Algorithm for conducting bacteriological research. Bacteriological method for diagnosing infectious diseases. Features of the analysis

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Bacteriological studies

Bacteriological research- intended for isolating bacteria and studying their properties in order to make a microbiological diagnosis.
The test material should be collected under aseptic conditions in sterile containers and delivered to the laboratory as soon as possible. If necessary, samples should be stored refrigerated. The sampling technique depends on the object, the nature of the disease and the properties of the microorganism. One of the common methods of bacteriological research is bacterioscopy.
To study unfixed bacteria, two methods are used: a crushed drop (between the slide and cover glass) and a hanging drop. It should be remembered that preparations of unfixed bacteria are infectious.
The most important elements of bacteriological research include inoculation and subculture of bacterial cultures performed with a bacterial loop or Pasteur pipette. The loop is sterilized by annealing in a flame, then it is cooled by touching an area of ​​uninoculated agar or by rinsing in a sterile liquid. When using a Pasteur pipette, break off the tip with tweezers, pass the pipette through the burner flame several times and allow it to cool. When sowing, liquid and solid nutrient media are used. When sown on a slanted agar, the bacterial culture is rubbed with a loop over the surface of the agar. When sowing into the thickness of an agar or gelatin column, the nutrient medium is pierced to the bottom of the tube with a loop or a special needle. When sowing in a liquid medium, care must be taken to ensure that the liquid does not spill out and wet the edges of the tubes and stoppers. Inoculations and subcultures should be carried out near the flame of a gas burner, the test tubes should not remain open for long, the loop or Pasteur pipette with the culture should not touch anything; Before closing the test tube, the edges should be burned. Inoculated tubes must be labeled immediately.

Microbiological research methods are divided into methods for direct detection of pathogens in the patient’s body - bacterioscopic and bacteriological studies; methods of indirect evidence of the presence of a pathogen in the patient’s body - serological studies aimed at detecting specific antigens in infected material or antibodies in the blood serum and various secretions of the patient’s body.
Bacterioscopic examination of blood, urine, cerebrospinal fluid, mucus from the throat and nose, feces and various pathological substrates for the presence of a pathogen is used for many infectious diseases. This method has rather limited applications, although it is very important. It is used, in particular, to detect pathogens of malaria, relapsing fever, and leptospirosis in the blood. Cerebrospinal fluid is examined for purulent and tuberculous meningitis, mucus from the throat and nose - for diphtheria, Vincent's angina, feces - for amoebiasis, balantidiasis, giardiasis; urine - for leptospirosis. Plague and anthrax bacilli can be seen in the punctate of the plague bubo and imprint smears taken from the anthrax carbuncle; microscopy of a punctate smear from the bone marrow and spleen, granulations from an ulcer can detect leishmania; in sputum - Mycobacterium tuberculosis. The advantage of the bacterioscopic method is its speed. Analysis results can be obtained within a few hours. In some diseases (malaria, epidemic cerebrospinal meningitis, etc.), pathogens can be detected in the patient’s body already on the 1st day of illness, which is very important for timely diagnosis and treatment. The possibilities of bacterioscopic diagnosis have significantly expanded due to the treatment of preparations with specific sera containing antibodies to a given pathogen, labeled with fluorochromes (immunofluorescence method) or enzymes (enzyme immunoassay method). In this case, labeled antibodies are combined with an antigen, which is detected using the immunofluorescent method under a fluorescent microscope, and with the enzyme immunoassay method - by staining the enzymatic reaction product after the introduction of a substrate-indicator mixture.
The bacteriological diagnostic method is based on the isolation of the pathogen from the blood, cerebrospinal fluid, sputum, mucus from the throat and nose, feces, urine, bile of the patient, and in case of death - from pieces of organs that are inoculated on special nutrient media. Bacteriological diagnostics are widely used to study mucus from the throat and nose for diphtheria bacteria, to isolate pathogens of intestinal infections (for example, cholera, dysentery, salmonellosis, escherichiosis) from the patient’s feces, pathogens of pneumonia from sputum, and in other cases. In this case, the characteristics of the suspected infection, the place of selective localization of the pathogen and the routes of its release into the environment are taken into account. The study gives positive results already in the first hours or days of illness. However, the laboratory reports the final answer for most infectious diseases only after 2-4 days, and for brucellosis and tuberculosis - after 3-4 weeks. This depends on the time required for the growth of microorganisms and their identification (biochemical and serological). Each microbe requires an appropriate nutrient medium for its growth. Depending on which microbe is supposed to be isolated (which is determined during a clinical and epidemiological examination of the patient), the appropriate nutrient medium is selected. Sometimes sowing is carried out simultaneously on several nutrient media. The value of the results of bacteriological studies is largely determined by whether the material is correctly taken from the patient and whether it is correctly delivered to the laboratory. Infectious material is collected in sterile containers, observing aseptic rules.

The most important stage of bacteriological research is identification- determination of the species or type of bacteria obtained in the form of a pure culture. When identifying bacteria, their physiological and biochemical properties and toxin formation are studied. Serological methods for identifying bacteria are widely used. In many cases, a biological method for identifying microorganisms is effective, based on infecting laboratory animals with the material being studied or the resulting bacterial culture and identifying characteristic pathological changes in the animals.
Mechanical and biological methods are used to isolate pure cultures. An example of a mechanical method: a drop of the test material is ground with the same sterile spatula or bacterial loop over the surface of a dense nutrient medium, sequentially in the first, second and third Petri dishes. Isolation of a pure culture is made from grown individual colonies and consists of their examination and screening on a fresh nutrient medium. Biological methods for isolating pure cultures are based on taking into account one or another property of the isolated microbe, which distinguishes it from other microbes found in the material under study.
The biological method uses this kind of nutrient media in which conditions are created that are favorable for the development of a certain type of microbe. Biological methods also include infection of laboratory animals that are sensitive to the isolated species of bacteria.
Bacteriological research is a set of methods for identifying pathogenic microorganisms in a patient, a carrier or on environmental objects.

Isolation of pure culture;

Methods of bacteriological research are increasingly being introduced into practice for a more detailed examination of patients, establishing the etiological factor of the inflammatory process, prescribing rational therapy and determining its effectiveness.

The diversity of clinical material and the uniqueness of the microflora of individual organs determine the features of bacteriological research methods, which require the use of special sampling techniques, inoculation on nutrient media and analysis.

Bacteriological examination of clinical material consists of several stages:

Sampling for research;

Sowing on nutrient media;

Isolation of pure culture;

Identification and differentiation of isolated microorganism cultures;

Analysis of the research results.

During bacteriological examination, the so-called sowing of the material collected from the patient is carried out on nutrient media, which promotes the growth and reproduction of the causative agent of the disease. During the study, it is possible to identify not only the fact of the presence, but also the concentration of pathogenic microorganisms in a particular biomaterial.
The method of bacteriological examination examines sputum, cerebrospinal fluid, blood, as well as discharge from the genitals, oral cavity, pharynx and wounds of the patient. Thus, microorganisms can be sown from almost any part of the human body.
The bacteriological culture technique is extremely convenient and effective for detecting and determining the type of bacteria and fungi. Detection of viruses presents a certain difficulty due to the peculiarities of their biology.
Bacteriological examination (culture) is of great importance not only for determining the specific type of microorganism, but also for establishing the degree of sensitivity of a particular antibiotic to it. Thus, the maximum effectiveness of a particular type of antibiotic therapy is determined.
When conducting an analysis, it is important to remember that some microorganisms, such as pneumococci, die quite quickly because they have an increased tendency to self-destruct. Thus, crops must be done in a short time.

The main method of microbiological diagnostics and the “gold standard” of microbiology is the bacteriological method.

The purpose of the bacteriological method consists in isolating a pure culture of the pathogen from the test material, accumulating a pure culture and identifying this culture by a set of properties: morphological, tinctorial, cultural, biochemical, antigenic, by the presence of pathogenicity factors, toxigenicity and determining its sensitivity to antimicrobial drugs and bacteriophages.

The bacteriological research method includes:

1. inoculation of the test material in nutrient media

2. isolation of pure culture

3. identification of microorganisms (determination of species).

Isolation and identification of pure cultures of aerobic and anaerobic bacteria involves the following studies:

Stage I (working with native material)

Goal: obtaining isolated colonies

1. Preliminary microscopy gives an approximate idea of ​​the microflora

2. Preparation of material for research

3. Sowing on solid nutrient media to obtain isolated colonies

4. Incubation at optimal temperature, most often 37°C, for 18-24 hours

Stage II

Goal: obtaining a pure culture

1. Macroscopic study of colonies in transmitted and reflected light (characteristics of size, shape, color, transparency, consistency, structure, contour, surface of colonies).

2. Microscopic examination of isolated colonies

3. Testing for aerotolerance (to confirm the presence of strict anaerobes in the test material).

4. Sowing of colonies characteristic of a particular species on pure culture accumulation media or selective media and incubation under optimal conditions.

Stage III

Goal: identification of isolated pure culture

1. To identify the selected culture based on a set of biological properties, the following is studied:

· morphology and tinctorial properties

· cultural properties (character of growth on nutrient media)

· biochemical properties (enzymatic activity of microorganisms)

Serological properties (antigenic)

· virulent properties (the ability to produce pathogenicity factors: toxins, enzymes, defense and aggression factors)

pathogenicity for animals

· phagolysability (sensitivity to diagnostic bacteriophages)

sensitivity to antibiotics

· other individual properties

Stage IV (Conclusion)

Based on the studied properties, a conclusion is made about the selected culture.

The first stage of research. The examination of pathological material begins with microscopy. Microscopy of stained native material makes it possible to establish approximately the composition of the microbial landscape of the object under study and some morphological features of microorganisms. The results of microscopy of the native material largely determine the course of further research; they are subsequently compared with the data obtained by inoculation on nutrient media.

If there is a sufficient content of pathogenic microorganisms in the sample, inoculation is carried out on solid nutrient media (to obtain isolated colonies). If there are few bacteria in the test material, then inoculation is carried out on liquid enrichment nutrient media. Nutrient media are selected according to the requirements of microorganisms.

Cultivation of microorganisms is possible only if optimal conditions for their life are created and rules are observed that exclude contamination (accidental contamination by foreign microbes) of the material being studied. Artificial conditions that would prevent contamination of the culture by other species can be created in a test tube, flask or Petri dish. All glassware and culture media must be sterile and, after inoculation of microbial material, protected from external contamination, which is achieved using stoppers or metal caps and lids. Manipulations with the test material should be carried out in the flame zone of an alcohol lamp to prevent contamination of the material from the external environment, as well as for the purpose of complying with safety regulations.

Inoculation of material on nutrient media must be done no later than 2 hours from the moment of collection.

Second stage of research. Study of colonies and isolation of pure cultures. After a day of incubation, colonies grow on the dishes, and on the first stroke the growth is continuous, and on the next strokes - isolated colonies. A colony is a collection of microbes of the same species growing from one cell. Since the material is most often a mixture of microbes, several types of colonies grow. Different colonies are marked with a pencil, outlining them in a circle from the bottom, and they are studied (Table 11). First of all, colonies are studied with the naked eye: macroscopic signs. The cup is viewed (without opening it) from the bottom in transmitted light, the transparency of the colonies is noted (transparent, if it does not block light; translucent, if it partially blocks light; opaque, if light does not pass through the colony), and the size of the colonies is measured (in mm). Then they study the colonies from the side of the lid, note the shape (regular round, irregular, flat, convex), the nature of the surface (smooth, shiny, dull, rough, wrinkled, wet, dry, slimy), color (colorless, colored).

Table 11. Scheme for studying colonies

№	Sign	Possible characteristics of colonies
1.	Form	Flat, convex, domed, depressed, round, rosette, star
2.	Size, mm	Large (4-5 mm), medium (2-4 mm), small (1-2 mm), dwarf (< 1 мм)
3.	Surface character	Smooth (S-shape), rough (R-shape), slimy (M-shape), striated, lumpy, matte, shiny
4.	Color	Colorless, colored
5.	Transparency	Transparent, opaque, translucent
6.	Character of the edges	Smooth, jagged, fringed, fibrous, scalloped
7.	Internal structure	Homogeneous, granular, heterogeneous
8.	Consistency	Viscous, slimy, crumbly
9.	Emulsification in a drop of water	Good bad

Note: points 5-7 are studied at low microscope magnification.

You can see the differences between colonies even better when viewing them with magnification. To do this, place a closed cup with the bottom up on the stage, slightly lower the condenser, use a slight magnification of the lens (x8), moving the cup, study the microscopic features of the colonies: the nature of the edge (smooth, wavy, jagged, scalloped), structure (homogeneous, granular, fibrous, homogeneous, or different in the center and periphery).

Next, the morphology of microbial cells from colonies is studied. To do this, smears are made from a part of each of the marked colonies and stained with Gram. When taking colonies, pay attention to the consistency (dry, if the colony crumbles and is difficult to pick up; soft, if it is taken easily with a loop; slimy, if the colony is pulled by a loop; hard, if part of the colony is not taken with a loop, you can only remove the entire colony) .

When viewing smears, it is established that the colony is represented by one type of microbe, therefore, pure cultures of bacteria can be isolated. To do this, the studied colonies are reseeded onto slanted agar. When reseeding from colonies, care must be taken to take exactly the intended colonies, without touching nearby colonies with the loop. The tubes are labeled and incubated in a thermostat at 37°C for 24 hours.

The third stage of research. Identification of the isolated culture. Identification of microbes - determination of the systematic position of a culture isolated from a material to species and variant. The first condition for reliable identification is the unconditional purity of the culture. To identify microbes, a set of characteristics is used: morphological (shape, size, presence of flagella, capsules, spores, relative position in a smear), tinctorial (relation to Gram staining or other methods), chemical (ratio of guanine + cytosine in DNA molecule), cultural (nutritional needs, cultivation conditions, rate and nature of growth on various nutrient media), enzymatic (cleavage of various substances with the formation of intermediate and final products), serological (antigenic structure, specificity), biological (virulence for animals, toxigenicity , allergenicity, influence of antibiotics, etc.).

For biochemical differentiation, the ability of bacteria to ferment carbohydrates with the formation of intermediate and end products, the ability to degrade proteins and peptones, and study redox enzymes.

To study saccharolytic enzymes, isolated cultures are inoculated into test tubes with semi-liquid media containing lactose, glucose and other carbohydrates and polyhydric alcohols. For semi-liquid media, inoculation is done by injecting into the depth of the medium. When sowing by injection, the test tube with the medium is held at an angle, the stopper is removed, and the edge of the test tube is burned. The material is taken with a sterile loop and the column of nutrient medium is pierced with it almost to the bottom.

To determine proteolytic enzymes, the isolated culture is inoculated on peptone water or MPB. To do this, take the test tube with the inoculation closer to you in your hand, and the test tube with the medium further away from you. Both test tubes are opened at the same time, grabbing their plugs with the little finger and the edge of the palm, burn the edges of the test tubes, grab a little culture with a calcined cooled loop and transfer it to the second test tube, grind it in a liquid medium on the wall of the test tube and wash it off with the medium.

When sowing and reseeding, attention should be paid to compliance with the rules of sterility, in order not to contaminate your crops with foreign microflora, and also not to pollute the environment. The tubes are labeled and placed in a thermostat for incubation at 37°C for 24 hours.

Conclusion

Accounting for results. Conclusion of the study. The identification results are taken into account and, based on the totality of the data obtained, based on the classification and characteristics of the typical strains described in the manual (Burgee's key, 1994-1996), the type of isolated crops is determined.

To examine samples for the presence of certain bacteria, various diagnostic methods are used, including the bacterioscopic method. The features of this method, as well as the bacteriological research method, will be discussed in this article.

The essence of the bacterioscopic diagnostic method

The bacterioscopic method of examining a sample is intended to identify microorganisms in the substance under study, as well as to study in detail the properties of these microorganisms, clarify their quantity and behavior in the environment under consideration. The advantages of this method of studying analyzes are simplicity, speed and accessibility. It can be carried out in almost any laboratory using a minimum number of instruments and chemical reagents. Its disadvantages include the impossibility of obtaining a complete picture of the behavior of microbes.

Materials required for research

To study analyzes using the bacterioscopic method, the following tools are required:

1. Metal loop.
2. Slide. This item must be clean, since any contamination can affect the condition of the sample.
3. Tweezers.
4. Filter paper.

New slides are cleaned using a 1% soda solution. After boiling in such a solution, the glass is washed with distilled water, a weak solution of hydrochloric acid, and then again with distilled water. Used glass is cleaned in several stages: first, they are placed in a sulfuric acid solution for 60-120 minutes, then boiled in a soda solution, after which the glass is washed with water. After this, the glass is dipped in medical alcohol.

Instrument glasses should be stored either in alcohol or in tightly closed containers. Moreover, in the latter case, the glass must be dry.

For this study you will also need the following chemical reagents:

• Alcohol;
• Lugol's solution;
• Dyes.

The most commonly used dyes are Ziehl fuchsin, methylene blue and carbolic gentian violet. The last dye is a solution of ordinary fuchsin in five percent carbolic water

Progress of the study

You can study the culture both in its original and colored form. In the latter case, both colonies of microorganisms and single bacteria will be dead, which allows us to become better acquainted with the structure of these simple organisms. When examining the drug in its original form, the laboratory technician, in turn, receives more information about the activity of microbes. In both cases, the preparation is examined using a microscope.

Staining of the medium is carried out in two stages: first, the preparation is prepared for the procedure and only then it is stained. Sample preparation proceeds as follows:

1. The sample intended for research is evenly distributed over the instrument glass in the shape of a circle or oval. If there are foreign impurities in the material (for example, pus), 1-2 drops of water are applied to the glass before adding the test sample.
2. After this, the resulting smear should be dried.
3. Once the smear has dried, it must be fixed using a flame from the burner.

To fix it in this way, the instrument glass is held over the flame three times. If the smear is fixed firmly enough, the laboratory assistant conducting the experiment will feel a burning sensation in his fingers.

At the end of this procedure, the sample is stained.

Sample staining methods

Coloring can be as simple as using a single dye. And complex, in which, in order to accurately differentiate bacterial cells according to one or another of their characteristic features, several different staining chemical reagents are sequentially applied to the sample. An example of complex staining is the Gram and Ziehl-Neelsen methods.

Gram method

The Gram method allows you to determine the chemical composition of the cell membrane. Thanks to this method, the Gram classification of bacteria was introduced: gram-positive bacteria (which include the causative agents of many diseases) after staining acquire a dark purple color, and gram-negative bacteria - a characteristic red tint. The Gram method consists of the following steps:

1. First, the drug is treated with Lugol's solution for one minute.
2. After treatment with Lugol, the sample is bleached with alcohol.
3. Then the preparation is washed with distilled water and additionally stained with fuchsin.

Ziehl-Neelsen method

The Ziehl-Neelsen method is used to determine acid-sensitive bacteria whose cell membranes contain a large amount of lipids, for example, the causative agents of tuberculosis. Work using this method is carried out in five stages:

1. Filter paper is placed on a glass slide, onto which a small amount of Ziehl fuchsin is then applied.
2. The slide is then heated until characteristic steam appears. After steam appears, the glass is allowed to cool. This procedure is repeated two more times, after which the glass is allowed to cool again.
3. After this, wash off the magenta from the instrument glass with distilled water, after removing the paper.
4. The glass is then placed in a solution of hydrochloric or sulfuric acid until the sample completely loses its color.
5. Finally, a solution of methylene blue is applied to the bleached preparation, the glass is washed with distilled water and allowed to dry for further examination of the resulting preparation under a microscope.

Leffler method

Simple staining methods include the Leffler method. With it, all bacteria turn blue. This method is carried out in two steps:

1. Filter paper is placed on the smear, onto which Loeffler's methylene blue solution is applied. The drug is left in this state for 3-5 minutes.
2. After this period of time, the preparation is washed with water and dried, after which it can be examined under a microscope.

With the bacterioscopic method, at least two preparations are stained, one of them is stained using a simple method, and one is stained using a complex method.

Bacteriological method

Often, when studying tests, it becomes necessary to determine the sensitivity of a pathogen to a particular drug. In this case, the sample is examined using the bacteriological method. This method consists of the following steps:

• First, it is necessary to purify the culture in order to identify specific types of microbes. To do this, the sample must be placed in an environment in which the development of the bacteria to be detected is most likely. It is also possible to mechanically separate the biomaterial during seeding.
• After obtaining a pure culture, samples taken from it are examined using the bacterioscopic method.

The following are examples of media used to detect microorganisms:

• Elschnig's medium, consisting of one third horse serum and two thirds broth.
• Loeffler's serum, which is used to identify diphtheria pathogens. This medium consists of three-quarters horse or bovine serum and one-fourth broth containing 1% glucose.
• Blood agar used to identify streptococci and test their sensitivity to antibacterial drugs. This medium consists of 5-10% animal blood serum and agar.

Types of bacterioscopy tests

Bacterioscopy of stool tests

For bacterioscopic examination of stool analysis, a patient sample is required. If it is necessary to analyze the intestinal microflora, disturbances in the composition of which indicate the presence of dysbacteriosis or infectious diseases of the digestive system, the sample is taken using a loop, which is inserted into the anus approximately 10 centimeters deep.

When examining a stool sample, the following dangerous microorganisms may be detected:

• Staphylococci.
• Klebsiella, the presence of which indicates damage to the colon.
• Pseudomonas aeruginosa, which produces toxins that are extremely dangerous to humans.

The presence of Pseudomonas aeruginosa in the human body can lead to blood poisoning and meningitis - diseases that can be fatal.

Bacterioscopy of urine tests

A urine test for bacterioscopic examination is given if there is a suspicion of inflammation of the urinary system and the presence of diseases such as pyelonephritis and cystitis, the causative agents of which are E. coli and some pathogens of sexually transmitted diseases, respectively. For such a test, 50 to 100 ml of urine is required, and the urine should be stored in a sterile container. Before taking the test, you need to wash yourself well so that there are fewer foreign impurities in the urine. It is not recommended to donate urine during menstruation.

Bacterioscopic urine analysis is indispensable for detecting diseases of the urinary system in infants. In this case, urine is collected through a catheter into a sterile container. The examination of a urine sample must be carried out as soon as possible, otherwise the detection of pathogens will be difficult.

Sputum bacterioscopy

When analyzing sputum, two smears are examined. One of them is being studied for the presence of Koch bacilli, the causative agent of tuberculosis. Based on the results of the second study, conclusions are drawn about the presence of other microbes in the sputum.

To analyze sputum for tuberculosis, the sample is stained using the Ziehl-Neelsen method.

To test for the presence of other microbes, the sample is Gram stained. Using the bacterioscopic method using Gram staining, it is possible to identify pneumococcus, a bacterium that causes pneumonia. Also, when working with a sample according to this scheme, it is possible to detect the presence of streptococci in the sputum - the causative agents of sore throat, as well as Staphylococcus aureus. The latter microorganism poses a particular danger to human health. It provokes purulent processes and the formation of abscesses, including on internal organs.

Let's sum it up

The bacterioscopic diagnostic method is one of the main research methods in microbiology. Despite its shortcomings, this method is widely used in modern medicine to diagnose a wide variety of diseases, some of which, for example, tuberculosis, pose a particular danger to humans.

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

If a positive result is obtained, the bacteriological method allows one to determine the sensitivity of the isolated pathogen to antimicrobial drugs. However, the effectiveness of this study depends on many parameters, in particular on conditions for collecting material and him transportation to the laboratory.

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

• taking material before the start of etiotropic treatment;
• compliance with sterile conditions when collecting material;
• technical correctness of material collection;
• sufficient amount of material;
• ensuring temperature conditions for storing and transporting material;
• minimizing the time interval between collecting material and sowing on solid nutrient media.

Transportation of the material to the laboratory should be carried out as soon as possible, but no more than within 1-2 hours after its collection. Material samples must be kept 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 (domestic refrigerator conditions). If it is impossible to deliver samples to the laboratory within the regulated time frame, it is recommended to use transport media designed to preserve the viability of pathogens under preservation conditions.

Blood for research should be taken from the patient during the period of rising body temperature, at the beginning of the onset of fever. It is recommended to examine 3-4 blood samples taken at intervals of 4-6 hours, which is justified from the point of view 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 detected in several venous samples. blood. A blood sample in the amount of 10 ml for an adult and 5 ml for children is inoculated into at least two bottles with a medium for aerobic and anaerobic microorganisms in a ratio of 1:10. A single study of arterial blood is advisable.

Take cerebrospinal fluid(CSF) is produced by a doctor during lumbar puncture in the amount of 1-2 ml into a dry sterile tube. The sample is immediately delivered to the laboratory, where its examination also begins 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 inoculating 1-2 drops of CSF into a test tube containing a semi-liquid medium with glucose and into a Petri dish with “blood” agar. To send the material, use isothermal boxes, heating pads, thermoses or any other packaging where the temperature is maintained at about 37 °C.

Excreta for bacteriological research, 3-5 g are taken using sterile wooden spatulas into a sterile vessel with a tight-fitting lid. The examination of the collected material should begin no later than 2 hours later. If it is not possible to begin the examination within this time, a small amount of material should be collected and placed in an appropriate transport medium. When collecting stool, one should strive to send pathological impurities (mucus, pus, epithelial particles, etc.) for examination, if any, avoiding the introduction of blood impurities, which have bactericidal properties, into the material.

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

I'm pissing(average portion of freely released urine) in an amount of 3-5 ml is collected in a sterile container after a thorough toilet of the external genitalia. It is preferable to collect urine in the morning.

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

Gastric lavage water collected in sterile jars in quantities of 20-50 ml. It should be borne in mind that in these cases, gastric lavage 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 attack 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 oral microflora.

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

Discharge from the pharynx, mouth and nose. Material from the oral cavity is taken on an empty stomach or 2 hours after a meal with a sterile cotton swab or spoon from the mucous membrane and its affected areas at the entrances of the ducts of the salivary glands, the surface of the tongue, and from ulcers. If there is a film, remove it with sterile tweezers. The 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, the tampon is not removed until the cough is over. To test for diphtheria, films and mucus from the nose and throat are examined simultaneously, taking the material with different swabs.

The test material is inoculated onto solid 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 selective media that inhibit 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. Recently, for identification in healthcare practice, microtest systems have been used, which are panels with a set of differential diagnostic environments, which speeds up the research. Microtest systems are also used to determine the sensitivity of microorganisms to antimicrobial drugs by diluting the antibiotic in a liquid nutrient medium.

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

Isolation from the blood, subject to all aseptic rules, of opportunistic microorganisms (staphylococcus epidermidis, Escherichia coli) and even saprophytes should be considered a manifestation of bacteremia, especially if these microbes are found in more than one sample of material or in different substrates (blood, urine), since By reducing the body’s immunoreactivity, these and other “non-pathogenic” microorganisms can be causative agents of infectious processes, including sepsis.

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

Yushchuk N.D., Vengerov Yu.Ya.

Bacteriological (cultural) method

a set of methods for the artificial cultivation of microorganisms on nutrient media in order to identify them when establishing an infectious disease or other process caused by microbes and to determine a number of physiological properties for other purposes, for example, when choosing a chemotherapy drug. Modern methods for studying most biol. properties of bacteria are possible only if there is clean water(cm.). Contamination of the herb with other types of microbes, as a rule, leads to distorted results and incorrect conclusions. Therefore, the first task of B.m. is to obtain pure grass of any type (var) from a microbial association and prevent contamination by foreign microflora of the material for research and microbial culture at all stages of the study. This is possible when collecting material under sterile conditions in sterile containers, inoculating the material into sterile media with a sterile instrument, preventing the entry of microbes from the air during delivery, storage and incubation of the material and inoculated nutrient media. The second task of B.m. - microbial identification(see) isolated pure herbs, the third is the determination of additional properties, for example, sensitivity to antibiotics, virulence. Stages of B.M.: 1) collection of material (see. Materials for research) and its delivery to bacteria. laboratory; 2) microscopy research. material (see Bacterioscopic method). This step is often not performed, although, despite the lower sensitivity, preliminary microscopy in some cases provides approximate information about the pathogen and allows you to select the media necessary for inoculation; 3) processing of research. physical material or chem. factors in order to remove or reduce foreign microflora. This measure is used, for example, when examining sputum, isolating acid-fast 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 expected species of the plant. Typically, crops are kept in a thermostat at 37°C for 1-2 days. If there is no growth, the incubation period can be extended for another 2 to 3 days. For longer incubation, it is necessary to prevent the medium from drying out, for which the crops are placed in a desiccator with water or the tube stoppers are filled with paraffin; 6) research colonies(cm.). For study, homogeneous isolated colonies located far from the edges of the dish and along the stroke of the loop are selected, surrounded by a line, and numbered. If the study is carried out in an undirected manner (polyetiological process, etc.), then based on the results of a visual inspection, 2-3 colonies of all types present on the plate are selected and smears are made from them; 7) reseeding from selected colonies onto accumulation media. To do this, from the remainder of the colony, in a smear of the cut, bacteria of uniform shape and color were identified; using a loop, trying not to touch the neighboring colonies, a part of the bacteria is taken and inoculated onto an MPA beveled in a test tube or a special medium with a streak; 8) incubation of crops in a thermostat until continuous growth appears (usually 1-2 days); 9) determination of the purity of the grass grown on slant media by macroscopic examination of the growth and microscopy of a smear from it; 10) identification of the isolated pure grass and, if necessary (at the request of the clinician, epidemiologist), determination of various biol. sv-v; 11) conclusion on the species identity of the selected species and its properties. The conclusion is given on official form with reference to the number, under the Crimea this plant is entered in the laboratory journal. B.m. is the main one in the diet of most bacteria. infections. It, as a rule, has high sensitivity, allowing it to be distinguished from, research. pure material, even if the seed dose of the material contained several dozen individuals. For B.m. characterized by high specificity. Isolation of one or another type of bacteria from pathological material subject to a number of conditions (see. The causative agent of the disease) reliably establishes the etiology of the pathological process. The exception is opportunistic bacteria, autochthonous for a particular biotope, to determine etiol. The role of which requires special criteria, as well as a distinction between carriage and disease (for example, diphtheria carriage in streptococcal sore throat). The value of B.m. also lies in the fact that with its help the medicines necessary for prescribing chemotherapy and serotherapy, identifying the source of infection and the mechanisms of transmission of the pathogen, and choosing anti-epidemic measures can be established. Finally, B.m. refers to the early methods of d-ki. Disadvantages of B. m. - danger of infection, complexity, labor intensity, duration. Reliable data on the composition of the microflora and its properties can only be obtained by studying a sample of 3-10 flora.