Nutrient media

Nutrient substrates used for cultivation in vitro different microorganisms. The nutrient medium are widely used in everyday microbiological practice for statement of laboratory diagnosis of infectious diseases, for selection of microbes of various subjects of external environment in order to clarify the sources of infection, and when epidemiological survey of people and animals to clarify the carriage of infectious diseases. P.F. apply also to obtain microbial mass used in the preparation of vaccines and diagnostic antigens in the production of antibiotics and other
Nutrient medium, intended for the cultivation of microbes, should have the optimal conditions for growth and reproduction. With this purpose, the creation of P.F. provide for the presence of compounds that are sources of nitrogen, carbon and hydrogen, a set of necessary ions in the form of inorganic salts. In some cases, on Wednesday add so-called growth factors, i.e. connections, which are not synthesized cultivated a microbe (see Bacterial growth factors). But the presence of P.F. all of these components are not yet able to provide optimal conditions for growth and reproduction of microbes, as these processes also depend on the physical-chemical properties of the medium. These include: pH, redox potential, viscosity, humidity and osmotic properties.
If P.F. meets the biological characteristics of the germ and ensure its growth and reproduction, it is called optimal or good, if it lacks any component, necessary for the growth and the reproduction of the organism, such an environment characterized as deficient. Since different types bacteria and even the strains belonging to the same species may differ greatly from each other in physiology and nature of metabolism, it is obvious that the growing use different media.
A carbon source in P.F. are carbohydrates, alcohols and some organic acids, nitrogen source - mineral or organic compounds. Very extensive use of gelatin products of enzyme or acid hydrolysis of proteins of different backgrounds and representing a mixture of amino acids and polypeptides of different complexity. Differences in amino acid and peptide structure peptone depend on the properties of the source of protein and extent of fission. The nutritional value peptone is determined by its amino acid and peptide structure.
Raw materials for manufacturing peptone are proteins of meat, fish, yeast, casein and other Along with gelatin as a source of nitrogen in microbiological practice also apply cold water to grow the number of bacteria prepare more meat animals. For cooking water meat meat released from the bones, tendons and fat and grind in a meat grinder. Received meat with cold tap water (1 teaspoon minced 2 hours of water), mix well and leave for days in a cool place. Meat infusion filtered through several layers of gauze, boil again filtered through filter paper, and then add water to the original volume.
In gelatin, meat infusion or tissue extracts, in addition to organic nitrogen contained bacterial growth factors, and therefore the environment prepared on their basis with addition of salt (NaCl), suitable for the cultivation of many organisms. However, for a growing number of pathogenic microbes (diphtheria Bacillus, the causative agent of whooping cough, Brucella and others) mesopatamia nutrient medium is unsuitable, and therefore in these cases, using special media, for example egg medium serum, blood, etc.
Depending on the properties, composition and destination of P.F. divided into groups. On a consistence distinguish nutrient medium solid, semi-solid and liquid. An example of solids are 1,5-2% agar medium (meat-peptone agar, agar of Hottinger), nutrient gelatin, rolled serum, rolled egg white, potatoes, etc. For preparation of solid P.F. most often used agar-agar, derived from seaweed. Agar-agar is a substance polysaccharide nature. In water swells, forming jelly. Liquefies at temperature 70 to 100 degrees and solidifies 40-50 degrees.
Semi-fluid medium is 0.5% agar meat-peptone (see).
To liquid media are broth meat-peptone (see), peptone water sugar soup, broth of Hottinger, representing diluted (in 4-9 times) meat filtrate affected by enzymatic hydrolysis by Pancreatin and containing 0.5-1% salt.

Nutrient can be simple or complex. Simple environments are peptone water, meat-peptone broth, meat-peptone agar, nutritious gelatin. On the basis of simple environments prepare complex (meat-peptone sugar broth, blood agar, ascites-agar and other). The complexity of this or that nutrient medium depends on the biological characteristics grown microbe, as well as to the research objectives. So, for example, for the cultivation of Haemophilus use complex environment Board - Zhang, in which defibrilatorun human blood or rabbit add to potato-glycerol broth or agar. Many complex nutrient media used for the detection of cultivated microbe specific properties, as expressed in their colony morphology, growth or in the peculiarities of metabolism. P.F. that allow you to set the specifics of the germ, called differential diagnostic (see Differential diagnostic environment). For example, to determine the ability of bacteria to break down glucose emitting acetyl methyl carbinol (reaction of Fogasa - Proskauer) use environment Clarke, which contains peptone, glucose, 2NRA4 and alkali (KOH). Acetyl methyl carbinol in the presence of alkali and oxygen oxidizes in diacetyl, and the last, connecting with guanidine under peptone, gives red color.
A special group comprises the selective environment in which to create the best conditions for growing a certain species of microbes and unfavorable for other species, such as environment Muller, Kaufman, Lifson, Shustova to highlight intestinal bacteria, alkaline agar - selective environment for cholera and kolerovochny vibrios environment with addition of antibiotics give the opportunity to selekcioniruut of antibiotic-resistant bacteria from sensitive. Of the listed environments is widespread environment Kaufman used as processing environment for a group of bacteria Salmonella and typhoid. The main ingredients are hyposulfite broth, brilliantly and bile. Selectivity for anaerobic microorganisms is created by the decline in the environment, concentrations of dissolved oxygen. One common nutrient media used for cultivation of anaerobic environment is the Kitt - Tarozzi, in which the role of the adsorbent of oxygen and reducing factor perform in the environment of the pieces of the liver of animals.
To the complex of P.F. also include synthetic (see Synthetic medium). The originality environments of this group is that all their components are chemically pure compounds, unlike the previously mentioned media containing as nutrient substrates products of natural origin (gelatin, blood, ascitic fluid, etc.). Minimum synthetic nutrient medium contains a set of salts and energy source is glucose or lactic acid, a source of nitrogen in ammonium salt. Such environment suitable for growing prototropic microbes. Auxotrophic microbes, unable to make any of vital compounds (amino acid, vitamins, purine or pyrimidine base), cultivated on media containing, in addition to the set of salts, this or that extra ingredient, on which the germ of auxotrophic. For example, if cultured bacteria are not able to synthesize tryptophan or another amino acid, salt minimal environment provide the appropriate amino acid. Usually 1-amino acids contribute at a concentration of 20 mg/ml and dl-amino acids - 50 ug/ml. Synthetic P.F. used to study the physiology of bacteria, in particular metabolism, and especially in the study of some problems of biochemical genetics. Synthetic P.F. used in the breeding of germs for selection of auxotrophic or prototropic strains.
In the laboratory for preparation, storage and bottling of nutrient media used for bacterial tubes, flat-bottom flasks, bottles, etc.
For plate of hard materials used in Petri dishes. In mass microbiological production of bacterial cultures grown in metal furnaces (reactors) high capacity (500-1000 l).
The utensils used for P. c., you cannot store disinfectants, antibiotics, as the slightest traces of them make the environment unsuitable for cultivation of microorganisms. Before use, the dishes are carefully washed and dried. New bowl pre boil in 1 % solution of hydrochloric acid and washed in distilled water flowing. The nutrient medium, usually should be transparent, so their use filtered or defend. Settled molten agar medium filtered through cotton-gauze filter (filtering through cotton wool quality of the environment can be reduced by adsorption on it compounds the environment, stimulating the growth of the germ). Liquid is filtered through filter paper or canvas. If agar after filtration or sedimentation remains murky, it is lighten by adding egg protein or whey per 1 l environment one egg or 20-25 ml of serum. Added proteins fold up, adsorb particles and, settling, illuminate the environment. Ready nutrient medium poured into the appropriate sterile glassware and sterilized.
Sterilization (see) is a very important stage of preparation of P.F. Mode it must not change the properties of the media. The most widespread and effective sterilization using high temperature in autoclaves (see) under pressure or fluid the ferry. Usually P.F. sterilized in an autoclave at 1 ATM. (120,6 degrees) within 15-30 minutes, in some cases, and with capacities greater than 1 l within 1-1,5 hour. This way is used for sterilization of environments, not containing carbohydrates and native proteins or other thermolabile substances. If the environment thermolabile substances are bacterial filters or fractional sterilization. When sterilization bouillon environments or environments containing other organic sources of power, you can change the pH in acidic side 0.1-0.2.
Ready nutrient store in a cool and dark place, with sufficient humidity. Long-store P.F. desirable: dense medium dry and liquid precipitation precipitation.
To standardize the conditions of cultivation of microorganisms and facilitate laboratory works apply dry P.F. - hygroscopic powder, soluble in water. Before use, such P.F. dissolved in distilled water and then sterilized.