Sterility

Nutritional value

Transparency

Isotonicity

Table 7. Reproduction of bacteria on nutrient media.

Table 7.1. Classification of culture media.

Table 7.2. Principles of isolation of pure cell culture.

Table 7.2.1. Stages of isolation of pure cell culture.

Table 7.3. Identification of bacteria.

Which of the processes is not related to the physiology of bacteria?

Reproduction

What substances make up 40 - 80% of the dry mass of a bacterial cell?

Carbohydrates

Nucleic acids

What classes of enzymes are synthesized by microorganisms?

Oxy reductase

All classes

Transferases

Enzymes, the concentration of which in the cell sharply increases in response to the appearance of an inducer substrate in the medium?

Iiducible

Constitutional

Repressive

Multi-enzyme complexes

An enzyme of pathogenicity secreted by Staphylococcus aureus?

Neuraminidase

Hyaluronidase

Lecithinase

Fibrinolysin

Do proteolytic enzymes perform a function?

Protein breakdown

Breaking down fats

Breakdown of carbohydrates

Alkali formation

Fermentation of Enterobacteriaceae?

Lactic acid

Formic acid

Propionic acid

Butyric acid

What mineral compounds are used to bind t-RNA to ribosomes?

Biological oxidation is ...?

1. Reproduction

2. Cell death

What substances themselves synthesize all carbon-containing components of the cell from CO 2.

Prototrophs

Heterotrophs

Autotrophs

Saprophytes

Culture media differ:

By composition

By consistency

By appointment

All of the above

The reproduction phase, which is characterized by a balance between the number of dead, newly formed and dormant cells?

2. Negative acceleration phase

   Stationary phase

Generation duration depends on?

Age

Populations

All of the above

In order to establish the species of bacteria, their antigenic structure is often studied, that is, identification is carried out, which one?

Biological

Morphological

Serological

Biochemical

Drygalski's surface sowing method is referred to as ...?

Mechanical principles of isolation of pure culture

Biological principles of isolation of pure culture

Bibliography

1. Borisov LB Medical microbiology, virology, immunology: a textbook for honey. universities. - M .: LLC "Medical Information Agency", 2005.

2. Pozdeev OK Medical microbiology: a textbook for honey. universities. - M .: GEOTAR-MED, 2005.

3. Korotyaev AI, Babichev SA Medical microbiology, immunology and virology / textbook for honey. universities. - SPb .: SpetsLit, 2000.

4. Vorobiev 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. Teza. - M .: Medicine, 2002.

Introduction 6

The composition of bacteria from the point of view of their physiology. 7

Metabolism 14

Nutrition (transport of nutrients) 25

Breath 31

Breeding 34

Microbial communities 37

APPENDICES 49

References 105

The antigenic structure of microorganisms is very diverse. In microorganisms, general, or group, and specific, or typical, antigens are distinguished.

Group antigens are shared by two or more types of microbes belonging to the same genus, and sometimes belonging to different genera. Thus, common group antigens are found in certain types of the genus Salmonella; the causative agents of typhoid fever have common group antigens with the causative agents of paratyphoid A and paratyphoid B (0-1.12).

Specific antigens are present only in a given type of microbe, or even only in a certain type (variant) or subtype within a species. Determination of specific antigens makes it possible to differentiate microbes within a genus, species, subspecies, and even type (subtype). So, within the genus Salmonella, more than 2000 types of Salmonella are differentiated by the combination of antigens, and in the subspecies Shigella Flexner - 5 serotypes (serovariants).

According to the localization of antigens in the microbial cell, somatic antigens associated with the body of the microbial cell are distinguished, capsular antigens - surface or envelope antigens and flagellar antigens located in flagella.

Somatic, O-antigens(from German ohne Hauch - without breathing), are associated with the body of the microbial cell. In gram-negative bacteria, the O-antigen is a complex complex of lipid-polysaccharide-protein nature. It is highly toxic and is the endotoxin of these bacteria. In causative agents of coccal infections, cholera vibrios, causative agents of brucellosis, tuberculosis and some anaerobes, polysaccharide antigens are isolated from the body of microbial cells, which determine the typical specificity of bacteria. As antigens, they can be active in pure form and in combination with lipids.

Flagellate, H antigens(from German. Hauch - respiration), are of a protein nature and are located in the flagella of motile microbes. Flagellate antigens are rapidly destroyed by heat and phenol. They are well preserved in the presence of formalin. This property is used in the manufacture of dead godfathers diagnosed for the agglutination reaction, when it is necessary to preserve the flagella.

Capsule, K - antigens, - are located on the surface of the microbial cell and are also called surface, or shell. They have been studied in most detail in microbes of the intestinal family, in which Vi-, M-, B-, L- and A-antigens are distinguished. Of these, the Vi antigen is of great importance. It was first discovered in strains of typhoid fever bacteria with high virulence, and was called virulence antigen. When a person is immunized with a complex of O- and Vi- antigens, a high degree of protection against typhoid fever is observed. Vi-antigen is destroyed at 60 ° C and is less toxic than O-antigen. It is also found in other intestinal microbes, such as E. coli.

Protective(from Lat. protectio - patronage, protection), or protective, antigen is formed by anthrax microbes in the body of animals and is found in various exudates with anthrax disease. The protective antigen is part of the exotoxin secreted by the anthrax microbe and is able to induce the development of immunity. In response to the introduction of this antigen, complement-binding antibodies are formed. A protective antigen can be obtained by growing the anthrax microbe on a complex synthetic medium. A highly effective chemical vaccine against anthrax has been prepared from the protective antigen. Protective protective antigens were also found in the causative agents of plague, brucellosis, tularemia, whooping cough.

Complete antigens cause synthesis of antibodies or sensitization of lymphocytes in the body and react with them both in vivo and in vitro. For high-grade antigens, strict specificity is characteristic, that is, they cause the body to produce only specific antibodies that react only with this antigen. These antigens include proteins of animal, plant and bacterial origin.

Defective antigens (haptens) are complex carbohydrates, lipids and other substances that are not capable of causing the formation of antibodies, but that enter into a specific reaction with them. Haptens acquire the properties of full-fledged antigens only if they are introduced into the body in combination with a protein.

Typical representatives of haptens are lipids, polysaccharides, nucleic acids, as well as simple substances: paints, amines, iodine, bromine, etc.

Vaccination as a method of preventing infectious diseases. The history of the development of vaccination. Vaccines. Requirements for vaccines. Factors determining the possibility of creating vaccines.

Vaccines are biologically active drugs that prevent the development of infectious diseases and other manifestations of immunopathology. The principle of using vaccines is to advance the creation of immunity and, as a result, resistance to the development of the disease. Vaccination refers to measures aimed at artificial immunization of the population by introducing vaccines to increase resistance to the disease. The goal of vaccination is to create an immunological memory against a specific pathogen.

Distinguish between passive and active immunization. The introduction of immunoglobulins obtained from other organisms is passive immunization. It is used for both therapeutic and prophylactic purposes. The administration of vaccines is active immunization. The main difference between active and passive immunization is the formation of immunological memory.

Immunological memory provides an accelerated and more efficient removal of foreign agents when they reappear in the body. The basis of immunological memory is memory T and B cells.

The first vaccine got its name from the word vaccinia(cowpox) is a viral disease of cattle. The English physician Edward Jenner first applied the smallpox vaccine to the boy James Phipps, obtained from the vesicles on the arm of a patient with vaccinia, in 1796. Only almost 100 years later (1876-1881) Louis Pasteur formulated the main principle of vaccination - the use of weakened preparations of microorganisms for formation of immunity against virulent strains.

Some of the live vaccines were created by Soviet scientists, for example, P.F.Zdrodovsky created a vaccine against typhus in 1957-59. The influenza vaccine was created by a group of scientists: A. A. Smorodintsev, V. D. Soloviev, V. M. Zhdanov in 1960. P. A. Vershilova in 1947-51 created a live vaccine for brucellosis.

The vaccine must meet the following requirements:

● activate cells involved in the processing and presentation of antigen;
● contain epitopes for T and T cells, providing a cellular and humoral response;
● easy to process with subsequent effective presentation of histocompatibility antigens;
● induce the formation of effector T-cells, antibody-producing cells and corresponding memory cells;
● prevent the development of the disease for a long time;
● be harmless, that is, do not cause serious illness and side effects.

The effectiveness of vaccination is actually the percentage of vaccinated who have responded to vaccination by the formation of specific immunity. Thus, if the effectiveness of a certain vaccine is 95%, then this means that out of 100 vaccinated, 95 are reliably protected, and 5 are still at risk of disease. The effectiveness of vaccination is determined by three groups of factors. Factors depending on the vaccine preparation: the properties of the vaccine itself, which determine its immunogenicity (live, inactivated, corpuscular, subunit, the amount of immunogen and adjuvants, etc.); the quality of the vaccine preparation, i.e., the immunogenicity is not lost due to the expiration of the vaccine's shelf life or due to the fact that it was not stored or transported correctly. Factors depending on the vaccinated: genetic factors that determine the fundamental possibility (or impossibility) of developing specific immunity; age, because the immune response is closely determined by the degree of maturity of the immune system; state of health "in general" (growth, development and malformations, nutrition, acute or chronic diseases, etc.); the background state of the immune system is primarily the presence of congenital or acquired immunodeficiencies.

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: guidelines for laboratory work on 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. un-t, BTI. - Biysk:

Publishing house Alt. state tech. University, 2007. - 36 p.

These guidelines consider the basic concepts, rules and principles of classification and identification of microorganisms. The laboratory work on the study of various properties of bacteria necessary for the description of the bacterial strain and its identification to the level of the genus is presented.

Reviewed and approved

at the meeting of the department

"Biotechnology".

Minutes No. 88 dated 01.01.2001

Reviewer:

Doctor of Biological Sciences, Professor, BPGU named after

© BTI AltSTU, 2007

1 BASIC CONCEPTS AND NAMING RULES

MICROORGANISMS

Several thousand species of microorganisms have been described, but it is believed that this is less than 1 % from the real ones. The study of the diversity of microorganisms is the subject of taxonomy. Its main task is to create a natural system that reflects the phylogenetic relationships of microorganisms. Until recently, the taxonomy of microorganisms was based mainly on phenotypic characters: morphological, physiological, biochemical, etc., therefore, the existing classification systems are largely artificial. However, they make it relatively easy to identify some newly isolated strains of microorganisms.

The taxonomy includes sections such as classification, nomenclature and eden tification . Classification determines the order of placing individuals with a given degree of homogeneity in certain groups (taxa). Nomenclature is a set of rules for naming taxa. Identification means the determination of the belonging of the studied organism to a particular taxon.

The term "taxonomy" is often used as a synonym for taxonomy, but sometimes it is understood as a branch of taxonomy that includes classification theory, the doctrine 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, molecular-genetic characteristics.

The term "strain" is understood as a pure culture of a microorganism isolated from a specific habitat (water, soil, animal organism, etc.). Different strains of the same type of microorganisms may differ in some traits, for example, sensitivity to antibiotics, the ability to synthesize certain metabolic products, etc., but these differences are less than those of the species. The concept of "strain" in microbiology and genetics is somewhat different: in microbiology, this concept is broader. The 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 rarely used.

The nomenclature of microorganisms is subject to international rules. So, there is an International Code of Nomenclature for Bacteria. For yeasts, 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, use binary or binomial (from lat. bis- twice) the nomenclature system, according to which each species has a name consisting of two Latin words. The first word means a 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 – with a lowercase even if the specific epithet is assigned in honor of the scientist, for example Clostridium pasteurianum. In the text, especially with Latin graphics, the entire phrase is italicized. When the name of the microorganism is mentioned again, the generic name can be shortened to one or more initial letters, for example WITH.pasteurianum. If the text contains the names of two microorganisms that begin with the same letter (for example, Clostridium pasteurianum and Citrobacterfreundii), then the abbreviations must be different (C. pasteurianum and Ct. freundii). If the microorganism is identified only to the genus, the word sp is written instead of the specific epithet. (species- view), for example Pseudomonas sp. In this case, when re-mentioning the name of the microorganism in the text, the generic name should always be written in full.

For the name of a subspecies, a phrase is used, consisting of the name of the genus, as well as the specific and subspecies epithets. To distinguish between these epithets, a letter combination is written between them, which is the abbreviated word subspecies - "subsp." or (less commonly) "ss." For instance, Lactobacillus delbrueckii subsp. bulgaricus.

For each strain, they also indicate the abbreviation of the name of the collection of cultures of microorganisms in which it is stored, and the number under which it appears there. For instance, Clostridium butyricum ATCC 19398 means that the strain is stored in the American Type Culture Collection (ATCC) under the number 19398. For a list of world-renowned microbial collections, see Bergeys Manual of Systematic Bacteriology (1984– 1989), in catalogs of cultures of microorganisms and other reference publications.

The description of any new type of microorganisms is based on a typical strain that is stored in one of the collections of microorganisms and on the basis of the combination of properties of which this species

characterized in the original article or qualifier. The type strain is the nomenclature type of the species, since the species name is assigned to it. If any strains, originally included in the same species, are later recognized as deserving to be separated into special species, they should be given new names, and the old species name is 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 coincide in their properties.

For a genus, the nomenclature type is a specially designated type species, which has a set of characteristics most characteristic of the representatives of this taxon. For example, in the genus Bacillus type is V.subtilis.

In some keys and catalogs, the old names of the renamed microorganisms are indicated, as well as the names of the authors who were the first to isolate this microorganism, and the year of publication in which this organism was first described. For example, one of the yeast species 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 1952 publication, then this species was named Torulopsis magnoliae. In 1978 g. Torulopsis magnoliae was renamed by explorers such as Meyer and Yarrow, to Candida magnoliae and is currently stored in the VKM under the number VKM Y-1685. The Y in front of the strain number stands for "the Yeasts" - yeast.

In addition to the concept of "strain" in microbiology, the terms "variant", "type", "form" are used. They are usually used to denote strains of microorganisms that differ in some way from the type strain. A strain that differs from the typical one in morphological features is called morfovar(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 the type of response to the introduction of bacteriophage - phagovar(phage, lysotype), antigenic characteristics - serovar(serotype)
etc.

In works on the genetics of microorganisms, the term is often used "clone", which means a population of genetically related cells obtained asexually from one parental cell. In molecular biology, a clone is called multiple

copies of identical DNA sequences obtained by their insertion into cloning vectors (for example, plasmids). The term "genetically modified" or "recombinant" strains means strains of microorganisms obtained as a result of genetically engineered 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 in order to obtain a complete set of data on the properties of the microorganism.
in pure culture. These data can be used, for example, for drawing up a passport of industrially valuable strains, as well as for their identification.

Purpose of identification - to establish the taxonomic position of the investigated strain on the basis of comparing its properties with the studied and accepted (officially registered) species. Therefore, the result of identification is usually the identification of the studied microorganism with some kind or assignment
to a certain genus. If the investigated strain or group of strains differ in their properties from the representatives of the known taxa, then they can be separated into a new taxon. For 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; characterization of each strain; list of properties considered essential
in a taxon; a list of properties that qualify a taxon for representation in the next higher taxon; a list of diagnostic characteristics that differentiate the proposed taxon from closely related taxa; a separate description of the typical (for the species) strain; photograph of the microorganism.

In order for a newly proposed taxon to be officially accepted, its description must be published in accordance with certain rules. For example, the actual or legal publication of a taxon of bacteria involves posting an article describing it in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). If a publication appears in another reputable scientific journal (effective publication), then a reprint of the article from that journal is sent to the IJSEM. Since 1980, the IJSEM has regularly published the so-called lists of legalized names of bacteria. They list all the names of bacteria that have been published in the IJSEM (valid or legal publication) or have been effectively published before in any

other reputable magazines. Once a bacteria name has been entered into the list of legalized IJSEM names, the name is valid, regardless of whether it was previously published in IJSEM or another journal. The date of appearance of the publication of the name of this taxon in the IJSEM or in the list of legal names of the IJSEM is priority for the taxon.

The culture of a typical strain of a new type of microorganisms is transferred for storage to one of the collections of microorganisms of world importance. In case of loss of the type strain, it can be replaced by the so-called non-type strain. In this case, it must be confirmed that the properties of the new strain are in good agreement with the description of the lost one. To indicate that the taxon is being proposed for the first time, the abbreviation “fam. nov. ", a new kind -" gen. nov. ", and the new species -" sp. nov. ". For instance,
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 certain taxonomic status, since it is not clear in which taxon of a higher order - a genus or a family - this species should be placed due to the lack of necessary experimental
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 features of the structure and methods of formation, in the first place, of the reproductive structures. In addition, the characteristics of asexual sporulation, the structure and degree of development of the mycelium (rudimentary, well-developed, septic or non-septic), cultural (colony) and physiological signs 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 identifier for identifying all mushrooms, therefore, the class or order of the identified mushroom is first determined and then the corresponding identifier 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 signs associated with 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. Determination of the systematic position of protozoa is carried out using morphological features and life cycles. Thus, the identification of eukaryotes is based mainly on the features of their morphology and developmental cycles.

The 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 traits. 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 sequence of nucleotides in the gene encoding the synthesis of 16S rRNA and other pheno - and genotypic traits. In this case, the following rules must be observed: work with pure cultures, apply 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 nutrient media

On the surface of solid nutrient media, depending on the sowing, microorganisms can grow in the form of a colony, a line or a solid lawn. Colony is called an isolated cluster of cells of the same species, which in most cases has grown from one 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 distinguish superficial, deep and bottom colonies.

Education overnostal colonies - the most essential feature of the growth of many microorganisms on a dense substrate. Such colonies are very diverse. When describing them, the following features are taken into account:

profile- flat, convex, crater-shaped, cone-shaped, etc. (Figure 1);

shape- rounded, amoeba, 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 point;

surface- smooth, rough, grooved, folded, wrinkled, with concentric circles or radially striated;

shine and transparency- the colony is shiny, dull, dull, mealy, transparent;

color- colorless (off-white colonies are referred to as colorless) or pigmented - white, yellow, golden, orange
wavy, lilac, red, black, etc .; highlight the

pigment substrate; when describing colonies of actinomycetes, pigmentation of the air and substrate mycelium is noted, as well as the release of pigments into the environment;

edge- smooth, wavy, jagged, fringed, etc. (Figure 3);

structure- homogeneous, fine - or coarse-grained, streaky, etc. (Figure 4); the edge and structure of the colony is determined with a magnifying glass or at low magnification of a microscope. For 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 easily be removed from the agar, be dense, soft or growing into the agar, mucous (sticks to the loop), stringy, have the appearance of a film (removed entirely), be fragile (easily breaks when touched by the loop).

1 - curved; 2 - crater-like; 3 - bumpy;

4 - growing into the substrate; 5 - flat; 6 - convex;

7 - drop-shaped; 8 - conical

Figure 1 - Colony profile

Deep colonies on the contrary, they are rather monotonous. Most often, they look like more or less flattened lentils,
in the projection have the shape of ovals with pointed ends. Only
in a few bacteria, deep colonies resemble bundles of cotton wool
with filamentous outgrowths into the nutrient medium. The formation of deep colonies is often accompanied by a rupture of the dense medium if the microorganisms release carbon dioxide or other gases.

Bottom colonies different microorganisms usually have the form of thin transparent films creeping 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 temperature of cultivation.

1 - round; 2 - round with a scalloped edge; 3 - round with a roller along the edge; 4, 5 - rhizoid; 6 - with a rhizoid edge; 7 - amoeba;
8 - threadlike; 9 - folded; 10 - wrong;

11 - concentric; 12 - complex

Figure 2 - Colony shape

/ - smooth; 2 - wavy; 3 - toothed; 4 - bladed; 5 - wrong; 6 - ciliated; 7 - filamentous; 8 - villous; 9 - branched

Figure 3 - The edge of the colony

1 - homogeneous; 2 - fine-grained; 3 - coarse-grained;

4 - streaky; 5 - fibrous

Figure 4 - Colony structure

When describing the growth of microorganisms by stroke note the following features: scanty, moderate or abundant, solid
with a smooth or wavy edge, beaded, resembling chains of isolated colonies, diffuse, feathery, treelike, or rhizoid (Figure 5). They characterize the optical properties of the plaque, its color, surface and consistency.

To describe colonies and growth by stroke, many microorganisms are often grown on mesopatamia agar. Mesopatamia gelatin is also used. For a better view of deep colonies, it is recommended to clarify media with agar or gelatin.

1 - solid with a smooth edge; 2 - solid with a wavy edge; 3 - beaded; 4 - diffuse; 5 - feathery; 6 - rhizoid

Figure 5 - Growth of bacteria along the stroke

2.1.2. Growth in liquid culture 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, note turbidity degree(weak, moderate, or strong), film features(thin, dense or loose, smooth or folded),
and when a sediment is formed, it is indicated that 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 gas evolution. The latter is detected by the formation of foam, bubbles, and also with the help of "floats" - small tubes sealed from one end. The float is placed
into a test tube with the sealed end up before sterilizing the medium and ensure that it is completely filled with the medium. If gas evolves, it accumulates in the float in the form of a bubble.

To describe the nature of the growth of microorganisms in liquid media, they are grown in mesopatamia broth (MPB) or on another medium that provides good growth.

2.2 Morphological characteristics

The morphological characteristics and organization of bacterial cells include such features as the shape and size of cells, their mobility, the presence of flagella and the type of flagellation, the ability to sporulate. Cellular detection may also be helpful.
characteristic membrane systems (chlorosomes, carboxisomes, phycobilisomes, gas vacuoles, etc.) inherent in individual groups of bacteria
ry, as well as inclusions (parasporal bodies, volutin granules,
poly-β-hydroxybutyrate, polysaccharides, etc.). Gram staining of cells is of paramount 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, the establishment of the way of feeding the studied bacterium (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 traits as the ratio of bacteria to molecular oxygen, temperature, pH of the environment, salinity, illumination and other environmental factors. 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, the presence of certain enzymes. For this, 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 formulation requires a significant investment of time, a large number of complex media and reagents, adherence to standard conditions, and accuracy. To speed up and facilitate the identification of some microorganisms, which 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. For example, the Enterotube II system, designed for the identification of enterobacteriaceae, it is a plastic chamber with 12 cells containing colored diagnostic media. Sowing of all media is carried out by forward-rotational movements through the needle chamber with the seed. The 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 agar (test for gas formation) or after the introduction of special reagents (test for the formation of indole, Voges-Proskau-er reaction). Each trait is designated by a specific number, so the data obtained can be entered into a computer with the appropriate program and an answer about the taxonomic position of the strain under study can be obtained.

Determination of 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 their 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 is the basis of immunochemical methods for their identification.

The lipid and fatty acid composition of bacterial cells is sometimes also used as a chemotaxonomic marker. An intensive study of fatty acids became possible with the development of the method 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 content of fatty acids in 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 carriers of electrons, as well as pigments.

Important information about the mutual relationship of bacteria can be obtained by studying cellular proteins - the products of gene translation. Based on the study of membrane, ribosomal, total cellular proteins, as well as individual enzymes, a new direction has been formed - protein taxonomy. The ribosomal protein spectra are among the most stable and are used to identify bacteria at the family or order level. The spectra of membrane proteins can reflect generic, species, and even intraspecific differences. However, the characteristics of the chemical compounds of the 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 traits.

Sometimes a method is used to identify bacteria or other microorganisms such as yeast numerical (or Adansonian) taxonomy... It is based on the ideas of the French botanist M. Adanson, who proposed various phenotypic traits that can be taken into account, to be considered equivalent, which makes it possible to quantitatively express taxonomic distances between organisms in the form of the ratio of the number of positive traits to the total number studied. The similarity between the two studied organisms is determined by quantifying the largest possible number (usually at least one hundred) phenotypic traits, which are selected so that their variants are alternative and can be indicated by the signs "minus" and "plus". The degree of similarity is established based on the number of matching features and is expressed as a match coefficient S:

where a + d- the sum of the traits by which strains A and B coincide;

a- both strains with positive traits;

d- both with negative;

b- the sum of the signs according to which the strain A is positive, B is negative;

With- the sum of the signs for which strain A is negative, strain B is positive.

The value of the coefficient of compliance can vary from 0 to 1. Coefficient 1 means complete identity, 0 - complete dissimilarity. Combinations of features are evaluated 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 to assess the similarity between taxa of microorganisms of only low rank (genera, species). It does not allow direct conclusions to be drawn regarding the genetic relationship of microorganisms, however, to a certain extent, it reflects their phylogenetic properties. Thus, it was found that the phenotypic traits of bacteria that can be studied at the present time reflect from 5 to 20% of the properties of their genotype.

2.4 Studying the genotype

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. The study of the genotype based on the analysis of nucleic acids, in principle, makes it possible to build over time a natural (phylogenetic) system of microorganisms. Phylogenetic relationships of bacteria are assessed determination of molar content guanine and cytosine (HC) in DNA, by 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 HZ molar content

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 HC 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 mutual arrangement, the same average GC content in the DNA of two bacterial species can be accompanied by their significant genotypic

division. If two organisms are very close in nucleotide composition, then this can be evidence of their evolutionary relationship only if they have a large number of common phenotypic traits or genetic similarities confirmed by other methods. At the same time, the discrepancy (more than 10 ... 15%) in the nucleotide composition of 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 relationship of bacteria. Careful experimentation can provide valuable information about the degree of genetic homology. Within one species of bacteria, the degree of genetic homology of the 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 the specific DNA – DNA reassociation becomes so weak that it cannot be measured. In this case, DNA – rRNA hybridization can 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 region of the bacterial genome encoding ribosomal RNAs, the original base sequence is much more complete than in other regions of the chromosome. As a result, the DNA – rRNA hybridization method often reveals a rather high homology of bacterial genomes, in which DNA – DNA reassociation does not reveal any noticeable homology.

2.4.3 DNA probes (gene probes) method

The DNA probe method is a variation of the DNA-DNA molecular hybridization method. The hybridization reaction is carried out in this case not between two preparations of total DNA, but between a fragment of the nucleotide sequence of DNA (probe), which includes a gene (genetic marker) responsible for some specific function (for example, resistance to some antibiotic), and DNA the bacteria under study. 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 cleaving its DNA with endonucleases

restriction, and then select the desired clone from the sum of DNA fragments by electrophoresis, followed by verification of the genetic properties of these fragments by transformation. Next, the selected DNA fragment is ligated into a suitable plasmid (vector),
and this combination plasmid is introduced into a convenient strain of bacteria (for example, Escherichia coli). Plasmid DNA is isolated from the biomass of a bacterium carrying a DNA probe and labeled, for example, with a radioisotope label. Then carry out the hybridization of the DNA probe
with bacterial DNA. The resulting hybrid areas are developed by autoradiography. According to the relative frequency of hybridization of the genetic marker with the chromosome of a particular bacterium,
conclusion on the genetic relationship of these bacteria with the investigated strain.

2.4.4 Method for analysis of nucleotide sequences

in ribosomal RNA

For the identification of bacteria and the creation of a phylogenetic system for their classification, the method of analysis of nucleotide sequences in ribosomal RNAs is the most widespread and important. 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 action 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 sequence of nucleotides, which in this molecule in prokaryotes is 120 nucleotides. When studying 16S and 23S rRNA containing 1500 and 2500 nucleotides, respectively, the analysis of oligonucleotides obtained from these molecules using specific restriction endonucleases is often carried out. 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 different microorganisms led to the identification of a group of archaea among prokaryotes. Similarity rate values SAB, separating the I6S rRNA of bacteria and archaea, lie 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 proposed for identifying bacteria, showing the relationship 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, the identification of bacteria before parturition on the basis of genetic methods only, without a preliminary study of their phenotypic characteristics, is often completely impossible. Therefore, the best approach in the work on the taxonomy of bacteria is considered to be the study of both genotypic and phenotypic properties. In the event of a discrepancy between phylogenetic and phenotypic data, the latter is temporarily given priority.

A particular problem is the identification of such bacteria and archaea, especially marine species, which are not able to grow on known laboratory culture media and for which it was therefore impossible to obtain a pure culture. 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 the microorganisms inhabiting this biotope without isolating them into a pure culture. The microorganism "uncultivated" in the laboratory identified in this way can even be described, but with the addition of the word "candidatus" (candidate). The word "candidatus" will accompany the new species until scientists find the conditions for the cultivation of this organism in the laboratory and its pure culture is obtained, which will make it possible to study all its properties and publish it as legalized.

Bacteria are usually identified using Bergey's Manual of Determinative Bacteriology, which was first published in 1923 under the guidance of the famous American bacteriologist D. Bergey (DH Bergey, 1860-1937). since then it is regularly reissued with the participation of leading microbiologists of the world.
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 the species of bacteria of some genera, for example, the genus Bacillus, not given, but the reader is referred to Burgey's Guide to the Systematics of Bacteria.

Bergey's four-volume Manual of Systematic Bacteriology (1984–1989) provides more complete information on the taxonomic position of bacteria.
and species, including those with unclear taxonomic status. In addition to a detailed phenotypic description, including morphology, organization and chemical composition of cells, antigenic properties, type of colonies, features of the life cycle and ecology, the characteristics of genera also provide information on the content of GC in DNA, the results of DNA – DNA and DNA – rRNA hybridization. Keys and tables make it possible to identify bacteria not only to the genus, but also to the species.

The second edition of the four-volume Bergcy's Manual of Systematic Bacteriology has now been published, and the first volume was published in 2002. In addition, there are a number of articles and books that offer original clues for identifying individual groups of bacteria, for example, bacilli, pseudomonads, actinomycetes, enterobacteria.

At present, a lot of new data has been accumulated, including those obtained as a result of the analysis of nucleotide sequences of ribosomal RNA, on previously studied and newly isolated species of bacteria. Based on this information, the species composition of some groups of bacteria, for example, the genus Bacillus, will be revised: some species will remain in the genus Bacillus, and some form new genera or will be attributed to other already existing genera of bacteria. It should also be noted that, as a rule, more traits are studied to describe new strains of bacteria than are necessary for their identification, since the keys and tables do not include all traits of identifiable bacteria, but only those that differ in different species (Table 1).

Table 1 - The minimum list of data required for

descriptions of new strains of bacteria (according to H. Truper, K. Schleifer, 1992)

Continuation of table 1

3 LABORATORY WORK "IDENTIFICATION
MICROORGANISMS "

Objective: acquaintance with the basic principles of determining microorganisms. In the process of performing laboratory work, each student studies the properties of bacteria necessary to describe a bacterial strain and identify it to the level of the genus.

Tasks

1. Determine the purity of the identified bacteria and study the morphology of its cells.

2. Describe the cultural properties.

3. To study the cytological properties of identified bacteria.

4. To study the physiological and biochemical properties of the identified bacteria.

5. Determine the sensitivity of bacteria to antibiotics.

6. Fill in the table and summarize.

3.1 Determination of the purity of the bacterium to be identified

and the study of the morphology of its cells

To carry out the work on the identification of microorganisms, each student receives one culture of bacteria (on an agar slant in a test tube), which is then checked for purity. This is done in several ways: visually, by seeding on nutrient media and microscopy.

Growth pattern the resulting bacteria is viewed by streak on the surface of the slant agar medium. If the growth along the stroke is heterogeneous, then the culture is contaminated. Then the culture is seeded into a test tube on a slant medium (mesopatamia agar) for use
in further work, and also do sifting on the surface of a solid medium in a Petri dish by the depletion streak method to check for purity (by the homogeneity of the grown colonies). 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 culture of bacteria in a test tube is used to check for purity by microscopy (according to the morphological homogeneity of the population), as well as to study the shape, relative position, mobility of cells and their size. Microscopic culture using preparations "crushed drop" and a preparation of fixed, fuchsin-stained cells. The results are entered in a table compiled in the form of table 2.

table 2 – Properties of the bacterium to be identified

3.2 Cultural properties

In the next lesson, a Petri dish inoculated with a suspension of the identified bacteria is examined. The criterion for the purity of the culture is the homogeneity of the grown colonies. Describe the cultural properties of bacterial colonies in accordance with the section
scrap 2.1 and the results are entered in table 2.

3.3 Study of the cytological properties of identifiable bacteria

3.3.1 Presence of endospores

A small number of cells from solid media are looped onto a glass slide in a drop of tap water and a smear is taken. 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 abundantly moistened with Tsil's carbolic fuchsin. Heat the preparation over the flame until vapors appear (not to boil), then take it aside and add a new portion of the dye. This procedure is carried out for 7 minutes. It is important that the dye evaporates, but that 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 done correctly, the color is contrasting, and the bright red spores stand out clearly against the blue background of the cytoplasm.

3.3.2 Gram stain

3.3.2.1 A thin smear is made on a defatted glass slide in a drop of water so that the cells are evenly distributed over the glass surface and do not form clumps.

3.3.2.2 The preparation is dried in air, fixed over the flame of a burner and stained for 1 ... 2 min with carbolic gentian or crystal violet.

3.3.2.3 Then the dye is poured off and the smears are treated for 1 ... 2 min with Lugol's solution until blackening.

3.3.2.4 Drain Lugol's solution, the preparation is decolorized for 0.5 ... 1.0 min with 96% ethyl alcohol and quickly washed with water.

3.3.2.5 In addition, it is stained for 1 ... 2 min with aqueous fuchsin.

3.3.2.6 The dye is poured off, the preparation is washed with water and dried.

3.3.2.7 Microscopy with an immersion system.

When stained correctly, gram-positive bacteria have a 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) cultures, since cells from old cultures sometimes give an unstable Gram reaction. Gram-negative bacteria may look like gram-positive bacteria if the bacterial film (smear) is too thick and alcohol decolorization has not been completed. Gram-positive bacteria may look like gram-negative bacteria if the smear is discolored with alcohol.

3.3.3 Coloring for acid resistance

A smear of the bacterium under investigation is prepared on a defatted glass slide in a drop of water. The preparation is dried in air and fixed over a burner flame. A filter bamaga is placed on the smear, the preparation is poured with Tsilya carbolic fuchsin and it is heated 2-3 times until vapors appear, 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 set aside.
drug aside. Allow the preparation to cool, remove the filter paper, discard the dye and wash the smear with water. Then
cells are discolored with 5% acid solution H https://pandia.ru/text/79/131/images/image009_42.gif "width =" 11 "height =" 23 src = ">. times in a glass of sulfuric acid,

without keeping him in her. The preparation is again thoroughly washed with water and stained from 3 to 5 min with methylene blue (according to Leffler). The paint is poured off, the preparation is washed with water, dried and examined with an immersion system. When stained correctly, the cells of acid-fast bacteria are red, and non-acid-fast – blue.

3.3.4 Determination of mobility

The test culture is sown in a column of 0.2 ... 0.5% semi-liquid agar by the injection method. In order for the features of growth to manifest themselves most clearly, a puncture is made in the immediate vicinity of the wall of the test tube. Sowing is placed in a thermostat for 24 hours. Sowing made in this way makes it possible to identify and separate mobile microorganisms from immobile ones.

Immobile forms of bacteria grow along the prick line, forming small outgrowths of a cylindrical or conical shape. At the same time, the environment remains completely transparent. Mobile microbes with such a sowing cause pronounced turbidity, which spreads more or less evenly throughout the entire thickness of the medium.

3.4 Study of physiological and biochemical properties

identifiable bacteria

3.4.1 Relationship 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
on the belonging of microorganisms to a particular group, the microbial suspension is inoculated into test tubes with a melted and agar nutrient medium cooled to a temperature of 45 ºС. Sowing can be done with an 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 - optional anaerobes;

4 - anaerobes

Figure 6 - The growth of microorganisms when sowing with an injection ( a) and when inoculated into a molten dense medium ( b)

3.4.2 Growth on medium with glucose and peptone

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 evidenced by its accumulation in the float. The observation results are compared with a sterile environment.

3.4.3 Growth on medium with gelatin

The activity of extracellular proteolytic enzymes in microorganisms is determined using gelatin, casein or other proteins as a substrate. Wednesday with gelatin consists of mesopatamia broth (MPB) and 10 ... 15% gelatin (MPF). Sowing is carried out with an injection.

With a bacteriological needle, the cells of microorganisms are sterilely selected from the jamb and the needle is inserted into the thickness of the MPG column to the bottom of the test tube.

Duration of cultivation is from 7 to 10 days at room temperature. The liquefaction of gelatin is observed visually. If gelatin liquefies, indicate the intensity and form of liquefaction - layer-by-layer, funnel-shaped, saccular, crater-shaped, rep-shaped, bubble-shaped.

3.4.4 Growth on medium with milk

Plating on "milk agar" in Petri dishes is performed 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. The bacteria are inoculated in a loop, drawing a stroke along the diameter of the dish or in the center of the sector into which the dish is divided. The duration of cultivation of bacteria in a thermostat at a temperature of 30 ° C is 7 days. Casein hydrolysis is detected by the zone of clarification of the medium around the colonies or the culture of microorganisms grown along the streak. The zone is especially clearly visible after treatment of the medium with the grown bacteria with a solution of 5% trichloroacetic acid. The zone of hydrolysis of casein is measured in millimeters from the edge of the line or colony to the border of the light zone. The larger the diameter of the bright zone, the higher the caseinolytic activity of bacteria.

3.4.5 Growth on medium with starch

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, produced to determine the formation of amylase by microorganisms. The bacteria are inoculated in a loop, drawing a stroke along the diameter of the dish or in the center of the sector into which the dish is divided. The bacteria were cultivated for 7 days in a thermostat at a temperature of 30 ° C. Starch hydrolysis is detected after treatment of the medium with the grown bacteria with Lugol's solution. For this, from 3 to 5 ml of Lugol's solution is poured onto the surface of the medium. The medium containing starch turns blue, and the hydrolysis zone remains colorless or becomes red-brown in color if the starch is hydrolyzed to dextrins. The zone of starch hydrolysis is measured from the edge of the streak (colony) to the border of the light zone (mm). The larger the diameter of the bright 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 evidenced 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 agar slant and observe the evolution of molecular oxygen.

3.4.7 Determination of the sensitivity of bacteria
to antibiotics

It is convenient to determine the sensitivity of microorganisms to antibiotics using ready-made paper disks impregnated with certain antibiotics. The investigated microorganisms are grown on an appropriate solid nutrient medium. A thick suspension of the studied microorganism is prepared in sterile tap water by washing the cells with water from the surface of a solid nutrient medium. Working near the flame of the burner, add 1 ml of the resulting suspension
into a test tube with 20 ml of agar medium, melted and cooled to a temperature of 50 ºС, for example, with mesopatamia agar (MPA). If the 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 hardens, paper is placed on its surface.
discs at equal distance 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 agar medium, and then, without inverting, are placed in a thermostat for 24 hours at a temperature of 30 ºС. After a day, 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 culture growth 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
about the high sensitivity of microorganisms to the antibiotic, and less than 12 mm - about the weak sensitivity.

When the experimenter has solutions
antibiotic substances or culture fluid containing

antibiotic, use the method using holes in the thickness of the agar.
In this case, holes are made at a distance of 1.5 ... 2.0 cm from the edge of the cup in a frozen agar medium inoculated with the test microorganism with a sterile cork drill (diameter from 6 to 8 mm).
Solutions of antibiotics or culture fluid are added to the wells. This method also makes it possible to reveal the ability of microorganisms grown in a liquid medium to form antibiotic substances.

Table 3 – The effect of antibiotics on bacterial growth

4 CONTROL 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 the existing systems of classification of microorganisms artificial?

5. What are the characteristics of different strains of the same type of microorganism?

6. Which taxonomic categories of microorganisms are required and which are optional?

7. List the basic rules for nomenclature of microorganisms.

8. What is the main purpose of identifying microorganisms?

9. What is the difference between the principles of classification and identification of different groups of prokaryotes and eukaryotes?

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 signs include the morphological characteristics and organization of bacterial cells?

15. What physiological and biochemical properties are studied when identifying bacteria?

16. When is it necessary to use chemotaxonomic methods?

17. What are some examples of substances used as chemo - taxonomic markers?

18. What are the features of protein taxonomy?

19. Describe the method of numerical taxonomy, what limitations does it have?

20. What methods are used to assess 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 the analysis of nucleotide sequences in ribosomal RNA?

23. What signs are used as the basis for the classification of bacteria in the "Bergey's Identifier of Bacteria"?

24. What properties and signs are studied when describing new strains of bacteria?

25. What methods are used to determine the purity of the identified bacteria?

26. What are the basic rules for the implementation of the Gram staining technique?

27. What groups are microorganisms divided into in relation to molecular oxygen?

28. What is used as a substrate for determining the activity of extracellular protolytic enzymes in microorganisms?

29. What methods of determining the sensitivity of microorganisms to antibiotics do you know, give their characteristics.

30. What method is used to determine the formation of amylase by microorganisms?

31. How does the performed sowing make it possible to identify and separate mobile microorganisms from immobile ones?

5 RECIPES FOR DYES AND NUTRITIONAL MEDIA

5.1 Fuchsin basic carbolic (fuchsin Tsilya)

- 5% aqueous solution of freshly distilled phenol - 100 ml;

- saturated alcoholic solution of basic fuchsin - 10 ml;

The prepared mixture is filtered off after 48 hours.

5.2 Methylene blue (according to Leffler)

- saturated alcoholic solution of methylene blue - 30 ml;

- distilled water - 100 ml;

- 1% aqueous solution of KOH - 1 ml.

5.3 Meat Peptone Broth (BCH)

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 37 ºС for 2 hours, and at 50 ºС for one hour. Then the meat is squeezed through cheesecloth, and the resulting infusion is boiled for 30 minutes. In this case, proteins are coagulated. The cooled mass is filtered through a cotton filter and added with water to the original volume. Next, 5 to 10 g of peptone and 5 g of sodium chloride are added to 1 liter of meat broth. The medium is heated until the peptone dissolves, stirring constantly. BCH is sterilized at a pressure of 2 atm for 20 minutes.

5.4 Meat Peptone Agar (MPA)

To 1 l of MPB add 20 g of agar. The medium is heated until the agar dissolves, then a slightly alkaline reaction of the medium is established

20% NaCO64 solution "height =" 52 "bgcolor =" white "style =" vertical-align: top; background: white ">