow different groups of microorganisms.
Liquid media They are solutions or suspensions of ingredients in water. As bulk media, sets of long-term stored dry components are used, which are dissolved or moistened with water before use. This can be grain, bran, solid waste from agriculture and the food industry. Currently, powdered synthetic and natural media are widely used. To obtain solid media, thickening agents are added to the liquid base. The most well-known hardeners are gelatin, agar and silica gel. Gelatin is a protein from animal connective tissue that forms a gel at 25 o C. The inconvenience of its use is that the growth temperature of many microorganisms is higher than the melting point of gelatin. The presence of proteolytic enzymes in many microorganisms leads to the breakdown and liquefaction of gelatin. The complex polysaccharide agar, obtained from sea brown algae, is more convenient as a sealant, since most microorganisms do not use it for nutrition. Agar can repeatedly melt at 100 o C and solidify at 45 o C. By adding 2% agar to a liquid base, the widely used meat-peptone agar (MPA), wort agar (SA) and broth-wort agar (BSA) are obtained. The inorganic silicon compound silica gel is often used as a solid base for synthetic media.
Based on their purpose, media are divided into universal, selective and indicator. Universal media are used for the accumulation of microbial cells and the initial identification of the species diversity of microorganisms in mixed populations. They allow the growth of a significant number of microorganisms to be maintained. At the same time, it should be remembered that there is no one environment that is universal for all microbial cultures. Elective media are used to obtain enrichment cultures as the first stage in isolating a pure culture from natural habitats. Creating conditions favorable for a certain group of microorganisms (elective conditions) leads to the predominance of the desired microorganisms in the mixed population. The growth and reproduction of other microorganisms under these conditions is not significant. To quickly identify certain groups of microorganisms or the characteristics of their metabolism, indicator media are used that contain an indicator substance that reacts by changing color to the manifestation of any property of the organism. Indicator media are most often used in sanitary and medical microbiology.
3. Methods for cultivating microorganisms
The growth characteristics of a microorganism (cultural properties) sometimes serve as one of the criteria in determining its systematic position. Depending on the conditions, microbial cells can grow in the form of a suspension, microcolonies or fouling in liquid media and form colonies, streaks or a lawn on solid media. Deep colonies form in the thickness of agar media in the form of lentils, thin films or bundles of cotton wool. Due to the release of gases by microorganisms during deep growth, ruptures in the agar medium may occur. Surface colonies are distinguished by a wide variety of shapes, sizes, colors, and profiles. The colony can be transparent, dense, soft, fragile, grow into agar, be removed entirely in the form of a film, stretch behind a loop, etc. Its surface can be shiny or matte, smooth or rough, have various convexities, striations, etc. Differences in edge shape and colony structure can be seen at low microscope magnification. The morphology of colonies can vary significantly depending on the composition of the medium, the age of the culture and the cultivation temperature. When sown with a streak (straight line on agar), growth can be abundant or sparse, continuous or in the form of chains of very small colonies, feathery, tree-like with a different edge shape. When a culture develops in liquid media, the development of a microorganism can lead to coloration of the medium and the appearance of an odor, the formation of foam and bubbles, the appearance of turbidity, a film on the surface of the medium or sediment at the bottom of the vessel.
There are two main methods of cultivating microorganisms - periodic and continuous. At batch cultivation the cells are placed in a closed vessel of a certain volume containing a nutrient medium, and the initial conditions are set. The population density gradually increases, the concentration of nutrients decreases and metabolic products accumulate, i.e. the conditions for the existence of microorganisms change. A periodic culture is usually viewed as a closed system experiencing different phases of development. Each phase is characterized by certain physiological parameters. The lag phase is the phase of “acclimation” of cells to the environment, during which an increase in the amount of DNA and RNA occurs and the induction of the synthesis of the corresponding enzymes. The lag phase is lengthened if you take old seed material and transfer the cells to a completely new medium. The lag phase is shortened (or may be completely absent) if active young cells are transferred to a fresh medium of the same composition and temperature. On media containing a mixture of substrates, diauxia is observed, in which, after the depletion of one substrate, the culture enters a second lag phase in preparation for the consumption of another substrate. In the exponential (logarithmic) phase, cells grow and divide at maximum speed, their growth is not limited. Typically, such cells are used in biochemical and physiological studies. As substrates are exhausted and metabolic products accumulate, the growth rate decreases (growth slowdown phase) and the culture enters the stationary phase, during which the processes of cell division and cell death in the population are in dynamic equilibrium. For bacteria, this phase is achieved at an average concentration of 10 9 cells/ml, for algae and protozoa - 10 6 cells/ml. When the depletion of nutrients and the accumulation of metabolic products overcome certain threshold concentrations, the death phase begins and the number of cells in the population gradually decreases.
Continuous (flow) cultivation allows you to fix a culture in a certain phase (usually exponential). At the same time, the composition of the medium and growth conditions remain constant. This is achieved by constantly adding new nutrient medium to the growing vessel and simultaneously removing the same amount of medium with cells. The simplest diagram of the organization of the duct is shown in Fig. 45. The supply of fresh medium and the removal of part of the suspension (duct) occurs at the same speed as the culture grows. In this case, dynamic equilibrium is established.
Some microorganisms are capable of remaining in a special physiological state in which living cells do not form colonies in laboratory media suitable for them, but are observed under a microscope as living. This uncultivable state (uncultivable form) is characteristic of a number of microorganisms in natural habitats, for example, the causative agents of salmonellosis and cholera found outside the human body. The mechanism of transition to an uncultivated form and back has not been studied, but there is evidence that this process is programmed in the genome of microorganisms and is triggered by a lack of nutrients in natural econiches. In natural samples, such microorganisms are studied by direct observation and by molecular analysis of the nucleic acid composition of the sample.
4. Mixed and pure cultures of microorganisms. Cumulative crops. Methods for obtaining pure cultures
Due to the small size of microorganisms, work in the laboratory is carried out not with one individual, but with a population of organisms, or a culture. A culture of microorganisms consisting of cells of one type is called a pure culture . If the number of species is two or more, then they speak of a mixed culture. To determine the systematic position, physiological and biochemical properties and characteristics of the development of microorganisms, it is necessary to obtain a pure culture. To do this, cells of a given species must be separated from cells of other species and subsequently exclude the possibility of foreign microorganisms entering. When isolating a pure culture from natural habitats, where microorganisms in most cases grow in the form of mixed populations, at the first stage they usually use the method proposed by S.N. Vinogradsky for obtaining enrichment cultures in which organisms of a certain group predominate. The accumulation of desired microorganisms occurs due to the creation of selective cultivation conditions favorable for this group. To do this, it is necessary to take into account the physiological and biochemical characteristics of the isolated culture. Selective inhibition of the growth of certain groups of microorganisms can be achieved by introducing antibiotics into the environment. The group of microorganisms for which the cultivation conditions created by the researcher are most acceptable will prevail. Other organisms, also present in the sample, do not reproduce under these conditions or are characterized by insignificant growth. For example, to obtain an enrichment culture of nitrogen-fixing microorganisms, a medium should be prepared without bound forms of nitrogen. To slow down the development of gram-positive bacteria, you can add penicillin, and filamentous fungi - nystatin or griseofulvin. To accumulate spore-forming microbes, short-term heating of the sample at high temperature (10 minutes at 80 o C) is often used, when the vegetative cells die and the endospores retain their viability. It is necessary to take into account that selective conditions are not always the best (optimal) for the growth of the isolated group, however, accompanying microorganisms tolerate them even worse. The receipt of an enrichment culture is judged by the characteristic microscopic picture, external changes in the environment, and the appearance of certain metabolic products. A pure culture can later be obtained from a single cell or from a separate colony. The cell is removed using a micropipette or microloop under microscopic control and transferred to a vessel with medium. Another method is to prepare a series of hanging drop preparations from a highly diluted suspension. The preparations are viewed under a microscope and those where one cell is present are selected. They are then placed in a humid chamber and microscoped again a day later. Drops in which cell multiplication has occurred are transferred to a nutrient medium. More often they use the method of isolating a pure culture from a separate colony, developed in the laboratory of R. Koch. A drop of an enrichment culture or its dilution is distributed over the surface or in the depth of a solid nutrient medium, achieving the separation of individual cells. Each such cell subsequently multiplies, forming a colony of cells of the same type. It is removed with a loop and transferred to a vessel with a nutrient medium. A sign of the purity of a culture is the uniformity of colonies during subcultures and the morphological uniformity of cells when viewing microscopic preparations.
Nutrient media in microbiology
Requirements for nutrient media: 1. Nutrient media must contain universal sources of carbon and nitrogen. 2. They must be a source of vitamins and minerals. 3. In media, pH must be maintained at a constant level, which is ensured by the presence of buffer systems in nutrient media. 4. The nutrient medium must be sterile. 5. The nutrient medium must be transparent. 6. It must have an optimal concentration of oxygen and carbon dioxide.
Classification of culture media for bacteria
1. By consistency. Based on consistency, all nutrient media are divided into liquid, semi-liquid and solid. For liquid nutrient media, the basis is meat water, protein hydrolysates, or natural products (blood, milk). The media acquire a dense consistency by adding agar to them. Agar is also added to semi-liquid media, but it is much less than in solid nutrient media. 2. By origin. Based on their origin, all environments are divided into artificial and natural. The basis of artificial nutrient media is peptones, while natural ones are represented by milk, blood, may include pieces of fruit, etc. 3. As intended. According to their purpose, all media are divided into universal, differential diagnostic, selective and special. All bacteria (more precisely, the majority) grow well on universal nutrient media. An example of universal nutrient media is meat-peptone broth and meat-peptone agar. Differential diagnostic media allow one species of bacteria to be distinguished from another species by their enzymatic activity or cultural properties. Differential diagnostic media are His, Clark, Endo, and Ploskirev media. Selective media allow you to select certain types of bacteria, since they contain substances that inhibit the growth of other bacteria, but at the same time promote the growth of this type of bacteria. Selective environments are also called selective, selective or enrichment. Examples of selective media are 1% peptone water and Mueller's medium. Special media are designed for the growth of bacteria that do not grow on universal nutrient media. Examples of special media are McCoy-Chapin medium (for the causative agent of tularemia), Levenstein-Jensen medium (for Mycobacterium tuberculosis), blood meat-peptone agar (for streptococci).
According to the nature of respiration, all microbes are divided into aerobes and anaerobes. Aerobes require oxygen to survive. Their breathing process proceeds according to the type of oxidative reaction. Anaerobes grow and reproduce in conditions that exclude access to air oxygen. Molecular oxygen has a toxic effect on them. Anaerobes obtain the energy they need for existence by breaking down organic and inorganic compounds that make up the nutrient medium. Between the extreme groups of obligate, i.e., strict aerobes and anaerobes, there are microorganisms that can change the aerobic type of respiration to anaerobic, depending on the environment. Such microorganisms are called facultative, i.e. conditional, anaerobes. These include the vast majority of pathogenic microorganisms. To grow anaerobes, it is necessary to create certain conditions, the essence of which is to remove molecular oxygen from the nutrient medium and the space surrounding these cultures.
Another mandatory condition that ensures the isolation of anaerobes from the test material is the introduction large quantity seed material into a nutrient medium. The only difference between the nutrient media used for growing anaerobes is their lower content of free oxygen. The easiest way to remove dissolved oxygen is boiling. Immediately before sowing the material, test tubes with nutrient media are boiled in a water bath for 10-20 minutes. When boiling, air is displaced from the medium and, therefore, oxygen is removed.
The freshly boiled nutrient medium is quickly cooled by immersing it in ice or placing it under running cold water to prevent it from being saturated with air oxygen, and is used for sowing. To reduce the diffusion of oxygen from the air, nutrient media are poured on top with sterile petroleum jelly or paraffin oil (layer thickness 1-1.5 cm). Inoculation of the medium is carried out with a pipette through the oil in an inclined position of the test tube. Glucose, ascorbic acid, cysteine, glycol, and glutathione are used as reducing substances. Animal tissues of parenchymal organs actively bind to oxygen. The preparation of the Kitta-Tarozzi nutrient medium (rec. 113), widely used for growing anaerobes, is based on this property of animal cells. Porous substances are sometimes placed in liquid nutrient media: cotton wool, pumice, which adsorb air bubbles on their surface. To create oxygen-free conditions, physical, chemical and biological factors are used. Physical methods for cultivating anaerobes.
1. Vignal-Veion method. Take 4-5 test tubes with 0.5% sugar agar (rec. 114) melted and cooled to a temperature of 40-45 °C. A small amount of the test material is pipetted into the contents of one of them and mixed thoroughly. To reduce the concentration of material in order to obtain isolated colonies, the inoculated medium in an amount corresponding to the volume of introduced material is transferred from the 1st tube to the 2nd, from the 2nd to the 3rd. Then the contents of each test tube are filled into the capillaries of three Pasteur pipettes. To prevent the nutrient medium from solidifying at the moment of sucking it into the pipettes, their tip, until it is broken off, is immersed for 3-5 minutes in sterile water at a temperature of 45-50 ° C. Once filled, the extended end of the tube is sealed and placed in a glass cylinder with cotton wool at the bottom. After 2-3 days, clearly visible colonies of anaerobic microbes grow in the agar column. Grown colonies are easy to isolate. To do this, the capillary is cut with a file above the level of the intended colony, broken, and the microbial colony located in the agar is removed with a loop and replanted in a fresh nutrient medium.
2. Growing anaerobes under vacuum conditions. Vacuum conditions for growing anaerobes are created in an anaerostat or desiccator. The test material or microbial culture is inoculated into test tubes with a liquid medium or into Petri dishes with a dense nutrient medium. The crops are placed in an anaerostat, then connected to a pump and the air is pumped out. The degree of air rarefaction is determined by the readings of a vacuum gauge. Colonies of anaerobes grow under vacuum conditions on the surface of a dense nutrient medium. Chemical methods for growing anaerobes (Aristovsky method).
The material to be tested for the presence of anaerobes is inoculated onto the medium in Petri dishes and placed in a desiccator, at the bottom of which a chemical oxygen absorber is placed: sodium hydrosulfite or pyrogallol. Cups with crops are placed on a stand in the expanded part of the vessel. The device is placed in a thermostat at a temperature of 37 ° C for 24-48 hours. Biological method of growing anaerobes (according to Fortner). A thick layer of 5% blood agar with 1-2% glucose is poured into a Petri dish. In the middle of the cup in the nutrient medium, a groove 1-1.5 cm wide is cut with a sterile scalpel, which divides the nutrient medium into two halves. One of them is inoculated with a culture of anaerobes or material tested for their presence, the other half is inoculated with a culture of aerobes: miraculous bacillus (Serratia marcescens) or Escherichia coli (E. coli).
Before sowing, the cups are dried in a thermostat so that aerobes, along with droplets of moisture, cannot get to the other side of the cup. The inoculated cups are closed, and the free space between the bottom and the lid is sealed with an adhesive plaster to prevent oxygen from entering the cup from the outside. In the thermostat, the cups are placed upside down. Fast-growing aerobes, absorbing the oxygen in the cup, thereby creating favorable conditions for the growth of anaerobes. Anaerostat for the cultivation of anaerobes. Anaerostat - a device for growing microbes under anaerobic conditions - is a thick-walled metal cylinder with a hermetically screwed lid, on which there is a vacuum gauge and two taps for connection to a vacuum pump.
Physiology and principles of cultivation of microorganisms.
Metabolism of microorganisms.
To grow and reproduce, microorganisms need substances used to build the structural components of the cell and obtain energy. Metabolism(i.e. metabolism and energy) has two components - anabolism And catabolism. Anabolism - synthesis of cell components ( constructive exchange). Catabolism is an energy metabolism associated with redox reactions, the breakdown of glucose and other organic compounds, and the synthesis of ATP. Nutrients can enter the cell in soluble form (this is typical for prokaryotes) - osmotrofy, or in the form of individual particles - phagotrophs.
The main regulator of the entry of substances into bacterial cell is the cytoplasmic membrane. There are four main mechanisms of substance entry: - passive diffusion- along a concentration gradient, energy-intensive, without substrate specificity;
- facilitated diffusion- along a concentration gradient, substrate-specific, energy-intensive, carried out with the participation of specialized proteins permease;
- active transport against a concentration gradient, substrate-specific (special binding proteins in complex with permeases), energy-consuming (due to ATP), substances enter the cell in a chemically unchanged form;
- translocation (group transfer) - against a concentration gradient, using the phosphotransferase system, energy-consuming, substances (mainly sugars) enter the cell in forphorylated form.
Basic chemical elements - organogens necessary for the synthesis of organic compounds - carbon, nitrogen, hydrogen, oxygen.
Depending on the source consumed carbon microbes are divided into autotrophs(use CO2) and heterotrophs(use ready-made organic compounds). Depending on the energy source microorganisms are divided into phototrophs(energy is obtained through photosynthesis - for example, cyanobacteria) and chemotrophs(energy is produced through chemical, redox reactions). If in this case the electron donors are inorganic compounds, then this lithotrophs, if organic- organotrophs. If a bacterial cell is able to synthesize all the substances necessary for life, then this prototrophs. If bacteria need additional substances (growth factors), then this auxotrophs. The main growth factors for difficult-to-cultivate bacteria are purine and pyrimidine bases, vitamins, some (usually essential) amino acids, blood factors (hemin), etc.
Respiration of microorganisms.
Microorganisms obtain energy through respiration. Respiration is the biological process of transferring electrons through the respiratory chain from donors to acceptors with the formation of ATP. Depending on what is the final electron acceptor, there are aerobic and anaerobic respiration. In aerobic respiration, the final electron acceptor is molecular oxygen (O 2), in anaerobic respiration, bound oxygen (-NO 3, =SO 4, =SO 3).
Aerobic respiration hydrogen donor H 2 O
Anaerobic respiration
nitrate oxidation of NO 3
(facultative anaerobes) hydrogen donor N 2
sulfate oxidation of SO 4
(obligate anaerobes) hydrogen donor H 2 S
By breathing type There are four groups of microorganisms.
1.Obligate(strict) aerobes. They need molecular (atmospheric) oxygen to breathe.
2.Microaerophiles require a reduced concentration (low partial pressure) of free oxygen. To create these conditions, CO 2 is usually added to the gas mixture for cultivation, for example up to a 10 percent concentration.
3.Facultative anaerobes can consume glucose and reproduce under aerobic and anaerobic conditions. Among them there are microorganisms that are tolerant to relatively high (close to atmospheric) concentrations of molecular oxygen - i.e. aerotolerant, as well as microorganisms that are capable of switching from anaerobic to aerobic respiration under certain conditions.
4.Strict anaerobes reproduce only under anaerobic conditions i.e. at very low concentrations of molecular oxygen, which in high concentrations is destructive for them. Biochemically, anaerobic respiration proceeds according to the type of fermentation processes; molecular oxygen is not used.
Aerobic respiration is energetically more efficient (more ATP is synthesized).
In the process of aerobic respiration, toxic oxidation products are formed (H 2 O 2 - hydrogen peroxide, -O 2 - free oxygen radicals), from which specific enzymes protect, primarily catalase, peroxidase, peroxide dismutase. Anaerobes lack these enzymes, as do redox potential regulation system (rH 2 ).
Basic methods for creating anaerobic conditions for the cultivation of microorganisms.
1. Physical - pumping out air, introducing a special oxygen-free gas mixture (usually N 2 - 85%, CO 2 - 10%, H 2 - 5%).
2. Chemical - chemical oxygen absorbers are used.
3. Biological - joint cultivation of strict aerobes and anaerobes (aerobes absorb oxygen and create conditions for the proliferation of anaerobes).
4. Mixed - they use several different approaches.
It should be noted that creating optimal conditions for strict anaerobes is a very difficult task. It is very difficult to ensure constant maintenance of oxygen-free cultivation conditions; special media without dissolved oxygen are required, maintenance of the necessary redox potential of nutrient media, collection and delivery, and sowing of material under anaerobic conditions.
There are a number of techniques that provide more suitable conditions for anaerobes - pre-boiling nutrient media, sowing in a deep agar column, filling the media with petroleum jelly to reduce the access of oxygen, using hermetically sealed bottles and test tubes, syringes and laboratory glassware with inert gas, using tightly closed desiccators with a burning candle. Special devices are used to create anaerobic conditions - anaerostats. However, at present, the simplest and most effective equipment for creating anaerobic and microaerophilic conditions is the Gazpak system with special gas regenerating packages operating on the principle of displacing atmospheric air gas mixtures in hermetically sealed containers.
Basic principles of cultivating microorganisms on nutrient media.
1.Use of all nutritional components necessary for the corresponding microbes.
2. Optimal temperature, pH, rH 2, ion concentration, degree of oxygen saturation, gas composition and pressure.
Microorganisms are cultivated on nutrient media at optimal temperatures in thermostats that provide incubation conditions.
By temperature optimum growth There are three main groups of microorganisms.
1.Psychrophiles - grow at temperatures below +20 degrees Celsius.
2. Mesophiles - grow in the temperature range from 20 to 45 degrees (often optimal at 37 degrees C).
3. Thermophiles - grow at temperatures above plus 45 degrees.
Brief characteristics of nutrient media.
By consistency secrete liquid, solid (1.5-3% agar) and semi-liquid (0.3-0.7% agar) media.
Agar- polysaccharide of complex composition from seaweed, the main hardener for dense (solid) media. Used as a universal source of carbon and nitrogen peptones- products of protein fermentation with pepsin, various hydrolysates- meat, fish, casein, yeast, etc.
By purpose environments are divided into a number of groups:
Universal (simple), suitable for various undemanding microorganisms (meat-peptone broth - MPB, meat-peptone agar - MPA);
Special media for microorganisms that do not grow on universal media (McCoy's medium for tularemia, Lowenstein-Jensen's medium for the causative agent of tuberculosis);
Differential diagnostic - for differentiating microorganisms by enzymatic activity and cultural properties (Endo, Ploskirev, Levin, Giss media);
Selective (elective) - for isolating certain types of microorganisms and suppressing the growth of associated ones - peptone water, selenite medium, Muller's medium.
By origin media are divided into natural, semi-synthetic and synthetic.
Growth and reproduction of microorganisms.
Bacterial cells multiply by division. The main stages of microbial reproduction in a liquid medium under stationary conditions:
Lag phase (initial stage of adaptation with a slow growth rate of bacterial biomass);
Exponential (geometric growth) phase with a sharp increase in the population of microorganisms (2 in power n);
Stationary phase (phase of equilibrium of reproduction and death of microbial cells);
The death stage is a decrease in population size due to a decrease and lack of conditions for the reproduction of microorganisms (nutrient deficiency, changes in pH, rH 2, ion concentrations and other cultivation conditions).
This dynamic is typical for periodic crops with gradual depletion of nutrients and accumulation of metabolites.
If conditions are created in the nutrient medium to maintain the microbial population in the exponential phase, this is chemostat (continuous) cultures.
Growth pattern bacteria on solid and liquid nutrient media: continuous growth, formation of colonies, sediment, film, turbidity.
Pure culture- a population of one type of microorganism.
The basic principles of obtaining pure cultures: mechanical separation, sieving, serial dilutions, the use of election media, special cultivation conditions (taking into account the resistance of some microbes to certain temperatures, acids, alkalis, partial pressure of oxygen, pH, and many others).
The main equipment of a microbiological laboratory is a thermostat, an oven, an autoclave and a scale.
Thermostat - a device for maintaining a constant temperature - is used for growing cultures of microorganisms. It is a cabinet (Fig. 14), in which a certain temperature is maintained for a long time. 
A drying cabinet (Fig. 15) is used to sterilize dishes, equipment, etc. with dry heat. The material to be sterilized is first wrapped in paper and placed in the cabinet so that it does not touch the walls. Sterilization is carried out at a temperature of 160 °C for 2 hours. The sterilized material is removed after turning off and cooling the cabinet
The Koch apparatus is used to sterilize culture media. It is a metal cylinder with a flat bottom and a cone-shaped lid that has a hole for steam to escape. The device is covered with heat-insulating material. Vessels with nutrient media are placed on a stand located inside the apparatus.
An autoclave (Fig. 16) is used to sterilize dishes and culture media with steam under pressure. This is a sealed cauldron with double metal walls and a lid. It is equipped with a pressure gauge, safety valves and a tap for draining water and steam. Used to sterilize culture media under pressure of 0.5-1.0 MPa for 20-30 minutes.
It is necessary to have technical and analytical scales in the laboratory. Technical ones have an accuracy of up to 0.01 g; analytical - up to 0.001 g.
In addition, centrifuges and stirrers, pH meters for determining the acidity of semi-finished products, a Koch apparatus, etc. are used. Glassware used in a microbiological laboratory include test tubes, graduated cylinders, flasks, Petri dishes, etc.
Petri dishes (Fig. 17) are used to grow cultures of microorganisms on solid nutrient media.
Using pipettes, liquid cultures of microorganisms are reseeded.
The equipment in the microbiological laboratory is as follows: bacteriological loops and dissecting needles (Fig. 18), spatulas, pipettes, stands for pipettes and test tubes, a glass pencil, a set of brushes for washing dishes.
Rice. 18. Bacteriological loop and dissecting needle
Test tubes and flasks are used for storing nutrient media and growing microorganism cultures. Fermentation tubes are used to determine fermentation activity by gas production. Petri dishes are used for growing cultures of microorganisms on solid nutrient media. Bacteriological needles and loops are used for inoculating microorganisms, spatulas are used for spreading liquid cultures on the surface of a solid nutrient medium. Pillettes are necessary for reseeding liquid cultures of microorganisms. Petri dishes, pipettes, spatulas, test tubes, flasks are wrapped in paper, placed in a drying cabinet without touching the walls, and sterilized at a temperature of 160 ° C for 2 hours. Loops and needles are sterilized by calcining them over a flame.
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Nutrient medium in microbiology refers to media containing various compounds of complex or simple composition that are used for the propagation of microorganisms in laboratory or
industrial conditions. Any nutrient medium must comply with the following
requirements: contain all the nutrients necessary for growth in an easily digestible form; have optimal humidity, viscosity, pH, be isotonic, balanced with high buffer
container and, if possible, transparent. Substances that cells are not able to synthesize on their own are called growth factors. Organisms that need their addition auxotrophic via the appropriate connections. A group capable of growth on simple media, prototrophic. Oligotrophic(for the poor), the opposite group for them are bacteria copiotrophic– capable of growing on rich food substrates.
By composition nutrient media are divided into natural And synthetic. Natural media are those that consist of products of plant or animal origin that have an uncertain chemical composition. Examples are a mixture of protein breakdown products 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 peptones. The main purpose of such nutrient media is isolation, cultivation, biomass production and maintenance of crops.
Media of uncertain composition also include media semi-synthetic. Known compounds are introduced into such an environment as clearly necessary; and small amounts of yeast or corn extract (or any other natural product) are added to meet unknown growth needs. Such media are often used in the case of 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 characteristics of physiology and metabolism.
microorganisms, isolation of genetic recombinants, etc.
By purpose: Elective environments ensure the development of one or an entire physiological group of microorganisms. Differential diagnostic media are used for rapid identification of closely related species of microorganisms, to determine species identity, in clinical bacteriology. The principle of constructing differential diagnostic environments is based on the fact that different types bacteria differ from each other in biochemical activity and have a different set of enzymes that break down the substrates that make up the nutrient medium.
The composition of the d.-d. includes: a) the main nutrient medium that ensures the proliferation of bacteria; b) a certain chemical substrate, the relationship to which is a diagnostic sign for a given microorganism; c) a color indicator, a change in color indicates a biochemical reaction and the presence of a given enzyme system in the microorganism under study.
By consistency environments can be liquid, semi-liquid,hard, friable. Liquids are obtained by dissolving a certain necessary set of nutrients in water V-c, macro- and microelements. The growth of microorganisms in a liquid medium can occur in periodic (closed) system, in this case, after inoculation of the medium, there is no addition or removal of any components other than the gas phase. At flow (continuous) cultivation is characterized by a constant supply of fresh nutritional components at a rate equal to the rate of removal of the medium ( open system). The preparation of solid media is achieved by adding certain compactors (agar, gelatin, silica gel, carrageenan) to liquid media. Bulk media are used in industrial microbiology for the cultivation of certain producers of physiologically active compounds. Such media include, for example, boiled millet and bran.
Part Hiss environment includes the main background (peptone and K2HPO4), an indicator (bromothymol blue, bromocresol purple, Andrede) and one of the carbohydrates or alcohols being studied. There are small and
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 Hiss includes, in addition to those that form a small series, other carbohydrates and alcohols, for example arabinose, rhamnose, sorbitol, dulcite, etc. Hiss media can be used in a liquid or semi-liquid state (0. 5% agar)
The release of metabolic products is recorded by changes in the pH of the environment. For example, if the Hiss medium contains the indicator bromothymol blue, then its color will change depending on the pH as follows: pH = 7.0 – green; pH > 7.0 – blue; pH< 7,0 – желтый.
Media for growing anaerobic microbes.Meat-peptone liver broth of China - Tarozzi (MPPB). Fresh or frozen liver (preferably from cattle) is cut into small pieces, poured with an equal amount of tap water, boiled for an hour, filtered through cotton wool and added to 1 part of the resulting extract 3 parts of meat-peptone broth. The mixture is heated to a boil, chemically pure table salt is added (1.25 g per 1 liter of medium) and the pH is adjusted to 7.6-7.8, then boiled for 15 minutes and filtered through a paper or moistened cotton filter. Finely chopped pieces (1.5-2 g) of liver are added to the filtered broth, at the rate of 100 g of liver per 1 liter of broth (the liver is first cleared of films and washed with water). Several such pieces are placed in a test tube, 7-10 ml of broth is poured into a high column, and Vaseline or paraffin oil is layered on its surface.
The broth with pieces of liver is sterilized under an excess pressure of 0.1 MPa V for 30 minutes. To remove oxygen from the test tube before inoculation, the medium is boiled for 10 minutes and quickly cooled with water.
Semi-solid agar for anaerobes. 0.25-0.75% agar-agar and 1% glucose are added to the MPB; pH of the environment is 7.4. The medium is poured into test tubes in high columns. Sterilize with fractionally flowing steam for 15-20 minutes for 3 days.
Media for growing lactic acid bacteria.Milk (whole). Heat to a boil. Pour into a tube bottle and place in a cold place for 10-20 hours to allow the cream to settle. After this time, the skimmed part of the milk is poured through the tube tap into test tubes and closed with cotton stoppers. Sterilize fractionally at 100°C for three days for 20 minutes or at 112°C once for 30 minutes.
Skimmed milk. To obtain skim milk, whole milk is separated and then proceeded in the same way as when using whole milk.
Hydrolyzed milk (according to Bogdanov). Take 1 liter of boiled And of skim milk cooled to 45° C, set the pH to 7.6-7.8, add 0.5 g of pancreatin powder (pre-diluted in a small amount warm water) or 2-3 g of crushed pancreas and after a few minutes 5 ml of chloroform. After this, the bottle is thoroughly shaken, tightly closed with a cork stopper and placed for 3 days in a thermostat at a temperature of 40 ° C with daily shaking of the liquid. After the specified period, in order to remove chloroform, the bottle is opened, the liquid is filtered and diluted 2-3 times with tap water. Adjust the pH of the medium to 7.0-7.2 and sterilize.
Hydrolyzed milk agar. 1.5-2% agar is added to hydrolyzed milk, melted, poured into test tubes and sterilized under an excess pressure of 0.1 MPa V for 15 minutes. Lactic acid bacilli grow well on this medium.
Whey agar. For 100 ml of tap water, take 7.5 g of agar, boil until completely dissolved, add water to the original volume (i.e. in a volume equal to the volume of evaporated water), add 400 ml of pre-prepared whey, expose to flowing steam for 30 min, filter through a layer of cotton wool, pour into test tubes And sterilize under pressure of 0.05 MPa for 30 minutes.
Cabbage Wednesday. 200 g of chopped cabbage (or alfalfa) are poured into 100 ml of water and boiled in a saucepan for 10 minutes, squeezed through a double layer of gauze. The resulting liquid is filtered and diluted 2 times with tap water. Add 2% glucose and 1% peptone, pour into test tubes and sterilize at three excess pressures of 0.05 MPa for 15 minutes.
Osmophilic yeast growth medium. Add 200 g of preheated honey, 1 g of potassium diphosphate, 0.5 g of magnesium sulfate, 0.5 g of ammonium tartrate, 0.1 g of sodium chloride and 0.1 g of potassium chloride to approximately 1 liter of distilled water. All components are mixed and sterilized under an excess pressure of 0.1 MPa for 20 minutes.
Halophile growing medium. Use ordinary meat-peptone media with the addition of 10-15 to 20-30% table salt. In addition, when producing solid nutrient media, the percentage of agar is increased. Sterilization is carried out under an excess pressure of 0.1 MPa for 20 minutes.
Enrichment media. Muller's environment. To 4.5 g of chemically pure chalk, previously sterilized with dry heat, add 90 ml of MPB and sterilize under an excess pressure of 0.1 MPa for 20 minutes. Prepare: a) hyposulfite solution (50 g of pure crystalline hyposulfite is poured to 100 ml with distilled water, sterilized with flowing steam for 30 minutes); b) iodine solution (20 g of metallic iodine and 25 g of potassium iodide are poured into 100 ml of distilled water). Before sowing, 10 ml of hyposulfite solution and 2 ml of iodine solution are sterilely added to the broth with chalk. Shake the mixture as each ingredient is added. Pour into sterile test tubes or flasks.
Wednesday Killian. To 100 ml of regular MPB sterilely add 1 ml before use aqueous solution(1:1000) brilliant green.
2. Classification of culture media
When preparing nutrient media, it is necessary to take into account the need of cultivated microorganisms for various nutrients. There are different classifications of culture media.
Classification of nutrient media by composition:
1. Simple Environments(MPB, MPA, gelatin, peptone water). Meat-peptone broth (MPB) is the protein basis of all media. There are several ways to prepare MPB:
a) in meat water with the addition of ready-made peptone (a product of incomplete digestion of protein) - this is the so-called meat-peptone broth;
b) on the digestion of hydrolysis products of raw materials using enzymes (trypsin - Hottinger broth, pepsin - Martin broth).
Meat-peptone agar (MPA) - obtained by adding arap-arapa (l.5-3%) to MPB. If the MPA is distributed diagonally in a test tube or bottle, it is a slanted agar. If the medium is distributed vertically in a test tube with a height of 5-7 cm, it is a columnar agar. MPA frozen in Petri dishes in the form of a plate - plate agar. If the medium has a vertical layer 2-3 cm high and a diagonal layer of the same size, it is semi-slope agar.
2. Complex environments prepared on a simple basis with certain additives (carbohydrates, blood, bile, eggs, whey, milk, salts, growth factors, etc.)
Classification of nutrient media according to initial components:
1.Natural culture media is a natural product of animal or plant origin. Can be:
Vegetable (initial products - soybeans, peas, potatoes, carrots, etc.)
Animals (initial products - meat, fish, eggs, milk, animal tissues, bile, blood serum, etc.)
Mixed (MPA, Levenshtein-Jensen environment, etc.)
2. Built 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 diverse group of media in composition. They are prepared according to certain recipes from various infusions or decoctions of animal or plant 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 established concentrations (with the addition of carbohydrates, salts, amino acids, vitamins, etc.). Based on these media, adding natural or artificial media to them, semi-synthetic media are obtained.
Classification of nutrient media by consistency: there are environments 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 and to accumulate biomass and metabolic products. Semi-liquid media are usually used for storing cultures, solid media for isolating microorganisms, studying the morphology of colonies, diagnostic purposes, quantitative recording, determining antagonistic properties, etc.
Classification of nutrient media according to intended purpose: universal (commonly used) 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 to them blood, sugar, milk, whey and other ingredients necessary for the reproduction of a particular 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.
The following types of special media are distinguished: enrichment media, selective media, differential diagnostic media, preservative media, and accumulation media.
1. Enrichment media. Many microorganisms do not grow on regular media, so carbohydrates (sugar broth or agar) or proteins (whey agar and broth, blood agar and broth) are added to increase the nutritional value of the medium. Blood agar or blood broth - prepared by adding to a nutrient medium 5-10% of warmed sterile defibrinated blood of a sheep, rabbit, horse, human. 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 plain media. The medium is used to isolate pneumococci and meningococci.
2. Elective (selective) environments. These media are designed to selectively isolate and accumulate microorganisms of a specific type 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 selective will appear first. The selectivity of the environment is achieved by creating conditions that are optimal for the cultivation of certain microbes (pH, Eh, salt concentration, nutrient composition), i.e. positive selection. Or by adding substances to the environment that inhibit other microorganisms (bile, high concentrations of NaCl, antibiotics, etc.), i.e. negative selection. This group includes:
Selenite environment- is better environment enrichment for salmonella and dysentery microbes Sonne. Sodium selenite contained in the medium stimulates the growth of these bacteria and inhibits the growth of associated flora.
Bismuth sulfite agar– contains bismuth salts, brilliant green. Salmonella grows on this medium in the form of black colonies. Other types of bacteria do not grow on this medium.
Yolk salt agar (YSA)– the medium for isolating staphylococci contains up to 10% sodium chloride, which suppresses most of the bacteria contained in the material. In addition, this medium is also differential diagnostic, since the presence of egg yolk makes it possible to detect the enzyme lecithinase (lecitovitellase), which is formed by pathogenic staphylococci. Lecithinase breaks down lecithin into phosphocholines and water-insoluble fatty acids, so the environment around lecithinase-positive colonies becomes cloudy and an opalescent zone appears in the form of a “rainbow corolla”.
Bile broth is selective for salmonella, the reproduction of which is stimulated by the added 10% bile, while simultaneously inhibiting the growth of accompanying microorganisms.
Alkaline agar or alkaline peptone water are selective for Vibrio cholerae; the alkaline reaction of the medium (pH 9.0) does not prevent the growth of Vibrio cholerae, but inhibits the growth of other microorganisms.
3. Differential diagnostic environments. Differential diagnostic media are used to study the biochemical properties and distinguish (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. The differentiating properties of these media are created by the addition of a substrate, to which the relationship of microbes, their enzymatic activity and the effect of toxins is determined (Hiss media, Endo, Levin, Ploskirev, Olkenitsky media, bismuth sulfite agar, etc.). According to their purpose, differential diagnostic nutrient media are divided as follows:
Media for identifying the proteolytic and hemolytic ability of microbes containing protein substances: blood, milk, gelatin, etc. The most common media are meat-peptone gelatin (MPG), coagulated horse serum, milk and blood agar (BA).
Environments with indifferent chemicals, which serve as a source of nutrition for some types of microbes and are not absorbed by other types. For example, media containing substances that are assimilated only by a certain group of bacteria. The most common media in this group are Simmons citrate agar and Coser citrate medium.
Media containing carbohydrates, polyhydric alcohols or indicators for the detection of appropriate enzymes and determination of the glycolytic activity of microorganisms. Enzymatic breakdown of substrates leads to a pH shift and a change in the color of the medium. The most common are colored media with various carbohydrates (for example, bromothymol blue, a BP indicator) and litmus milk (Minkevich's medium). Also widely used are Hiss media, which take into account differences in the ability to ferment various carbohydrates with the formation of acid, or acid and gas. To differentiate enterobacteria, peptone water is used with a set of various carbohydrates, Andrede's indicator and floats, which facilitate the detection of gas formation and help visually determine the change in pH characteristic of various microorganisms. In particular, a shift to the acidic side causes the medium with Andrede's reagent to turn red or yellow when using a medium with bromothymol blue, whereas when alkalized, Andrede's indicator and bromothymol blue do not change the color of the medium. For example, to isolate pathogenic bacteria from the intestine, media are used that make it possible to differentiate pathogenic microorganisms from permanent inhabitants of the intestine - microorganisms that decompose lactose. Such a medium is Endo medium, which contains lactose. The main components of the Endo medium are MPA, lactose and basic fuchsin, decolorized with sodium sulfite. The initial nutrient medium is colored light pink. When lactose is fermented, acetaldehyde is formed, which reacts with sulfite and, when released, fuchsin colors the colonies bright red. Therefore, E. coli, which ferments lactose, when growing on this medium, forms red colonies with a metallic sheen, while Salmonella and Shigella are colorless, since they do not ferment lactose.
Media for determining the reducing ability of microorganisms. This group includes media with paints that become discolored when reduced by redox enzymes (for example, methylene blue, acid fuchsin, bromothymol blue), as well as media with nitrates for determining the denitrifying activity of bacteria (if the result is positive, the media are colored in Blue colour). By changing its color at different pH values, the indicator indicates the presence or absence of splitting, oxidation or reduction of the ingredient introduced into the medium. However, the indicator is not a mandatory component of media intended for the detection of enzymes. Thus, the presence of gelatinase and other proteolytic enzymes in a culture is determined by the liquefaction of gelatin, coagulated egg or whey protein.
4. Storage media, on which the rapid growth of certain types of microorganisms occurs.
5. Preservative media. Designed to preserve microorganisms during transportation to the research site. These media contain additives that prevent the proliferation and death of microbes, which helps maintain their viability. The most widely used are glycerin mixture (Tig's medium), hypertonic solution, and phosphate-buffer mixture.
Sterilization of culture media. All nutrient media, regardless of their purpose, are poured into clean containers 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 atmospheres.
2. Media with carbohydrates and milk (which includes lactose), nutritious gelatin are sterilized with flowing steam at a temperature of 100°C fractionally or in an autoclave at 112°C and a pressure of up to 1 atmosphere.
3. Media containing protein substances (blood serum, ascitic fluid) are decontaminated by tindalization or filtration.
4. To sterilize culture media containing native proteins, filtration through Seitz membrane filters is used.
To control the sterility of the medium after sterilization, place it in a thermostat at 37°C for 3-5 days. Liquid media should remain clear, and no signs of growth should appear on the surface or in the thickness of solid culture media. In addition to sterility control, chemical control of prepared media is carried out, which consists of determining the pH, the amount of total and amine nitrogen and chlorides in several samples of each series.
There is also biological control of 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.
Research on bacteria requires meticulous work with numerous equipment and instruments. In order for microorganisms to multiply as quickly as possible in laboratory conditions and be able to maintain normal life functions, 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 are grown in laboratory conditions 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 high-quality growth of the crop.
For example, such an selective environment is only suitable for the reproduction of Escherichia coli. Then, from sowing many bacteria on a Petri dish, we will see only colonies of that same E. coli and no more. Before starting work, it is necessary to have a good knowledge of the metabolism of the bacterium being studied in order to successfully select it from a mixture of other species.
Solid, semi-liquid and liquid nutrient media
Bacteria can be grown not only on solid substrates. Nutrient media differ in state of aggregation, which depends on the composition during manufacture. Initially, they all have a liquid consistency, and when adding gelatin or agar in a certain percentage the mixture hardens.
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 forms, a film appears, small flakes float, or a cloudy solution forms.
Solid nutrient medium 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 microorganism culture.
Preparation of culture media
Substrates such as meat-peptone mixtures based on broth, gelatin or agar are very easily prepared. If you need to make a solid or semi-liquid substrate, add 2-3% or 0.2-0.3% gelatin or agar to the liquid, respectively. They are playing main role in the hardening of the mixture, but are in no way a source of nutrients. Thus, nutrient media are obtained that are suitable for the growth of bacterial cultures.