IDENTIFICATION OF MICROBES (late lat. to identify identificare) — definition of specific or standard accessory of microbes. And. m — the major stage mikrobiol, researches, necessary for definition of an etiology of an infectious disease; it is of great importance for epidemiol, the analysis of outbreaks of infectious diseases and holding effective actions for their elimination. And. the m is also widely used at a dignity. - a gigabyte. to assessment of the soil, air, water and foodstuff.
And. the m is carried out by studying of a complex morfol., cultural, biochemical, antigenic, pathogenic and other properties of this culture that allows to establish its identity (identity) to typical representatives, a certain type (type) of microorganisms. For these researches, as a rule, it is necessary to have pure growth as presence of foreign microbes can be the cause for the wrong conclusions.
The choice of methods of a research for And. the m considerably is defined by a source of allocation of a microbe (e.g., the material received from the patient from a corpse or objects of the environment).
Definition of properties of microorganisms
General schemes I. the m applied in practice do not exist. For each group of microorganisms identification is carried out on the basis of them biol, features. So, for identifications of viruses (see) importance reckon cellular cultures in which there is their reproduction, character of a cytopathic effect, formation of inclusions, an antigenic structure, in certain cases morphology of viruses, and also pathogenicity of viruses for experimental animals.
In identification rickettsiae (see) studying of their morphological properties, features of intracellular parasitism, antigenic properties etc. matter. At identification of representatives of fungi, actinomycetes and protozoa features of the activator matter morfol (see. Actinomycetes , Fungi parasitic , Protozoa ). The main signs in identification of mycoplasmas are them morfol., cultural and antigenic properties (see. Mycoplasmataceae ). Bacteria identify on the basis of complex studying them morfol., tinktorialny, cultural, biochemical, antigenic, fagolizabelny, bakteriotsinogenny and pathogenic properties.
The proposal of some researchers deserves attention [Cowen and Steel (S. T. Cowan, To. I. Steel), 1961, 1965; Sealy and Van-Demark (H. W. Seeley, V. of I. Van Demark), 1972] to use as starting point of identification of bacteria coloring across Gram. At the first stage of differentiation of gram-positive bacteria authors consider a form of a cell, acid resistance, sporogenesis, mobility, products of a catalase, oxidase, the relation to glucose, and gram-negative bacteria — a form of a cell, mobility, products of a catalase, oxidase and the relation to glucose. At the subsequent stages of a research, using the tables characterizing bacteria, belonging to a certain sort, find a key to definition of types, subspecies and types.
Morphological and tinktorialnye properties
Studying morfol, and tinktorialny signs of a microbe is usually only an initial stage of its identification. The morphology of microorganisms is studied by microscopy of the fixed and stained preparations, and also live uncolored microorganisms in the trailing or crushed drop.
Live bacteria use special cameras (Peshkova, Fonbryun) to long overseeing. Microscopic examination allows to define a form, the sizes and a structure of microorganisms, their relative positioning, mobility, quantity and distribution of flagellums, a form and situation a dispute, and also formation of capsules. For studying of mobility young people (6 — 8 hours are not more senior) take fast-growing bouillon cultures. Flagellums are found in young agar cultures, disputes, on the contrary, in the cultures which are grown up within several days easier, and capsules — in patol, exudates. At microscopy of a hanging drop it is better to use a dark field or the phase and contrast device. At the same time it is necessary to consider that forms and the sizes of microorganisms change depending on features of a strain, age of culture, structure of the environment, temperature of an incubation and other factors.
Tinktorialny properties of microbes define during the coloring of the fixed drugs. Coloring across Gram allows to divide all bacteria into 2 groups: gram-negative and gram-positive (see. Grama method ). Coloring across Tsil — to Nelsen gives the chance to differentiate acid resisting bacteria from not acid resisting (see. Tsilya-Nelsena method ). By means of special methods reveal separate elements of a bacterial cell: nucleoid, protoplasm and inclusions (Romanovsky's methods — Gimza, Feylgen, Robino, etc.), metachromatic granules (see. Neicer methods etc.), flagellums, capsules and disputes. The method of fluorescent antibodies does possible preliminary identification of species and even like a microbe (see. Immunofluorescence ).
In cases of specificity of morphology of a microbe by microscopic examination it is possible to identify it presumably. In medical microbiology such identification is proved only when it corresponds a wedge, to the diagnosis. So, e.g., acid resisting sticks in cerebrospinal liquid of the patient about a wedge, symptoms of meningitis it is possible to carry to tubercular mycobacteria previously. The painted ovoidny sticks gram-negative bipolyarno in juice limf, nodes of the patient with inguinal buboes in the area where plague is widespread, it is possible to consider presumably as plague bacteria.
Cultural properties indicate belonging of a microbe to a certain group and plan the direction of further researches for its final identification. They are defined by crops of the studied culture on mediums (an agar, broth, a prick in gelatin, etc.). From cultural signs of bacteria and fungi importance have outward and interior of the colonies forming during the seeding of culture on dense mediums. If the microbe does not give growth on a usual beef-extract agar, then another shall be applied, Wednesday, optimum for it. Colonies are usually looked through in 24 hours of an incubation at t ° 37 °, and then repeatedly at an interval of 1 — 3 day. At the description of colonies pay attention to their sizes, color (chromogenesis), a form, a profile, a surface, edges, density. If bacteria show a tendency to dissociation on phase options (see. Dissociation of bacteria ), they are divided by sowing on Petri dishes with a medium. With a growth on liquid mediums note a pridonnost of growth, growth in the form of a film or uniform opacification of the environment. Growth on special environments, such as Leffler's serum, glyceric potatoes, the Wednesdays containing blood, etc. is in certain cases studied. Cultural properties of a microbe are essential addition to it morfol, to signs.
Resistance of microbes to various environmental factors
Resistance of microbes to various environmental factors is used at And. m since in some cases microbes considerably differ on this sign. So, e.g., not sporiferous bacteria and vegetative forms of sporiferous bacteria are temperature-sensitive also to small concentration of antiseptic agents. They perish at t ° 60 ° within half an hour and in 1% solution of phenol within 1 hour. Acid resisting bacteria are temperature-sensitive, but concerning a rezistentna to disinfectants; they perish at t ° 60 ° within half an hour, but in the cold resist to antiseptic agents often within several hours. Disputes of bacteria have especially high stability (see. Disputes, bacteria ). They perish or from a pressured steam (at t ° 120 ° within half an hour) or from high concentration of antiseptic agents, napr, under the influence of 5% of phenol within several hours. Therefore at suspicion on education by a microbe the dispute is put by tests on resistance to temperature.
For certain species of bacteria their stability to a nek-eye to antibiotics and chemotherapeutic drugs is indicative. So, e.g., one of the tests allowing to differentiate a classical cholera vibrio from a vibrio El-Tor and also Proteus mirabilis from other colibacilli, serves ability of a vibrio El-Tor and Proteus mirabilis grow in the presence of polymyxin B (50 units in 1 ml and above).
Features of physiology and biochemical activity
During the definition of biochemical activity of microbes consider their relation to oxygen, carbonic acid and various substrates, optimum temperature of growth, hemolitic ability, and also influence on their growth of various substances, including bacterial growth factors (see). In relation to free oxygen microbes usually divide into strict aerobes (see), strict and optional anaerobe bacterias (see). Therefore to allocation and identification of the activator apply the special methods and mediums promoting growth only of aerobic, optional and aerobic or anaerobic representatives.
For the majority of pathogenic microbes the optimum temperature of cultivation 37 ° (see. Bacteria ).
Hemolitic activity of microbes decides at their cultivation in cups on a blood agar or way of addition of various cultivations of bouillon culture to a suspension of the washed erythrocytes.
Studying of influence on growth of bacteria various biol, substrates and chemical connections (blood, serum, glucose, nitrates, salts bilious to - t, vitamins, amino acids, etc.) often matters for differentiation of this group of microorganisms.
For And. m the features of enzymatic activity of microbes revealed on the Wednesdays containing sugar and alcohols, proteinaceous substrates and fats (lipolytic properties) that allows to reveal the thinnest distinctions between closely related microbes are of great importance. Important also definition of the reducing properties of bacteria and their ability to form an indole, ammonia and hydrogen sulfide, to use citrates and tartrates (see. Differential and diagnostic environments ).
The antigenic structure and the relation to a bacteriophage
the Antigenic structure and the relation to a bacteriophage and bakteritsina are studied at the final stage And. m. Identification of an antigenic structure of microbes is carried out by means of various serol, reactions, napr, by reactions agglutinations (see), reactions of binding complement (see), etc.
If in the developed agglutination test the tested microbe is agglutinated to a caption of immune serum or a half of a caption, then in practice it can be considered belonging to that look (type) by what this serum is designated. For full identification the allocated activator shall be agglutinated to a caption by the immune serum prepared against a reference microbe: the examinee a microbe shall adsorb all agglutinins from this serum. On the other hand, the reference microbe shall be agglutinated to a caption by the serum prepared against the studied microbe and also to adsorb all agglutinins from this serum. In other words, there has to be a full cross agglutination and cross adsorption between both serums and both microbes. The agglutination test is sometimes supplemented or replaced with reaction precipitations (see), and also reaction of indirect hemagglutination (with the erythrocytes loaded with antibodies). Serol, a method finds the thinnest distinctions between related microbes. It often is only an available method for differentiation of subspecies or types of this look. In laboratory practice
the agglutinating monoreceptor serums for identification of salmonellas, shigellas and other microbes were widely used. Very effectively also use of a method immunofluorescence (see) which allows quickly (1 — 2 hour) to carry out And. m.
By a sensitive method I. the m is typing of the identifying culture bacteriophage (see). This method is used, e.g., during the studying of a typroid stick (see. Vi-typroid phage ), since allows to distinguish fagotip within a look. Specific a phage apply to differentiation of shigellas, cholera vibrioes from choleroid, a classical cholera vibrio from a vibrio El-Tor, a plague bacillus from bacteria of a pseudotuberculosis and other bacteria.
For differentiation of some bacteria within a look use a phenomenon bakteriotsinogeniya (see), and also testing of sensitivity of bacteria to bakteritsina of various types (colicines, vibriotsina, pestitsina, difteriotsina, etc.). Found for Kolitsinotipirovaniye broad application for definition of belonging of the marked-out culture of shigellas to a certain kolitsinotip.
Pathogenicity for animals
Pathogenicity of microbes is usually defined in experiences on white mice, Guinea pigs and rabbits. Animals are infected subcutaneously, vnutrikozhno, intramusculary, intravenously, intraperitoneally, orally, intranazalno or intratserebralno (see. Bioassey ).
During the studying of pathogenic microorganisms sometimes it is required to define whether they form exotoxins. For this purpose on sensitive animals the filtrate of the bacterial culture which is grown up during a certain term on the corresponding fluid medium is tested. Exotoxins of high-toxicogenic bacteria (a diphtheritic stick, a tetanic bacillus, a botulinic bacillus, etc.) cause a disease of animals with characteristic a wedge, a picture and the subsequent their death with typical patol oho anatomic changes. Apply to detection of some microbic exotoxins cultures of fabrics, sensitive to them, and also chicken embryos. Neutralization of exotoxins specific antitoxins plays an essential role at And. m.
Bibliography: Krasilnikov N. A. Determinant of bacteria and actinomycetes, M. — L., 1949, bibliogr.; The guide to microbiological diagnosis of infectious diseases, under the editorship of K. I. Matveev, M., 1973; Tim and V. D.'s k and D. M Goldfarb. Fundamentals of experimental medical bacteriology, M., 1958, bibliogr.; Bergey’s manual of determinative bacteriology, ed. by R. E. Buchanan a. N. E. Gibbons, Baltimore, 1975, bibliogr.; Cowan S. T. a. Steel K. J. Manual for the identification of medical bacteria, Cambridge, 1974; Identification methods for microbiology, ed. by B. M. Gibbs a. F. A. Skinner, v. 1—2, L. — N. Y., 1966 — 1968; International code of nomenclature of bacteria, ed. by S. P. Lapage a. o., Washington, 1975; M e at n e 1 1 G. G. a. M e y n e 1 1 E. Theory and practice in experimental bacteriology, Cambridge, 1970, bibliogr.; Nomura M. Colicins and related bacteriocins, Ann. Rev. Microbiol., v. 21, p. 257, 1967, bibliogr.; W i 1-s o n G. S. a. M i 1 e s A. A. Topley and Wilson’s principles of bacteriology and immunity, v. 1—2, L., 1964.
A. V. Ponomarev.