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Definition of a specific or sample accessory of a microbe is the major final stage of microbiologic research. It is carried out by studying of a complex of morphological and biological properties of the yielded culture that allows positioning its identity (sameness) with the typical representative of a certain kind (and phylum) microorganisms and by that to carry a discharged microbe to this kind (and to phylum). For these researches, as a rule, it is necessary to have pure growth as presence of ectogenic microorganisms can deform results of researches and become the cause for the erroneous conclusions. Culture identification is expedient for beginning with studying of some features of its growth. Almost all bacteria grow on liquid and dense nutrient mediums at 37 °, and also at 21-25 °. Some microbes facultative anaerobes, do not yield growth in dense, fluid mediums, and educe in mediums of semifluid, keeping 0,05-0,1 %. Identification of discharged culture is routinely carried out on the basis of set of its properties.
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Widely increasing quantity of the tests demanded at studying bacteria, has led to drawing up enough the complex cards which filling is necessary at research works. Usual practice allows confining more narrow researches which results are brought in a card. At m. it is necessary to be guided by the certain schemas providing uniformity of methods, applied to their characteristics; however the general schemas applied to identification of all microbes, and does not exist. At regular studying of properties of a microbe routinely are guided by the following schema. Morphology and chemical properties.
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The morphology of microorganisms is routinely studied by microscopy fixed and stained preparations, and also alive uncolored microorganisms in pendulous or in the crushed drop. Special chambers are applied to long observation of alive and propagating bacteria (Peshkov, Fabrena). Microscopic examinations allow defining the form, the dimensions and a constitution of microorganisms, their relative positioning, motility, quantity and allocation of flagellas, the form and position of spores, and also formation of sheaths.
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For motility studying it is necessary to take young (is not more senior 6-8 hours) quickly growing cultures. Flagellas are easier found in young agarinic cultures, disputes, on the contrary, in the cultures grown within several days, and sheaths – in the pathological exsudates received from infected animals. The form and the dimensions of microbes are exposed to appreciable changes depending on features of the strain, age of culture, composition medium, temperatures of incubation and other factors which should be noted in a card. Studying of motility of microbes is effected by means of microscopy crushed and a hanging drop prepared from young cultures, grown at various temperatures (from 20 to 37 °). For microscopy of the crushed drop it is better to use a dark field or the fazovo-contrast device. At studying of motility of bacteria in a hanging drop sometimes there are microbes which, being are supplied by flagellas, appear motionless. These bacteria fall into – so-called with the paralyzed flagellas.
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As good check of motility of microbes stab culture, in the semifluid can serve. Tittsler, L. A. Sandholz. Mobile microbes form the diffuse region of growth propagating on a course of a nyxis, and motionless do not yield this region. This method demands, however, a lot of time (the incubation at 30 ° proceeds sometimes 6 days). At studying of morphology of microbes the microscopy fixed and stained preparations in which features of the chemical constitution of various microbes are taped has great value. Among the special methods of a coloration used for identification of bacteria, the coloration on gramme allows to part all bacteria on two bunches: Gram-negative and Gram-positive.
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The coloration method on Tsil-Nilsenu has similar value as it gives the chance to differentiate acid-resistant from not acid-resistant bacteria. The great number of other special methods of a coloration is applied to the differentiated staining of head elements, protoplasm and includings (methods Romanovsky – Giza, Filkina, Rubinova and others), metachromatic beads (a method It sulphur and others), and also flagellas, sheaths and spores. The special cytochemical methods of a coloration taping the chemical nature of intrinsic structure of cells, are widely applied also in studying of morphology of microorganisms.
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Fazovo-contrast, electron, ultra-violet and fluorescent microscopy allow to inpour more penetrating into intrinsic structure of microorganisms and to use the obtained data for their differentiation. Probably that the characteristic regions of adsorption found by an infra-red spectophotometery. With success will be applied at identification of various kinds, but also variants of a kind of microbes. In certain cases, when the microbe parentage is known, by microscopic examination it is possible to make the presumable diagnosis. In medical microbiology such pre-award identification is proved only in that case when there is a conformity with the clinical diagnosis. So, for example acid steady rods in a neurolymph of the patient with clinical symptoms of a meningitis can be defined preliminarily as tubercular bacteria Gram-negative bipolar imbued bypass rods in juice of lymph nodes of the patient with inguinal buboes in district where the plague is propagated, it is possible to survey preliminarily as plague bacteria. However, as a rule, to identify a microbe only on its morphological and to chemical properties it is not obviously possible. Cultural properties are studied by sowing of the yielded strain on a slanting hangar, in a broth and a nyxis in gelatine.
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From cultural signs of bacteria especially outward and interior of their colonies which are formed at culture seeding on Petri dishes have great value. If the microbe does not yield growth on usual other medium approaching for it should be applied. Colonies are looked through later 24 hours of incubation at 37 °, and then is repeated with an interval in a week. At the description of colonies the special attention should be reversed for their dimensions, the form, a profile, a surface, edges, density. Colonies of microbes are expedient for photographing or sketching. The obtained data is brought in a descriptive card. If microbes show a tendency to dissociation on phase variants they are necessary for parting a dispersion method on Petri dishes or others it is useful for receptions of Property of each individual strain to fix on a separate card.
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Repeated researches are thus demanded to show that noted morphological and biological signs are bound to a phase in which specifies phylum of colonies. At m. it is necessary to study also growth on a slanting process, in a broth and in gelatin, noting thus character of growth. At a colliquation gelatines note prominent features of this process. In some researches growth on special mediums, such as Serum of Leffera, a glyceric potato, egg mediums and others Studying of signs specifies only bunch which the yielded microbe concerns is studied also. They do not allow to differentiate various representatives of this bunch, and serve mainly for acknowledgement of the data received at research of morphology and chemical properties of a microbe. Resistance. Microbes it is considerably separated on their resistance to Temperature and other unfavorable factors. Nesporonosnye bacteria and vegetative forms a trichidium of bacteria are sensitive to temperature and to small concentration of chemical antiseptics.
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They perish at 60 ° during a half an hour and in 1 % solution of Phenolum within an hour. Acid-resistant bacteria are sensitive to temperature, but rather. To refractory and to disinfectants. They perish at 60 ° during a half an hour, but on a cold stand up to to antiseptics often within several hours. Especially high resistance disputes of bacteria possess. That them to kill, it is necessary to apply a steam under pressure at 120 ° during a half an hour or high concentration of antiseptics, for example, 5 % Phenolum within several hours. Studying of resistance yielded a tank – theories, as a rule, is only auxiliary sign for acknowledgement of the conclusions made on the establishment before described researches, but sometimes it and in itself has diagnostic value. So, some not disputes forming bacteria, an enterococcus and others – die at 60 ° degrees. For half an hour; a necessary condition for this purpose is rise in temperature to 65 °.
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This unusual resistance to a thermal factor is of interest at differentiation of this kind of a streptococcus from others. In all cases when it is possible to suspect that the microbe forms disputes, it is necessary to supply assay on resistance to temperature. Before many forms were surveyed as true disputes, but at the further studying appeared deprived of this characteristic property a resistance term. Some features of a metabolism and biochemical activity, studying of the attitude of microbes here concerns oxygen and Carbonei dioxydum, definition of optimum temperature, a pigment of formation and hemolitic ability, and also influence on growth of various materials, including bacteriemic growth factors in relation to free oxygen. Microbes routinely share on three bunches: 1) strict aerobes which grow only in the presence of free oxygen; 2) the strict anaerobes yielding growth only in absence of free oxygen; 3) facultative anaerobes which grow in aerobic conditions is better, but are formative and under anaerobic conditions.
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It is possible to carry the fourth bunch to them – mikra-aeroforces which is better grow at the depressed fractional pressure of oxygen. On a temperature optimum of cultivation of a bacterium divide on 5: 1) a psihrofilen-bast, normally educe more low 20 °; 2) mezo-fily which is better thermophiles for which the optimum temperature lies within 50-60 ° grow between 20 both 40 ° and 3. The majority of pathogenic microorganisms is cultivated at 37 ° and does not grow at ambient temperature. Hemolitic activity of microbes is defined at a bacterial cultivation on cups with bloody charcoal fumes or by gaining of various delutions of culture to cloud of the washed erythrocytes. This property is used as paramount criterion, to differentiation of representatives of bunch of streptococci.
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Studying of influence on growth of various biological substrates and chemical combinations (blood, Serum, a glucose, Sodium nitritums, salts of cholic acids, vitamins, amino acids and others) often matters for differentiation of bacteria. Formation of pigments is studied on various nutrient mediums and at various temperatures. A pigment is better it is formed on a surface of dense medium at 25-30 °. Some pigments vary in the shade depending on substrate reaction. Final reading of results is effected only in some days of stay of culture at ambient temperature. Often strengthens, when the culture contains in a diffused light.
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Studying of various metabolic functions of a microbe considerably dilates the data received on the basis of invited methods of research, and is often used. For differential diagnostics of microbial kinds. Studying of biochemical properties of microorganisms is carried out by test of their enzymatic activity for some carbohydrates and alcohols (Saccharum lytic ability), proteinaceous substrates (proteolytic properties) and fats (or – lytic properties). The Great value has also definition of reducing properties of bacteria and ability to produce a catalase, to form ammonia and hydrogen sulphide, to use Citras. The fermentation microbes of carbohydrates is shown by fermentation end products (organic acids, gases). Acid presence is registered by means of the indicator, and gas production – by means of a float.
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For fast detection of some bacteriemic ferments the accelerated methods or micro-methods S are offered. Cowan, 1993 and others. At differentiation of bacteria biochemical properties often play rather important role. At many bunches of microbes classification is carried out on the basis of a fermentation of carbohydrates, proteolytic activity or both these tests. Assays on Saccharums and other carbohydrates allows to tap the most thin differences between closely related to microbes. Antigenic structure and the attitude to a bacteriophage. Studying of an antigenic constitution of microbes is carried out by means of various serological tests from which bacteria the agglutination test and a reaction of binding complement are applied to identification with success (by Berde to the Genre reaction).
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These reactions, in particular an agglutination test, often represent the fastest and reliable method of identification of discharged culture. For completeness of identification the microbe should agglutinability to titer Serum prepared against that microbe which is presumably similar to the studied. The examinee a microbe should adsorb all agglutinins from this Serum. On the other hand, the typical microbe should agglutinability to titer Serum prepared against an unknown microbe, and also to exhaust from this Serum all agglutinins. Differently, there should be a full two-dimensional agglutination and two-dimensional adsorption between both Serums and to both microbes. The agglutination test is sometimes supplemented or changed with a precipitation test.
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The serological method as the fastest and satisfactoriest mean of differentiation is widely used for identification of some microbes. This method not only allows to identify many microorganisms, but finds at the same time and the most thin differences between closely related microbes. In this respect, it even is more valuable, than biochemical assays. At the same time it often is is unique an accessible method for differentiation of subspecies or types of the yielded kind. In an ice crust, time wide application in laboratory practice was received by mono-receptor Serums for identification of salmonellas, dysenteric bacteria and other microorganisms.
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Even more sensitive the method of an identification of culture a bacteriophage appears a research mean sometimes. This method is used, for example, at studying of a typroid rod (typroid), allowing to distinguish numerous so-called fago-types within a kind. Specific phages with success are applied to differentiation of a choleraic vibrio from a cholera similar, a plague bacillus from bacteria of a false tuberculosis of gnawers, and also for identification of other microorganisms. Pathogenicity for animals. Pathogenicity routinely is tested in experiences on white mice, Guinea pigs, rabbits and other laboratory animals. Infestation of animals effect subcutaneously, intradermal, it is intramuscular, intravenously, Sodium nitritum-tonalno, perorally, infra-cerebral and others to methods. At studying of pathogenic microorganisms sometimes it is necessary to define, whether they form solvable ectotoxin. With that end in view on sensitive animals the sterile filtrate of the culture grown during certain term on the conforming fluid medium is tested.
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Ectotoxins of highly bacteria (a diphtheritic rod, a tetanic bacillus and others) cause disease of animals with a characteristic clinical pattern and their subsequent destruction. With typical pathoanatomical to changes. At the microbial kinds developing such ectotoxins, experience of neutralisation of the last by a specific antitoxin plays an essential role greetings identification. Cultures are applied to detection of some microbial toxines recently with success sensitive to them of tissues, and also chicken embryos.
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With doctrine development about the variated reactance, immunologic and allergic, and also about reflex, including conditionally reflex activity. The phenomena began to explain from a position of the modern doctrine about allergic responses, an anaphylaxis, reactions an antigen an antibody and so on. Tried to delimit from an allergy and it is more accurate the studied anaphylaxis following features quite often is. To congenital property which shows already after the first contact by the intolerable. Under material the anaphylaxis and an allergy arise, as it is considered to be only after repeated influence of a stimulus-antigene on an organism.
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Congenital or seeming to the congenital speaks, apparently, only transferring of allergens through a placenta during pregnancy or with milk of mother, or at the expense of the remained unknown person of a sensibilization most sick of usual paths. It is known also, as other implications of an allergy in a broad sense. For example, a serum disease, can educe and at the first application of Serum can cause the simple chemical combinations which are not possessing antigenic properties (Sulfanilamidums, pyramidon); however, being bridged to protein in an organism (or protein of tissues), they can react as antigens (Land Shtejner and others). At damage of cells of an organism (blood cells, parenchymatous organs) such path can arise and an antibody miss. Antibodies at medicinal intolerance and other kinds are found changeably and in a low body. 3.
