Identification of bacteria by antigenic structure. Serological reactions. Study of physiological and biochemical properties
IDENTIFICATION OF MICROBES(Late Lat. identificare to identify) - determination of the species or type of microbes. I. m. is the most important stage of microbiol, research necessary to determine the etiology of an infectious disease; it is of great importance for epidemiol, analysis of outbreaks of infectious diseases and carrying out effective measures to eliminate them. I. m. is also widely used for dignity. assessment of soil, air, water and food.
I. m. is carried out by studying the complex of morphological, cultural, biochemical, antigenic, pathogenic and other properties of a given culture, which makes it possible to establish its identity (identity) to typical representatives of a certain species (type) of microorganisms. For these studies, it is usually necessary to have a pure culture, since the presence of foreign microbes can lead to erroneous conclusions.
The choice of research methods for I. m. is largely determined by the source of isolation of the microbe (for example, material obtained from a patient, from a corpse or environmental objects).
Determination of the properties of microorganisms
There are no general I. m. schemes used in practice. For each group of microorganisms, identification is carried out on the basis of their biol characteristics. Thus, for the identification of viruses (see), the types of cell cultures in which their reproduction occurs, the nature of the cytopathic action, the formation of inclusions, the antigenic structure, in some cases the morphology of the viruses, as well as the pathogenicity of the viruses for experimental animals are important.
The proposal of some researchers deserves attention [Cowan and Steel (S. T. Cowan, K. I. Steel), 1961, 1965; Seeley and Van Demark (H. W. Seeley, V. I. Van Demark), 1972] use Gram staining as the starting point for identifying bacteria. At the first stage of differentiation of gram-positive bacteria, the authors take into account cell shape, acid resistance, spore formation, motility, production of catalase, oxidase, and relationship to glucose, and of gram-negative bacteria - cell shape, motility, production of catalase, oxidase, and relationship to glucose. At subsequent stages of research, using tables characterizing bacteria belonging to a particular genus, they find the key to identifying species, subspecies and types.
Morphological and tinctorial properties
The study of morphol and tinctorial signs of a microbe is usually only the initial stage of its identification. The morphology of microorganisms is studied by microscopy of fixed and stained preparations, as well as living unstained microorganisms in a hanging or crushed drop.
For long-term observation of living bacteria, special cameras are used (Peshkova, Fontbrune). Microscopic examination makes it possible to determine the shape, size and structure of microorganisms, their relative position, motility, number and distribution of flagella, shape and position of spores, as well as the formation of capsules. To study motility, young (no older than 6-8 hours) fast-growing broth cultures are taken. Flagella are more easily detected in young agar cultures, spores, on the contrary, in cultures grown for several days, and capsules in patol and exudates. For hanging drop microscopy, it is better to use a darkfield or phase contrast device. It should be taken into account that the shapes and sizes of microorganisms change depending on the characteristics of the strain, the age of the culture, the composition of the medium, the incubation temperature and other factors.
The tinctorial properties of microbes are determined by staining fixed preparations. Gram staining allows you to divide all bacteria into 2 groups: gram-negative and gram-positive (see Gram method). Ziehl-Neelsen staining makes it possible to differentiate acid-fast bacteria from non-acid-fast bacteria (see Ziehl-Neelsen method). Using special methods, individual elements of a bacterial cell are identified: nucleoid, protoplasm and inclusions (methods of Romanovsky-Giemsa, Feilgen, Robineau, etc.), metachromatic granules (see Neisser methods, etc.), flagella, capsules and spores. The method of fluorescent antibodies makes it possible to preliminary determine the type and even type of microbe (see Immunofluorescence).
In cases where the morphology of a microbe is specific, microscopic examination can presumptively identify it. In medical In microbiology, this kind of identification is justified only when it corresponds to the wedge and diagnosis. So, for example, acid-fast bacilli in the cerebrospinal fluid of a patient with wedge, symptoms of meningitis can be tentatively attributed to tuberculous mycobacteria. Gram-negative bipolar staining ovoid rods in the juice of lymph nodes of a patient with inguinal buboes in areas where plague is widespread can be considered presumably as plague bacteria.
Cultural properties indicate that a microbe belongs to a certain group and outline the direction of further research in order to finally identify it. They are determined by sowing the culture under study on nutrient media (agar, broth, injection into gelatin, etc.). Of the cultural characteristics of bacteria and fungi, the appearance and internal structure of the colonies formed when the culture is sown on solid nutrient media are important. If a microbe does not grow on regular meat peptone agar, then another medium that is optimal for it should be used. Colonies are usually examined after 24 hours of incubation at t° 37°, and then again at intervals of 1 - 3 days. When describing colonies, attention is paid to their size, color (pigmentation), shape, profile, surface, edges, and density. If bacteria tend to dissociate into phase variants (see Dissociation of bacteria), then they are separated by sieving on Petri dishes with a nutrient medium. When growing on liquid nutrient media, growth is near the bottom, growth in the form of a film, or uniform turbidity of the medium. In some cases, growth is studied on special media, such as Loeffler's serum, glycerin potatoes, media containing blood, etc. The cultural properties of the microbe are an essential addition to its morphological characteristics.
Resistance of microbes to various environmental factors
The resistance of microbes to various environmental factors is used in I. m., since in some cases microbes differ significantly in this characteristic. So, for example, non-spore-bearing bacteria and vegetative forms of spore-bearing bacteria are sensitive to temperature and to low concentrations of antiseptics. They die at a temperature of 60° within half an hour and in a 1% phenol solution within 1 hour. Acid-fast bacteria are temperature sensitive but relatively resistant to disinfectants; they die at a temperature of 60° within half an hour, but in the cold they resist antiseptics often for several hours. Bacterial spores are especially highly resistant (see Spores, bacteria). They die either from steam under pressure (at a temperature of 120° for half an hour) or from high concentrations of antiseptics, for example, under the influence of 5% phenol for several hours. Therefore, if the formation of spores by a microbe is suspected, temperature resistance tests are performed.
For certain types of bacteria, their resistance to certain antibiotics and chemotherapeutic drugs is indicative. So, for example, one of the tests that makes it possible to differentiate the classic cholera vibrio from the El-Tor vibrio, as well as Proteus mirabilis from other intestinal bacteria, is the ability of the El-Tor vibrio and Proteus mirabilis to grow in the presence of polymyxin B (50 units in 1 ml and higher).
Features of physiology and biochemical activity
When determining the biochemical activity of microbes, their relationship to oxygen, carbon dioxide and various substrates, the optimal growth temperature, hemolytic ability, as well as the influence of various substances on their growth, including bacterial growth factors, are taken into account (see). In relation to free oxygen, microbes are usually divided into strict aerobes (see), strict and facultative anaerobes (see). Therefore, to isolate and identify the pathogen, special methods and nutrient media are used that promote the growth of only aerobic, facultative aerobic or anaerobic representatives.
For most pathogenic microbes, the optimal cultivation temperature is 37° (see Bacteria).
The hemolytic activity of microbes is determined by growing them in blood agar plates or by adding various dilutions of a broth culture to a suspension of washed erythrocytes.
Study of the influence of various biol, substrates and chemicals on the growth of bacteria. compounds (blood, serum, glucose, nitrates, bile salts, vitamins, amino acids, etc.) is often important for differentiating this group of microorganisms.
For I. m., the characteristics of the enzymatic activity of microbes, detected on media containing sugars and alcohols, protein substrates and fats (lipolytic properties), are of great importance, which makes it possible to identify subtle differences between closely related microbes. It is also important to determine the reducing properties of bacteria and their ability to form indole, ammonia and hydrogen sulfide, and use citrates and tartrates (see Differential diagnostic media).
Antigenic structure and relationship to bacteriophage
The antigenic structure and relationship to the bacteriophage and bactericins are studied at the final stage of I. m. Identification of the antigenic structure of microbes is carried out using various serols, reactions, for example, the agglutination reaction (see), the complement fixation reaction (see), etc.
If, in an extensive agglutination reaction, the tested microbe agglutinates to the titer of the immune serum or half the titer, then in practice it can be considered to belong to the species (type) with which this serum is designated. For complete identification, the isolated pathogen must be agglutinated to titer with an immune serum prepared against the reference microbe: the test microbe must adsorb all agglutinins from this serum. On the other hand, the reference microbe must be agglutinated to titer by the serum prepared against the microbe under study, and also adsorb all agglutinins from this serum. In other words, there must be complete cross-agglutination and cross-adsorption between both sera and both microbes. The agglutination reaction is sometimes supplemented or replaced by the precipitation reaction (see), as well as the indirect hemagglutination reaction (with red blood cells loaded with antibodies). Serol, the method reveals subtle differences between related microbes. It is often the only available method for differentiating subspecies or types of a given species.
Agglutinating monoreceptor sera are widely used in laboratory practice for the identification of Salmonella, Shigella and other microbes. The use of the immunofluorescence method (see), which allows you to quickly (1 - 2 hours) carry out I. m., is also very effective.
A sensitive method of I. m. is typing the identifying culture with a bacteriophage (see). This method is used, for example, in the study of typhoid bacillus (see Vi-typhoid phages), since it allows one to recognize the phagotype within a species. Specific phages are used to differentiate Shigella, cholera vibrios from cholera-like ones, classical cholera vibrios from El Tor vibrios, plague bacillus from pseudotuberculosis bacteria and other bacteria.
To differentiate some bacteria within a species, the phenomenon of bacteriocinogeny is used (see), as well as testing the sensitivity of bacteria to bactericins of various types (colicins, vibriocins, pesticins, diphtheriocins, etc.). Colicinotyping has found wide application to determine whether an isolated Shigella culture belongs to a specific colicinotype.
Pathogenicity for animals
The pathogenicity of microbes is usually determined in experiments on white mice, guinea pigs and rabbits. Animals are infected subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, orally, intranasally or intracerebrally (see Biological Assay).
When studying pathogenic microorganisms, it is sometimes necessary to determine whether they produce exotoxins. For this purpose, a filtrate of a bacterial culture grown for a certain period of time in an appropriate liquid medium is tested on sensitive animals. Exotoxins of highly toxic bacteria (diphtheria bacillus, tetanus bacillus, botulinum bacillus, etc.) cause disease in animals with a characteristic clinical picture and their subsequent death with typical pathological anatomical changes. To detect some microbial exotoxins, cultures of tissues sensitive to them, as well as chicken embryos, are used. Neutralization of exotoxins with specific antitoxins plays a significant role in I. m.
Bibliography: Krasilnikov N.A. Key to bacteria and actinomycetes, M.-L., 1949, bibliogr.; Guide to microbiological diagnosis of infectious diseases, ed. K. I. Matveeva, M., 1973; Tim and kov V.D. and Goldfarb D.M. 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 W. 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 u 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.
Federal Agency for Education
Biysk Technological Institute (branch)
state educational institution
in the courses “General Biology and Microbiology”, “Microbiology” for students of specialties 240901 “Biotechnology”,
260204 “Technology of fermentation production and winemaking”
all forms of education
UDC 579.118:579.22
Kamenskaya, microorganisms: methodological recommendations for laboratory work in the courses “General Biology”
and microbiology", "Microbiology" for students of specialties 240901 "Biotechnology", 260204 "Technology of fermentation production and winemaking" of all forms of education / ,
.
Alt. state tech. University, BTI. – Biysk:
Publishing house Alt. state tech. University, 2007. – 36 p.
These guidelines discuss the basic concepts, rules and principles of classification and identification of microorganisms. Laboratory work is presented to study the various properties of bacteria necessary to describe a bacterial strain and identify it to the genus level.
Reviewed and approved
at a department meeting
"Biotechnology".
Protocol No. 88 of 01/01/2001
Reviewer:
Doctor of Biological Sciences, Professor, BPGU named after.
© BTI AltSTU, 2007
1 BASIC CONCEPTS AND NAME RULES
MICROORGANISMS
Several thousand species of microorganisms have been described, but it is believed that this represents less than 1 % from those that actually exist. The study of the diversity of microorganisms is the subject of taxonomy. Its main task is to create a natural system reflecting the phylogenetic relationships of microorganisms. Until recently, the taxonomy of microorganisms was based primarily on phenotypic characteristics: morphological, physiological, biochemical, etc., therefore existing classification systems are largely artificial. However, they make it relatively easy to identify some newly isolated strains of microorganisms.
The taxonomy includes such sections as classification, nomenclature And Eden tification . Classification determines the order in which individuals with a given degree of homogeneity are placed into certain groups (taxa). Nomenclature is a set of rules for naming taxa. Identification means determining whether the organism under study belongs to a particular taxon.
The term “taxonomy” is often used as a synonym for systematics, but sometimes it is understood as a section of systematics, including the theory of classification, the study of the system of taxonomic categories, boundaries and subordination of taxa. The main taxonomic category in microbiology, as in other biological sciences, is view- a set of individuals characterized by a number of common morphological, physiological, biochemical, and molecular genetic characteristics.
The term “strain” refers to a pure culture of a microorganism isolated from a specific habitat (water, soil, animal body, etc.). Different strains of the same type of microorganisms may differ in some characteristics, for example, sensitivity to antibiotics, the ability to synthesize certain metabolic products, etc., but these differences are less than species-specific. The concept of “strain” in microbiology and genetics is somewhat different: in microbiology this concept is broader. Types of microorganisms are grouped into taxonomic categories of a higher order: genera, families, orders, classes, divisions, kingdoms. These categories are called mandatory. There are also optional categories: subclass, suborder, subfamily, tribe, subtribe, subgenus, subspecies. However, in taxonomy, optional categories are used quite rarely.
The nomenclature of microorganisms is subject to international rules. Thus, there is the International Code of Nomenclature of Bacteria. For yeast fungi, the main guide is “The Yeasts. A Taxonomic Study", for filamentous fungi and algae - International Code of Botanical Nomenclature.
To name objects in microbiology, as in zoology and botany, they use binary or binomial (from lat. bis- twice) a system of nomenclature, according to which each species has a name consisting of two Latin words. The first word means genus, and the second defines a specific species of this genus and is called a specific epithet. The generic name is always written with a capital letter, and the specific name – with lowercase even if the specific epithet is assigned in honor of a scientist, for example Clostridium pasteurianum. In the text, especially with Latin graphics, all phrases are highlighted in italics. When mentioning the name of a microorganism repeatedly, the generic name may be shortened to one or more initial letters, e.g. WITH.pasteurianum. If the text contains the names of two microorganisms that begin with the same letter (for example, Clostridium pasteurianum And Citrobacter freundii), then the abbreviations must be different (S. pasteurianum And Ct. freundii). If a microorganism is identified only to the genus, the word sp is written instead of the specific epithet. (species– type), for example Pseudomonas sp. In this case, when the name of a microorganism is mentioned again in the text, the generic name should always be written in full.
To name a subspecies, use a phrase consisting of the name of the genus, as well as specific and subspecific epithets. To distinguish between these epithets, a letter combination is written between them, which is an abbreviated word subspecies - “subsp.” or (less commonly) "ss." For example, Lactobacillus delbrueckii subsp. bulgaricus.
For each strain, indicate also the abbreviation of the name of the collection of microorganism cultures in which it is stored, and the number under which it is listed there. For example, Clostridium butyricum ATCC 19398 means that the strain is held in the American Type Culture Collection (ATCC) under number 19398. A list of world-famous collections of microorganisms is given in the Bergeys Manual of Systematic Bacteriology, 1984– 1989), in catalogs of microorganism cultures and other reference publications.
The description of any new species of microorganisms is based on a standard strain, which is stored in one of the collections of microorganisms and based on the totality of properties of which this species
characterized in the original article or definition. The type strain is the nomenclatural type of the species because the species name is assigned to it. If any strains originally included in the same species are subsequently found worthy of being designated as special species, they should be given new names, the old species name being retained for the type and related strains. In this case, the number of the renamed strain remains the same. Authentic strains are those that completely match their properties.
For a genus, a nomenclatural type is a specially designated type species that has a set of characters that are most characteristic of representatives of a given taxon. For example, in kind Bacillus the type species is IN.subtilis.
Some guides and catalogs indicate the old names of renamed microorganisms, as well as the names of the authors who first isolated this microorganism, and the year of publication in which this organism was first described. For example, one of the types of yeast is indicated in the catalog of the All-Russian Collection of Microorganisms (VKM) as Candida magnoliae(Lodder et Kreger van Rij, 1952) Meyer et Yarrow 1978, BKM Y-1685. This means that it was first described by Lodder and Kreger van Rij in a publication in 1952, at which time the species was named Torulopsis magnoliae. In 1978 Torulopsis magnoliae was renamed by researchers such as Meyer and Yarrow to Candida magnoliae and is currently stored in VKM under VKM number Y-1685. The letter Y in front of the strain number means “the Yeasts”.
In addition to the concept of “strain,” the terms “variant,” “type,” and “form” are used in microbiology. They are usually used to designate strains of microorganisms that differ in some characteristics from the type strain. A strain that differs from the typical one in morphological characteristics is called morphovar(morphotype), physiological and biochemical characteristics – biovar(biotype, physiological type), according to the ability to synthesize certain chemical compounds - hemovar(chemoform, chemotype), cultivation conditions – cultivar, by type of response to the introduction of a bacteriophage - phagovar(phagotype, lysotype), antigenic characteristics – serovar(serotype)
and so on.
In works on the genetics of microorganisms the term is often used "clone", by which we mean a population of genetically related cells obtained asexually from a single parent cell. In molecular biology, a clone refers to multiple
copies of identical DNA sequences obtained by inserting them into cloning vectors (for example, plasmids). The term “genetically modified” or “recombinant” strains refers to strains of microorganisms obtained as a result of genetic engineering manipulations. Often new strains of microorganisms are obtained using mutagens.
Each new strain of microorganisms isolated from natural or man-made sources must be characterized to obtain a complete set of data on the properties of the microorganism
in pure culture. These data can be used, for example, to compile a passport of industrially valuable strains, as well as for their identification.
Purpose of identification
– establish the taxonomic position of the studied strain based on a comparison of its properties with the studied and accepted (officially registered) species. Therefore, the result of identification is usually the identification of the microorganism under study with some species or assignment
to a certain genus. If the studied strain or group of strains differs in their properties from representatives of known taxa, then they can be separated into a new taxon. To do this, a description of the new taxon is given, including, for example, in the case of bacteria, the following: a list of strains included in the taxon; characteristics of each strain; list of properties considered as essential
in taxon; a list of properties that qualify a taxon for representation in the nearest higher taxon; a list of diagnostic characteristics that differentiate the proposed taxon from closely related taxa; a separate description of the typical (species) strain; photograph of a microorganism.
For a newly proposed taxon to be officially accepted, its description must be published according to certain rules. For example, valid or authorized publication of a bacterial taxon requires publication of an article describing it in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). If the publication appears in another reputable scientific journal (effective publication), then a reprint of the article from that journal is sent to IJSEM. Since 1980, the IJSEM has regularly published so-called lists of legal names of bacteria. They list all bacterial names that have been published in IJSEM (actual or authorized publication) or effectively published before in any
other reputable journals. Once a bacterial name is included in the IJSEM list of authorized names, the name is considered valid, regardless of whether it has previously been published in IJSEM or another journal. The date of publication of the name of a given taxon in IJSEM or in the list of legalized names of IJSEM is a priority for the taxon.
The culture of a type strain of a new type of microorganism is transferred for storage to one of the collections of microorganisms of world importance. If a type strain is lost, it can be replaced with a so-called neotype strain. In this case, it must be confirmed that the properties of the new strain coincide well with the description of the lost one. To indicate that a taxon is being proposed for the first time, the abbreviated combination "fam." is added after the name of the new family. nov.”, a new genus – “gen. nov.”, and the new species – “sp. nov." For example,
in 2000, with co-authors, a new family of bacteria was proposed - Oscillochloridaceae,
fam. nov. The expression “species insertac sedis” means that we are talking about a species that temporarily does not have a definite taxonomic status, since it is not clear in which taxon of a higher order - genus or family - the given species should be placed due to the lack of experimental data necessary for this
data.
2 DESCRIPTION AND IDENTIFICATION
MICROORGANISMS
As already noted, the principles of classification and identification of different groups of prokaryotes and eukaryotic microorganisms have significant differences. Identification of mushrooms to classes, orders
and families is based on the characteristic structural features and methods of formation, first of all, of sexual structures. In addition, the characteristics of asexual sporulation, the structure and degree of development of the mycelium (rudimentary, well-developed, septate or nonseptate), cultural (colony) and physiological characteristics are used. Differentiation of genera within families and identification of species are carried out using morphological characters obtained
using electron microscopy, as well as physiological and cultural features. There is no single determinant for identifying all mushrooms, so first the class or order of the mushroom being identified is determined and then the appropriate determinant for this class or order is used.
Identification of yeast fungi, which are among the widely used objects of various microbiological studies, is based on cultural (macromorphological), cytological, physiological and biochemical characteristics, characteristics of life cycles and the sexual process, specific characteristics related to ecology, and is carried out using special determinants for yeast.
The taxonomy of microscopic forms of algae is based on the structure of their cells and the composition of pigments. The systematic position of protozoa is determined using morphological features and life cycles. Thus, the identification of eukaryotes is based mainly on the features of their morphology and development cycles.
Identification of prokaryotes, which are morphologically less diverse than eukaryotes, is based on the use of a wide range of phenotypic and, in many cases, genotypic characters. To a greater extent than the identification of eukaryotes, it is based on functional characteristics, since most bacteria can be identified not by their appearance, but only by finding out what processes they are capable of carrying out.
When describing and identifying bacteria, their cultural properties, morphology, cell organization, physiological and biochemical characteristics, chemical composition of cells, content
guanine and cytosine (GC) in DNA, the nucleotide sequence in the gene encoding the synthesis of 16S rRNA and other pheno- and genotypic characteristics. In this case, the following rules must be observed: work with pure cultures, use standard research methods, and also use cells that are in an active physiological state for inoculation.
2.1 Cultural properties
Cultural, or macromorphological, properties include the characteristic features of the growth of microorganisms on solid and liquid nutrient media.
2.1.1 Growth on solid media
On the surface of dense nutrient media, depending on the seeding, microorganisms can grow in the form of a colony, streak or continuous lawn. Colony called an isolated cluster of cells of the same type, growing in most cases from a single cell. Depending on where the cells developed (on the surface of a dense nutrient medium, in its thickness or at the bottom of the vessel), they are distinguished superficial, deep And bottom colonies.
Education on topnal Colonies are the most significant feature of the growth of many microorganisms on a dense substrate. Such colonies are very diverse. When describing them, the following characteristics are taken into account:
profile– flat, convex, crater-shaped, cone-shaped, etc. (Figure 1);
form– round, amoeboid, irregular, rhizoid, etc. (Figure 2);
size (diameter)– measured in millimeters; if the size of the colony does not exceed 1 mm, then they are called dotted;
surface– smooth, rough, grooved, folded, wrinkled, with concentric circles or radially striated;
shine And transparency– the colony is shiny, matte, dull, powdery, transparent;
color– colorless (dirty white colonies are classified as colorless) or pigmented – white, yellow, golden, orange
vaya, lilac, red, black, etc.; especially note the allocation in
pigment substrate; when describing colonies of actinomycetes, the pigmentation of aerial and substrate mycelium is noted, as well as the release of pigments into the medium;
edge– smooth, wavy, jagged, fringed, etc. (Figure 3);
structure– homogeneous, fine- or coarse-grained, streamy, etc. (Figure 4); the edge and structure of the colony are determined using a magnifying glass or at low magnification of a microscope. To do this, the Petri dish is placed on the microscope stage with the lid down;
consistency determined by touching the surface of the colony with a loop. The colony can be easily removed from the agar, be dense, soft or growing into the agar, mucous (sticks to the loop), viscous, look like a film (removed entirely), be fragile (easily breaks when touched by the loop).
1 - curved; 2 – crater-shaped; 3 – lumpy;
4 – growing into the substrate; 5 – flat; 6 – convex;
7 – teardrop-shaped; 8 – cone-shaped
Figure 1 – Colony profile
deep colonies, on the contrary, they are quite monotonous. Most often they look like more or less flattened lentils,
in projection they have the shape of ovals with pointed ends. Only
in a few bacteria, deep colonies resemble tufts of cotton wool
with filamentous outgrowths into the nutrient medium. The formation of deep colonies is often accompanied by rupture of the dense medium if microorganisms release carbon dioxide or other gases.
Bottom colonies of various microorganisms usually take the form of thin transparent films spreading along the bottom.
The size and many other features of the colony can change with age and depend on the composition of the environment. Therefore, when describing them, indicate the age of the culture, the composition of the medium and the cultivation temperature.
1
– round; 2
– round with scalloped edge; 3
– round with a roll around the edge; 4, 5
– rhizoid; 6
– with a rhizoid edge; 7 – amoeboid;
8
– thread-like; 9
– folded; 10
– incorrect;
11 – concentric; 12 - complex
Figure 2 – Colony shape
/ – smooth; 2 – wavy; 3 – toothed; 4 – bladed; 5 – incorrect; 6 – ciliated; 7 – filamentous; 8 – villous; 9 - branchy
Figure 3 – Colony edge
1 – homogeneous; 2 – fine-grained; 3 – coarse-grained;
4 – streamy; 5 – fibrous
Figure 4 – Colony structure
When describing the growth of microorganisms by stroke note the following features: scanty, moderate or abundant, continuous
with a smooth or wavy edge, clear-shaped, resembling chains of isolated colonies, diffuse, pinnate, tree-like, or rhizoid (Figure 5). Characterize the optical properties of plaque, its color, surface and consistency.
To describe colonies and streak growth, many microorganisms are often grown on meat peptone agar. Meat-peptone gelatin is also used. To better view deep colonies, media containing agar or gelatin are recommended to be clarified.
1 – solid with a smooth edge; 2 – solid with a wavy edge; 3 – clearly visible; 4 – diffuse; 5 – feathery; 6 – rhizoid
Figure 5 – Growth of bacteria along the line
2.1.2. Growth in liquid media
The growth of microorganisms in liquid nutrient media is more uniform and is accompanied by turbidity of the medium, the formation of a film or sediment. Characterizing the growth of microorganisms in a liquid medium, it is noted degree of turbidity(weak, moderate or strong), film features(thin, dense or loose, smooth or folded),
and when a sediment forms, it is indicated whether it is scanty or abundant, dense, loose, slimy or flaky.
Often the growth of microorganisms is accompanied by the appearance of odor, pigmentation of the environment, and the release of gas. The latter is detected by the formation of foam, bubbles, and also with the help of “floats” - small tubes sealed at one end. The float is placed
into the test tube with the sealed end up before sterilizing the medium and make sure that it is completely filled with the medium. If gas is released, it accumulates in the float in the form of a bubble.
To describe the growth pattern of microorganisms in liquid media, they are grown in meat peptone broth (MPB) or in another medium that ensures good growth.
2.2 Morphological characteristics
The morphological characteristics and organization of bacterial cells include such characteristics as the shape and size of cells, their motility, the presence of flagella and the type of flagellation, and the ability to form spores. Detection in cells may also be useful.
characteristic membrane systems (chlorosomes, carboxysomes, phycobilisomes, gas vacuoles, etc.) inherent in individual groups of bacteria
rium, as well as inclusions (parasporal bodies, volutin granules,
poly-β-hydroxybutyrate, polysaccharides, etc.). Gram staining of cells is of primary importance for the taxonomy of bacteria.
and the structure of their cell walls.
2.3 Physiological and biochemical properties
The study of physiological and biochemical properties includes, first of all, establishing the method of nutrition of the bacterium under study (photo/chemo-, auto/heterotrophy) and the type of energy metabolism (ability for fermentation, aerobic or anaerobic respiration or photosynthesis). It is important to determine such characteristics as the ratio of bacteria to molecular oxygen, temperature, pH of the environment, salinity, light and other environmental factors. In this group of signs
also includes a list of substrates utilized as sources of carbon, nitrogen and sulfur, the need for vitamins and other growth factors, the formation of characteristic metabolic products, and the presence of certain enzymes. For this purpose, special tests are used.
Many of the tests used to detect these signs (sometimes called routine tests) are important for diagnosis and are widely used in medical microbiology. Their production requires a significant investment of time, a large number of complex media and reagents, compliance with standard conditions, and careful execution. To speed up and facilitate the process of identifying some microorganisms that are mainly of medical importance, various test systems have been developed, for example, the Oxi/Ferm Tube, Mycotube and Enterotube II systems from Hoffmann-La Roche (Switzerland), etc. Thus, the Enterotube II system, designed for the identification of enterobacteria, it is a plastic chamber with 12 cells containing colored diagnostic media. Inoculation of all media is carried out by translational-rotational movements through the chamber of the needle with seed material. Incubation is carried out for 24 hours at a temperature of 37 ºС. A positive or negative test result is judged by a change in the color of the medium, rupture of the agar (gas formation test) or after the introduction of special reagents (indole formation test, Voges-Proskauer reaction). Each characteristic is designated by a specific number, so the obtained data can be entered into a computer with the appropriate program and receive an answer about the taxonomic position of the strain under study.
Determining the composition of bacterial cells is also important for their systematics (chemosystematics). Chemotaxonomic methods can be important, in particular, for those groups of bacteria in which morphological and physiological characteristics vary widely and are insufficient for satisfactory identification. The cell walls of different prokaryotes include several classes of unique heteropolymers: murein (or pseudomurein), lipopolysaccharides, mycolic and teichoic acids. The composition of the cell wall also determines the serological properties of bacteria. This underlies immunochemical methods for their identification.
The lipid and fatty acid composition of bacterial cells is sometimes also used as a chemotaxonomic marker. Intensive study of fatty acids became possible with the development of gas chromatographic analysis. Differences in lipid composition are used to identify bacteria at the genus and even species level. This method, however, has certain limitations, since the fatty acid content of the cells may depend on the culture conditions and the age of the culture.
The taxonomy of some bacteria takes into account the composition of quinones
and other electron carriers, as well as pigments.
Important information about the mutual relationship of bacteria can be obtained by studying cellular proteins - products of gene translation. Based on the study of membrane, ribosomal, total cellular proteins, as well as individual enzymes, a new direction was formed - protein taxonomy. The spectra of ribosomal proteins are among the most stable and are used to identify bacteria at the family or order level. The spectra of membrane proteins can reflect genus, species, and even intraspecific differences. However, the characteristics of the chemical compounds of a cell cannot be used to identify bacteria in isolation from other data describing the phenotype, since there is no criterion for assessing the significance of phenotypic characteristics.
Sometimes a method is used to identify bacteria or other microorganisms, such as yeast. numeric (or Adansonian) taxonomy. It is based on the ideas of the French botanist M. Adanson, who proposed that various phenotypic traits that can be taken into account should be considered equivalent, which makes it possible to quantify the taxonomic distances between organisms in the form of the ratio of the number of positive traits to the total number of those studied. The similarity between two organisms under study is determined by quantitatively assessing the largest possible number (usually at least one hundred) of phenotypic characteristics, which are selected so that their options are alternative and can be indicated by “minus” and “plus” signs. The degree of similarity is established based on the number of matching features and is expressed as a correspondence coefficient S:
Where a + d– the sum of characteristics for which strains A and B coincide;
A– both strains with positive characteristics;
d– both with negative;
b– the sum of characteristics for which strain A is positive, strain B is negative;
With– the sum of characteristics for which strain A is negative and strain B is positive.
The value of the correspondence coefficient can vary from 0 to 1. Coefficient 1 means complete identity, 0 means complete dissimilarity. Evaluations of combinations of characteristics are made using a computer. The results obtained are presented in the form of a similarity matrix and/or in the form of a dendrogram. Numerical taxonomy can be used when assessing the similarity between taxa of microorganisms only of low rank (genus, species). It does not allow one to draw direct conclusions regarding the genetic relationship of microorganisms, but to a certain extent reflects their phylogenetic properties. Thus, it has been established that the phenotypic characteristics of bacteria that can currently be studied reflect from 5 to 20% of the properties of their genotype.
2.4 Genotype study
The study of the genotype of microorganisms became possible as a result of the successful development of molecular biology and led to the emergence of genosystematics. Genotype research based on nucleic acid analysis, in principle, makes it possible to construct over time a natural (phylogenetic) system of microorganisms. Phylogenetic relationships of bacteria are assessed determination of molar content guanine and cytosine (GC) in DNA, DNA methods–DNA and DNA–rRNA hybridization using DNA probes, as well as studying the nucleotide sequence in 5S,
J6
SAnd
23
S rRNA.
2.4.1 Determination of the molar content of GC
The determination of the molar content of GCs from the total number of DNA bases in prokaryotes, as already indicated, ranges from 25 to 75%. Each bacterial species has DNA with a characteristic average GC content. However, since the genetic code is degenerate, and genetic coding is based not only on the content of nucleotide bases in coding units (triplets), but also on the relative position, the same average content of GCs in the DNA of two bacterial species can be accompanied by their significant genotypic
division. If two organisms are very similar in nucleotide composition, then this can be evidence of their evolutionary relationship only if they have a large number of common phenotypic characteristics or genetic similarity confirmed by other methods. At the same time, the discrepancy (more than 10...15%) in the nucleotide composition of the DNA of two strains of bacteria with common phenotypic properties shows that they belong, at least, to different species.
2.4.2 DNA method –DNA hybridization
This method is more important for assessing the genetic relatedness of bacteria. When experiments are carried out carefully, valuable information can be obtained about the degree of their genetic homology. Within one bacterial species, the degree of genetic homology of strains reaches from 70 to 100%. However, if, as a result of evolutionary divergence, the nucleotide base sequences of the genomes of two bacteria differ to a greater extent, then specific DNA-DNA reassociation becomes so weak that it cannot be measured. In this case, DNA–rRNA hybridization makes it possible to significantly increase the range of organisms in which the degree of genetic homology can be determined due to the fact that in a relatively small section of the bacterial genome encoding ribosomal RNA, the original base sequence is preserved much more completely than in other sections of the chromosome. As a result, DNA–rRNA hybridization often reveals a fairly high homology of the genomes of bacteria in which DNA–DNA reassociation does not reveal noticeable homology.
2.4.3 DNA probe (gene probe) method
The DNA probe method is a variation of the DNA–DNA molecular hybridization method. In this case, the hybridization reaction is carried out not between two total DNA preparations, but between a fragment of the DNA nucleotide sequence (probe), including a gene (genetic marker) responsible for a specific function (for example, resistance to some antibiotic), and DNA the bacteria being studied. The most common way to create gene probes is to isolate specific fragments by molecular cloning. To do this, first create a gene bank of the studied bacterium by splitting its DNA with endonucleases.
restriction, and then select the desired clone from the sum of DNA fragments by electrophoresis, followed by checking the genetic properties of these fragments by transformation. Next, the selected DNA fragment is ligated into a suitable plasmid (vector),
and this combined plasmid is introduced into a bacterial strain convenient for work (for example, Escherichia coli).
Plasmid DNA is isolated from the bacterial biomass carrying the DNA probe and labeled, for example, with a radioisotope label. Then the DNA probe is hybridized
with bacterial DNA. The resulting hybrid areas are demonstrated by autoradiography. Based on the relative frequency of hybridization of a genetic marker with the chromosome of a particular bacterium, they make
conclusion about the genetic relationship of these bacteria with the strain under study.
2.4.4 Nucleotide sequence analysis method
in ribosomal RNA
To identify bacteria and create a phylogenetic system for their classification, the most widespread and important method is the analysis of nucleotide sequences in ribosomal RNA. The 5S, 16S and 23S rRNA molecules contain regions with the highest degree of genetic stability. It is believed that they are outside the mechanism of natural selection and evolve only as a result of spontaneous mutations occurring at a constant rate. The accumulation of mutations depends only on time, therefore information on the nucleotide sequence of these molecules is considered the most objective for determining the phylogenetic relationship of organisms at the level from subspecies to kingdom. In case of analysis
5S rRNA usually determines the complete nucleotide sequence, which in this molecule in prokaryotes is 120 nucleotides. When studying 16S and 23S rRNA, containing 1500 and 2500 nucleotides, respectively, oligonucleotides obtained from these molecules are often analyzed using specific restriction endonucleases. The most widespread study is the study of the nucleotide sequence in 16S rRNA. The study of the structure of 16S rRNA of representatives of various microorganisms led to the identification of a group of archaea among prokaryotes. Similarity coefficient values SAB,
separating the I6S rRNA of bacteria and archaea are within 0.1, while the value SAB,
equal to 1.0 corresponds to complete homology of nucleotide sequences, and 0.02 to the level of random coincidence.
Increasingly, dendrograms are being proposed for the identification of bacteria, showing the relationships between bacterial genera, species or strains based on the study of the sequence of nucleotides (or oligonucleotides) in rRNA, as well as DNA-DNA
and DNA–rRNA hybridization. However, identification of bacteria before birth based only on genetic methods without first studying their phenotypic characteristics is often completely impossible. Therefore, the best approach to working on bacterial taxonomy is considered to be the study of both genotypic and phenotypic properties. In case of inconsistency between phylogenetic and phenotypic data, priority is temporarily given to the latter.
A particular challenge is the identification of those bacteria and archaea, especially marine species, that are unable to grow on known laboratory nutrient media and for which pure cultures could therefore not be obtained. Until recently, this problem seemed insoluble. However, about 15 years ago, methods were developed that made it possible to extract, clone, sequence
and compare ribosomal RNAs directly from the environment. This made it possible to accurately count and identify microorganisms inhabiting a given biotope without isolating them into a pure culture. A microorganism thus identified that is “uncultivable” in the laboratory can even be described, but with the addition of the word “candidatus” (candidate). The word “candidatus” will accompany the new species until scientists find conditions for cultivating this organism in the laboratory and obtain a pure culture of it, which will allow all its properties to be studied and published as legal.
Identification of bacteria is usually carried out using Bergey's Manual of Determinative Bacteriology. The first edition of this manual was published in 1923 under the leadership of the famous American bacteriologist D. H. Bergey (1860–1937). Since then Since then, it has been regularly republished with the participation of the world's leading microbiologists. In the latest, ninth edition, all bacteria are divided into 35 groups according to easily identifiable phenotypic characteristics.
in the group names. The taxonomic position of bacteria within groups is determined using tables and keys compiled on the basis of a small number of phenotypic characters. Differentiation tables for differentiating species of bacteria of certain genera, for example the genus Bacillus,
are not given, and the reader is referred to Burgee's Guide to the Taxonomy of Bacteria.
The four-volume Bergey's Manual of Systematic Bacteriology, 1984–1989 contains more complete information about the taxonomic position of bacteria. For each group of bacteria, a description of the genera included in it is given
and species, including those with unclear taxonomic status. In addition to a detailed phenotypic description, including the morphology, organization and chemical composition of cells, antigenic properties, type of colonies, features of the life cycle and ecology, the characteristics of the genera also provide information on the content of GCs in DNA, the results of DNA-DNA and DNA-rRNA hybridization. Keys and tables allow you to identify bacteria not only to genus, but also to species.
Currently, the second edition of the four-volume Bergcy's Manual of Systematic Bacteriology has been published. The first volume was published in 2002. In addition, there are a number of articles and books that offer original keys for identifying individual groups of bacteria, for example, bacilli, pseudomonads, actinomycetes, enterobacteria.
Currently, a lot of new data has accumulated, including those obtained from the analysis of nucleotide sequences of ribosomal RNA, about previously studied and newly isolated bacterial species. Based on this information, the species composition of some groups of bacteria, for example the genus Bacillus, will be revised: some species will remain within the genus Bacillus, and some will form new genera or will be assigned to other, already existing genera of bacteria. It should also be noted that to describe new strains of bacteria, as a rule, more characteristics are studied than are necessary for their identification, since the keys and tables do not include all the characteristics of the identified bacteria, but only those that differ in different species (Table 1).
Table 1 - Minimum list of data required for
descriptions of new strains of bacteria (according to H. Truper, K. Schleifer, 1992)
Properties | Main features | Additional signs |
Cell morphology | Form; size; mobility; intra- and extracellular structures; mutual arrangement of cells; cellular differentiation; type of cell division; cell ultrastructure | Color; nature of flagellation; disputes; capsules; covers; outgrowths; life cycle; heterocysts; ultrastructure of flagella, membrane and cell wall |
Continuation of Table 1
Growth pattern | Features of growth on solid and liquid nutrient media; Colony morphology | Colony color, suspension |
Acid resistance; color of spores, flagella |
||
Cell composition | DNA composition; spare substances | Homology of nucleic acids; cellular pigments; cell wall composition; typical enzymes |
Physiology | Relation to temperature; to the pH of the environment; type of metabolism (phototroph, chemotroph, lithotroph, organotroph); relation to molecular oxygen; electron acceptors; carbon sources; nitrogen sources; sulfur sources | Requirement for salts or osmotic factors; need for growth factors; typical metabolic products (acids, pigments, antibiotics, toxins); antibiotic resistance |
Ecology | Living conditions | Pathogenicity; circle of hosts; formation of antigens; serology; susceptibility to phages; symbiosis |
3 LABORATORY WORK “IDENTIFICATION
MICROORGANISMS"
Goal of the work: familiarization with the basic principles of identifying microorganisms. During laboratory work, each student studies the properties of bacteria necessary to describe a bacterial strain and identify it to the genus level.
Tasks
1. Determine the purity of the identified bacterium and study the morphology of its cells.
2. Describe cultural properties.
3. Study the cytological properties of the identified bacteria.
4. Study the physiological and biochemical properties of identifiable bacteria.
5. Determine the sensitivity of bacteria to antibiotics.
6. Fill out the table and summarize.
3.1 Determination of the purity of the identified bacterium
and studying the morphology of its cells
To carry out work on identifying microorganisms, each student receives one bacterial culture (on a slanted agar medium in a test tube), which is then tested for purity. This is done in several ways: visually, by sowing on nutrient media and microscopy.
Growth pattern The resulting bacterium is viewed by a streak on the surface of the slanted agar medium. If the growth along the line is not uniform, then the crop is contaminated. Then the culture is screened into a test tube onto a slant medium (meat peptone agar) for use
in further work, and also sift on the surface of a solid medium in a Petri dish using the exhaustion streak method to check for purity (by the uniformity of the grown colonies). The inoculated test tubes and dishes are placed in a thermostat at a temperature of 30 ºС for a period of 2 to 3 days. The remainder of the original bacterial culture in a test tube is used to check for purity using microscopy (based on the morphological homogeneity of the population), as well as to study the shape, relative position, mobility of cells and their sizes. The culture is microscoped using “crushed drop” preparations and a preparation of fixed, fuchsin-stained cells. The results are entered into a table compiled in the form of Table 2.
table 2 – Properties of the identified bacterium
Properties | Signs | results |
Cultural properties | ||
Size, mm | ||
Surface | ||
Structure | ||
Consistency | ||
Cell morphology and | Shape and arrangement of cells | |
Mobility | ||
Presence of endospores | ||
Gram stain | ||
Acid resistance painting | ||
Physiological and biochemical properties | Relation to molecular oxygen | |
Growth on glucose medium | ||
Growth on gelatin medium | ||
Growth on medium with milk | ||
Growth on starch medium | ||
Catalase test | ||
Antibiotic sensitivity |
3.2 Cultural properties
In the next lesson, a Petri dish seeded with a suspension of the identifiable bacterium is examined. The criterion for the purity of a culture is the uniformity of the grown colonies. Describe the cultural properties of bacterial colonies in accordance with section
scrap 2.1 and the results are entered into table 2.
3.3 Study of the cytological properties of identified bacteria
3.3.1 Presence of endospores
A small number of cells from a solid medium is placed in a loop on a glass slide in a drop of tap water and a smear is made. The smear is dried in air, fixed in a burner flame and a 5% solution of chromic acid is applied to it. After 5...10 minutes it is washed off with water. The preparation is covered with a strip of filter paper and the paper is generously moistened with Ziehl’s carbolic fuchsin. Heat the preparation over a flame until vapor appears (not to a boil), then take it aside and add a new portion of dye. This procedure is carried out for 7 minutes. It is important that the dye evaporates, but the paper does not dry out. After cooling, it is removed, the preparation is washed with water and thoroughly blotted with filter paper.
If all operations are performed correctly, the coloring turns out to be contrasting, and bright red spores stand out clearly against the blue background of the cytoplasm.
3.3.2 Gram stain
3.3.2.1 Make a thin smear on a degreased glass slide in a drop of water so that the cells are evenly distributed over the surface of the glass and do not form clumps.
3.3.2.2 The preparation is dried in air, fixed over a burner flame and stained for 1...2 minutes with carbolic gentian or crystal violet.
3.3.2.3 Then the dye is drained and the smears are treated for 1...2 minutes with Lugol’s solution until they turn black.
3.3.2.4 Drain Lugol’s solution, decolorize the preparation for 0.5...1.0 min with 96% ethyl alcohol and quickly wash with water.
3.3.2.5 Additionally stained for 1…2 minutes with water fuchsin.
3.3.2.6 The dye is drained, the preparation is washed with water and dried.
3.3.2.7 Microscope with an immersion system.
When stained correctly, gram-positive bacteria are blue-violet, gram-negative – pink-red color.
To obtain reliable results, it is necessary to prepare smears for Gram staining from young, actively growing (usually one-day old) cultures, since cells from old cultures sometimes give an unstable Gram reaction. Gram-negative bacteria may appear as gram-positive bacteria if the bacterial film (smear) is too thick and alcohol bleaching is not completed completely. Gram-positive bacteria may appear as gram-negative bacteria if the smear is bleached with alcohol.
3.3.3 Painting for acid resistance
A smear of the bacteria being studied is prepared on a fat-free glass slide in a drop of water. The preparation is dried in air and fixed over a burner flame. Filter paper is placed on the smear, the preparation is filled with Ziehl’s carbol fuchsin and heated 2-3 times until vapor appears, holding the slide with tweezers high above the burner flame. The appearance of vapors is observed by looking at the smear from the side, and when they appear, they are immediately put aside.
drug to the side. Allow the preparation to cool, remove the filter paper, drain the dye and wash the smear with water. Then
the cells are decolorized with a 5% solution of acid Hhttps://pandia.ru/text/79/131/images/image009_42.gif" width="11" height="23 src=">. To do this, the glass slide is immersed 2–3 times in a glass of sulfuric acid,
without keeping him in it. The preparation is again thoroughly washed with water and stained for 3 to 5 minutes with methylene blue (according to Leffler). The paint is drained, the preparation is washed with water, dried and examined with an immersion system. When stained correctly, cells of acid-fast bacteria appear red, while cells of non-acid-fast bacteria – blue.
3.3.4 Determination of mobility
The culture under study is inoculated into a column of 0.2...0.5% semi-liquid agar using the prick method. In order for the growth characteristics to appear most clearly, the puncture is made in close proximity to the wall of the test tube. Sowing is placed in a thermostat for 24 hours. Sowing done in this way makes it possible to identify and separate mobile microorganisms from immobile ones.
Stationary forms of bacteria grow along the puncture line, forming small cylindrical or conical outgrowths. The environment remains completely transparent. Mobile microbes during such inoculation cause pronounced turbidity, spreading more or less evenly throughout the entire thickness of the medium.
3.4 Study of physiological and biochemical properties
identifiable bacteria
3.4.1 Relation to molecular oxygen
In relation to molecular oxygen, microorganisms are divided into four groups: obligate aerobes, microaerophiles, facultative aerobes (anaerobes) and obligate anaerobes. To judge
to determine whether microorganisms belong to one group or another, the microbial suspension is inoculated into test tubes with melted and cooled to a temperature of 45 ºC agar nutrient medium. Sowing can also be done by injection. Strict aerobes grow on the surface of the medium and in the upper layer, microaerophiles– at some distance from the surface. Facultative anaerobes usually develop throughout the entire thickness of the medium. Strict anaerobes grow only in the depths of the medium, at the very bottom of the test tube (Figure 6).
|
1 – aerobes; 2 – microaerophiles; 3 – facultative anaerobes;
4 – anaerobes
Figure 6 – Growth of microorganisms during sowing by injection ( A) and when sowing in a molten dense medium ( b)
3.4.2 Growth on glucose and peptone media
The culture is introduced with a sterile loop into a liquid medium containing: 5.0 g/l peptone, 1.0 g/l K2HP04, 10.0 g/l glucose, 2 ml bromothymol blue (1.6% alcohol solution), distilled water poured into test tubes (8...10 ml each) with floats. The duration of cultivation is 7 days in a thermostat at a temperature of 30 °C. The growth of microorganisms or its absence is determined by the turbidity of the medium, the formation of a film or sediment. A change in the color of the indicator (bromothymol blue) indicates the formation of acidic (yellow color of the medium) or alkaline (blue color of the medium) metabolic products. The formation of gas is indicated by its accumulation in the float. The observation results are compared with a sterile environment.
3.4.3 Growth on gelatin medium
The activity of extracellular proteolytic enzymes in microorganisms is determined using gelatin, casein or other proteins as a substrate. The medium with gelatin consists of meat-peptone broth (MPB) and 10...15% gelatin (MPB). Sowing is carried out by injection.
Using a bacteriological needle, microbial cells are sterilely selected from the shoal and the needle is inserted into the thickness of the MPZ column to the bottom of the tube.
The duration of cultivation is from 7 to 10 days at room temperature. Liquefaction of gelatin is noted visually. If gelatin liquefies, indicate the intensity and form of liquefaction - layer-by-layer, funnel-shaped, bag-shaped, crater-shaped, turnip-shaped, bubble-shaped.
3.4.4 Growth on medium with milk
Sowing on “milk agar” in Petri dishes is carried out to determine the ability of bacteria to decompose milk casein. The medium consists of equal parts of sterile skim milk and sterile 3% aqueous agar agar. Bacteria are inoculated with a loop, drawing a stroke along the diameter of the cup or along the center of the sector into which the cup is divided. The duration of bacterial cultivation in a thermostat at 30 °C is 7 days. Casein hydrolysis is detected by the zone of clearing of the medium around colonies or a culture of microorganisms grown along the line. The zone is especially clearly visible after treating the medium with grown bacteria with a solution of 5% trichloroacetic acid. The hydrolysis zone of casein is measured in millimeters from the edge of the streak or colony to the border of the light zone. The larger the diameter of the light zone, the higher the caseinolytic activity of bacteria.
3.4.5 Growth on starch medium
Sowing on agar medium with starch (in Petri dishes) containing (g/l): peptone – 10.0; KN2R04 – 5.0; soluble starch – 2.0; agar – 15.0; pH 6.8 – 7.0, is produced to determine the formation of amylase by microorganisms. Bacteria are inoculated with a loop, drawing a stroke along the diameter of the cup or along the center of the sector into which the cup is divided. The duration of bacterial cultivation is 7 days in a thermostat at a temperature of 30 °C. Starch hydrolysis is detected after treating the medium with grown bacteria with Lugol's solution. To do this, pour 3 to 5 ml of Lugol's solution onto the surface of the medium. The medium containing starch turns blue, and the hydrolysis zone remains colorless or acquires a red-brown color if the starch has been hydrolyzed to dextrins. The starch hydrolysis zone is measured from the edge of the streak (colony) to the border of the light zone (mm). The larger the diameter of the light zone, the higher the amylase activity.
3.4.6 Catalase test
Part of the grown culture is suspended using a bacteriological loop in a drop of 3% hydrogen peroxide on a glass slide. The presence of catalase is indicated by the formation of gas bubbles, observed 1...5 minutes after the introduction of bacteria with the naked eye or under a microscope at low magnification. You can apply a few drops of hydrogen peroxide directly to a colony or culture grown on an agar slant and observe the release of molecular oxygen.
3.4.7 Determination of bacterial sensitivity
to antibiotics
The sensitivity of microorganisms to antibiotics can be conveniently determined using ready-made paper disks impregnated with certain antibiotics. The microorganisms under study are grown on an appropriate solid nutrient medium. A thick suspension of the microorganism being studied is prepared in sterile tap water by washing the cells with water from the surface of the solid nutrient medium. Working near the burner flame, add 1 ml of the resulting suspension
into a test tube with 20 ml of melted and cooled to a temperature of 50 ºC agar medium, for example, meat peptone agar (MPA). If microorganisms were grown in a liquid nutrient medium, then the appropriate volume of culture is added to the agar. The contents of the tube are quickly and thoroughly mixed and poured into a sterile Petri dish.
When the medium has hardened, paper is placed on its surface.
ny disks at equal distances from each other and at a distance
1.5...2.0 cm from the edge of the cup. Petri dishes are kept for 2 hours at room temperature for better diffusion of antibiotics into the thickness of the agar medium, and then, without inverting, they are placed in a thermostat for 24 hours at a temperature of 30 ºC. A day later, the formation of zones of suppression of the growth of the studied microorganisms around the discs is noted. If the bacterium under study is sensitive to certain antibiotics, then zones of no growth of the culture are found around the discs. The diameter of the growth inhibition zone is measured with a millimeter ruler and the results are recorded in Table 3. A zone of more than 30 mm indicates
indicates high sensitivity of microorganisms to the antibiotic, and less than 12 mm indicates weak sensitivity.
When the experimenter has solutions at his disposal
antibiotic substances or culture fluids containing
antibiotic, use a method using wells in the thickness of agar.
In this case, in a frozen agar medium inoculated with the test microorganism, holes are made with a sterile plug drill (diameter from 6 to 8 mm) at a distance of 1.5...2.0 cm from the edge of the dish.
Antibiotic solutions or culture liquid are added to the wells. This method also makes it possible to identify the ability of microorganisms grown in a liquid medium to form antibiotic substances.
Table 3 – Effect of antibiotics on bacterial growth
Antibiotic | Diameter of growth inhibition zones, mm |
Penicillin disc | |
Disc with chloramphenicol |
4 TEST QUESTIONS
1. Define the following terms:
- strain; authentic strain; type strain;
- the colony;
– cultural properties;
– taxonomy;
– classification;
– nomenclature;
– plasmid;
– phage typing.
2. What sections does the taxonomy of microorganisms include? Give their characteristics.
3. Why are existing systems of classification of microorganisms artificial?
5. What are the characteristics that distinguish different strains of the same type of microorganism?
6. Which taxonomic categories of microorganisms are classified as obligatory and which are classified as optional?
7. List the basic rules for the nomenclature of microorganisms.
8. What is the main purpose of identifying microorganisms?
9. How do the principles of classification and identification of different groups of prokaryotes and eukaryotes differ?
10. What properties are studied when describing and identifying bacteria?
11. What signs are taken into account when describing surface, deep and bottom colonies of microorganisms?
12. What features are noted when describing the growth of microorganisms by stroke?
13. What is noted when characterizing the growth of microorganisms in a liquid nutrient medium?
14. What features do the morphological characteristics and organization of bacterial cells include?
15. What physiological and biochemical properties are studied when identifying bacteria?
16. In what cases is it necessary to use chemotaxonomic methods?
17. Give examples of substances used as chemo-taxonomic markers?
18. What are the features of protein taxonomy?
19. Describe the method of numeric taxonomy, what limitations does it have?
20. What methods are used to evaluate the phylogenetic relationships of bacteria?
21. What is the essence of the DNA probe method and its difference from the method
DNA–DNA hybridization?
22. What are the features of the method for analyzing nucleotide sequences in ribosomal RNA?
23. What signs are the basis for the classification of bacteria in the “Burgee's Guide to Bacteria”?
24. What properties and characteristics are studied when describing new strains of bacteria?
25. What are the methods for determining the purity of an identified bacterium?
26. What are the basic rules for performing the Gram staining technique?
27. What groups are microorganisms divided into in relation to molecular oxygen?
28. What is used as a substrate when determining the activity of extracellular protolytic enzymes in microorganisms?
29. What methods do you know for determining the sensitivity of microorganisms to antibiotics? Give their characteristics.
30. What method is used to determine the production of amylase by microorganisms?
31. How does seeding make it possible to identify and separate motile microorganisms from immobile ones?
5 RECIPES OF DYES AND NUTRIENTS
5.1 Basic carbolic fuchsin (Tsilya fuchsin)
– 5% aqueous solution of freshly distilled phenol – 100 ml;
– saturated alcohol solution of basic fuchsin – 10 ml;
The prepared mixture is filtered after 48 hours.
5.2 Methylene blue (Leffler)
– saturated alcohol solution of methylene blue – 30 ml;
– distilled water – 100 ml;
– 1% aqueous solution of KOH – 1 ml.
5.3 Meat peptone broth (MPB)
500 g of minced meat without fat and tendons is poured into 1 liter of tap water and extracted at room temperature for 12 hours or in a thermostat at a temperature of 37 ºC for 2 hours, and at a temperature of 50 ºC for one hour. Then the meat is squeezed through cheesecloth, and the resulting infusion is boiled for 30 minutes. In this case, proteins coagulate. The cooled mass is filtered through a cotton filter and added with water to the original volume. Next, add 5 to 10 g of peptone and 5 g of table salt to 1 liter of meat broth. The medium is heated until the peptone dissolves, stirring constantly. MPB is sterilized at a pressure of 2 atm for 20 minutes.
5.4 Meat peptone agar (MPA)
Add 20 g of agar to 1 liter of MPB. The medium is heated until the agar dissolves, then the medium is slightly alkaline.
20% NaCO64 solution" height="52" bgcolor="white" style="vertical-align:top;background: white">
Each microorganism, no matter how primitive it is, contains several antigens. The more complex its structure, the more antigens can be found in its composition. Different microorganisms belonging to the same systematic categories are distinguished
group-specific antigens - found in different species of the same genus or family, species-specific - in different representatives of the same species, and type-specific (variant) antigens - in different variants within the same species. The latter are divided into serological variants, or serovars. Among bacterial antigens, there are H, O, K, etc. Antigens of different types of microorganisms differ sharply from each other in structure and composition. The best studied is the antigenic mosaic of bacteria, which include somatic O- and Vi-antigens, envelope, capsular (K), flagellar (H), protective and ribosomal. As a rule, they are all complex protein compounds. Thus, somatic O- and Vi-antigens are contained in the surface structures of bacterial cells and are closely associated with lipopolysaccharides. Envelope antigens are formed by O-antigens, but unlike the latter they consist of thermolabile and thermostable fractions. Capsular K-antigens are represented by protein substances (anthrax bacillus) or complex polysaccharides (streptococcus, Klebsiella). Flagellar H-antigens are proteins, while ribosomal and protective antigens are complex compounds of proteins and nucleic acids. Antigens are also endo- and exotoxins of bacteria.
Knowledge of the antigenic structure of bacteria has made it possible to obtain a number of diagnostic and therapeutic sera, used respectively to determine the species of microbes and treat infectious diseases.
diseases.
Flagellar H-antigens. These antigens are part of bacterial flagella. The H antigen is a flagellin protein. It is destroyed when heated, and after treatment with phenol it retains its antigenic properties.
Somatic O-antigen. Previously, it was believed that the O-antigen was contained in the contents of the cell, its soma, and therefore it was called the somatic antigen. This antigen was subsequently found to be associated with the bacterial cell wall. O-antigen of gram-negative bacteria is associated with LPS of the cell wall. The determinant groups of this complex antigen are the terminal repeating units of polysaccharide chains attached to its main part. The composition of sugars in determinant groups, as well as their number, varies among different bacteria. Most often they contain hexoses (galactose, glucose, rhamnose, etc.), amino sugar (N-acetylglucosamine). O-antigen is thermostable: it is preserved by boiling for 1-2 hours, and is not destroyed after treatment with formaldehyde and ethanol. When animals are immunized with live cultures that have flagella, antibodies to the O- and H-antigens are formed, and when immunized with a boiled culture, antibodies are formed only to the O-antigen.
K-antigens (capsule). These antigens have been well studied in Escherichia and Salmonella. They, like O-antigens, are closely associated with LPS of the cell wall and capsule, but unlike O-antigen they contain mainly acidic polysaccharides: glucuronic, galacturonic and other uronic acids. Based on sensitivity to temperature, K-antigens are divided into A-, B- and L-antigens. The most thermostable are A-antigens, which can withstand boiling for more than 2 hours, B-antigens can withstand heating at a temperature of 60 °C for an hour, and L-antigens are destroyed when heated to 60 °C. K antigens are located more superficially than O-antigens and often mask the latter. Therefore, to identify O-antigens, it is necessary to first destroy K-antigens, which is achieved by boiling the cultures. Capsule antigens include the so-called Vi antigen. It is found in typhoid and some other enterobacteria that are highly virulent, and therefore this antigen is called the virulence antigen. Capsular antigens of a polysaccharide nature have been identified in pneumococci, Klebsiella and other bacteria that form a pronounced capsule. Unlike group-specific O-antigens, they often characterize the antigenic characteristics of certain strains (variants) of a given species, which on this basis are divided into serovars. In anthrax bacilli, the capsular antigen consists of polypeptides.
Antigens of bacterial toxins. Bacterial toxins have full antigenic properties if they are soluble compounds of a protein nature. Enzymes produced by bacteria, including pathogenicity factors, have the properties of full-fledged antigens. Protective antigens that have low toxicity and ensure the production of numerous blocking antibodies deserve serious attention. Good antigens are toxoids obtained from exotoxins by neutralizing them with formaldehyde.
Protective antigens. First discovered in the exudate of affected tissue during anthrax. They have strongly expressed antigenic properties, providing immunity to the corresponding infectious agent. Protective antigens are also formed by some other microorganisms when they enter the host’s body, although these antigens are not their permanent components.
Antigens of viruses. Each virion of any virus contains different antigens. Some of them are virus-specific. Other antigens include host cell components (lipids, carbohydrates), which are included in its outer shell. Antigens of simple virions are associated with their nucleocapsids. According to their chemical composition, they belong to ribonucleoproteins or deoxyribonucleoproteins, which are soluble compounds and are therefore designated as S-antgens (solutio-solution). In complex virions, some antigenic components are associated with nucleocapsids, others with glycoproteins of the outer shell. Many simple and complex virions contain special surface V-antigens - hemagglutinin and the enzyme neuraminidase. The antigenic specificity of hemagglutinin varies among different viruses. This antigen is detected in the hemagglutination reaction or its variation - the hemadsorption reaction. Another feature of hemagglutinin is manifested in the antigenic function of causing the formation of antibodies - antihemagglutinins and interacting with them in the hemagglutination inhibition reaction (HRI).
Viral antigens can be group-specific, if they are found in different species of the same genus or family, and type-specific, inherent in individual strains of the same species. These differences are taken into account when identifying viruses. Along with the listed antigens, host cell antigens may be present in viral particles. For example, an influenza virus grown on the allantoic membrane of a chicken embryo reacts with the antiserum obtained for the allantoic fluid. The same virus, taken from the lungs of infected mice, reacts with antiserum to the lungs of these animals and does not react with antiserum to allantoic fluid.
Heterogeneous antigens (heteroantigens). These are common or interspecies (similar in specificity) antigens. They were first discovered by J. Forssman. By immunizing a rabbit with an aqueous extract from guinea pig kidneys, he caused the formation of group antibodies in its serum that reacted with sheep erythrocytes. It was further revealed that the Forssman antigen is a lipopolysaccharide and is found in the organs of horses, cats, dogs, and turtles. Common antigens are found in human erythrocytes and pyogenic cocci, enterobacteria, smallpox viruses, influenza and other microorganisms. The group commonality of antigenic structure in different types of cells is called antigenic mimicry . In cases of antigenic mimicry, the human immune system loses the ability to quickly recognize a foreign mark and develop immunity, as a result of which pathogenic microbes can multiply unhindered in the body for some time. Antigenic mimicry is used to justify the long-term survival of pathogenic microbes in the patient’s body, or persistence, resident (stable) microbial carriage, and even post-vaccination complications. Common antigens found in representatives of various types of microorganisms, animals and plants are called heterogeneous. For example, the heterogeneous Forsman antigen is found in the protein structures of guinea pig organs, in sheep erythrocytes and salmonella.
Based on specificity, bacterial antigens are divided into homologous - species- and type-specific and heterogeneous - group, interspecific.
Species and especially type antigens are highly specific. In response to their introduction, the animals' bodies produce only those antibodies that react with antigens of a certain type or variety of microbe.
Antigens of microorganisms
Each microorganism, no matter how primitive it is, contains several antigens. The more complex its structure, the more antigens can be found in its composition.
In different microorganisms belonging to the same systematic categories, group-specific antigens are distinguished - they are found in different species of the same genus or family, species-specific - in different representatives of the same species, and type-specific (variant) antigens - in different variants within the same and the same type. The latter are divided into serological variants, or serovars. Among bacterial antigens there are H, O, K, etc.
Flagellar H-antigens. As the name implies, these antigens are part of bacterial flagella. The H-antgen is a flagellin protein. It is destroyed when heated, and after treatment with phenol it retains its antigenic properties.
Somatic O-antigen. Previously, it was believed that the O-antigen was contained in the contents of the cell, its soma, and therefore it was called the somatic antigen. This antigen was subsequently found to be associated with the bacterial cell wall.
O-antigen of gram-negative bacteria is associated with LPS of the cell wall. The determinant groups of this contiguous complex antigen are the terminal repeating units of the polysaccharide chains connected to its main part. The composition of sugars in determinant groups, as well as their number, varies among different bacteria. Most often they contain hexoses (galactose, glucose, rhamnose, etc.), amino sugar (M-acetylglucosamine). O-antigen is thermally stable: it is preserved when boiled for 1-2 hours, and is not destroyed after treatment with formaldehyde and ethanol. When animals are immunized with live cultures that have flagella, antibodies to the O- and H-antigens are formed, and when immunized with a boiled culture, antibodies are formed only to the O-antgen.
K-antigens (capsule). These antigens have been well studied in Escherichia and Salmonella. They, like O-antigens, are closely associated with LPS of the cell wall and capsule, but unlike O-antigen they contain mainly acidic nolysaccharides: glucuronic, galacturonic and other uronic acids. Based on their sensitivity to temperature, K-antigens are divided into A-, B- and L-antigens. The most thermostable are A-antigens, which can withstand boiling for more than 2 hours. B-antigens can withstand heating at a temperature of 60°C for an hour, and L-antigens are destroyed when heated to 60°C.
K-antigens are located more superficially than O-antigens and often mask the latter. Therefore, to identify O-antigens, it is necessary to first destroy K-antigens, which is achieved by boiling the cultures. Capsule antigens include the so-called Vi antigen. It is found in typhoid and some other enterobacteria that are highly virulent, and therefore this antigen is called the virulence antigen.
Capsular antigens of a polysaccharide nature have been identified in pneumococci, Klebsiella and other bacteria that form a pronounced capsule. Unlike group-specific O-antigens, they often characterize the antigenic characteristics of certain strains (variants) of a given species, which on this basis are divided into serovars. In anthrax bacilli, the capsular antigen consists of polypeptides.
Antigens of bacterial toxins. Bacterial toxins have full antigenic properties if they are soluble compounds of a protein nature.
Enzymes produced by bacteria, including pathogenicity factors, have the properties of full-fledged antigens.
Protective antigens. First discovered in the exudate of affected tissue during anthrax. They have strongly expressed antigenic properties, providing immunity to the corresponding infectious agent. Some other microorganisms also form protective antigens when they enter the host’s body, although these antigens are not their permanent components.
Antigens of viruses. Each virion of any virus contains different antigens. Some of them are virus-specific. Other antigens include host cell components (lipids, carbohydrates), which are included in its outer shell. Antigens of simple virions are associated with their nucleocapsids. According to their chemical composition, they belong to ribonucleoproteins or deoxyribonucleoproteins, which are soluble compounds and are therefore designated as S-antigens (solutio-solution). In complex virions, some antigenic components are associated with nucleocapsids, others with glycoproteins of the outer shell. Many simple and complex virions contain special surface V-antigens - hemagglutinin and the enzyme neuraminidase. The antigenic specificity of hemagglutinin varies among different viruses. This antigen is detected in the hemagglutination reaction or its variation - the hemadsorption reaction. Another feature of hemagglutinin is manifested in the antigenic function of causing the formation of antibodies - antihemashpotinins and interacting with them in the hemagglutination inhibition reaction (HRI).
Viral antigens can be group-specific, if they are found in different species of the same genus or family, and type-specific, inherent in individual strains of the same species. These differences are taken into account when identifying viruses.
Along with the listed antigens, host cell antigens may be present in viral particles. For example, an influenza virus grown on the allantoic membrane of a chicken embryo reacts with the antiserum obtained for the allantoic fluid. The same virus, taken from the lungs of infected mice, reacts with antiserum to the lungs of these animals and does not react with antiserum to allantoic fluid.
Heterogeneous antigens (heteroantigens). Common antigens found in representatives of different types of microorganisms, animals and plants are called heterogeneous. For example, the heterogeneous Forsman antigen is found in the protein structures of guinea pig organs, in sheep erythrocytes and salmonella.
Antigens of the human body
All tissues and cells of the human body have antigenic properties. Some antigens are specific for all mammals, others are species-specific for humans, and others are for certain groups; they are called isoantigens (for example, blood group antigens). Antigens characteristic only of a given organism are called alloantigens (Greek allos - other). These include histocompatibility antigens - products of the genes of the major histocompatibility complex MHC (Major Histocompatibiliti Complex), characteristic of each individual. Antigens from different individuals that have no differences are called syngeneic. Organs and tissues, in addition to other antigens, have organ and tissue antigens specific to them. Human and animal tissues of the same name have antigenic similarity. There are stage-specific antigens that appear and disappear at individual stages of tissue or cell development. Each cell contains antigens characteristic of the outer membrane, cytoplasm, nucleus and other components.
Antigens of each organism normally do not cause immunological reactions in it, since the body is tolerant to them. However, under certain conditions they acquire signs of foreignness and become autoantigens, and the reaction that arises against them is called autoimmune.
Tumor antigens and antitumor immunity. Malignant tumor cells are variants of normal cells in the body. Therefore, they are characterized by antigens of those tissues from
from which they originated, as well as tumor-specific antigens that make up a small proportion of all cell antigens. During carcinogenesis, dedifferentiation of cells occurs, so the loss of some antigens and the appearance of antigens characteristic of immature cells, including embryonic ones (fetoproteins), may occur. Tumor-specific antigens are specific only to a given type of tumor, and often to a tumor in a given person. Tumors induced by viruses may have viral antigens that are the same in all tumors induced by a given virus. Under the influence of antibodies, a growing tumor may change its antigenic composition.
Laboratory diagnosis of tumor disease includes the identification of antigens characteristic of the tumor in blood serum. For this purpose, the medical industry is currently preparing diagnostic kits containing all the necessary ingredients for detecting antigens using enzyme immunoassay, radioimmunoassay, and immunoluminescence analysis.
The body's resistance to tumor growth is ensured by the action of natural killer cells, which make up 15% of all lymphocytes constantly circulating in the blood and all tissues of the body. Natural killer cells (NK) have the ability to distinguish any cells that have signs of foreignness, including tumor cells, from normal cells of the body and destroy foreign cells. In stressful situations, illnesses, immunosuppressive influences and some other situations, the number and activity of NK decrease and this is one of the reasons for the onset of tumor growth. During the development of a tumor, its antigens cause an immunological reaction, but it is usually insufficient to stop tumor growth. The reasons for this phenomenon are numerous and not well understood. These include:
low immunogenicity of tumor antigens due to their proximity to normal antigens of the body, to which the body is tolerant;
development of tolerance instead of a positive response;
development of an immune response of the humoral type, whereas only cellular mechanisms can suppress the tumor;
immunosuppressive factors produced by a malignant tumor.
Chemotherapy and radiotherapy of tumors, stressful situations during surgical interventions can be additional factors that reduce the body's immune defense. Measures to increase the level of antitumor resistance include the use of immunostimulating agents, cytokine preparations, stimulation of the patient's immunocytes in vitro with return to the patient's bloodstream.
Isoantigens. These are antigens by which individuals or groups of individuals of the same species differ from each other.
Several dozen types of isoantigens have been discovered in erythrocytes, leukocytes, platelets, as well as in human blood plasma.
Isoantigens, genetically related, are combined into groups called: LBO system, Rhesus, etc. The division of people into groups according to the ABO system is based on the presence or absence of antigens on red blood cells, designated A and B. In accordance with this, all people are divided into 4 groups. Group I (0) - no antigens, group II (A) - erythrocytes contain antigen A, group
III (B) - erythrocytes have antigen B, group IV (AB) - erythrocytes have both antigens. Since there are microorganisms in the environment that have the same antigens (they are called cross-reacting), a person has antibodies to these antigens, but only to those that he does not have. The body is tolerant to its own antigens. Consequently, the blood of persons of group I contains antibodies to antigens A and B, the blood of persons of group II contains anti-B, the blood of persons of group III contains anti-A, and the blood of persons of group III contains anti-A.
Group IV antibodies to A and Vantigens are not contained. When blood or red blood cells are transfused into a recipient whose blood contains antibodies to the corresponding antigen, agglutination of the transfused incompatible red blood cells occurs in the vessels, which can cause shock and death of the recipient. Accordingly, people of group I (0) are called universal donors, and people of group IV (AB) are called universal recipients. In addition to antigens A and B, human erythrocytes may also have other isoantigens (M, M2, N, N2), etc. There are no isoantibodies to these antigens, and therefore, their presence is not taken into account during blood transfusion.
Antigens of the major histocompatibility complex. In addition to antigens common to all people and group antigens, each organism has a unique set of antigens that are unique to itself. These antigens are encoded by a group of genes located on human chromosome 6 and are called major histocompatibility complex antigens and are designated MHC antigens (Major histocompatibility complex). Human MHC antigens were first discovered on leukocytes and therefore have another name HLA (Human leucocyte antigens). MHC antigens belong to glycoproteins and are contained on the membranes of the body's cells, determining its individual properties and inducing transplantation reactions, for which they received a third name - transplantation antigens. In addition, MHC antigens play a mandatory role in inducing an immune response to any antigen.
MHC genes encode three classes of proteins, two of which are directly related to the functioning of the immune system and are discussed below, and class III proteins include complement components, TNF cytokines, and heat shock proteins.
Class I proteins are found on the surface of almost all cells of the body. They consist of two polypeptide chains: the heavy chain is non-covalently linked to the second chain. The heavy chain exists in three variants, which determines the division of class antigens into three serological groups A, B and C. The heavy chain determines the contact of the entire structure with the cell membrane and its activity. The chain is a microglobulin that is the same for all groups. Each class I antigen is designated by a Latin letter and the serial number of this antigen.
Class I antigens ensure the presentation of antigens to cytotoxic CO8+ lymphocytes, and recognition of this antigen by antigen-presenting cells of another organism during transplantation leads to the development of transplantation immunity.
MHC class II antigens are located predominantly on antigen-presenting cells - dendritic cells, macrophages, and B lymphocytes. On macrophages and Blymphocytes, their expression increases sharply after cell activation. Class II antigens are divided into 5 groups, each of which contains from 3 to 20 antigens. Unlike class I antigens, which are detected in serological tests using sera containing antibodies to them, class II antigens are best detected in cell tests - cell activation by co-cultivating the test cells with standard lymphocytes.
Isolation of microorganisms from various materials and obtaining their cultures is widely used in laboratory practice for microbiological diagnosis of infectious diseases, in research work and in the microbiological production of vaccines, antibiotics and other biologically active products of microbial life.
Cultivation conditions also depend on the properties of the relevant microorganisms. Most pathogenic microbes are grown on nutrient media at a temperature of 37°C for 12 days. However, some of them require longer periods. For example, whooping cough bacteria - in 2-3 days, and mycobacterium tuberculosis - in 3-4 weeks.
To stimulate the processes of growth and reproduction of aerobic microbes, as well as to reduce the time required for their cultivation, the method of deep cultivation is used, which consists of continuous aeration and mixing of the nutrient medium. The deep method has found wide application in biotechnology.
For the cultivation of anaerobes, special methods are used, the essence of which is to remove air or replace it with inert gases in sealed thermostats - anaerobes. Anaerobes are grown on nutrient media containing reducing substances (glucose, sodium formic acid, etc.) that reduce the redox potential.
In diagnostic practice, pure cultures of bacteria that are isolated from the test material taken from a patient or environmental objects are of particular importance. For this purpose, artificial nutrient media are used, which are divided into basic, differential diagnostic and elective media of the most diverse composition. The choice of nutrient medium for isolating a pure culture is essential for bacteriological diagnostics.
In most cases, solid nutrient media are used, previously poured into Petri dishes. The test material is placed on the surface of the medium in a loop and rubbed with a spatula to obtain isolated colonies grown from a single cell. Reseeding an isolated colony on a slanted agar medium in a test tube results in a pure culture.
For identification, i.e. To determine the generic and species affiliation of an isolated crop, phenotypic characteristics are most often studied:
a) morphology of bacterial cells in stained smears or native preparations;
b) biochemical characteristics of the culture according to its ability to ferment carbohydrates (glucose, lactose, sucrose, maltose, mannitol, etc.), to form indole, ammonia and hydrogen sulfide, which are products of the proteolytic activity of bacteria.
For a more complete analysis, gas-liquid chromatography and other methods are used.
Along with bacteriological methods, immunological research methods, which are aimed at studying the antigenic structure of the isolated culture, are widely used to identify pure cultures. For this purpose, serological reactions are used: agglutanation, immunofluorescence precipitation, complement fixation, enzyme immunoassay, radioimmune methods, etc.
Methods for isolating pure culture
In order to isolate a pure culture of microorganisms, it is necessary to separate the numerous bacteria that are found in the material from one another. This can be achieved using methods that are based on two principles − mechanical And biological separation of bacteria.
Methods for isolating pure cultures based on the mechanical principle
Serial dilution method , proposed by L. Pasteur, was one of the very first, which was used for the mechanical separation of microorganisms. It consists of carrying out successive serial dilutions of material that contains microbes in a sterile liquid nutrient medium. This technique is quite painstaking and imperfect in operation, since it does not allow controlling the number of microbial cells that enter the test tubes during dilutions.
Doesn't have this drawback Koch method (plate dilution method ). R. Koch used solid nutrient media based on gelatin or agar-agar. Material with associations of different types of bacteria was diluted in several test tubes with melted and slightly cooled gelatin, the contents of which were later poured onto sterile glass plates. After the medium had gelled, it was cultivated at the optimal temperature. Isolated colonies of microorganisms formed in its thickness, which can easily be transferred to a fresh nutrient medium using a platinum loop to obtain a pure culture of bacteria.
Drigalski method is a more advanced method that is widely used in everyday microbiological practice. First, the material to be tested is applied to the surface of the medium in a Petri dish using a pipette or loop. Using a metal or glass spatula, thoroughly rub it into the medium. The cup is kept open during sowing and gently rotated to evenly distribute the material. Without sterilizing the spatula, apply it to the material in another Petri dish, and if necessary, in a third one. Only after this is the spatula dipped in a disinfectant solution or fried in a burner flame. On the surface of the medium in the first cup, we usually observe continuous growth of bacteria, in the second - dense growth, and in the third - growth in the form of isolated colonies.
Colonies using the Drigalsky method
Line seeding method Today it is most often used in microbiology laboratories. The material that contains microorganisms is collected with a bacteriological loop and applied to the surface of the nutrient medium near the edge of the dish. Remove excess material and apply it in parallel strokes from edge to edge of the cup. After a day of incubation of the crops at the optimal temperature, isolated colonies of microbes grow on the surface of the dish.
Stroke method
To obtain isolated colonies, you can use a swab used to collect the test material. Open the Petri dish with the nutrient medium slightly, insert a tampon into it and carefully rub the material into the surface of the dish, gradually returning the tampon and the dish.
Thus, a significant advantage of the Koch, Drygalski plate dilution and streak culture methods is that they create isolated colonies of microorganisms, which, when inoculated onto another nutrient medium, turn into a pure culture
Methods for isolating pure cultures based on biological principles
The biological principle of bacterial separation involves a focused search for methods that take into account the numerous characteristics of microbial cells. Among the most common methods are the following:
1. By type of breathing. All microorganisms according to the type of respiration are divided into two main groups: aerobic (Corynebacterium diphtheriae, Vibrio сholeraeetc) And anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringensand etc.). If the material from which anaerobic pathogens are to be isolated is preheated and then cultivated under anaerobic conditions, then these bacteria will grow.
2. By sporulation . It is known that some microbes (bacillus and clostridia) are capable of sporulation. Among them Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Bacillus subtilis, Bacillus cereus. The spores are resistant to environmental factors. Consequently, the material under study can be subjected to the action of a thermal factor, and then inoculatively transferred into a nutrient medium. After some time, exactly those bacteria that are capable of sporulation will grow on it.
3. Resistance of microbes to acids and alkalis. Some microbes (Mycobacterium tuberculosis, Mycobacterium bovis) As a result of the peculiarities of their chemical structure, they are resistant to acids. That is why the material that contains them, for example, sputum from tuberculosis, is pre-treated with an equal volume of 10% sulfuric acid solution and then sown on nutrient media. Foreign flora dies, and mycobacteria grow as a result of their resistance to acids.
Vibrio cholerae (Vibrio сholerae) , on the contrary, is a halophilic bacterium, therefore, to create optimal growth conditions, it is sown on media that contain alkali (1% alkaline peptone water). Within 4-6 hours, characteristic signs of growth appear on the surface of the medium in the form of a delicate bluish film.
4. Motility of bacteria. Some microbes (Proteus vulgaris) have a tendency to creeping growth and are able to quickly spread over the surface of some moist environment. To isolate such pathogens, they are inoculated into a droplet of condensation liquid, which is formed when a column of slanted agar is cooled. After 16-18 years they spread to the entire surface of the medium. If we take material from the top of the agar, we will have a pure culture of pathogens.
5. The sensitivity of microbes to the action of chemicals, antibiotics and other antimicrobial agents. As a result of the metabolic characteristics of bacteria, they may have different sensitivity to certain chemical factors. It is known that staphylococci, aerobic bacilli that form spores, are resistant to the action of 7.5–10% sodium chloride. That is why, to isolate these pathogens, selective nutrient media (yolk-salt agar, mannitol-salt agar) that contain this particular substance are used. Other bacteria practically do not grow at this concentration of sodium chloride.
6. Administration of certain antibiotics (nystatin) is used to inhibit the growth of fungi in material that is heavily contaminated with them. Conversely, adding the antibiotic penicillin to the medium promotes the growth of bacterial flora if fungi are to be isolated. The addition of furazolidone in certain concentrations to the nutrient medium creates selective conditions for the growth of corynebacteria and micrococci.
7. The ability of microorganisms to penetrate intact skin. Some pathogenic bacteria (Yersinia pestis) As a result of the presence of a large number of aggressive enzymes, they are able to penetrate through intact skin. To do this, the hair on the body of a laboratory animal is shaved and the test material, which contains a pathogen and a large amount of third-party microflora, is rubbed into this area. After some time, the animal is slaughtered, and microbes are isolated from the blood or internal organs.
8. Sensitivity of laboratory animals to pathogens of infectious diseases. Some animals exhibit high sensitivity to various microorganisms.
For example, with any method of administration Streptococcus pneumoniae white mice develop a generalized pneumococcal infection. A similar picture is observed when Guinea pigs are infected with tuberculosis pathogens. (Mycobacterium tuberculosis) .
In everyday practice, bacteriologists use such concepts as strain And pure culture microorganisms. A strain refers to microbes of the same species that are isolated from different sources, or from the same source, but at different times. A pure culture of bacteria is microorganisms of one species, descendants of one microbial cell, which grew on (in) a nutrient medium.
Isolation of pure culture aerobic microorganisms consists of a number of stages.
First day (Stage 1 of the study) Pathological material is taken into a sterile container (test tube, flask, bottle). It is studied - appearance, consistency, color, smell and other signs, a smear is prepared, painted and examined under a microscope. In some cases (acute gonorrhea, plague), at this stage it is possible to make a preliminary diagnosis, and in addition, select the media on which the material will be inoculated. Then it is carried out with a bacteriological loop (used most often), using a spatula - the Drigalsky method, and a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimal temperature (37 ° C) for 18-48 hours. The purpose of this stage is to obtain isolated colonies of microorganisms.
However, sometimes, in order to accumulate material, it is sown on liquid nutrient media.
On the second day (Stage 2 of the study) on the surface of a dense nutrient medium, microorganisms form continuous, dense growth or isolated colonies. The colony– these are accumulations of bacteria visible to the naked eye on the surface or in the thickness of the nutrient medium. As a rule, each colony is formed from the descendants of one microbial cell (clones), therefore their composition is quite homogeneous. The growth characteristics of bacteria on nutrient media are a manifestation of their cultural properties.
The plates are carefully examined and isolated colonies that have grown on the surface of the agar are studied. Pay attention to the size, shape, color, nature of the edges and surface of the colonies, their consistency and other characteristics. If necessary, examine the colonies under a magnifying glass, low or high magnification microscope. The structure of the colonies is examined in transmitted light at low microscope magnification. They can be hyaline, granular, filamentous or fibrous, which are characterized by the presence of intertwined filaments in the thickness of the colonies.
Characteristics of colonies is an important part of the work of a bacteriologist and laboratory assistant, because microorganisms of each species have their own special colonies.
On the third day (Stage 3 of the study) study the growth pattern of a pure culture of microorganisms and carry out its identification.
First, they pay attention to the characteristics of the growth of microorganisms on the medium and make a smear, staining it with the Gram method, in order to check the culture for purity. If bacteria of the same type of morphology, size and tinctorial (ability to stain) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, just by the appearance and characteristics of their growth, one can draw a conclusion about the type of pathogens isolated. Determining the type of bacteria based on their morphological characteristics is called morphological identification. Determining the type of pathogens based on their cultural characteristics is called cultural identification.
However, these studies are not enough to make a definitive conclusion about the type of microbes isolated. Therefore, the biochemical properties of bacteria are studied. They are quite diverse.
Identification of bacteria.
Determining the type of pathogen by its biochemical properties is called biochemical identification.
In order to establish the species of bacteria, their antigenic structure is often studied, that is, identification is carried out by antigenic properties. Each microorganism contains different antigenic substances. In particular, representatives of the Enterobacteriaceae family (Escherichia, Salmonella, Shigela) contain envelope O-antigen, flagellar H-antigen and capsular K-antigen. They are heterogeneous in their chemical composition, therefore they exist in many variants. They can be determined using specific agglutinating sera. This determination of the type of bacteria is called serological identification.
Sometimes identification of bacteria is carried out by infecting laboratory animals with a pure culture and observing the changes that pathogens cause in the body (tuberculosis, botulism, tetanus, salmonellosis, etc.). This method is called identification by biological properties. The objects most often used are Guinea pigs, white mice and rats.
APPLICATIONS
(tables and diagrams)
Physiology of bacteria
Scheme 1. Physiology of bacteria.
reproduction
growing on nutrient media
Table 1. General table of bacterial physiology.
|
Characteristic |
||
|
The process of acquiring energy and substances. |
||
|
A set of biochemical processes that result in the release of energy necessary for the life of microbial cells. |
||
|
Coordinated reproduction of all cellular components and structures, ultimately leading to an increase in cell mass |
||
|
Reproduction |
Increasing the number of cells in a population |
|
|
Growing on nutrient media. |
In laboratory conditions, microorganisms are grown on nutrient media, which must be sterile, transparent, moist, contain certain nutrients (proteins, carbohydrates, vitamins, microelements, etc.), have a certain buffering capacity, have an appropriate pH, and redox potential. |
Table 1.1 Chemical composition and physiological functions of elements.
|
composition element |
Characteristics and role in cell physiology. |
||
|
The main component of a bacterial cell, accounting for about 80% of its mass. It is in a free or bound state with the structural elements of the cell. In spores, the amount of water decreases to 18.20%. Water is a solvent for many substances, and also plays a mechanical role in providing turgor. During plasmolysis—the loss of water by a cell in a hypertonic solution—protoplasm is detached from the cell membrane. |
|||
|
Removing water from the cell and drying it out stop metabolic processes. |
|||
|
Most microorganisms tolerate drying well. When there is a lack of water, microorganisms do not multiply. |
Drying in a vacuum from a frozen state (lyophilization) stops reproduction and promotes long-term preservation of microbial individuals. |
||
|
40 – 80% dry weight. They determine the most important biological properties of bacteria and usually consist of combinations of 20 amino acids. The bacteria contain diaminopimelic acid (DAP), which is absent in human and animal cells. Bacteria contain more than 2,000 different proteins, located in their structural components and involved in metabolic processes. Most proteins have enzymatic activity. Proteins of a bacterial cell determine the antigenicity and immunogenicity, virulence, and species of bacteria. |
composition element |
||
|
Characteristics and role in cell physiology. |
Nucleic acids |
||
|
They perform functions similar to nucleic acids of eukaryotic cells: the DNA molecule in the form of a chromosome is responsible for heredity, ribonucleic acids (information, or matrix, transport and ribosomal) are involved in protein biosynthesis. |
|||
|
Carbohydrates |
Found in the ash after cells are burned. Phosphorus, potassium, sodium, sulfur, iron, calcium, magnesium, as well as microelements (zinc, copper, cobalt, barium, manganese, etc.) are detected in large quantities. They participate in the regulation of osmotic pressure, pH of the environment, redox potential , activate enzymes, are part of enzymes, vitamins and structural components of microbial cells. |
||
Table 1.2. Nitrogenous bases.
Table 1.2.1 Enzymes
|
Characteristic |
|||
|
Definition |
Specific and efficient protein catalysts present in all living cells. |
||
|
Enzymes reduce the activation energy, ensuring the occurrence of chemical reactions that without them could only take place at high temperature, excess pressure and other non-physiological conditions unacceptable for a living cell. |
|||
|
Enzymes increase the rate of reaction by about 10 orders of magnitude, which reduces the half-life of any reaction from 300 years to one second. |
|||
|
Enzymes “recognize” the substrate by the spatial arrangement of its molecule and the distribution of charges in it. A certain part of the enzymatic protein molecule, its catalytic center, is responsible for binding to the substrate. In this case, an intermediate enzyme-substrate complex is formed, which then decomposes to form the reaction product and free enzyme. |
|||
|
Varieties |
Regulatory (allosteric) enzymes perceive various metabolic signals and change their catalytic activity in accordance with them. |
Effector enzymes are enzymes that catalyze certain reactions (more details in Table 1.2.2.) |
|
|
Functional activity |
The functional activity of enzymes and the rate of enzymatic reactions depend on the conditions in which a given microorganism is located and, above all, on the temperature of the environment and its pH. For many pathogenic microorganisms, the optimal temperature is 37°C and pH 7.2-7.4. |
||
CLASSES OF ENZYME:
microorganisms synthesize various enzymes belonging to all six known classes.
Table 1.2.2. Effector enzyme classes
|
Enzyme class |
Catalyzes: |
|
|
Oxidoreductases |
Electron transfer |
|
|
Transferases |
Transfer of various chemical groups |
|
|
Hydrolases |
Transfer of functional groups to a water molecule |
|
|
Addition of double bond groups and reverse reactions |
||
|
Isomerases |
Transfer of groups within a molecule to form isomeric forms |
|
|
Formation of C-C, C-S, C-O, C-N bonds due to condensation reactions associated with the breakdown of adenosine triphosphate (ATP) |
Table 1.2.3. Types of enzymes according to formation in a bacterial cell
|
Characteristic |
Notes |
||
|
Inducible (adaptive) enzymes "substrate induction" |
Enzymes whose concentration in the cell increases sharply in response to the appearance of an inducer substrate in the environment. Synthesized by a bacterial cell only if the substrate of this enzyme is present in the medium | ||
|
Repressible enzymes |
The synthesis of these enzymes is inhibited as a result of excessive accumulation of the reaction product catalyzed by this enzyme. |
An example of enzyme repression is the synthesis of tryptophan, which is formed from anthranilic acid with the participation of anthranilate synthetase. |
|
|
Constitutive enzymes |
Enzymes synthesized regardless of environmental conditions |
Glycolytic enzymes |
|
|
Multienzyme complexes |
Intracellular enzymes combined structurally and functionally |
Respiratory chain enzymes localized on the cytoplasmic membrane. |
Table 1.2.4. Specific enzymes
|
Enzymes |
Identification of bacteria |
|
|
Superoxide dismutase and catalase |
All aerobes or facultative anaerobes possess superoxide dismutase and catalase, enzymes that protect the cell from toxic products of oxygen metabolism. Almost all obligate anaerobes do not synthesize these enzymes. |
|
|
Only one group of aerobic bacteria, lactic acid bacteria, are catalase-negative. |
Peroxidase |
|
|
Lactic acid bacteria accumulate peroxidase, an enzyme that catalyzes the oxidation of organic compounds under the influence of H2O2 (reduced to water). |
Arginine dihydrolase |
|
|
A diagnostic feature that allows one to distinguish saprophytic Pseudomonas species from phytopathogenic ones. |
Among the five main groups of the Enterobacteriaceae family, only two - Escherichiae and Erwiniae - do not synthesize urease.
|
Table 1.2.5. Application of bacterial enzymes in industrial microbiology. |
Enzymes |
|
|
Application |
Amylase, cellulase, protease, lipase |
|
|
To improve digestion, ready-made enzyme preparations are used, which facilitate the hydrolysis of starch, cellulose, protein and lipids, respectively. |
Yeast invertase |
|
|
In the manufacture of sweets to prevent crystallization of sucrose |
Pectinase |
|
|
Used to clarify fruit juices |
Clostridia collagenase and streptococcal streptokinase |
|
|
Hydrolyze proteins, promote healing of wounds and burns |
They are secreted into the environment, act on the cell walls of pathogenic microorganisms and serve as an effective means of combating the latter, even if they have multiple resistance to antibiotics |
|
|
Ribonucleases, deoxyribonucleases, polymerases, DNA ligases and other enzymes that specifically modify nucleic acids |
Used as a tool in bioorganic chemistry, genetic engineering and gene therapy |
Table 1.2.6. Classification of enzymes by localization.
|
Localization | |||
|
Endoenzymes |
In the cytoplasm In the cytoplasmic membrane In the periplasmic space |
They function only inside the cell. They catalyze reactions of biosynthesis and energy metabolism. |
|
|
Exoenzymes |
Released into the environment. |
They are released into the environment by the cell and catalyze reactions of hydrolysis of complex organic compounds into simpler ones that are available for assimilation by the microbial cell. These include hydrolytic enzymes, which play an extremely important role in the nutrition of microorganisms. |
Table 1.2.7. Enzymes of pathogenic microbes (aggression enzymes)
|
Enzymes | |||
|
Lecitovitellase Lecithinase |
Destroys cell membranes |
Inoculation of the test material on the ZhSA nutrient medium Result: a zone of turbidity around the colonies on the LSA. |
|
|
Hemolysin |
Destroys red blood cells |
Inoculation of the test material on a blood agar nutrient medium. Result: a complete zone of hemolysis around the colonies on blood agar. |
|
|
Coagulase-positive cultures |
Causes blood plasma clotting |
Inoculation of the test material on sterile citrated blood plasma. Result: plasma coagulation |
|
|
Coagulase-negative cultures |
Mannitol production |
Sowing mannitol on a nutrient medium under anaerobic conditions. Result: Appearance of colored colonies (in the color of the indicator) |
|
|
Enzymes |
Formation of some enzymes in vitro |
||
|
Hyaluronidase |
Hydrolyzes hyaluronic acid - the main component of connective tissue |
Inoculation of the test material on a nutrient medium containing hyaluronic acid. Result: in test tubes containing hyaluronidase, no clot formation occurs. |
|
|
Neuraminidase |
It splits off sialic (neuraminic) acid from various glycoproteins, glycolipids, polysaccharides, increasing the permeability of various tissues. |
Detection: reaction for determining antibodies to neuraminidase (RINA) and others (immunodiffusion, immunoenzyme and radioimmune methods). |
Table 1.2.8. Classification of enzymes according to biochemical properties.
|
Enzymes |
Detection |
||
|
Saccharolytic |
Breakdown of sugars |
Differential diagnostic media such as Hiss's environment, Olkenitsky's environment, Endo's environment, Levin's environment, Ploskirev's environment. |
|
|
Proteolytic |
Protein breakdown |
Microbes are inoculated by injection into a column of gelatin, and after 3-5 days of incubation at room temperature, the nature of gelatin liquefaction is noted. |
|
|
Proteolytic activity is also determined by the formation of protein decomposition products: indole, hydrogen sulfide, ammonia. To determine them, microorganisms are inoculated into meat-peptone broth. |
Enzymes identified by final products Formation of alkalis Acid formation Hydrogen sulfide formation |
Ammonia formation, etc. To distinguish one species of bacteria from others based on their enzymatic activity, they use |
differential diagnostic environments
Scheme 1.2.8. Enzyme composition.
ENZYME COMPOSITION OF ANY MICROORGANISM:
Determined by its genome
Is a stable sign
Widely used for their identification
Determination of saccharolytic, proteolytic and other properties.
|
Table 1.3. Pigments |
Pigments |
|
|
Synthesis by microorganism |
Fat-soluble carotenoid pigments that are red, orange, or yellow. |
|
|
They form sarcina, mycobacterium tuberculosis, and some actinomycetes. These pigments protect them from UV rays. |
Black or brown pigments - melanins |
|
|
Synthesized by obligate anaerobes Bacteroides niger and others. Insoluble in water and even strong acids |
A bright red pyrrole pigment called prodigiosin. |
|
|
Formed by some serata |
Water-soluble phenosine pigment - pyocyanin. Produced by Pseudomonas bacteria |
(Pseudomonas aeruginosa). In this case, a nutrient medium with a neutral or alkaline pH turns blue-green.
|
Table 1.4. Glowing and aroma-producing microorganisms |
||
|
Condition and characteristics |
Glow (luminescence) |
|
|
Bacteria cause the glow of those substrates, such as fish scales, higher fungi, rotting trees, and food products, on the surface of which they multiply. |
Some microorganisms produce volatile aromatic substances, for example, ethyl acetate and amyl acetate, which add flavor to wine, beer, lactic acid and other food products, and are therefore used in their production. |
Table 2.1.1.Metabolism
|
Definition |
|||
|
Metabolism |
The biochemical processes occurring in the cell are united by one word - metabolism (Greek metabole - transformation). This term is equivalent to the concept of “metabolism and energy”. There are two sides of metabolism: anabolism and catabolism. |
||
|
Anabolism is a set of biochemical reactions that carry out the synthesis of cell components, i.e. that side of metabolism, which is called constructive metabolism. |
Catabolism is a set of reactions that provide the cell with energy necessary, in particular, for constructive exchange reactions. Therefore, catabolism is also defined as the energy metabolism of a cell. |
||
|
Amphibolism |
Intermediate metabolism that converts low molecular weight fragments of nutrients into a series of organic acids and phosphorus esters is called |
||
Scheme 2.1.1. Metabolism
METABOLISM –
a combination of two opposite but interacting processes: catabolism and anabolism
Anabolism= assimilation = plastic metabolism = constructive metabolism
Catabolism= dissimilation = energy metabolism = breakdown = providing energy to the cell
Synthesis (of cell components)
Enzymatic catabolic reactions that result in release of energy, which accumulated in ATP molecules.
Biosynthesis of monomers:
amino acids nucleotides monosaccharides fatty acids
Biosynthesis of polymers:
proteins nucleic acids polysaccharides lipids
As a result of enzymatic anabolic reactions, the energy released in the process of catabolism is spent on the synthesis of macromolecules of organic compounds, from which biopolymers are then assembled - components of the microbial cell.
Energy is spent on the synthesis of cell components
Table 2.1.3. Metabolism and transformation of cell energy.
|
Metabolism |
Characteristic |
Notes |
|
|
Metabolism ensures the dynamic balance inherent in a living organism as a system, in which synthesis and destruction, reproduction and death are mutually balanced. |
Metabolism is the main sign of life |
||
|
Plastic exchange Synthesis of proteins, fats, carbohydrates. |
This is a set of biological synthesis reactions. |
From substances entering the cell from the outside, molecules similar to cell compounds are formed, that is, assimilation occurs. |
|
|
Energy metabolism |
The process is the opposite of synthesis. This is a set of splitting reactions. |
When high-molecular compounds are broken down, the energy necessary for the biosynthesis reaction is released, that is, dissimilation occurs. When glucose is broken down, energy is released in stages with the participation of a number of enzymes. |
Table 2.1.2. Difference in metabolism for identification.
Table 2.2 Anabolism (constructive metabolism)
Scheme 2.2.2. Biosynthesis of amino acids in prokaryotes.
Scheme 2.2.1. Biosynthesis of carbohydrates in microorganisms.
Figure 2.2.3. Lipid biosynthesis
Table 2.2.4. Stages of energy metabolism - Catabolism.
|
Stages |
Characteristic |
Note |
|
|
Preparatory |
Molecules of disaccharides and polysaccharides, proteins break down into small molecules - glucose, glycerol and fatty acids, amino acids. Large molecules of nucleic acids into nucleotides. |
At this stage, a small amount of energy is released and dissipated as heat. |
|
|
Anoxic or incomplete or anaerobic or fermentation or dissimilation. |
The substances formed at this stage undergo further breakdown with the participation of enzymes. For example: glucose breaks down into two molecules of lactic acid and two molecules of ATP. |
ATP and H 3 PO 4 are involved in the breakdown of glucose. During the oxygen-free breakdown of glucose in the form of a chemical bond in the ATP molecule, 40% of the energy is retained, the rest is dissipated as heat. |
|
|
In all cases of the breakdown of one glucose molecule, two ATP molecules are formed. |
The stage of aerobic respiration or oxygen breakdown. With oxygen access to the cell, the substances formed during the previous stage are oxidized (broken down) to final products 2 COAnd 2 H. |
O
|
The overall equation for aerobic respiration is:
Scheme 2.2.4. Fermentation. Fermentative metabolism –
characterized by the formation of ATP through phosphorylation of substrates.
First (oxidation) = splitting
Second (recovery)
Includes the conversion of glucose to pyruvic acid.
Includes hydrogen utilization to restore pyruvic acid.
Pathways for the formation of pyruvic acid from carbohydrates
Scheme 2.2.5. Pyruvic acid.
Glycolytic pathway (Embden-Meyerhof-Parnas pathway)
Entner-Doudoroff path
Pentose phosphate pathway
|
Table 2.2.5. Fermentation. |
Fermentation type |
Representatives |
Notes |
|
|
Final product |
|
Lactic acid |
In some cases (homoenzyme fermentation) only lactic acid is formed, in others also by-products. |
|
|
Formic acid |
Enterobacteriaceae |
Formic acid is one of the final products. (along with it - side effects) |
Some types of enterobacteria break down formic acid to H 2 and CO 2/ |
|
|
Butyric acid |
Butyric acid and by-products |
Some types of clostridia, along with butyric and other acids, form butanol, acetone, etc. (then it is called acetone-butyl fermentation). |
||
|
Propionic acid |
Propionobacterium |
Form propionic acid from pyruvate |
Many bacteria, when fermenting carbohydrates, along with other products, form ethyl alcohol. However, it is not the main product. |
Table 2.3.1. Protein synthesis system, ion exchange.
|
Item name |
Characteristic |
|
|
Ribosomal subunits 30S and 50S |
In the case of bacterial 70S ribosomes, the 50S subunit contains 23S rRNA (~3000 nucleotides in length) and the 30S subunit contains 16S rRNA (~1500 nucleotides in length); In addition to the “long” rRNA, the large ribosomal subunit also contains one or two “short” rRNAs (5S rRNA of bacterial ribosomal subunits 50S or 5S and 5.8S rRNA of large ribosomal subunits of eukaryotes). |
|
|
(for more details, see Fig. 2.3.1.) | ||
|
Messenger RNA (mRNA) |
A complete set of twenty aminoacyl-tRNAs, the formation of which requires the corresponding amino acids, aminoacyl-tRNA synthetases, tRNA and ATP |
|
|
This is an amino acid charged with energy and bound to tRNA, ready to be transported to the ribosome and included in the polypeptide synthesized on it. |
Transfer RNA (tRNA) |
|
|
Ribonucleic acid, the function of which is to transport amino acids to the site of protein synthesis. |
Protein initiation factors |
|
|
(in prokaryotes - IF-1, IF-2, IF-3) They got their name because they participate in the organization of the active complex (708 complex) of subunits 30S and 50S, mRNA and initiator aminoacyl-tRNA (in prokaryotes - formylmethionyl -tRNA), which “starts” (initiates) the work of ribosomes - the translation of mRNA. |
Protein elongation factors |
|
|
Item name |
Characteristic |
|
|
(in prokaryotes - EF-Tu, EF-Ts, EF-G) Participate in the lengthening (elongation) of the synthesized polypeptide chain (peptidyl). Protein termination or release factors (RF) ensure codon-specific separation of the polypeptide from the ribosome and the end of protein synthesis. |
Protein termination factors |
|
|
(in prokaryotes - RF-1, RF-2, RF-3) |
Protein translation factors necessary for the functioning of the system |
|
|
Guanosine triphosphate (GTP) |
To carry out translation, the participation of GTP is necessary. The requirement of the protein synthesizing system for GTP is very specific: it cannot be replaced by any of the other triphosphates. The cell spends more energy on protein biosynthesis than on the synthesis of any other biopolymer. |
|
|
The formation of each new peptide bond requires the cleavage of four high-energy bonds (ATP and GTP): two in order to load the tRNA molecule with an amino acid, and two more during elongation - one during aa-tRNA binding and the other during translocation. |
Inorganic cations in a certain concentration. |
To maintain the pH of the system within physiological limits. Ammonium ions are used by some bacteria to synthesize amino acids, and potassium ions are used to bind tRNA to ribosomes. Iron and magnesium ions act as a cofactor in a number of enzymatic processes
Figure 2.3.1. Schematic representation of the structures of prokaryotic and eukaryotic ribosomes.
|
Table 2.3.2. Features of ion exchange in bacteria. |
Peculiarity |
||
|
Characterized by: |
High osmotic pressure |
||
|
Due to the significant intracellular concentration of potassium ions in bacteria, high osmotic pressure is maintained. |
Iron intake |
For a number of pathogenic and opportunistic bacteria (Escherichia, Shigella, etc.), the consumption of iron in the host body is difficult due to its insolubility at neutral and slightly alkaline pH values Siderophores – |
|
|
special substances that, by binding iron, make it soluble and transportable. |
Assimilation |
||
|
Bacteria actively assimilate SO2/ and P034+ anions from the environment to synthesize compounds containing these elements (sulfur-containing amino acids, phospholipids, etc.). |
|||
For the growth and reproduction of bacteria, mineral compounds are required - ions NH4+, K+, Mg2+, etc. (for more details, see Table 2.3.1.)
|
Table 2.3.3. Ion exchange |
Name of mineral compounds |
|
|
Function |
NH 4 + (ammonium ions) |
|
|
Used by some bacteria to synthesize amino acids |
K+ (potassium ions) Used to bind tRNA to ribosomes |
|
|
Maintain high osmotic pressure |
Fe 2+ (iron ions) Act as cofactors in a number of enzymatic processes |
|
|
Part of cytochromes and other hemoproteins |
||
|
Mg 2+ (magnesium ions) |
SO 4 2 - (sulfate anion) |
|
|
Necessary for the synthesis of compounds containing these elements (sulfur-containing amino acids, phospholipids, etc.) |
Scheme 2.4.1. Energy metabolism.
To synthesize, bacteria need...
Nutrients
Table 2.4.1. Energy metabolism (biological oxidation).
|
Process |
Necessary: |
|
|
Synthesis of structural components of microbial cells and maintenance of vital processes |
Sufficient amount of energy. This need is satisfied through biological oxidation, which results in the synthesis of ATP molecules. |
|
|
Energy (ATP) |
Iron bacteria receive energy released during the direct oxidation of iron (Fe2+ to Fe3+), which is used to fix CO2; bacteria that metabolize sulfur provide themselves with energy through the oxidation of sulfur-containing compounds. However, the vast majority of prokaryotes obtain energy through dehydrogenation. Energy is also obtained during the breathing process (see the detailed table in the corresponding section). |
Scheme 2.4. Biological oxidation in prokaryotes.
Breakdown of polymers into monomers
Characteristics and role in cell physiology.
glycerol and fatty acids
amino acids
monosaccharides
Decomposition under oxygen-free conditions
Formation of intermediates
Oxidation under oxygen conditions to final products
Table 2.4.2. Energy metabolism.
|
Concept |
Characteristic |
|
|
The essence of energy metabolism |
Providing the energy cells need to manifest life. |
|
|
The ATP molecule is synthesized as a result of the transfer of an electron from its primary donor to its final acceptor. |
||
|
Respiration is biological oxidation (breakdown). Depending on what is the final electron acceptor, they distinguish breath: Aerobic - in aerobic respiration, the final electron acceptor is molecular oxygen O 2. Anaerobic - the final electron acceptor is inorganic compounds: NO 3 -, SO 3 -, SO 4 2- |
||
|
Mobilization of energy |
Energy is mobilized in oxidation and reduction reactions. |
|
|
Oxidation Reaction |
The ability of a substance to donate electrons (oxidize) |
|
|
Recovery Response |
The ability of a substance to gain electrons. |
|
|
Redox potential |
The ability of a substance to donate (oxidize) or accept (recover) electrons. |
(quantitative expression)
Scheme 2.5. Synthesis.
carbohydrates
Table 2.5.1. Synthesis
|
Table 2.5.1. Synthesis |
Biosynthesis |
Notes |
|
|
Of what |
Biosynthesis of carbohydrates |
Autotrophs synthesize glucose from CO 2 . |
|
|
Heterotrophs synthesize glucose from carbon-containing compounds. |
Calvin cycle (see diagram 2.2.1.) Biosynthesis of amino acids α-ketoglutorate fumorate |
The energy source is ATP. Pyruvate is formed in the glycolytic cycle. Auxotrophic microorganisms consume ready-made microorganisms in the host’s body. |
|
|
Lipid biosynthesis |
Lipids are synthesized from simpler compounds - metabolic products of proteins and carbohydrates |
Acetyl transfer proteins play an important role. Auxotrophic microorganisms consume ready-made microorganisms in the host body or from nutrient media. |
Table 2.5.2. The main stages of protein biosynthesis.
|
Stages |
Characteristic |
Notes |
|
|
Transcription |
The process of RNA synthesis on genes. This is the process of rewriting information from DNA - gene to mRNA - gene. |
It is carried out using DNA-dependent RNA polymerase. The transfer of information about protein structure to ribosomes occurs using mRNA. |
|
|
Broadcast (transmission) |
The process of self-protein biosynthesis. The process of deciphering the genetic code in mRNA and implementing it in the form of a polypeptide chain. |
Because each codon contains three nucleotides, the same genetic text can be read in three different ways (starting at the first, second, and third nucleotides), that is, in three different reading frames. |
Note to the table: The primary structure of each protein is the sequence of amino acids in it.
Scheme 2.5.2. Electron transfer chains from the primary donor of hydrogen (electrons) to its final acceptor O 2.
organic matter
(primary electron donor)
Flavoprotein (- 0.20)
Quinone (-0.07)
Cytochrome (+0.01)
Cytochrome C(+0.22)
Cytochrome A(+0.34)
final acceptor
Table 3.1. Classification of organisms by type of nutrition.
|
Organogen element |
Power types |
Characteristic |
|
|
Carbon (C) |
Autotrophs |
The cells themselves synthesize all the carbon-containing components from CO 2 . |
|
|
Heterotrophs |
They cannot satisfy their needs with CO 2; they use ready-made organic compounds. |
||
|
Saprophytes |
The food source is dead organic substrates. |
||
|
The source of nutrition is living tissues of animals and plants. |
|||
|
Prototrophs |
Meet your needs with atmospheric and mineral nitrogen |
||
|
Auxotrophs |
They require ready-made organic nitrogen compounds. |
||
|
Hydrogen (H) |
The main source is H 2 O |
||
|
Oxygen (O) |
|||
Table 3.1.2. Conversion of energy
Table 3.1.3. Carbon Nutrition Methods
|
Energy source |
Electron donor |
Carbon nutrition method |
|
|
Energy from sunlight |
Inorganic compounds |
Photolithoheterotrophs |
|
|
Organic compounds |
Photoorganoheterotrophs |
||
|
Redox reactions |
Inorganic compounds |
Chemolithoheterotrophs |
|
|
Organic compounds |
Chemoorganoheterotrophs |
Table 3.2. Power Mechanisms:
|
Mechanism |
Conditions |
Concentration gradient |
Energy costs |
Substrate specificity |
|
|
Passive diffusion |
The concentration of nutrients in the environment exceeds the concentration in the cell. |
By concentration gradient | |||
|
Facilitated diffusion |
Permease proteins are involved. |
By concentration gradient | |||
|
Active transport |
Permease proteins are involved. | ||||
|
Translocation of chemical groups |
During the transfer process, chemical modification of nutrients occurs. |
Against a concentration gradient |
Table 3.3. Transport of nutrients from the bacterial cell.
|
Name |
Characteristic |
|
|
Phosphotransferase reaction |
Occurs when the transported molecule is phosphorylated. |
|
|
Translational secretion |
In this case, the synthesized molecules must have a specific leading sequence of amino acids in order to attach to the membrane and form a channel through which the protein molecules can escape into the environment. In this way, tetanus, diphtheria and other toxins are released from the cells of the corresponding bacteria. |
|
|
Membrane budding |
Molecules formed in the cell are surrounded by a membrane vesicle, which is released into the environment. |
Table 4. Growth.
|
Concept |
Definition of the concept. |
|
|
An irreversible increase in the amount of living matter, most often caused by cell division. If multicellular organisms usually experience an increase in body size, then in multicellular organisms the number of cells increases. But in bacteria, an increase in the number of cells and an increase in cell mass should also be noted. |
||
|
Factors influencing bacterial growth in vitro. |
Culture media: Mycobacterium leprae is not capable of in vitro Temperature (increasing in range): Mesophilic bacteria (20-40 o C) Thermophilic bacteria (50-60 o C) Psychrophilic (0-10 o C) |
|
|
Bacterial growth assessment |
Quantification of growth is usually carried out in liquid media where the growing bacteria form a homogeneous suspension. The increase in the number of cells is determined by determining the concentration of bacteria in 1 ml, or the increase in cell mass is determined in weight units per unit volume. |
Growth factors
Amino acids
Vitamins
Nitrogenous bases
Table 4.1. Growth factors
|
Growth factors |
Characteristic |
Name of mineral compounds |
||
|
Amino acids |
|
Many microorganisms, especially bacteria, need certain amino acids (one or more), since they cannot synthesize them on their own. |
||
|
Such microorganisms are called auxotrophic for those amino acids or other compounds that they are not able to synthesize. |
Purine bases and their derivatives |
They are bacterial growth factors. Some types of mycoplasmas require nucleotides. |
||
|
Required for the construction of nucleic acids. |
Pyrimidine bases and their derivatives |
|||
|
Growth factors |
Characteristic |
Name of mineral compounds |
||
|
Nucleotides |
Neutral lipids |
|||
|
Contains membrane lipids |
||||
|
Phospholipids |
Fatty acid |
|||
|
They are components of phospholipids |
Glycolipids |
|||
|
Vitamins In mycoplasmas they are part of the cytoplasmic membrane |
(mostly group B) |
Thiamine (B1) |
||
|
Staphylococcus aureus, pneumococcus, Brucella |
Nicotinic acid (B3) |
|||
|
All types of rod-shaped bacteria |
Folic acid (B9) |
|||
|
Bifidobacteria and propionic acid |
Pantothenic acid (B5) |
|||
|
Some types of streptococci, tetanus bacilli |
Biotin (B7) |
|||
|
Yeast and nitrogen-fixing bacteria Rhizobium |
Hemes are components of cytochromes |
|||
Haemophilus influenzae bacteria, Mycobacterium tuberculosis
|
Name |
Characteristic |
|
|
Table 5. Breathing. |
||
|
Biological oxidation (enzymatic reactions) |
Base |
|
|
Respiration is based on redox reactions that occur with the formation of ATP, a universal accumulator of chemical energy. |
Processes During breathing the following processes occur: Oxidation is the giving away of hydrogen or electrons by donors. |
|
|
Reduction is the addition of hydrogen or electrons to an acceptor. |
Aerobic respiration |
|
|
The final acceptor of hydrogen or electrons is molecular oxygen. |
Anaerobic respiration |
|
|
The hydrogen or electron acceptor is an inorganic compound - NO 3 -, SO 4 2-, SO 3 2-. |
Fermentation |
Organic compounds are hydrogen or electron acceptors.
|
Table 5.1. Classification by type of breathing. |
Characteristic |
Notes |
|
|
Bacteria |
Strict anaerobes Energy exchange occurs without the participation of free oxygen. ATP synthesis during glucose consumption under anaerobic conditions (glycolysis) occurs due to phosphorylation of the substrate. |
Oxygen does not serve as the final electron acceptor for anaerobes. Moreover, molecular oxygen has a toxic effect on them Strict anaerobes lack the enzyme catalase, so it accumulates in the presence of oxygen and has a bactericidal effect on them; |
|
|
Strict anaerobes lack a system for regulating redox potential (redox potential). |
Strict aerobes Organisms that obtain energy and form ATP using only oxidative phosphorylation of the substrate, where only molecular oxygen can act as an oxidizing agent. The growth of most aerobic bacteria stops at oxygen concentrations of 40-50% or higher. |
Strict aerobes include, for example, representatives of the genus Pseudomonas |
|
|
Table 5.1. Classification by type of breathing. |
Characteristic |
Notes |
|
|
Facultative anaerobes |
Grows in both the presence and absence of molecular oxygen Aerobic organisms most often contain three cytochromes, facultative anaerobes - one or two, obligate anaerobes do not contain cytochromes. |
Facultative anaerobes include enterobacteria and many yeasts that can switch from respiration in the presence of 0 2 to fermentation in the absence of 0 2 . |
|
|
Microaerophiles |
A microorganism that, unlike strict anaerobes, requires for its growth the presence of oxygen in the atmosphere or nutrient medium, but in lower concentrations compared to the oxygen content in ordinary air or in normal tissues of the host body (unlike aerobes, whose growth requires normal oxygen content in the atmosphere or nutrient medium). Many microaerophiles are also capnophiles, meaning they require increased concentrations of carbon dioxide. |
In the laboratory, such organisms can be easily cultured in a “candle jar.” |
A “candle jar” is a container into which a burning candle is placed before sealing with an airtight lid. The candle flame will burn until it is extinguished from lack of oxygen, resulting in a carbon dioxide-rich, oxygen-depleted atmosphere in the jar.
Table 6. Reproduction characteristics.
Scheme 6. Dependence of generation duration on various factors.
Generation duration
Type of bacteria
Population
Temperature
Composition of the nutrient medium
|
Table 6.1. Bacterial reproduction phases. |
Characteristic |
|
|
Phase |
Initial stationary phase |
|
|
Lasts 1-2 hours. During this phase, the number of bacterial cells does not increase. |
Lag phase (phase of delayed reproduction) |
|
|
It is characterized by the beginning of intensive cell growth, but the rate of their division remains low. |
Log phase (logarithmic) |
|
|
Characterized by a maximum rate of cell reproduction and an exponential increase in the size of the bacterial population |
Characterized by less activity of bacterial cells and longer generation period. |
|
|
This occurs as a result of depletion of the nutrient medium, accumulation of metabolic products in it and oxygen deficiency. |
Stationary phase |
|
|
It is characterized by a balance between the number of dead, newly formed and dormant cells. |
Death phase |
Occurs at a constant speed and is replaced by UP-US phases of decreasing rate of cell death.
Scheme 7. Requirements for nutrient media.
Requirements
Viscosity
Humidity
Sterility 
Nutritional value
Transparency
Isotonicity
|
Table 7. Reproduction of bacteria on nutrient media. |
Characteristic |
||
|
Nutrient medium |
Solid culture media |
||
|
SOn solid nutrient media, bacteria form colonies - clusters of cells.- type (smooth – smooth and shiny) |
Round, with a smooth edge, smooth, convex.On solid nutrient media, bacteria form colonies - clusters of cells. R (rough – rough, unequal) |
||
|
Irregular in shape with jagged edges, rough, dented. |
Liquid culture media Bottom growth (sediment) Surface growth (film) |
||
Diffuse growth (uniform cloudiness)
|
Classification |
Table 7.1. Classification of nutrient media. |
Kinds |
|
|
Examples |
By composition MPA – meat-peptone agar MPB - meat-peptone broth |
||
|
PV – peptone water Blood agar JSA – yolk salt agar |
|||
|
Hiss media |
By purpose | ||
|
Basic |
Elective Alkaline agar |
||
|
Alkaline peptone water |
|
||
|
Ploskireva |
Special Wilson-Blair Kitta-Tarozzi Thioglycol broth |
||
|
Milk according to Tukaev |
PV – peptone water By consistency |
||
|
Alkaline agar |
Semi-liquid |
||
|
Semi-solid agar |
By origin | ||
|
Natural | |||
|
Semi-synthetic |
|
Simmonson
|
Table 7.2. Principles of isolating pure cell culture. |
Mechanical principle |
|
Biological principle 1. Fractional dilutions of L. Pasteur 2. Plate dilutions of R. Koch 3. Surface crops of Drigalsky |
4. Surface strokes Take into account: a - type of breathing (Fortner method); b - mobility (Shukevich method); c - acid resistance; g - sporulation; d - temperature optimum; |
e - selective sensitivity of laboratory animals to bacteria
|
Table 7.2.1. Stages of isolating a pure cell culture. |
Characteristic |
|
|
Stage |
Stage 1 of the study |
|
|
Pathological material is collected. It is studied - appearance, consistency, color, smell and other signs, a smear is prepared, painted and examined under a microscope. |
Stage 2 of the study The colony– these are accumulations of bacteria visible to the naked eye on the surface or in the thickness of the nutrient medium. As a rule, each colony is formed from the descendants of one microbial cell (clones), therefore their composition is quite homogeneous. The growth characteristics of bacteria on nutrient media are a manifestation of their cultural properties. |
|
|
Stage 3 of the study |
The growth pattern of a pure culture of microorganisms is studied and its identification is carried out. |
Table 7.3. Identification of bacteria.
|
Name |
Characteristic |
|
|
Biochemical identification |
Determining the type of pathogen by its biochemical properties |
|
|
Serological identification |
In order to establish the species of bacteria, their antigenic structure is often studied, that is, identification is carried out by antigenic properties |
|
|
Identification by biological properties |
Sometimes bacteria are identified by infecting laboratory animals with a pure culture and observing the changes that pathogens cause in the body. |
|
|
Cultural identification |
Determining the type of pathogens based on their cultural characteristics |
|
|
Morphological identification |
Determining the type of bacteria by their morphological characteristics |
Which process is not related to the physiology of bacteria?
Reproduction
What substances make up 40–80% of the dry mass of a bacterial cell?
Characteristics and role in cell physiology.
Nucleic acids
What classes of enzymes are synthesized by microorganisms?
Oxyreductases
All classes
Transferases
Enzymes whose concentration in the cell increases sharply in response to the appearance of an inducer substrate in the environment?
Iducible
Constitutional
Repressive
Multienzyme complexes
Pathogenicity enzyme secreted by Staphylococcus aureus?
Neuraminidase
Hyaluronidase
Lecithinase
Fibrinolysin
Do proteolytic enzymes have a function?
Protein breakdown
Breakdown of fats
Breakdown of carbohydrates
Formation of alkalis
Fermentation of enterobacteria?
Final product
Formic acid
Propionic acid
Butyric acid
What mineral compounds are used to bind tRNA to ribosomes?
Biological oxidation is...?
Reproduction
Cell death
What substances themselves synthesize all the carbon-containing components of the cell from CO 2.
Prototrophs
Heterotrophs
Autotrophs
Saprophytes
Nutrient media vary:
Examples
Milk according to Tukaev
Hiss media
For all of the above
The reproduction phase, which is characterized by a balance between the number of dead, newly formed and dormant cells?
Negative acceleration phase
Stationary phase
The duration of generation depends on?
Age
Populations
All of the above
In order to establish the species identity of bacteria, their antigenic structure is often studied, that is, identification is carried out, which one?
Biological
Morphological
Serological
Biochemical
The Drigalski method of surface seeding is referred to as...?
Mechanical principles of pure culture isolation
Biological principles of isolating pure culture
Bibliography
1. Borisov L. B. Medical microbiology, virology, immunology: a textbook for honey. universities – M.: Medical Information Agency LLC, 2005.
2. Pozdeev O.K. Medical microbiology: a textbook for honey. universities – M.: GEOTAR-MED, 2005.
3. Korotyaev A.I., Babichev S.A. Medical microbiology, immunology and virology / textbook for medical professionals. universities – St. Petersburg: SpetsLit, 2000.
4. Vorobyov A. A., Bykov A. S., Pashkov E. P., Rybakova A. M. Microbiology: textbook. – M.: Medicine, 2003.
5. Medical microbiology, virology and immunology: textbook / ed. V. V. Zvereva, M. N. Boychenko. – M.: GEOTar-Media, 2014.
6. Guide to practical training in medical microbiology, virology and immunology / ed. V.V. Tetsa. – M.: Medicine, 2002.
Introduction 6
Composition of bacteria from the point of view of their physiology. 7
Metabolism 14
Nutrition (nutrient transport) 25
Breathing 31
Reproduction 34
Microbial communities 37
APPLICATIONS 49
References 105