FUNDAMENTALS OF MICROBIOLOGY - V. M. Samygin - 2015

CHAPTER 11. METHODS FOR ISOLATION AND IDENTIFICATION OF MICROORGANISMS

In laboratory practice, various Methods are used to detect and identify microorganisms. These methods are based on studying microbial Cells, growth characteristics on nutrient media, biochemical activity, antigenic Structure, and the Specificity of DNA nucleotide sequences. These include bacteriological, microscopic, immunological, and molecular-Genetic Methods.

11.1. Microscopic Research Methods

Microscopic examination of a specimen typically begins with the Cell/15.html">Microscopy of native smears. Light microscopy results alone are insufficient for identifying microorganisms, but they provide a preliminary idea of their quantity in the sample and whether they belong to rod-shaped or coccoid microflora. When Gram staining is applied, it determines the further course of the study, the goal of which is to isolate and establish the species identity of the microorganism. At the same time, some microorganisms are capable of forming capsules, endospores, and exospores, or transforming into cysts. Depending on cultivation conditions and staining methods, one can observe the Morphology of the cells themselves or individual cellular structures.

To determine the shape and motility of Bacteria, "crushed drop" or "hanging drop" preparations are used, i.e., bacteria are examined in a living state. However, to see clear contours of microorganisms, smears are prepared and examined in a stained state, meaning various biological staining techniques are applied. These include methods aimed at detecting acid-fast microorganisms, spores, capsules, cell walls, flagella, cytoplasmic inclusions, and others.

For light microscopy, biological objects are usually stained after preparing fixed smears. Phase-contrast and dark-field microscopy are used to examine living and unstained biological preparations. Stained microbial cells stand out sharply against the silvery-white Background of the preparation, creating a vivid picture of the microbial landscape. The Use of special staining methods reveals morphological objects and structures of the microbial cells contained in the sample (such as spores, capsules, flagella, etc.).

Smears for microscopy are prepared from microbial cultures grown on solid or liquid nutrient media, as well as from various clinical specimens. When preparing smears from solid nutrient media, a drop of tap Water or physiological NaCl solution is placed in the center of a clean, degreased Glass slide. A small amount of the test culture is added to it using a bacteriological loop. Excess microbial material on the loop is incinerated in an alcohol burner flame. When preparing smears from liquid nutrient media, a drop of broth culture is applied to the glass slide using a Pasteur pipette or a bacteriological loop and spread in a circular motion into a uniform layer 8-10 mm in diameter. Smears should contain enough material for a thorough study, but should not be excessively thick, as the cells within them will be difficult to differentiate.

The smears prepared on the glass slide are air-dried and fixed. Smear fixation serves several purposes:

- to inactivate the microorganisms contained in the smear;

- to anchor the microbial cells to the glass surface, preventing them from washing off during subsequent staining and rinsing of the preparation;

- to increase the cells' affinity for stains, since killed microbial cells stain significantly better than living ones.

There are Physical and Chemical methods of fixation. The physical method is based on the Effect of Temperature on the microbial cell, while the chemical method relies on chemical agents that cause the coagulation of cytoplasmic Proteins.

In the physical method of fixation, the glass slide with the smear facing upward is passed 2-3 times through the burner flame. The entire fixation process takes 2-3 seconds. To fix microbes using chemicals, methyl and ethyl alcohols, acetone, Nikiforov, Peshkov, Carnoy, and Bouin fluids, as well as commercial formalin, are used. In these cases, depending on the substance used, the fixation time ranges from 5 to 20 minutes.

Methods of microbial staining are divided into simple and complex differential methods. Simple methods are used to detect microbes in a sample, determine their quantity, shape, and arrangement. Simple staining techniques use a single dye, most commonly methylene blue or an aqueous-alcoholic solution of fuchsin.

When using complex differential methods, the smear is first stained with a dye intended to reveal the target microorganism based on its physicochemical characteristics. This is followed by treating the smear with a mordant (chemical compounds such as acids, alkalis, alcohol, phenol, etc.), which results in the decolorization of all other microorganisms present in the smear. After mordanting, a counterstain of a contrasting color is applied.

Many complex differential-diagnostic staining methods can be considered one of the Initial Stages of microorganism identification. The most common among them are the following methods.

Gram staining. Proposed in the late 19th century by the Danish bacteriologist Gram, this differential staining method made it possible to divide the majority of known microorganisms into two groups: Gram-positive and Gram-negative. Susceptibility to Gram staining is determined by the thickness of The Cell wall and its chemical structure. The Cell wall of Gram-positive microorganisms consists of multi-layered peptidoglycan structures (20-60 nm) covalently linked to teichoic and lipoteichoic acids, which protrude to the cell surface through Pores in the peptidoglycan framework. The ability of Gram-positive cells to retain the iodine-crystal violet complex is related to the capacity of peptidoglycan to interact with the dye, as well as The structure of the peptidoglycan pores, which prevents the dye from washing out when the bacterial smear is treated with alcohol.

The cell wall of Gram-negative microorganisms is significantly thinner (10-20 nm) than that of Gram-positive ones, but more complex in structure. It additionally includes an outer membrane linked by a bimolecular lipid layer overlying the peptidoglycan layer. When treated with alcohol, Gram-negative bacteria lose the iodine-crystal violet complex due to the insufficient amount of peptidoglycan in their cell wall, become decolorized, and then take on the contrasting color of the counterstain (fuchsin or safranin).

To stain bacteria using the Gram method, the smear prepared on a glass slide is air-dried and fixed over a burner flame. The smear is flooded with a solution of the primary dye—crystal violet—for 1-2 minutes. The excess stain is drained, and Lugol's solution is applied until the preparation darkens (usually for 1-2 minutes). Then Lugol's solution is drained, and the preparation is treated with ethyl alcohol for 20-30 seconds, thoroughly rinsed with water, and counterstained with an aqueous-alcoholic solution of fuchsin for 2 minutes. The stained smear is dried and examined under a Microscope.

It is important to control the decolorization time of the smear, because prolonged Treatment with alcohol will decolorize Gram-positive microbes, while insufficient decolorization time will fail to decolorize the cells, causing them to retain the purple color of the primary dye.

Acid-fast staining. Acid-fast microbes are those that, once stained with carbol fuchsin, are not decolorized by the action of concentrated inorganic acids. A characteristic feature of this group of microbes is that they take up stains poorly. This property is due to the presence of long-chain Fatty acids with 50-100 carbon atoms (mycolic, tuberculostearic acids) in the cell wall and Cytoplasm of acid-fast bacteria, which make the microorganism's cell wall impermeable to crystal violet and other Dyes. Therefore, to ensure the dye penetrates the cell wall of these bacteria, more concentrated dye solutions are used in a heated state, detergents are added, and staining times are extended. Once the dye has entered the cell, it cannot be washed out with ordinary acids and alcohol Solvents.

The Ziehl-Neelsen method and certain other modified techniques are commonly used to stain acid-fast bacteria.

In the Ziehl-Neelsen staining method, the fixed smear is stained with Ziehl's carbol fuchsin for 3-5 minutes. The preparation is allowed to cool, the excess dye is drained off, and the preparation is rinsed with water. The stained preparation is decolorized with a 5% sulfuric acid solution for 3-5 seconds or with 95% ethyl alcohol containing 3% (v/v) Hydrochloric acid (HCl) by repeatedly dipping the slide into a vessel containing the acid-alcohol. After decolorization, the remaining acid is drained, and the preparation is thoroughly rinsed with water. It is then counterstained with Löffler's alkaline methylene blue for 3-5 minutes. The stained preparation is rinsed with water, dried, and examined under a microscope.

When staining preparations using the Ziehl-Neelsen method, acid-fast bacteria stain ruby red with fuchsin and are not decolorized by acid. Non-acid-fast bacteria are decolorized by the acid and take on the color of the counterstain.

Methods for visualization and staining of capsules. Some microorganisms produce a capsular substance that concentrates around the microbial cell to form a capsule. Under standard staining conditions, capsules remain unstained, which makes it possible to use simple staining techniques for their detection. When stained with methylene blue, bacterial cells appear blue, whereas a colorless zone—the capsule—is preserved around the bacteria and stands out against a dark background. Special staining Procedures are employed to visualize the capsular material. In the Burri method for capsule detection, a drop of India ink is placed at one end of a glass slide, and a broth culture or a loopful of colonies from a solid medium is emulsified in it. After thorough mixing with an inoculation loop, a smear is prepared, air-dried, and examined microscopically using an immersion system without prior fixation. The background of the slide becomes dark smoky-gray, while the microbial cells and capsules remain unstained by the ink, retaining their transparency; hence, this technique is referred to as a negative stain. The Burri method can be utilized not only to identify microbial capsules but also to detect spirochetes in test samples.

Cell wall staining. Standard staining methods fail to reveal the cell wall. To render it visible, the smear requires specific pretreatment prior to staining. The preparation is made from a heavily diluted microbial suspension to ensure adequate interspaces between cells, preventing them from overlapping.

When staining cell walls using Gutstein's method, the smear prepared from the microbial suspension is air-dried, fixed in Bouin's fluid (a saturated picric acid solution), mordanted with an aqueous tannin solution, and rinsed with tap water. It is then stained with an aqueous-alcoholic crystal violet solution, the excess stain is poured off, and the slide is dried without water rinsing.

Under microscopic examination, cell walls appear as long, thin, dark-violet or black filaments outlining cells with white or light-blue cytoplasm.

Endospore staining. Spores appear as round or oval structures. Their shape, size, and intracellular Location are characteristic of each bacterial species. Unlike the vegetative portion of the cell, spores contain significantly less free water and high concentrations of Lipids and calcium. The tough spore coat is impermeable to water and highly resistant to staining; consequently, under conventional staining conditions, spores appear as colorless unstained vacuole-like spaces within the cell. Special techniques involving mordants (acids or alkalis) are required to stain spores. Mordants loosen the spore coat, facilitating dye penetration. Once stained, spores exhibit acid-fastness, unlike vegetative cells, which are decolorized by acid treatment. Therefore, the underlying principle of staining spores and acid-fast bacteria is identical: the smear is stained with a primary dye, subsequently decolorized with acid, and counterstained with a contrasting color.

When staining spores by OžDžeczko's method, several drops of 0.5% HCl are applied to an air-dried, unfunded smear (prepared as a thick layer at the edge of the slide) and heated gently over a burner flame for 1–2 minutes until boiling, after which the residual acid is discarded. The cooled slide is washed with water, air-dried, and heat-fixed over a burner flame. It is then stained with Ziehl's carbol fuchsin with heating until steam rises, decolorized with a 5% sulfuric acid solution for a few seconds, and rinsed with water. Counterstaining is performed for 3–5 minutes using Loeffler's methylene blue or a 1% aqueous malachite green solution. Stained spores appear ruby-red, while vegetative cells acquire the color of the counterstain—blue when methylene blue is used, or green with malachite green.

When staining spores by Peshkov's method, Loeffler's methylene blue is poured onto a fixed smear and brought to a boil. Staining with the boiling dye is carried out for 20–30 seconds. The slide is rinsed with water and counterstained with a 0.5% neutral red solution for 30–60 seconds, followed by water washing and air-drying. Spores stained with Loeffler's methylene blue appear blue, whereas vegetative bacteria appear red.

In addition to light microscopy, phase-contrast and Fluorescence Microscopy, as well as Bacteriological examination methods, are frequently employed for the detection and identification of microorganisms.

11.2. Bacteriological Method

The bacteriological method involves inoculating test material onto artificial nutrient media, isolating pure cultures, and subsequently identifying them. This approach is of paramount importance and currently serves as the "gold standard." To obtain comprehensive data, it is necessary to isolate all bacterial species present in the sample. Therefore, appropriate environmental conditions must be established, including adequate nutrients and a suitable physicochemical milieu.

Most natural habitats harbor a multitude of diverse microbial species. Their Abundance depends on a range of factors, such as the structure and Chemical composition of the natural substrate. Samples for inoculation may include water, soil, food products, biological specimens, and others. Upon inoculation onto artificial nutrient media, microorganisms encounter an entirely novel and unfamiliar environment. To ensure successful microbial proliferation during early generations, providing adequate nutrients and growth factors is insufficient on its own. Replicating the physicochemical factors characteristic of the microorganisms' natural habitat is equally crucial for isolating a specific culture. These factors encompass temperature, the pH of the growth environment, the gaseous COMPOSITION OF THE cultivation atmosphere (a defined ratio of oxygen, carbon dioxide, and nitrogen), and humidity. Fulfilling all these conditions guarantees the growth of microorganisms present in the sample and yields a pure culture in sufficient quantity for identification.

Microbial growth requires nutrients from which cells are constructed and energy is derived. In principle, culture media must meet minimal requirements: they must contain all essential building blocks in a form assimilable by the microorganisms. Many less fastidious microbes, such as most aquatic and soil pseudomonads as well as *Escherichia coli*, thrive on glucose-mineral media. More demanding species require numerous growth factors, and their specific nutritional requirements are not always fully understood.

To isolate a target microorganism from natural samples, obtaining appropriate material from its specific ecological niche is essential. For instance, thermophilic bacteria are most likely to be isolated from hot spring waters, whereas anaerobes originate from sludge deposits. Similarly, the enrichment culture technique relies on selecting a selective (enrichment) medium favorable to the Organism of interest and inhibitory to others, combined with selective physicochemical cultivation conditions. Unfortunately, using this approach often leads to the dominance of fast-growing species within the enrichment culture, which outcompete all other forms. Slow-growing species can only be successfully isolated if samples from the enrichment culture are timely subcultured onto solid media in Petri dishes. Alternatively, natural samples can be directly inoculated onto solid selective media, where slow-growing species form visible colonies upon prolonged incubation. Selective conditions are engineered by combining various Energy Sources, carbon, and other elements (N, P, S, etc.).

To inhibit concomitant microflora while accommodating the physiological traits of the target microorganism, the test sample may undergo physical, chemical, or biological treatment. For example, when isolating spore-forming bacteria, the material is heated at 80 °C for 10 minutes to eliminate vegetative forms.

Laboratory practice frequently utilizes culture media designed for the selective cultivation of specific microbial groups. Accordingly, media are classified into elective (selective), differential-diagnostic (indicator), enrichment, and transport media.

Elective (selective) media are primarily utilized for isolating microorganisms from their natural habitats or for obtaining enrichment cultures. The terms "elective" and "selective" denote the preferential action of a medium toward particular microorganisms, promoting the robust growth of a single species or group. The selectivity of a nutrient medium for specific microbes is achieved either by establishing optimal conditions (pH, salt concentration, nutrient composition)—known as positive Selection (elective media)—or by supplementing the medium with agents that suppress other microorganisms (Bile, sodium azide, potassium tellurite, Antibiotics, etc.)—known as negative selection (selective media). Concomitant microorganisms either fail to grow on such media or their proliferation is markedly suppressed. For instance, egg-yolk-salt Agar serves as an elective medium for staphylococci, whereas alkaline agar is used for *Vibrio cholerae*.

To impart selectivity, various chemical compounds—including antibiotics, dyes, salts, and others—are incorporated into nutrient media as growth inhibitors of extraneous microflora. These agents include ampicillin, polymyxin, erythromycin, nystatin, penicillin, novobiocin, methicillin, gentian violet, ethyl violet, sodium and zinc azides, cycloheximide, potassium tellurite, boric acid and its derivatives, bile salts, and others. When deriving a pure culture from isolated colonies, plating test samples onto non-selective media is generally preferred; otherwise, viable concomitant microorganisms that fail to grow on selective media may still persist within or adjacent to the target colony and be co-isolated. On non-selective media, conversely, concomitant microorganisms form readily discernible colonies.

The inoculation technique used to obtain a pure culture depends on The Nature of the test sample, the consistency of the culture medium, and the objective of the study. Liquid samples are collected using an inoculation loop or pipette. When using a loop, the liquid should form a thin, transparent film across the loop's aperture. Pipettes are employed when large or precisely measured volumes must be inoculated. The sampling method for solid Materials is determined by their consistency, with the bacteriological loop being the most frequently used tool.

Various methods are employed to isolate pure microbial cultures. Treating test material with acid permits the isolation of acid-fast mycobacteria, whereas pre-heating the sample destroys all vegetative bacterial forms, facilitating the isolation of spore-forming pure cultures. The most widespread techniques for obtaining pure cultures are outlined below.

- Pasteur's method: serial dilution of the test material in liquid nutrient medium until a concentration of a single cell per volume is reached.

- Koch's method ("dilution plate method"): serial dilution of the test material in molten agar followed by pouring into Petri dishes. To isolate a pure culture via the streak-plate method, three test tubes containing 15 ml of meat-peptone agar each are placed in a water bath to melt the medium. The molten agar is cooled to 48–50 °C. A single loopful of the test sample is introduced into the first tube. To ensure thorough mixing, the inoculated tube is rolled several times between the palms. Subsequently, one loopful (flamed and cooled) of the contents from the first tube is transferred to the second, and similarly from the second to the third. The resulting microbial dilutions are poured from the test tubes into sterile Petri dishes labeled to correspond with the tube numbers. Once the inoculated medium solidifies, the plates are placed in an incubator. Colony counts on the agar plates decrease correspondingly with sample dilution. Originally proposed by R. Koch, this method is currently utilized to determine total bacterial counts in water and food products.

- Shukevich's method is applied to isolate pure cultures of microorganisms exhibiting "swarming" growth (such as *Proteus* species). For this purpose, the sample is inoculated into the Condensation water at the base of a agar slant. *Proteus* species migrate upward along the slanted agar, whereas non-motile forms remain restricted to the bottom at the inoculation site.

- Drigalski's method involves diluting the test sample contained in a test tube with sterile saline or broth, followed by plating onto Petri dishes. To isolate a pure culture, molten nutrient medium is poured into three Petri dishes. The solidified agar must be thoroughly dried, as moisture On the surface promotes confluent growth. A single drop of the test sample is placed onto the first plate and spread evenly across the agar surface using a sterile glass spreader. Without acquiring additional inoculum or flaming the spreader, the same tool is transferred to the second and subsequently the third plate, rubbing the remaining material across their surfaces. When inoculating samples heavily laden with microflora, isolated colonies develop on the second and third plates. These individual colonies are subsequently subcultured into tubes containing liquid or solid media to establish pure cultures.

Alternatively, inoculation can be performed using a bacteriological loop instead of a spreader. The sample is streaked across four quadrants of the nutrient medium in parallel lines without flaming the loop between quadrants (Fig. 12).

Class="center">Fig. 12. Drigalski plating method

The Petri dishes are incubated for 18–24 hours, after which the number of microbial colonies is counted in different sectors. With this inoculation method, the material on the loop is depleted gradually, and isolated microbial colonies grow along the streak lines made at the end of the Procedure. If slow-growing microorganisms are suspected, the dishes are left for a longer period (up to 5–7 days or more).

- Weinberg method. The Essence of this method is that dilutions of the test material are prepared in a nutrient agar medium melted and cooled to 45–50 °C. Following 6–10 serial dilutions, the tubes are rapidly cooled, and their surfaces are sealed with a layer of paraffin and liquid petrolatum mixture to prevent oxygen from penetrating into the depth of the nutrient medium. To retrieve isolated anaerobic colonies formed within the medium, the tube is slightly heated by rotating it over a flame; this melts the agar adjacent to the walls, allowing the agar Column to slide out into a sterile Petri dish. The agar column is then cut with sterile forceps, and colonies are extracted using a loop and transferred to a liquid medium favorable for these microorganisms (e.g., Kitt-Tarozzi medium). This method is used to isolate anaerobic microorganisms.

- Hungate method is used to obtain isolated colonies of strict anaerobes that are highly sensitive to oxygen. For this purpose, the molten agar medium is inoculated with bacteria under a continuous flow of inert gas through the tube. Using a thin layer in a tube filled with inert gas makes it possible to obtain isolated microbial colonies.

- Isolation of single cells using a micromanipulator—a device that allows individual cells to be extracted from a suspension using a micropipette or microloop. The procedure is performed under a microscope, with a moist chamber mounted on the microscope stage containing a "hanging drop" preparation. The micropipettes (microloops) are secured in stand holders, and using a system of screws and levers while looking through the microscope, the researcher extracts individual cells with micron precision and transfers them into tubes with sterile liquid medium to obtain a cell clone.

Thus, to isolate pure cultures, the material is streaked to obtain isolated colonies. The ISOLATION OF PURE cultures is achieved through mechanical Separation on The surface of a solid nutrient medium (using streak plate methods, agar dilutions, surface spreading with a Drigalski spatula), the use of selective media, and the creation of conditions favorable for the growth of a single species (genus) of bacteria (enrichment media). Cultural characteristics of microbes are determined by their growth patterns on nutrient media. Being consistent for each species, these serve as important diagnostic features.

To characterize growth on a solid nutrient medium, Petri dishes are first examined with the unaided eye or a magnifying glass, and then placed upside down on the microscope stage to inspect the colonies in transmitted light at low magnification. Examining the dishes can provide preliminary information regarding the inoculation results. The size, shape, edge contours, relief (elevation above the surface, drop-like, dome-shaped, flat-convex), surface texture (matte, glossy, dry, moist), color, structure (fine- or coarse-grained, filamentous, fibrous), and consistency of the colony are determined.

The growth patterns of microorganisms in liquid nutrient media are less diverse than those on solid media. In liquid media, bacterial growth may appear as uniform turbidity, sediment at the bottom, growth along the tube walls, or surface growth forming a pellicle.

To reveal the Specific features of microbial growth in a semi-solid nutrient medium—primarily active bacterial motility—the test culture is inoculated by stabbing the medium in close proximity to the tube wall. Motile microbes in the semi-solid agar column cause pronounced uniform turbidity throughout the entire thickness of the medium.

11.3. Biochemical Identification Tests

The study begins with the phenotypic identification of freshly isolated pure cultures. The oxidation-reduction Enzymes, hydrolytic properties, ability to decarboxylate specific Amino Acids, and capacity to assimilate sugars and other Organic compounds are determined. The most common tests include:

- oxidase tests, based on the principle that in certain bacteria, Biological Oxidation is carried out by cytochrome oxidase or indoloxidase enzymes. The visual manifestation of this process involves introducing artificial colorless Reagents in a reduced state into the nutrient medium, which become oxidized in the presence of microbial oxidase and develop a characteristic color;

- litmus milk test, based on a complex of enzymatic reactions whose manifestation corresponds to characteristic Changes in the initial Properties of the nutrient medium (lactose Fermentation, synthesis of Proteolytic Enzymes, production of Decarboxylases by growing bacteria, etc.);

- methylene blue reduction test, based on the ability of milk supplemented with methylene blue to become decolorized if the microorganisms possess reducing properties;

- TTC reduction test (triphenyltetrazolium chloride - C19H15ClN4), based on the reduction of TTC—a colorless compound—to triphenylformazan, which in organic solvents and under the action of metabolic products from certain microbial species, notably enterococci, is reduced to a cherry-red color;

- catalase test—an enzyme that promotes The breakdown of hydrogen peroxide into water and molecular oxygen. The test is based on the fact that from the very first seconds of contact between hydrogen peroxide and catalase, its decomposition begins, accompanied by the release of oxygen bubbles;

- saccharolytic activity test, based on the ability of the test microorganism to utilize a carbohydrate added to the nutrient medium, resulting in The formation of acid or acid and gas, which are detected by A change in the indicator color of the medium and the formation of bubbles;

- starch Hydrolysis test, used to detect the microorganism's ability to synthesize the enzyme amylase, which breaks down starch into glucose;

- oxidation-fermentation test (OF test), based on the principle that during oxidative processes, oxygen molecules serve as the final acceptors of H (protons) and electrons. Carbohydrate fermentation occurs under both anaerobic and aerobic conditions with the participation of saccharolytic enzymes;

- ONPG test (β-galactosidase test with nitrophenyl-β-D-galactopyranoside), based on the hydrolysis of lactose by bacteria and determined by the fact that the enzyme β-galactosidase (lactase) catalyzes the breakdown of the disaccharide lactose into two Monosaccharides—glucose and galactose. During ONPG hydrolysis, 0-

nitrophenyl is released, coloring the solution yellow, which serves as an indicator of the presence of the β-galactosidase enzyme in the culture;

- acetylmethylcarbinol production test (Voges-Proskauer reaction), based on the fermentation of glucose (in Clark's medium) to pyruvic acid, its conversion into acetoin or acetylmethylcarbinol, with the formation of cherry-red nitrogen-containing complexes;

- esculin hydrolysis test (a chemical compound belonging to Glycosides), based on the breakdown of esculin into glucose and esculetin, which reacts with iron citrate to precipitate, forming brownish-black zones around the colonies;

- citrate utilization test (on Simmons or Christensen medium), used for the Cytology/practical/136.html">Differential Diagnosis OF Shigella and Escherichia;

- AMINO ACID DECARBOXYLATION test (removal of СО₂ from СООН). The process takes place with the participation of enzymes in an acidic environment, where the resulting amines (ammonia derivatives) yield an alkaline reaction upon breakdown;

- indole test (tryptophanase test) is based on the principle that during microbial multiplication, the Tryptophan present in the nutrient medium is degraded to form indole, the presence of which is detected by turning the compounds pink or red;

- urease test (test for Enzymatic Cleavage of urea) is based on the bacterial hydrolysis of urea resulting in Ammonia Production, a shift in pH toward alkalinity, and a change in the indicator color within the medium;

- nitrate reduction test (nitrate reductase test). This test relies on the ability of certain microorganisms to reduce nitrates to nitrites and the subsequent detection of nitrites using Griess reagent, The addition of which to the nitrite-containing medium turns the Contents of the test tube red;

- proteolytic gelatin liquefaction test (using a mixture of protein substrates) is based on the irreversible complete or partial liquefaction of gelatin under the action of microbial proteases;

- enzymatic casein hydrolysis test is based on The production of a proteolytic enzyme by certain microorganisms that hydrolyzes milk casein down to Polypeptides, resulting in clear zones forming around the colonies;

- hemolysin test is based on the ability of certain bacteria to produce protein-based substances capable of enzymatically destroying red Blood Cells with the release of Hemoglobin. The hemolytic activity of bacteria is observed on blood agar;

- hydrogen sulfide (H₂S) production test is based on cultivating bacteria capable of enzymatically releasing sulfur compounds on nutrient media containing sources of H₂S (Sulfur-Containing Amino Acids or inorganic sulfur compounds such as sulfates and thiosulfates). In this process, H₂S in an acidic medium reacts with an indicator to form a brownish-black precipitate—iron sulfide or lead sulfide;

- plasma coagulation test is based on plasma clotting resulting from the production of a Thrombin-like substance by the coagulase-positive microbe;

- sporulation test is based on artificially creating unfavorable conditions for the test microorganism, leading to spore formation.

11.4. Methods of Immunological (Serological) Diagnostics

Immunological methods used for the detection and identification of microorganisms are based on the antigen-antibody interaction reaction. Antigens (from anti — against, genos — birth, origin) are macromolecular substances foreign to a specific organism that are capable of eliciting an Immune Response. Most commonly, these include various microbial cells, their structural elements, and metabolic products. The primary properties of an antigen are immunogenicity and specificity. Immunogenicity is The ability to induce an immune response. Specificity is the ability of an antigen to interact with Antibodies specific to it. Depending on their chemical nature and molecular weight, antigens can be complete (immunogenic) or incomplete (haptens). Existing antigens are classified based on their origin (natural, artificial, synthetic), chemical nature (proteins, CARBOHYDRATES, Nucleic Acids, lipids), and several other features.

Depending on their localization, capsular, flagellar, somatic, cytoplasmic, and extracellular antigens are distinguished. The complex of antigens from whole microbial cells and bacterial exotoxins are complete antigens and stimulate immune response reactions. According to their degree of specificity, microbial antigens are divided into cross-reacting, group-specific, species-specific, and type-specific. The most characteristic antigens serve as the basis for designing immunobiological preparations for detecting microorganisms and specific IMMUNOGLOBULINS.

Antibodies, or immunoglobulins, are a group of structurally related Serum proteins with characteristic physicochemical and biological properties. An important property of immunoglobulins is their ability to contact and bind whole, native antigen molecules in an unaltered state. Compounds formed by an antigen and an antibody are called immune complexes.

Serological diagnostic methods can be conventionally divided into two groups:

- direct methods of interaction and visual determination of Antigen-Antibody Reaction results, which include agglutination, precipitation, lysis, and Complement fixation reactions;

- methods utilizing "markers" or carriers, which include antigen or antibody carriers (erythrocytes, Liposomes) or various labels attached to one of the interaction components (enzymatic, fluorescent, radioisotopic, etc.). Depending on the label used, these methods are referred to as enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), etc.

The agglutination reaction is driven by the clumping of whole microbial cells (agglutinogens) by homologous antibodies (agglutinins) in the presence of electrolytes, manifested by the formation of clearly visible clumps of agglutinate that precipitate out of solution. There are various modifications of this reaction.

Precipitation reactions are characterized by the sedimentation of an antigen (precipitinogen) from solution in the form of fine flakes As a result of interaction with an immune serum containing precipitating antibodies (precipitins). The antigen can be a polysaccharide (hapten) or a bacterial protein (complete antigen) contained, for example, in microbial cell extracts. Precipitation reactions are qualitative research methods characterized by high specificity, and they are mainly used to identify an antigen and its nature.

The complement fixation test (CFT) is based on the principle that the interaction of an antigen with an antibody forms a complex that adsorbs (fixes) complement (a complex system of proteolytic enzymes, regulatory proteins, and cell-lysing proteins). However, this phenomenon occurs in a test tube without visible manifestations. To detect the results of the reaction, a hemolysis test is used as an indicator system.

The indirect (passive) hemagglutination assay (IHA or HIHA) is based on the ability of erythrocytes from various animal species, birds, and humans (type 1(O) blood) to adsorb bacterial antigens or antibodies on their surface. IHA is used either to detect an unknown antigen based on its interaction with a known antibody diagnosticum, or to detect antibodies in blood serum using a known erythrocytic antigen diagnosticum. In positive IHA results, a sediment shaped like an inverted "umbrella" forms in the wells of a polystyrene plate. In a negative reaction, the erythrocytes slide down to the bottom of the well, forming a sediment shaped like a "button."

The latex agglutination test (LAT) is a type of agglutination reaction in which synthetic polymer (latex) particles are used as carriers for the antigen or antibody.

The coagglutination (CoA) reaction is manifested by the clumping of antibody-sensitized Staphylococcus aureus cells (Cowan I strain) as a result of interaction with homologous antigens.

The immunofluorescence assay (IFA) is used as a rapid diagnostic method for detecting antigens in test specimens. The method involves treating a glass-fixed specimen containing the antigen with a homologous immune serum conjugated with a fluorochrome, such as fluorescein isothiocyanate (FITC). The specimen is examined using a fluorescence microscope. In positive cases, a glowing green border appears around the periphery of the object.

Enzyme-linked immunosorbent assay (ELISA) is an immunological reaction of specific antigen-antibody interaction that employs enzyme molecules (such as horseradish peroxidase, alkaline phosphatase, etc.) as indicators. The ability of the marker enzyme to cleave the substrate leads to a color change in the reaction medium, which forms The basis of this test.

Radioimmunoassay (RIA) is based on the DETECTION OF IMMUNE complexes using a radioactive label introduced into one of the reaction components—either the antigen or the antibody. Quantitative determination is performed using radioactivity counters. RIA is considered one of the most sensitive immunochemical methods and allows the detection of 0.1–10 ng of protein in a sample.

Serological methods enable the precise and rapid identification of the species of a test object, as well as the detection of microorganisms in both pure cultures and contaminated samples, even in relatively small quantities.

11.5. Molecular-Genetic Analysis Methods. Polymerase Chain Reaction

The Introduction of the polymerase chain reaction (PCR) into laboratory practice has been one of The most significant milestones in microbiology, molecular biology, and medicine over recent decades. PCR makes it possible to detect extremely small amounts of nucleic acids. This method has elevated Laboratory Diagnostics to an entirely new level by enabling the direct detection of Viruses and bacteria, as well as the identification of any alterations in genetic material, down to single Mutations.

PCR was invented in 1984 by the American biochemist Kary Mullis, who first demonstrated the feasibility of Amplification (the exponential Replication) of DNA segments during the reaction. The synthesis of DNA copies (the polymerase reaction) occurs in All living organisms (in vivo) during Cell Division through the action of DNA polymerase enzymes. PCR is the in vitro generation of multiple copies of a specific DNA fragment. Thus, THE PRINCIPLE OF PCR relies on amplifying a genomic region flanked by a pair of specific primers (short single-stranded DNA fragments) using a DNA polymerase enzyme, followed by the detection of the reaction products via Electrophoresis or other methods.

Today, PCR is widely applied across various fields, notably in paleontology for DNA diagnostics, the production of recombinant Vaccines, the monitoring of Gene Therapy, research involving Transgenic Animals, the diagnosis of bacterial and viral infectious diseases, and the assessment of treatment efficacy for various disorders. Furthermore, PCR has found application in the food industry, including quality control of raw materials of animal and plant origin.