The purpose and stages of bacteriological research. Bacteriological research

The possibilities of using bacteriological examination in eye diseases are limited by the limits of accessibility pathological focus. When it is located in the posterior segment of the eye, it is often very difficult to obtain material for research, and the possibility of bacteriological diagnosis in such cases, of course, is excluded. The main area of ​​application of bacteriological diagnostics in eye practice is diseases of the outer eye, as well as penetrating wounds, in which material for research can be obtained during surgical treatment of a wound or when a foreign body is removed. In general, it should be noted that during surgical interventions, the range of possibilities for obtaining bacteriological material is significantly expanded.

Obtaining material for research

To obtain material from the conjunctival sac, a platinum loop is used (if it is not available, you can use a loop made from an electric stove spiral thread, or another instrument (glass rod, spatula, etc.), which is pre-sterilized by calcination in the flame of an alcohol burner. Pulling the lower eyelid , with a cooled loop, a lump of conjunctival discharge is captured from the depth of the lower fornix.It is necessary to avoid touching the loop to the skin and the edges of the eyelids, so as not to bring foreign microflora into the material.It is advisable to take the material before the start of treatment and in the morning, when the discharge is more abundant.When in the absence of mucus and pus (for example, when examining a healthy conjunctiva before surgery), you should use lacrimal fluid or slightly scrape the surface of the connective sheath (according to Lindner).But it is better in such cases to apply the Elynnig method: let one drop of sterile saline solution into the conjunctival sac from a Pasteur pipette or broth and after a few seconds suck this drop.

According to the same general rules, material is obtained from the lacrimal sac, squeezing out its contents, and from the edge of the eyelid with ulcerative blepharitis, having previously removed the crust from the ulcer.

Great care is required when taking material from the cornea, especially in the presence of a creeping corneal ulcer. The loop (or other blunt instrument) should be directed obliquely to the surface of the ulcer and take material from its progressive edge. This requires anesthesia (3 drops of 1% dicaine) and fixation of the eyeball.

As a material for research in case of penetrating wounds of the eye, you can use the wound discharge (if any), capturing it with a loop or gauze swab, punctate of the anterior chamber or an extracted foreign body, which is placed in a sterile tube with 1-2 ml of saline. After shaking the tubes, the solution is used for research.

From the anterior chamber, the material for research can be obtained from a cutting instrument during paracentesis or by puncture. After anesthetizing the cornea (3 drops of 1% dicaine solution) and fixing the eyeball obliquely at the limbus, a syringe needle is injected, inserted into the anterior chamber (be careful not to injure the iris and lens capsule!) and aspirate 0.3 ml of the contents of the anterior chamber.

With endophthalmitis, there may be a need to obtain material from the vitreous body. Puncture of the eyeball is performed after anesthesia (1 ml of 2% solution of novocaine under the conjunctiva) with a sharp and fairly wide lumen needle, injected closer to the equator. Aspirated 0.3-0.5 ml of intraocular contents.

The resulting material is subjected to study in order to identify the microbe and determine its type (microbiological diagnosis). Material can be studied:

1. on a glass slide (bacterioscopic method);
2. in crops (actual bacteriological method);
3. in an animal experiment (biological method).

Bacterioscopic method

can be used in any eye hospital with simple laboratory equipment and some reagents.

For this you need to have the following:

1. metal loop;
2. clean glass slides (on a clean glass, a drop of water spreads, and does not take on a spherical shape);
3. a tray with stands for glass slides (glass rods or thick copper wire);
4. distilled water;
5. alcohol;
6. tweezers;
7. filter paper;
8. Lugol's solution (iodine 1 g, potassium iodide 2 g, distilled water 300 ml);
9. paint solutions (preferably in bottles closed with pipettes with rubber cans).

For cleaning, new glasses are boiled in a 1% soda solution (ash can be used), and then washed with water, weak hydrochloric acid and again with water. Glasses that were in use are placed in sulfuric acid for 1-2 hours, after which they are boiled in a soda solution, washed with water and immersed in 96 ° alcohol. Glasses are stored either in alcohol, or, after wiping the alcohol, dry, in jars with ground stoppers.

The most commonly used paints are: Tsilya carbolic fuchsin (basic fuchsin 1 g, crystalline carbolic acid 5 g, alcohol 96 ° 10 ml, glycerin - a few drops, distilled water 100 ml). For coloring, Tsilya magenta is usually diluted 10 times with distilled water (diluted, or water magenta). Methylene blue (methylene blue 10 g, alcohol 96 ° 100 ml). Alkaline blue Leffler is prepared from it (alcohol solution of blue 30 ml, 1% potassium or sodium alkali 1 ml, distilled water 100 ml). Carbolic gentian violet (prepared in the same way as Tsilya carbolic fuchsin).

Microbes are studied either in a living form (methods of "crushed" and "hanging" drops), or killed (in a colored preparation). A live study mainly reveals the ability of microbes to actively move, while stained preparations make it possible to study their morphology well.

There are simple methods of coloring, when one paint is usually used, and difficult ways stains that reveal some features of the physicochemical structure of the microbial cell and therefore have a differential diagnostic value.

The technique for preparing stained preparations is as follows. The material is applied to a clean glass slide and evenly distributed over the surface in the form of a circle or oval. The smear should be thin enough. In the presence of thick pus, one drop of distilled water should first be applied to the glass slide and the material should be stirred in this drop. The smear is dried in air. The glass with the dried preparation is taken by the edges with the thumb and forefinger with a stroke up and fixed by passing it through the flame of an alcohol burner three times (on the border of its light and dark parts). With sufficient fixation, a slight burning sensation in the fingers is felt. The fixed smear is stained. Several preparations are prepared, at least two, one of which is stained in a simple way(Leffler's methylene blue, diluted Ziel's fuchsin), the other - according to the Gram method.

Leffler staining:

1. a piece of filter paper is placed on the smear and a solution of Leffler's blue is poured for 3-5 minutes;
2. the drug is washed with distilled water and dried.

Gram stain:

1. a piece of filter paper is placed on the smear, onto which a solution of gentian violet is poured for 1-2 minutes;
2. the paint is drained, the paper is removed and, without rinsing with water, the Lugol solution is poured into the preparation for 1 minute; while the smear turns black;
3. drain the Lugol solution and discolor the smear with alcohol ( better way immersing the drug in a glass of alcohol until the discharge of purple streams stops);
4. washed thoroughly with water;
5. pour a smear with diluted fuchsin for 1-2 minutes;
6. drain the paint, wash the drug with water and dry.

A convenient Gram method modified by Sinev, who suggested using pre-prepared pieces of filter paper soaked in a solution of gentian violet and dried. 2-3 drops of water are preliminarily applied to the smear and then these pieces of paper are placed. Then proceed as described above.

Microscopy of smears is performed with an immersion lens. When stained according to Loeffler, all microbes are stained in Blue colour; when stained according to Gram - either blue-violet (gram-positive microbes) or red (gram-negative microbes).

Limited number of microbes involved in diseases of the outer eye and their characteristics morphological features quite often allow to put the correct microbiological diagnosis already by means of one only bacterioscopic research.

Bacteriological method

becomes necessary in cases where the study in smears is insufficient to establish the type of microbe or there is a need to determine the sensitivity of the isolated pathogen to antibacterial drugs.

Referring for a detailed acquaintance with the bacteriological method to the special manuals on microbiology, we dwell here only on the principles of this method. The first stage of the work is to select certain types microbes, i.e., to obtain pure cultures. To do this, they resort to mechanical separation of the material during sowing and to the use of media that are most suitable for the development of the alleged pathogens (elective media). By reseeding individual colonies grown on the medium, a pure culture is obtained, which is examined under a microscope (smears are prepared) and subcultured onto differential diagnostic media for study. biochemical properties pathogen.

The high demands of most microbes pathogenic for the eye under cultivation conditions led to the preferential use of elective media containing native protein for their isolation. These are, for example, Elschnig's medium (one part horse serum or ascitic fluid and two parts broth), clotted Loeffler's serum (three parts whey and one part broth with 1% peptone, 0.5% NaCl and 1% grape sugar), blood agar Leventhal (agar and 5-10% defibrinated blood).

With regard to determining the sensitivity of microbes to antibiotics, the simplest way to do this is to use commercially manufactured filter paper discs soaked in antibiotic solutions and dried in a vacuum. Material for research (pus, punctate, extracted foreign body) is placed in a sterile tube with saline (1.5-2 ml). After shaking, the liquid is poured into Petri dishes, preferably two - one with sugar, the other with blood agar - and evenly distributed over the surface of the medium by shaking. Then discs with various antibiotics are placed on the surface of the agar at a distance of 2 cm from each other and from the edge of the cups. The cups are placed in a thermostat (37°) and the next day, the degree of sensitivity of the microbe to the tested antibiotics is judged by the size of the growth retardation zone around the discs. The absence of a zone of growth inhibition indicates the insensitivity of the microbe to this antibiotic.

biological method

in ophthalmic practice, it is used only in cases where, with difficult diagnosis, the toxigenic property may be decisive in determining the type of microbe (for example, in the differential diagnosis of diphtheria bacillus and xerosis bacillus).

Laboratory diagnosis of viral diseases of the eye

At present, a special study of viruses, including the trachoma virus (Fig. 77), is carried out by culturing them in tissue cultures (chicken embryo, mouse ascites carcinoma, etc.) and electron microscopy.

Rice. 77. Cell inclusions in trachoma.

In clinical practice, for the diagnosis of trachoma, the method of cytological examination of conjunctival scrapings for the presence or absence of bodies (inclusions) of Provachek-Halberstedter is used. To do this, with a blunt scalpel or the edge of a glass slide, the epithelial cover of the conjunctiva (without blood) is scraped off, which is applied in a thin layer on the glass slide, dried in air for 5 minutes and fixed by lowering for 15-20 minutes into a glass with methyl alcohol or Nikiforov's mixture ( ethyl ether and absolute alcohol in equal amounts). Coloring is carried out within 3-4 hours with a freshly prepared solution of Romanovsky-Giemsa paint (at the rate of one drop of paint per 1 ml of distilled water). Then the preparation is washed with flowing water, dried in air and examined under a microscope. In this case, the nuclei of the epithelial cells turn out to be colored pink, the protoplasm - in light blue, the inclusions - in blue, blue-violet (Fig. 77). The latter appear as coccoid formations located among the fine-grained mass, and are recognized as carriers of the trachomatous virus. They are more often found in fresh cases of untreated trachoma, but can also occur with paratrachoma conjunctivitis.

Research recent years revealed new form contagious viral eye disease - epidemic keratoconjunctivitis, and a cytological study of conjunctival scrapings in this disease made it possible to detect peculiar inclusions in epithelial cells that are completely different from Prsvachek's bodies (B. L. Polyak, N. V. Ploshinskaya).

Cultural (bacteriological) research method - a set of methods aimed at isolating and identifying pure cultures of microorganisms (bacteria) by culturing on nutrient media.

A pure culture is a collection of microorganisms of the same species. Most often, a pure culture is obtained by selecting and cultivating an isolated colony (the offspring of a single microbial cell).

Method steps:

1. Collection of material for research.

2. Isolation of pure culture and its identification.

3. Conclusion.

Collection of material for research. The type of the studied material depends on the purpose of the study (diagnosis - from the patient; epidemiological analysis - from external environment, food, patient and (or) bacteria carrier).

Isolation of pure culture. Includes 3 or 4 stages:

1. Sowing the material (after preliminary microscopy) on a cup with a dense nutrient medium (preferably differential diagnostic or selective) in order to obtain isolated colonies. It is produced most often by the method of mechanical separation. In some cases (for example, blood), the material is pre-seeded in a liquid enrichment medium, followed by re-plating on a plate with agar medium. Sometimes a selective treatment of the material is carried out before inoculation (taking into account the properties of the isolated microorganism; for example, treatment with an acid or alkali to isolate resistant bacteria). Cultivated at a temperature of 37°C for 18-24 hours. Cultivation time for different types bacteria can fluctuate.

2(3): a) study of colonies on an agar plate (cultural traits), selection of the most typical ones; b) preparation of smears from these colonies with staining (according to Gram or other methods); a) screening out the rest of the studied colony on the accumulation medium and growing in a thermostat at the optimum temperature.

3(4). Study of the purity of the culture obtained on the accumulation medium. With this

the purpose is to prepare a smear, stain (usually according to Gram), microscopically study

morphological and tinctorial homogeneity (in different fields of view).

4(5). Pure culture identification.

Conclusion. According to the totality of features in comparison with the properties of reference (typical) strains, the type of microorganism isolated from the material is indicated.

Method evaluation:

advantages: relatively high sensitivity and accuracy, the ability to determine the number of microbes in the test material, as well as sensitivity to antibiotics; flaws: relative duration, the method is expensive.

21. Nutrient media for aerobes and anaerobes. Requirements for nutrient media, classification.

Requirements:

media must be nutritious

must have certain ph

must be isotonic, i.e. the osmotic pressure in the medium must be the same as in the cell.

should be moist and not too runny

must have a certain redox potential

must be sterile

must be unified, i.e. contain constant amounts of individual ingredients.

Nutrient media can be divided:

A) Origin

1) natural - natural food (meat, milk, potatoes);

2) artificial - specially prepared for the cultivation of microbes: - environments from natural products(meat water, meat-peptone broth (MPB), meat-peptone agar (MPA), - do not have a constant composition; - synthetic nutrient media - solutions of strictly defined amounts of salts, amino acids, nitrogenous bases, vitamins in distilled water - have a constant composition, are used for growing microorganisms and cell cultures in the production of vaccines, immune sera and antibiotics;

B) By appointment:

1) general purpose (MPB, MPA) - most microbes grow on them;

2) elective - selectively promote the growth of one type of microbes from a mixture (for example, yolk-salt agar for staphylococci);

3) differential diagnostic - allow one type of microbe to be differentiated by the appearance of the environment from others (for example, Endo, Levin media for the intestinal group of microbes).

In addition, depending on purposes of use In the scheme for isolating pure cultures, the following media can be distinguished by purpose:

1) enrichment - inhibit the growth of microbes associated with the pathogen;

2) to obtain isolated colonies;

3) accumulation of pure culture;

B) Consistency:

1) liquid;

2) semi-liquid (with the addition of agar-agar at a concentration of 0.5-0.7%);

3) dense - above 1%.

Bacteriological (cultural) method

a set of techniques for the artificial cultivation of microorganisms on nutrient media in order to identify them when establishing the cause of an infectious disease or other process caused by microbes and to determine a number of physiological St. in k-ry for other purposes, for example, when choosing a chemotherapeutic drug. Modern methods studying of the majority biol. sv-in bacteria are possible only in the presence of clean to-ry(cm.). Contamination to-ry by another type of microbes, as a rule, leads to distortion of results and the wrong conclusion. Therefore, the first task of B.m. is to obtain a pure to-ry of any type (var) from a microbial association and to prevent contamination of material for research with foreign microflora and microbial to-ry at all stages of the study. This is possible when taking the material under sterile conditions into sterile dishes, inoculating the material into sterile media with a sterile instrument, preventing the ingress of microbes from the air during the delivery, storage and incubation of the material and inoculated nutrient media. The second task of B.m. - microbial identification(see) isolated pure to-ry, the third - the definition of additional sv-in, for example, sensitivity to antibiotics, virulence. Stages of B.m.: 1) material sampling (see. Materials for research) and its delivery to bacterial. laboratory; 2) microscopy research. material (see bacterioscopic method). This step is often not performed, although, despite the lower sensitivity, preliminary microscopy in a number of cases gives indicative information about the pathogen and allows you to select the media necessary for sowing; 3) processing research. physical material. or chem. factors in order to remove or reduce extraneous microflora. This measure is used, for example, in the study of sputum, the isolation of acid-resistant and spore-forming microbes; 4) sowing research. material (see Bacteriological cultures) on nutrient media to obtain isolated colonies; 5) incubation of inoculated nutrient media. The timing and temperature of incubation depend on the intended species to-ry. Usually crops are kept in a thermostat at 37°C for 1-2 days. If there is no growth, the incubation period can be extended by another 2 to 3 days. With a longer incubation, it is necessary to prevent the drying of the medium, for which the crops are placed in a desiccator with water or the stoppers of the test tubes are filled with paraffin; 6) research colonies(cm.). For study, homogeneous isolated colonies are selected, located far from the edges of the cup and along the stroke of the loop, surround them with a line, and number them. If the study is conducted non-directionally (polyetiological process, etc.), then, based on the results of a visual examination, 2-3 colonies of all types available on the plate are selected and smears are made from them; 7) transfer from the selected colonies to the accumulation media. To do this, from the remainder of the colony, in a smear, bacteria of a uniform shape and color are revealed, with a loop, trying not to hurt neighboring colonies, a part of the to-ry is taken and inoculated on a MPA slanted in a test tube or a special medium with a stroke; 8) incubation of crops in a thermostat until continuous growth appears (usually 1-2 days); 9) determination of the purity of the to-ry grown on sloping media by macroscopic examination of growth and microscopy of a smear from it; 10) identification of the allocated pure to-ry and in case of need (at the request of the clinician, epidemiologist) definition of various biol. sv-in; 11) a conclusion about the species affiliation of the selected to-ry and its holy trees. The conclusion is given on an official form with reference to the number, under the Crimea, this to-ra is listed in the laboratory journal. B.m. is the main one in the d-ke of the majority of bacterium. infections. He usually has high sensitivity, allowing to select from, research. material pure to-ru, even if the sowing dose of the material contained several tens of individuals. For B.m. characterized by high specificity. Isolation of one or another type of bacteria from pathological material under a number of conditions (see. causative agent) reliably establishes the etiology of the pathological process. The exception is conditionally pathogenic bacteria, autochthonous for this or that biotope, for definition etiol. the role of to-rykh needs special criteria, as well as the distinction between carriage and disease (eg, diphtheria carriage with streptococcal angina). The value of B.m. It also consists in the fact that it can be used to establish the Holy Islands to-ry necessary for the appointment of chemotherapy and serotherapy, identifying the source of infection and the mechanisms of transmission of the pathogen, the choice of anti-epidemic measures. Finally, B.m. refers to the early methods of d-ki. Disadvantages B. m. - the risk of infection, complexity, laboriousness, duration. Reliable data on the composition of the microflora and its properties can only be obtained by examining a sample of 3-10 k-r.

This is the main method used in laboratory diagnosis. infectious diseases. The essence of the bacteriological research method is the sowing of pathological material from patients and the isolation of a pure culture of the pathogen, followed by its identification by morphological, cultural, tinctorial, biochemical and antigenic characteristics.

The method of isolating pure cultures, which makes it possible to isolate certain types of microbes from one or another natural habitat, is the most important method of microbiological research. The first to propose a method for isolating a pure culture was L. Pasteur.

The Pasteur method, based on the use of liquid nutrient media, ensured the isolation of a pure culture mainly from a material containing one type of microbe (for example, from blood in septicemia, etc.). It was less effective in cases where it was necessary to isolate certain types of microorganisms from their mixture. Meanwhile, under natural conditions, the material for bacteriological examination (sputum, pus, soil, water, etc.) most often contains a mixture of various microorganisms.

The successful isolation of bacterial mixtures and the isolated study of individual species became possible due to the improvement of the method for isolating pure cultures by Robert Koch (R. Koch), who used for this purpose in 1881 solid nutrient media on which during sowing it is possible to distribute the material in such a way that individual microbial cells are isolated from each other. Under appropriate conditions (nutrient medium, optimal temperature), the reproduction of isolated cells gives offspring of the same species, i.e. a pure culture of a given microbial species.

After a certain period (most often in a day), in those places of a dense nutrient medium on which isolated cells are located, populations of multiplied microbes are formed - the so-called colony, visible to the naked eye. They do not represent a chaotic accumulation of microbes, which can already be judged by the fact that for many types of microbes the colonies have a characteristic structure, due to which it is possible to roughly determine the flora of the material under study and select those colonies that are subject to further study. Reseeding such colonies on an appropriate nutrient medium is the isolation of a pure culture.

The isolation of pure cultures with their subsequent identification is of paramount importance in the diagnosis of infectious diseases, ensuring their rapid recognition, timely treatment and prevention. It is no less important in determining the microflora in the study of the sanitary and hygienic state of environmental objects (air, water, soil, etc.), as well as in scientific research.

Numerous methods for isolating pure cultures are now available. Some of them are used to a limited extent, others are widely used. The most universal are the methods described below for isolating pure bacterial cultures (Drigalsky's method). While other microorganisms - spirochetes, protozoa - require the use of special methods isolation or cannot be isolated at all on artificial nutrient media (some protozoa, rickettsia, viruses).

Methods for isolating pure cultures from microbial mixtures are usually divided into two main groups: methods based on the principle of mechanical separation of microbes in a nutrient medium, and methods based on the use of the biological properties of microbes. The first group includes methods for isolating individual cells: 1) in the depth of the nutrient medium; 2) on the surface of the medium and 3) under the control of the eye. The second group uses such properties of microbes as their mobility, attitude to temperature, oxygen and their pathogenic properties.

Seeding and reseeding technique

by sowing in microbiological practice, it is called the introduction of some test material into a sterile nutrient medium to detect microorganisms.

Reseeding is the transfer of cultured microorganisms to a sterile environment. Cultures and subcultures of microbes are one of the most common methods in microbiological practice.

Reseedings are carried out in such a way that foreign microorganisms do not get into the nutrient medium from the air or from the surface of surrounding objects. For this, the following steps must be strictly observed:

1) crops are made directly near a lit burner, in the flame of which loops, tweezers, cotton plugs, edges of test tubes are sterilized;

2) one test tube with a culture to be crossed is taken in the left hand, the other (with a sterile nutrient medium) is held in an inclined position between the thumb and forefinger;

3) the loop is held in a vertical position over the flame of the burner and its metal part is calcined red-hot, and then tilted horizontally and the loop holder is sterilized;

4) take out cotton plugs and hold them with the ring finger and little finger of the right hand; putting corks on the table or on any object is not recommended;

5) burn the edges of both tubes;

6) introduce a loop into the test tube with the culture to be subcultured carefully, without touching the walls, capture a drop of liquid or a small amount of plaque on the solid medium and transfer, trying not to touch the walls, into the second test tube with a depleted nutrient medium;

7) the loop is taken out, the edges of the test tubes and the inner ends of the stoppers are burned. If the cotton plug catches fire, then close the test tube with it, and extinguish the outer end with a hand or tweezers;

8) the loop is fired again in a flame, an appropriate inscription is made on the test tube: the name of the culture and the date of sowing.

Sowing in a liquid medium can be produced with a Pasteur or graduated pipette. When using a Pasteur pipette, use the burnt tweezers to break the sealed end and lightly burn the entire pipette. The test tube with the culture under study is placed in the left hand, and the pipette is placed in the right hand between the thumb and middle fingers, holding its upper opening with the index finger.

After removing the cotton plug from the test tube, burn the edges of the latter. Carefully lower the pipette into the test tube and remove the index finger. Then close index finger top hole of the pipette, remove it from the test tube. The cotton plug and the edges of the test tube are fired before being closed. The test material is transferred into a liquid medium. After inoculation, the pipettes are placed in a disinfectant solution.

Seeding on solid media. When sowing on oblique agar, a straight or zigzag stroke is applied. To do this, the loop with the inoculated material is introduced into the test tube until the condensation water accumulated at the bottom and carefully, without loosening the agar, a stroke is applied. Solid inoculation is obtained by smearing the inoculum over the entire surface of the slant agar.

On a dense medium in a Petri dish, inoculation is carried out as follows. The nutrient medium in test tubes is melted in a boiling water bath, cooled to 48-50°C and poured into sterile cups in an even layer 3-5 mm high. The solidified medium is dried in a thermostat in closed cups for 20-30 minutes. Open cups are placed upside down on the thermostat shelves covered with sterile paper. Lids are placed next to the cups. During drying, condensation water evaporates from the surface of the nutrient medium and the inner surface of the cups. Sowing is done with a loop in the form of parallel strokes or with a glass spatula.

When determining the type of microbe and for growing anaerobes, sowing by injection in a column of agar or gelatin. To do this, the test tube is turned upside down and a column of medium is pierced from top to bottom to the very bottom with a long straight needle with inoculum. Then the needle is carefully removed and the test tube is closed with a burnt cotton plug. If special precautions against infection are needed, crops are carried out in a special cabinet for reseeding pure cultures. The inoculated tubes and Petri dishes are placed in a growth incubator.

Microorganisms and spores located inside the nutrient media or on their surface cannot move, but remain in the place where they were at the time of solidification. Each cell or spore begins to multiply and after 2-3 days forms colony - great amount cells of the same type. If a colony was formed from a single cell, then it will be a pure culture of the microorganism from whose cell it grew.

Growing colonies are viewed first with the naked eye, and then with a magnifying glass or under a microscope. At the same time, it is impossible not to notice that the colonies differ in appearance, color, structure, etc.

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The use of the bacteriological method makes it possible to isolate the pathogen in a pure culture from the material obtained from the patient, and to identify it based on the study of a complex of properties. Most bacteria are capable of cultivation on various artificial nutrient media (except for chlamydia and rickettsia), so the bacteriological method is important in the diagnosis of many infectious diseases.

If a positive result is obtained, the bacteriological method makes it possible to determine the sensitivity of the isolated pathogen to antimicrobial drugs. However, the effectiveness of this study depends on many parameters, in particular on collection conditions and his transportation to the laboratory.

TO basic requirements requirements for the selection and transportation of material for bacteriological examination include:

• taking material before the start of etiotropic treatment;
• observance of sterility conditions when collecting material;
• technical correctness of material collection;
• a sufficient amount of material;
• ensuring the temperature regime of storage and transportation of the material;
• reduction to the minimum time interval between the collection of material and sowing on dense nutrient media.

Transportation of the material to the laboratory should be carried out as soon as possible, but not more than within 1-2 hours after its taking. Samples of the material must be at a certain temperature; in particular, normally sterile materials (blood, cerebrospinal fluid) are stored and delivered to the laboratory at 37 °C. Non-sterile materials (urine, respiratory secretions, etc.) are stored at room temperature for no more than 1-2 hours or no more than a day at 4 ° C (household refrigerator conditions). If it is impossible to deliver samples to the laboratory within the prescribed timeframe, it is recommended to use transport media designed to preserve the viability of pathogens under conservation conditions.

Blood for research should be taken from the patient during the rise in body temperature, at the beginning of the onset of fever. It is recommended to examine 3-4 blood samples taken with an interval of 4-6 hours, which is reasonable in terms of reducing the risk of "missing" transient bacteremia and increasing the ability to confirm the etiological role of opportunistic microflora isolated from the blood if this microflora is found in several samples of the venous blood. A blood sample in the amount of 10 ml in an adult and 5 ml in children is inoculated into at least two vials with a medium for aerobic and anaerobic microorganisms in a ratio of 1:10. A single study of arterial blood is also desirable.

Take cerebrospinal fluid(CSJ) is produced by a doctor with a lumbar puncture in the amount of 1-2 ml in a dry sterile tube. The sample is immediately delivered to the laboratory, where its study is also started immediately. If this is not possible, the material is stored at 37 °C for several hours. Significantly increases the number of positive results of bacteriological examination by sowing 1-2 drops of CSF in a test tube containing a semi-liquid medium with glucose, and in a Petri dish with "blood" agar. To send the material, isothermal boxes, heating pads, thermoses or any other packaging where the temperature is maintained at about 37 ° C is used.

Excreta for bacteriological examination, they are taken with sterile wooden spatulas in the amount of 3-5 g into a sterile vessel with a tightly closed lid. The study of the taken material should be started no later than 2 hours later. If it is impossible to start the study during this time, a small amount of material should be selected and placed in an appropriate transport medium. When selecting feces, one should strive to send pathological impurities (mucus, pus, epithelial particles, etc.) for research, if any, avoiding blood impurities with bactericidal properties entering the material.

To take the material, rectal swabs (with a cotton tip) can be used. The swab should be moistened with sterile isotonic sodium chloride solution or transport medium (not oil gel). It is introduced per rectum to a depth of 5-6 cm and, turning the tampon, carefully remove it, controlling the appearance of fecal color on the tampon. The swab is placed in a dry test tube if the study of the material is started within 2 hours, otherwise - in the transport medium.

Urine(an average portion of freely released urine) in the amount of 3-5 ml is collected in a sterile dish after a thorough toilet of the external genital organs. It is preferable to take morning portions of urine.

Bile collected during duodenal sounding in the treatment room separately in portions A, B and C in three sterile test tubes, observing the rules of asepsis.

Wash water of the stomach collected in sterile jars in the amount of 20-50 ml. It should be borne in mind that gastric lavage in these cases is carried out only with indifferent (not having a bacteriostatic or bactericidal effect on microorganisms) solutions - preferably boiled water (without adding soda, potassium permanganate, etc.).

Sputum. Morning sputum released during a coughing fit is collected in a sterile jar. Before coughing, the patient brushes his teeth and rinses his mouth with boiled water in order to mechanically remove food debris, desquamated epithelium and microflora of the oral cavity.

Flushing water of the bronchi. During bronchoscopy, no more than 5 ml of isotonic sodium chloride solution is injected, followed by suction into a sterile tube.

Discharge of the pharynx, oral cavity and nose. Material from the oral cavity is taken on an empty stomach or 2 hours after eating with a sterile cotton swab or a spoon from the mucous membrane and its affected areas at the entrances of the ducts of the salivary glands, the surface of the tongue, from sores. If there is a film, the latter is removed with sterile tweezers. Material from the nasal cavity is taken with a dry sterile cotton swab, which is inserted deep into the nasal cavity. Material from the nasopharynx is taken with a sterile posterior pharyngeal cotton swab, which is carefully inserted through the nasal opening into the nasopharynx. If a cough begins at the same time, the swab is not removed until the cough ends. To conduct an analysis for diphtheria, films and mucus from the nose and pharynx are simultaneously examined, taking the material with different swabs.

The test material is inoculated on dense nutrient media using special techniques to obtain the growth of individual colonies of microorganisms, which are then screened out in order to isolate a pure culture of the pathogen.

Certain types of bacteria are isolated using elective (selective) media that retard the growth of foreign microorganisms or contain substances that stimulate the growth of certain pathogenic microbes.

Microorganisms isolated on nutrient media identify, i.e. determine their species or type affiliation. IN Lately for identification in healthcare practice, microtest systems are used, which are panels with a set of differential diagnostic environments, which speeds up the study. Microtest systems are also used to determine the sensitivity of microorganisms to antimicrobial drugs by diluting an antibiotic in a liquid nutrient medium.

When evaluating the results of a bacteriological study, the doctor should take into account that a negative result does not always mean the absence of a pathogen and may be associated with the use of antimicrobials, high blood microcidal activity, and technical errors. Detection of a pathogenic microbe in the material from a patient, regardless of the clinical picture, is possible in the case of convalescent, healthy or transient bacteriocarrier.

Isolation from the blood, subject to all the rules of asepsis, of conditionally pathogenic microorganisms (epidermal staphylococcus aureus, coli) and even saprophytes should be considered a manifestation of bacteremia, especially if these microbes are found in more than one material sample or in different substrates (blood, urine), since with a decrease in the body's immunoreactivity, these and other "non-pathogenic" microorganisms can be pathogens of infectious processes, including number and sepsis.

A certain difficulty is interpretation of the results of bacteriological examination of non-sterile media, namely the proof of the etiological role of opportunistic microorganisms. In this case, such indicators as the type of isolated crops, the number of microbial cells of this species in the material, their repeated isolation during the course of the disease, the presence of a monoculture or association of a microorganism.

Yushchuk N.D., Vengerov Yu.Ya.