Equipment and operating mode of a bacteriological laboratory. Bacteriological laboratory. Modern general laboratory equipment necessary for the operation of a microbiological laboratory

Working with pathogens of infectious diseases requires that the laboratory premises be located in a separate building or isolated from hospital wards and food units. Diagnostic laboratories must have two entrances: one for employees, the other for delivery of material for testing. It is allowed to receive material through the transfer window. The laboratory premises are divided into “contaminated” and “clean” zones and are located as tests are performed.

The “clean” area of ​​the laboratory includes:

1. Room for outerwear.
2. Preparatory work room (preparation room, washing room for preparing and bottling nutrient media, etc.)
3. Sterilization.
4. A room with a refrigerator for storing culture media and diagnostic preparations.
5. Room for rest and eating.
6. Room for working with documentation.
7. Utility rooms.
8. Toilet.

The following are placed in the “contagious” zone:

1.A room for receiving and registering material submitted for research.

3.Rooms for conducting bacteriological research.

4.Rooms for conducting serological studies.

5. Room for luminescence microscopy.

6. Room for zooentomological work.

7.Thermostat room and autoclave.

Premises where work with living organisms is carried out are equipped with bactericidal lamps.

The task of a medical microbiology laboratory - diagnosis of infectious diseases. To do this, the pathogen is isolated and the body’s immune response to the introduction of microorganisms is determined ( serological diagnostics). In addition, carriers of pathogenic (disease-causing) microorganisms are identified. There are laboratories that conduct virological studies. In special sanitary and bacteriological laboratories, research is carried out to identify the degree of microbial contamination of the external environment and various objects.

Laboratory room intended for microbiological research. It should be spacious and bright. The walls are painted with light oil paint, the floor is covered with linoleum, laboratory tables are covered with plastic or glass, which is convenient for wet cleaning and disinfection. The laboratory room is equipped with work tables for the doctor and laboratory assistant, a place for staining preparations, a thermostat, a refrigerator, a centrifuge, a microscope, cabinets, a sink with hot water and cold water, gas burners (in the absence of gas, they work with alcohol burners).

The number of laboratory rooms is determined by the volume of laboratory work. In large laboratories, separate rooms are allocated for working with different types of pathogens.

The desktop is placed near the window so that the light falls from the side or directly. A burner, bacteriological loops, jars with a disinfectant solution and cotton wool are placed on the table.

Before starting work, everything necessary for the study is placed on the table. The burner is installed at a distance equal to the worker’s forearm, i.e. in a position that eliminates unnecessary movements during work. The size of the flame in the burner and the correct glow are adjusted before starting work.

The temperature in the thermostat during routine research should be 37°C. In large laboratories, a special thermal room can be equipped. Temperatures are recorded daily.

Rice. 1 Drying cabinet

Some nutrient media, diagnostic preparations, blood, bile, etc. are kept in the refrigerator.

A centrifuge is used to separate dense particles from a liquid (for example, red blood cells from serum).

The cabinets contain tripods, dishes, dry nutrient media, reagents, etc.

Near the sink there should be a vessel with a disinfectant solution for treating hands and a first aid kit with a set of items for first aid.

Boxing is a strictly isolated room for conducting microbiological work under conditions requiring special sterility. Air defertilization is carried out using bactericidal lamps. The supply of disinfected air of a certain temperature and humidity into the box through supply and exhaust ventilation is the best way provision necessary conditions for work. Usually two people work in the box. They enter the box through the front box, in which they change clothes (robe, slippers, cap, mask) and enter the box through the second door.

In boxing there is no talking and unnecessary movements are avoided.

The room for preparing nutrient media should be located next to the washing and sterilization room. This room should have a sink with hot and cold water supply, a distiller, a stove (gas or electric), cabinets or shelving for storing dry nutrient media, chemical reagents, and sterile utensils.

Washing– a room for washing and processing dishes, which must have a sink (with cold and hot water) and a stove. The washing room is equipped with tables, shelving, and equipment for washing dishes: detergents, brushes, rags.

The sterilization room contains devices for sterilizing clean dishes, culture media and disinfecting waste material: autoclaves, drying cabinets, etc.

If there is a separate preparation room, it is used for preparation, packaging of dishes and other auxiliary work.

In the registry or part of the premises that replaces it, material received for research is received and registered and microbiological research conclusions are issued.

Vivarium– a room for keeping experimental animals, available only in large laboratories.

All microbiological, biochemical and molecular biological studies of microorganisms are carried out in special laboratories, the structure and equipment of which depend on the objects of study (bacteria, viruses, fungi, protozoa), as well as on their target orientation (scientific research, disease diagnosis) . The study of the immune response and serodiagnosis of human and animal diseases is carried out in immunological and serological (serum - blood serum) laboratories.

Bacteriological, virological, mycological and serological (immunological) laboratories are part of sanitary-epidemiological stations (SES), diagnostic centers and large hospitals. In the laboratories of the SES, they perform bacteriological, virological and serological analyzes of materials obtained from patients and persons in contact with them, examine bacteria carriers and conduct sanitary and microbiological studies of water, air, soil, food products etc.

In bacteriological and serological laboratories of hospitals and diagnostic centers, they conduct research to diagnose intestinal, purulent, respiratory and other infectious diseases, and carry out microbiological control over sterilization and disinfection.

Diagnostics especially dangerous infections(plague, tularemia, anthrax, etc.) are carried out in special sensitive laboratories, the organization and procedure of which are strictly regulated.

Virology laboratories diagnose diseases caused by viruses (influenza, hepatitis, polio, etc.), some bacteria - chlamydia(ornithosis, etc.) and rickettsia(typhus, Q fever, etc.). When organizing and equipping virology laboratories, the specifics of working with viruses, cell cultures and chicken embryos, which require the strictest asepsis, are taken into account.

In mycological laboratories, they diagnose diseases caused by pathogenic fungi and pathogens of mycoses.

Laboratories are usually located in several rooms, the area of ​​which is determined by the volume of work and the intended purpose.

Each laboratory provides:

a) boxes for working with individual groups of pathogens;

b) premises for serological research;

c) premises for washing and sterilizing dishes, preparing
lenition of nutrient media;

d) a vivarium with boxes for healthy and experimental animals;
nykh;

e) registry for receiving and issuing tests.

Along with these premises, virology laboratories have boxes for special processing of research material and work with cell cultures.

Equipment for microbiological laboratories

Laboratories are equipped with a number of mandatory instruments and apparatus.

1. Instruments for microscopy: biological immersion microscope with additional devices (oscillator, phase-contrast device, dark-field condenser, etc.), fluorescent microscope.

2. Thermostats and refrigerators.

3. Equipment for preparing nutrient media, solutions, etc.: apparatus for producing distilled water (distiller), technical and analytical balances, pH meters, filtration equipment, water baths, centrifuges.

4. A set of tools for manipulating microbes: bacteriological loops, spatulas, needles, tweezers, etc.

5. Laboratory glassware: test tubes, flasks, Petri dishes, mattresses, vials, ampoules, Pasteur and graduated pipettes, etc., apparatus for making cotton-gauze plugs.

Large diagnostic complexes have automatic analyzers and a computerized system for assessing the information received.

The laboratory has a designated area for staining microscopic preparations, where there are solutions of special dyes, alcohol, acids, filter paper, etc. Each workplace is equipped with a gas burner or alcohol lamp and a container with a disinfectant solution. For daily work, the laboratory must have the necessary nutrient media, chemical reagents, diagnostic drugs and other materials.

Large laboratories have thermostatic rooms for mass cultivation of microorganisms and serological reactions. The following equipment is used for growing, storing cultures, sterilizing laboratory glassware and other purposes.

1. Thermostat. A device in which a constant temperature is maintained. The optimal temperature for the reproduction of most pathogenic microorganisms is 37 "C. Thermostats are air and water.

2. Microanaerostat. Apparatus for growing microorganisms under anaerobic conditions.

3. C0 2 -incubator. An apparatus for creating a constant temperature and atmosphere of a certain gas composition. Designed for the cultivation of microorganisms that are demanding on the gas composition of the atmosphere.

4. Refrigerators. Used in microbiological laboratories for storing cultures of microorganisms, nutrient media, blood, vaccines, serums and other biologically active preparations at a temperature of about 4 °C. To store drugs at temperatures below 0 °C, low-temperature refrigerators are used, in which the temperature is maintained at -20 °C or -75 °C.

5. Centrifuges. Used for sedimentation of microorganisms, erythrocytes and other cells, for separation of heterogeneous liquids (emulsions, suspensions). Laboratories use centrifuges with different operating modes.

6. Drying and sterilization cabinet(Pasteur oven). Designed for dry-air sterilization of laboratory glassware and other heat-resistant materials.

7. Steam sterilizer (autoclave). Designed for sterilization with superheated water steam (under pressure). Autoclaves are used in microbiological laboratories different models(vertical, horizontal, stationary, portable).

BACTERIOLOGICAL, VIRUSOLOGICAL, MYCOLOGICAL, IMMUNOLOGICAL LABORATORIES AND THEIR EQUIPMENT. DEVICE OF MODERN MICROSCOPE. METHODS OF MICROSCOPY. METHODS FOR STUDYING THE MORPHOLOGY OF MICROORGANISMS

Program

1. Rules of work and organization of microbiological (bacteriological, virological, mycological) laboratories.

2. Basic instruments and equipment of a microbiological laboratory.

3. Microscopes and microscopic equipment. Rules for working with an immersion microscope (objectives).

Demonstration

1. Design and use of basic instruments and equipment used in microbiological laboratories: thermostat, centrifuges, autoclave, drying cabinet, instruments and utensils.

2. Design of a biological microscope. Various microscopy methods: dark-field, phase-contrast, fluorescent, electron.

3. Preparations of microbes (yeast and bacteria) using various microscopy methods.

Assignment for students

1. Microscope and sketch preparations of yeast-like fungi of the genus Candida using different kinds microscopy.

Guidelines

Rules for working in microbiological laboratories.

Work in a microbiological laboratory medical institution carried out with pathogens of infectious diseases - pathogenic microorganisms.

Therefore, to protect against infection, personnel must strictly adhere to the internal regulations:

1. All employees must work in medical gowns, caps and removable shoes. Entry into the laboratory without a gown is strictly prohibited. If necessary, workers put a gauze mask on their face. Work with especially dangerous microbes is regulated by special instructions and is carried out in sensitive laboratories.

2. Smoking and eating are prohibited in the laboratory.

3. Workplace must be maintained in exemplary order. Personal belongings of employees should be stored in a specially designated place.

4. If infected material accidentally gets on the table, floor or other surfaces, this place must be thoroughly treated with a disinfectant solution.

5. Storage, observation of microbial cultures and their destruction must be carried out in accordance with special instructions. Cultures of pathogenic microbes are registered in a special journal.

6. Upon completion of work, hands should be washed thoroughly and, if necessary, treated with a disinfectant solution.

Microscopes and microscopy methods

Rice. 1.1. Microscopes.

A - general form microscope "Biolam"; b — MBR-1 microscope: 1 — microscope base; 2 - object table; 3 — screws for moving the object stage; 4 — terminals that press the drug; 5 - condenser; 6 — condenser bracket; 7 - screw securing the condenser in the sleeve; 8 — handle for moving the condenser; 9 — handle of the iris diaphragm of the condenser; 10 - mirror; 11 — tube holder; 12 — macrometric screw handle; 13 — micrometer screw handle; 14 — lens revolver; 15 — lenses; 16 — inclined tube; 17 — screw for fastening the tube bead; 18 - eyepiece.

For microbiological studies, several types of microscopes (biological, fluorescent, electronic) and special microscopy methods (phase contrast, dark field) are used.

In microbiological practice, microscopes of domestic brands are used: MBR-1, MBI-2, MBI-3, MBI-6, "Bio-lam" R-1, etc. (Fig. 1.1). They are designed to study the shape, structure, size and other characteristics of various microbes, the size of which is at least 0.2-0.3 microns.

Immersion microscopy

Used to increase the resolution of the method light microscopy. The resolution of a light-optical microscopy system is determined by the wavelength visible light and the numerical aperture of the system. The numerical aperture measures the angle of maximum cone of light entering the lens and depends on the optical properties (refractive power) of the medium between the object and the objective lens. Immersing the lens in a medium (mineral oil, water) that has a high refractive index, close to that of glass, prevents light from scattering from the object.

Rice. 1.2. Path of rays in an immersion system, n is the refractive index.

Rice. 1.3. Ray path in dark-field condensers, a - paraboloid-condenser; b — cardioid condenser; 1 - lens; 2 - immersion oil; 3 - drug; 4 - mirror surface; 5 - diaphragm.

In this way, an increase in the numerical aperture and, accordingly, resolution is achieved. For immersion microscopy, special immersion objectives equipped with a mark are used (MI - oil immersion, VI - water immersion). The maximum resolution of an immersion microscope does not exceed 0.2 microns. The path of rays in the immersion system is shown in Fig. 1.2.

The overall magnification of a microscope is determined by the product of the objective magnification and the eyepiece magnification. For example, the magnification of a microscope with an immersion objective of 90 and an eyepiece of 10 is: 90 x 10 = 900.

Microscopy in transmitted light (bright-field microscopy) used to study colored objects in fixed preparations.

Dark-field microscopy. It is used for intravital study of microbes in native unstained preparations. Dark field microscopy is based on the phenomenon of light diffraction under lateral illumination of particles suspended in a liquid ( Tyndall effect). The effect is achieved using a paraboloid or cardioid condenser, which replaces a conventional condenser in a biological microscope (Fig. 1.3). With this lighting method, only rays reflected from the surface of the object enter the lens. As a result, brightly luminous particles are visible against a dark background (unlit field of view). The drug in this case has the form shown in Fig. 1.4, b (on the inset).

Phase contrast microscopy. Designed for the study of native drugs. A phase-contrast device makes it possible to see transparent objects through a microscope. Light passes through different biological structures at different speeds, which depend on the optical density of the object. As a result, a change in the phase of the light wave occurs that is not perceived by the eye. A phase device, including a special condenser and lens, ensures the conversion of changes in the phase of a light wave into visible changes in amplitude. In this way, an increase in the difference in the optical density of objects is achieved. They acquire high contrast, which can be positive or negative. Positive phase contrast is a dark image of an object in a bright field of view, negative phase contrast is a light image of an object on a dark background (see Fig. 1.4; inset).

For phase-contrast microscopy, a conventional microscope and an additional phase-contrast device KF-1 or KF-4 (Fig. 1.5), as well as special illuminators, are used.

Luminescent (or fluorescent) microscopy. Based on the phenomenon of photoluminescence.

Luminescence- glow of substances that occurs under the influence of external radiation: light, ultraviolet, ionizing, etc. Photoluminescence - luminescence of an object under the influence of light. If you illuminate a luminescent object with blue light, it emits rays of red, orange, yellow or green. The result is a color image of the object.

Rice. 1.5. Phase contrast device, a - phase lenses; b - auxiliary microscope; c - phase capacitor.

The wavelength of the emitted light (luminescence color) depends on the physicochemical structure of the luminescent substance.

Primary luminescence of biological objects (own, or bioluminescence) is observed without preliminary staining due to the presence of its own luminescent substances, secondary (induced) - occurs as a result of staining preparations with special luminescent dyes - fluorochromes(acridine orange, auromin, coryphosphine, etc.). Luminescent microscopy has a number of advantages compared to conventional methods: the ability to examine living microbes and detect them in the material under study in small concentrations due to high degree contrast.

In laboratory practice, fluorescence microscopy is widely used to identify and study many microbes.

Electron microscopy. Allows you to observe objects whose dimensions lie beyond the resolution of a light microscope (0.2 microns). The electron microscope is used to study viruses, thin structure various microorganisms, macromolecular structures and other sub-microscopic objects. Light rays in such microscopes are replaced by a stream of electrons, which at certain accelerations has a wavelength of about 0.005 nm, i.e. almost 100,000 times shorter than the wavelength of visible light. The high resolution of the electron microscope, reaching 0.1-0.2 nm, allows a total useful magnification of up to 1,000,000.

Along with translucent type devices, they use scanning electron microscopes, providing a relief image of the surface of an object. The resolution of these devices is significantly lower than that of transmission electron microscopes.

Rules for working with a microscope

Working with any light microscope includes setting the correct illumination for the field of view and the specimen and microscopying it with various lenses. Lighting can be natural (daylight) or artificial, for which special light sources are used - illuminators of different brands.

When microscopying specimens with an immersion lens, you should strictly adhere to a certain order:

1) apply a drop of immersion oil to the smear prepared on a glass slide and stained and place it on the stage, securing it with clamps;

2) turn the revolver to the immersion lens mark 90x or 10Ох;

3) carefully lower the microscope tube until the lens is immersed in a drop of oil;

4) set the approximate focus using a macrometric screw;

5) carry out final focusing of the preparation with a micrometer screw, rotating it within only one turn. Do not allow the lens to come into contact with the
paratomy, as this may lead to breakage of the cover glass or the front lens of the objective lens (free distance of the immersion objective 0.1-1 mm).

After finishing the microscope operation, it is necessary remove oil from the immersion lens and transfer the revolver to the small 8x lens.

For dark-field and phase-contrast microscopy, native preparations are used ("crushed" drop, etc., see topic 2.1); microscope with a 40x objective or a special immersion objective with an iris diaphragm, which allows you to adjust the numerical aperture from 1.25 to 0.85. The thickness of glass slides should not exceed 1 - 1.5 mm, cover glasses - 0.15-0.2 mm.

Regulation of working conditions with pathogens of infectious diseases is carried out in accordance with the degree of danger of microorganisms to humans. Based on this criterion, four groups of pathogenic biological agents (PBA) have been identified:

Group I: pathogens of particularly dangerous infections (plague, smallpox, etc.)

Group II: highly contagious bacterial, fungal and viral infections(anthrax, cholera, rabies, etc.)

Group III: pathogens of bacterial, fungal, viral and protozoal infections, isolated into independent nosological forms (whooping cough, tetanus, tuberculosis, etc.)

Group IV: pathogens of bacterial, fungal, viral septicemia, meningitis, pneumonia, enteritis, toxic infections, acute poisoning(pseudomonas infection, etc.).

Most microbiological laboratories work with pathogenic pathogens of groups III and IV, and only specialized laboratories study pathogens of especially dangerous infections (groups I and II).

Basic laboratories working with group III and IV pathogenic biological agents must meet a number of requirements (a separate building or a separate entrance, the presence of water and electricity supply systems, heating, ventilation, etc.) and have the necessary set of premises in accordance with the production capacity and range of studies performed . Each laboratory must have a “clean” and “dirty” area.

The “dirty” zone includes rooms for receiving and recording material, boxes and rooms for conducting microbiological studies, a thermostat, and an autoclave for disinfecting material. Windows and doors of all rooms must be tightly closed. Premises for working with live microorganisms must be equipped with bactericidal lamps or have biological safety cabinets. Supply and exhaust ventilation of the “dirty” area must be equipped with filters for fine purification of the exhaust air. Marking of tables, autoclaves, containers with disinfectant solutions, and racks for clean and infected material is mandatory. The laboratory must be equipped with special furniture and have smooth floor and wall surfaces that are resistant to detergents and disinfectants.

The “clean” zone includes rooms for preliminary work (washing room, preparation room, room for preparing and bottling culture media, etc.), a room for working with documentation, rooms with refrigerators for storing culture media and diagnostic drugs, a wardrobe for outerwear, a rest room . In the “clean” zone it is possible to work with non-living pathogens (serological, molecular genetic, biochemical studies).

Ensuring the safety of working with pathogenic microorganisms includes two main factors: technical and human. The technical factor is the availability of “clean” and “dirty” zones, equipment, protective systems, etc. necessary for work. The human factor is the correctness of a person’s actions to ensure safety, the level of mastery of professional equipment, knowledge of possible sources and mechanisms of infection, appropriate preparation and training.

Work in a training bacteriological laboratory is also associated with two dangerous factors - microorganisms that cause infectious diseases and open fire, which requires compliance with anti-epidemic and fire safety measures. Students are required to familiarize themselves with the safety rules and strictly follow them:

Necessary:

· work in long-sleeved medical gowns, medical caps and shoe covers;

· store personal belongings in a specially designated place, leave outerwear in the wardrobe;

· each type of activity is carried out in a certain area: work with microorganisms - on a specially equipped laboratory table, filling out protocols - on a desktop;

· if infected material gets on the table, floor or other objects, immediately inform the teacher and carry out disinfection;

· Infected materials should be placed in durable, waterproof containers or containers of disinfectant solution that are sealed before removal from the laboratory.

· Laboratory doors must be kept closed during work.

· After finishing work, wash your hands thoroughly and, if necessary, treat them with a disinfectant solution.

· eat in the laboratory

· draw liquid into a pipette with your mouth

· light one alcohol lamp from another

· carry a burning alcohol lamp

· leave the alcohol lamp burning after finishing its intended use

· leave unfixed preparations, Petri dishes with cultures and other dishes with infectious material at the workplace

touch the test material in seeded Petri dishes with your hands

Bacteriological laboratory and rules of work in it. Classification of microorganisms. Morphology of bacteria. Methods for determining the type of microbes. Bacterioscopic method. Immersion microscopy technique

Lesson summary

Medicine and veterinary medicine

LESSON 1 LESSON TOPIC: Bacteriological laboratory and rules of work in it. Classification of microorganisms. Morphology of bacteria. Methods for determining the type of microbes. Bacterioscopic method. Microscopy technique with immersion system. LEARNING OBJECTIVE OF ACTIVITY...

LESSON 1

LESSON TOPIC : Bacteriological laboratory and rules of work in it. Classification of microorganisms. Morphology of bacteria. Methods for determining the type of microbes. Bacterioscopic method. Microscopy technique with immersion system.

LEARNING OBJECTIVE OF THE LESSON: Familiarize yourself with the structure of a bacteriological laboratory and the rules of work in it. Familiarize yourself with the principles of classification of microorganisms. Study the morphological characteristics of bacteria and methods for determining the type of microbes. Master the bacterioscopic method of examination and microscopy techniques with an immersion system.

OBJECTIVES OF THE LESSON:

1. Familiarize yourself with the structure of the bacteriological laboratory and the rules of work in it.

2. Get acquainted with the principles of classification of microorganisms.

3. Study the morphological characteristics of bacteria and methods for determining the type of microbes.

4. Master the technique of microscopy with an immersion system.

Construction of a bacteriological laboratory

The bacteriological laboratory is designed to study materials containing pathogens bacterial infections, to determine sanitary and microbiological indicators, monitor the state and intensity of specific immunity and other microbiological studies. The bacteriological laboratory should be located in rooms isolated from other laboratories with necessary equipment and furniture. The laboratory must have a separate entrance, wardrobe and shower. The bacteriological laboratory should include the following premises:

Room for receiving and registering materials;

Boxed rooms for microbiological research;

Autoclave;

Washing;

Vivarium.

Rooms for microbiological research are equipped with thermostats, refrigerators, centrifuges, scales, water baths, and electromagnetic stirrers. The necessary equipment is placed on the tables. Work with infected material is carried out in boxing with prebox . At the entrance to the box there should be a mat soaked in a disinfectant solution. In the box, received samples are sorted, fingerprint smears are prepared and recorded, and microorganisms are inoculated on nutrient media. Therefore, in the box there are tables on which the tools necessary for work are placed: containers with disinfectant solutions for used glassware, racks for test tubes, test tubes and Petri dishes with nutrient media, sterile pipettes, mortars, etc. In the pre-box in the bins, it is necessary to have sterile gowns , hats, masks, and also in the pre-boxing area there should be removable shoes. The prebox can accommodate thermostats, refrigerators, centrifuges and other equipment. In boxes and pre-boxing areas, wet cleaning, disinfection treatment and irradiation with bactericidal lamps are carried out daily for 30-40 minutes before starting work and after work.

In an autoclave it is necessary to have two autoclaves: one autoclave for clean materials (for sterilizing dishes, culture media, instruments); another autoclave for infected materials (for neutralizing infected instruments and materials).

Washing designed for washing dishes. Dishes, pipettes and instruments contaminated with infected material are washed only after sterilization. It houses drying cabinets.

Vivarium called a room used for keeping laboratory animals. The vivarium must have a quarantine department, rooms for experimental and healthy animals, rooms for washing and disinfecting cages, equipment and protective clothing, a kitchen for preparing food, a pantry, a fodder room, and an incinerator. All rooms of the vivarium must be isolated from each other.

Rules for working in a bacteriological laboratory

Laboratory employees are required to comply with the following rules:

1. You are allowed to work in special clothing: a robe and a cap. In boxing, they wear a sterile gown, mask, cap, and, if necessary, wear rubber gloves and goggles. Be sure to change your shoes.

2. It is prohibited to leave the laboratory in gowns or put outer clothing over a gown.

3. Smoking and eating are prohibited in the laboratory.

4. All material entering the laboratory for analysis must be considered infected. Therefore, care must be taken when unpacking the material. Containers should be wiped from the outside with a disinfectant solution and placed on trays or in cuvettes.

5. If infected material gets on a gown, hands, table, or shoes, it is necessary to carry out disinfection and report this to the head of the laboratory.

6. Contaminated material must be destroyed by autoclaving. Tools, as well as the surface of the work table, are disinfected after work.

7. It is prohibited to remove equipment, inventory, and materials from the laboratory without first disinfecting them.

8. Pipettes, slides, cover glasses and other used glassware are disinfected by immersing them in a disinfectant solution.

9. At the end of work, the workplace is tidied up and thoroughly disinfected. Cultures of microorganisms necessary for further work are stored in the refrigerator.

The following documentation is maintained in the bacteriological laboratory:

1. Inventory book of museum strains of cultures.

2. Journal of material movement in the laboratory.

3. Logbook for sterilization and destruction of infected material.

4. Logbook of infected experimental animals.

5. Journal of research (expertise).

Classification of microorganisms

Classification - this is the distribution of organisms based on their accounting common features into groups or taxa . Classification is based on external signs organisms (phenotype) andgenetic characteristicsorganisms (genotype).

Currently mmicroorganisms are divided into the following forms:

1. Non-cellular forms:

Prions;

Viroids;

Viruses.

2. Cellular forms:

2.1. Prokaryotes:

Domain Bacteria:

Bacteria with a thin cell wall (Gram-negative);

Bacteria with a thick cell wall (Gram-positive);

Bacteria without a cell wall (mycoplasma).

Domain Archaea:

Archaebacteria.

2.2. Eukaryotes:

Protozoa;

Mushrooms.

The classification of the living world is based on the type of cell structure: eukaryotic or prokaryotic. The main differences between a prokaryotic (bacterial) cell and a eukaryotic one are: the absence of the following structures: a formed nucleus (i.e. nuclear membrane), intracellular membranes, nucleoli, Golgi complex, lysosomes, mitochondria.

The following are used in the classification of microbes:taxonomic categories: kingdom, department, class, order, family, genus, species. ABOUTThe basic taxonomic unit is the species.Microorganisms are named in accordance with the rules of the International Code of Nomenclature of Bacteria. To designate types of bacteria, it is accepteddouble (binary) nomenclature, proposed back in XVIII century by Carl Linnaeus. According to the nomenclature, in Latin letters it is first written genus name (generic name), and then - species name (species name). If a microorganism is identified only to the genus, then instead of the species name the word is written sp. (species - view). The generic affiliation of a microbe is indicated by some morphological characteristic or the name of the scientist who discovered the microbe, and the species affiliation is denoted by either the type of colony or the habitat of the microorganism. For example, Escherichia coli indicates that the microbe was discovered by T. Escherich, and the microbe lives in the intestines.The formation and use of scientific names for microorganisms is regulated by the International Code of Nomenclature of Bacteria, the International Code of Botanical Nomenclature (fungi), the International Code of Zoological Nomenclature (protozoa) and decisions of the International Committee on Taxonomy of Viruses.

Bacteria are highly variable. For intraspecific differentiation of bacteria that differ in a certain characteristic, the concept of “variant” (abbreviated as “var”) is used. There are variants that differ in antigenic characteristics ( serovars ), variants resistant to bacteriophages ( phageware ), as well as variants that differ in biochemical ( chemovars ), biological or cultural characteristics ( biovars).

In microbiology, specialized terms are used: pure culture, mixed culture, strain, clone.

Culture is a collection of microorganisms grown on a solid or liquid nutrient medium in a laboratory. A culture of microorganisms consisting of individuals of one species is calledpure culture. Mixed culturecalled a mixture of microorganisms different types, grown in a nutrient medium when sowing the material under study or when other types of microorganisms from the external environment enter a nutrient medium inoculated with one type of microbe.

Strain (German: stammen to occur) - this is a pure culture of a certain type of microorganism, isolated from the material being studied, taken at a certain moment from a specific object.

Clone (Greek klon layering) - this is the offspring (culture) of one mother cell (viral particle) of a certain type of microorganism.

Principles of classification of microorganisms

The minimum list of data required to describe bacteria includes the following characteristics.

1. Morphological and tinctorial properties -size, shape, cells, presence of capsule, spores, flagella, ability to be stained with dyes.

2. Type of breathing need for oxygen gas.

3. Biochemical properties -the ability to ferment carbohydrates and break down proteins.

4. Antigenic structure presence of antigens.

5. Sensitivity to bacteriophages.

6. Chemical composition - content and composition of carbohydrates, lipids, proteins.

7. Genetic relationship with other bacteria.

In microbiology, determinants have been created for identifying microorganisms: “Identifier of bacteria and actinomycetes” by N.A. Krasilnikova (1949), “Identifier of microbes” R.A. Zion (1948) and “Identifier of bacteria” by D.Kh. Burgee. The most common is the classification of the American bacteriologist D.H. Burgee.Bergey's key systematizes all known bacteria into 4 sections:

Division I. Gracilicutes (lat. gracilis - elegant, thin, cutis skin) - species with a thin cell wall, staining gram-negatively.

Division II. Firmicutes (lat. firmus - strong, cutis skin) - bacteria with a thick cell wall, staining gram-positive.

Division III. Tenericutes (lat. tener - tender, cutis skin) - bacteria that do not have a cell wall mycoplasma.

Division IV. Mendosicutes (lat. mendosus - incorrect, cutis skin) - archaebacteria. This department includesmethane-forming, sulfur-oxidizing, mycoplasma-like, thermoacidophilic and other bacteria of the most ancient origin.

Morphology of bacteria

Bacteria are not visible to the naked eye. Light and electron microscopes are used to study them. Bacterial cells are measured in micrometers (1 micron equals 10-3 mm), and the elements of the fine structure of bacteria are measured in nanometers (1 nm is equal to 10-3 µm). The average size of bacteria is 0.5-3 microns.

Based on the shape of their cells, bacteria are divided into 3 main groups:

Globular forms or cocci;

Rod-shaped;

Twisted forms.

Cocci have a spherical shape in the form of a regular ball, ellipse, bean. Depending on the relative position of cells after division, they are distinguished the following types cocci:

Micrococci divided in different planes and arranged singly, in pairs or randomly;

Staphylococcus divided in different planes and arranged in clusters;

Diplococcus divided in one plane, arranged in pairs;

Streptococci divided in one plane, arranged in the form of a chain;

Tetracocci divided in two mutually perpendicular planes, arranged in groups of four;

Sarcins are divided in three mutually perpendicular planes and form regular packages of 8-16 cells.

Rod-shaped bacteriahave a cylindrical shape with rounded, pointed or blunt ends. Rod-shaped bacteria are divided into 2 groups:

Bacteria non-spore forming rods;

bacilli - rods that form spores. Rods in which the diameter of the spore exceeds the width of the vegetative cell are called clostridia.

By size rod-shaped bacteria are divided into groups:

Small up to 1.5 microns;

Medium size (1.5 3 microns);

Large (more than 3 microns).

Based on the shape of the ends, they are distinguished:

Rounded (E. coli);

Chopped (causative agent of anthrax);

Pointed (Caulobacter);

Thickened (causative agent of diphtheria);

Split (bifidobacteria).

According to the relative arrangement of cells:

Randomly located (salmonella);

Arranged in pairs (diplobacteria);

Chains (streptobacteria);

In the form of brushwood (mycobacterium tuberculosis);

In the form of packs of cigarettes (the causative agent of leprosy);

At an angle (the causative agent of diphtheria).

Twisted bacteria combine:

Vibrios - have a cylindrical curved shape, forming 1/2-1/4 of a spiral curl, resembling a comma in shape;

Spirilla have the shape of spirally twisted sticks with 4-6 turns;

Spirochetes spirally convoluted forms in which there are 2 types of turns: primary turns formed by the bends of the protoplasmic cylinder, and secondary turns representing the bends of the whole body.

Methods for determining the type of microbes

Determination of the type of microorganisms is carried out using the following research methods:

- bacterioscopic methodstudy of microorganisms by microscopying them in a living or stained state;

- bacteriological methodstudy of the nature of microbial growth on solid and liquid nutrient media, determination of the enzymatic activity of microbes, identification of microbes (species identification);

- serological methodstudy of the antigenic structure of microbes;

- biological method (experimental)study of the pathogenic properties of bacteria using laboratory animals;

- molecular biological methodstudy of the genetic characteristics of microbes.

Using these methods, the following properties of microbes are studied:

- morphologicalproperties shape and size of bacteria;

Tinctorial properties ratio of bacteria to dyes;

Cultural properties growth pattern on nutrient media;

Biochemical activity fermentation of carbohydrates, proteins and other compounds;

Antigenic structure of bacteria;

Pathogenicity;

Genetic characteristics of microbes.

Bacterioscopic research method

Microbial cells can be studied both in a living state (crushed drop method and hanging drop method), and in a fixed and stained state.

Crushed drop method. A drop of the test material or a suspension of bacteria is applied to the surface of a degreased glass slide and covered with a coverslip. The drop should not extend beyond the edges of the cover glass. Microscope the specimen with a x40 lens. The crushed drop method is convenient for studying the motility of bacterial cells, as well as for studying large microorganisms - molds, yeasts.

Hanging drop method. The drug is prepared on a cover glass, in the center of which a drop of bacterial suspension is applied. Then a special glass slide with a well, the edges of which are pre-lubricated with Vaseline, is pressed against the cover glass so that the drop is in the center of the well. The preparation is turned over with the cover glass facing up. In a properly prepared preparation, the drop should hang freely above the well, without touching its bottom or edges. For microscopy, first use a dry x8 lens, under the magnification of which the edges of the drop are found, and then install an x40 lens and examine the preparation.

Preparation of fixed preparations. To prepare the preparation, a drop of water or an isotonic sodium chloride solution is applied to a fat-free glass slide, into which the test material is introduced using a bacteriological loop and the loops are distributed in a circular motion so as to obtain a thin and uniform smear with a diameter of 1-1.5 cm. If liquid material is examined , then it is applied directly to a glass slide with a loop and a smear is prepared. The smears are air dried.

For fixation use physical and chemical methods. To fix the smear physical method The slide is slowly passed through the burner flame 3 times. Blood smears, smears of organs and tissues are recorded chemical method by immersing them for 5-20 minutes in methyl or ethanol, Nikiforov's mixture and other fixing liquids.

For coloring microbes use simple and complex methods. At simple method a fixed smear is stained with any one dye, for example, aqueous solution fuchsin (1-2 minutes) or methylene blue (3-5 minutes), washed with water, dried and microscopically. Complex dyeing methods involve the sequential use of several dyes. This makes it possible to identify certain cell structures and differentiate some types of microorganisms from others.

Immersion microscopy technique

Immersion objectives are most often used for bacterioscopic examination of microorganisms. Unlike dry objectives, when working with which there is air between the preparation and the objective lens, when using immersion objectives, a liquid having a refractive index close to the refractive index of glass is placed between the objective lens and the preparation. The role of such a liquid is performed by immersion oil, most often cedar oil. Light rays passing through a homogeneous optical medium (glass and oil) do not change their direction. This allows you to significantly improve image clarity. Immersion lenses differ from dry lenses in their design (movable front lens) and in appearance: on their frame there is a black circular cut and the designation MI (oil immersion) is engraved.

Oil immersion microscopy requires good illumination of the object. For this purpose it is used additional system lenses located under the stage condenser. When preparing the microscope for use, the condenser is moved up until it stops using a special screw. A drop of immersion oil is applied to the stained smear and the glass is placed on the stage. Under visual control, the lens is lowered from the side until it comes into contact with the drop. After immersing the lens in a drop of oil, the contours of the object are determined by rotating the macrometric screw, and then a clear image of the object is established using the micrometric screw.

After completion of microscopy, the immersion objective is raised, the specimen is removed, and the front lens of the objective is wiped of oil residues with a soft cloth. The lens is then set to low magnification or neutral and the condenser is lowered.

Test questions on the topic of the lesson:

1. Construction of a bacteriological laboratory.

2. Rules for working in a bacteriological laboratory.

3. Principles of classification of microorganisms.

4. Shapes of bacterial cells.

5. Methods for determining the type of microbes.

6. Microscopy technique with immersion system.

Literature to prepare for the lesson:

Main literature:

1. Medical microbiology, virology and immunology. Ed. A.A. Vorobyova. M., 2004.

Additional literature:

1. L.B. Borisov. Medical microbiology, virology, immunology. M., 2002.

2. O.K. Pozdeev. Medical microbiology. M., GEOTAR-MEDIA, 2005.

3. Medical microbiology. Directory. Ed. IN AND. Pokrovsky and O.K. Pozdeeva. M., GEOTAR-MED, 1998.

General information

Bacteriological laboratories as independent structural units organized at sanitary-epidemiological stations (SES), in infectious diseases hospitals, hospitals general type, some specialized hospitals (for example, in tuberculosis, rheumatology, dermatovenerology) and in clinics.

Bacteriological laboratories at the SES examine environmental objects for general bacterial contamination, as well as contamination of environmental objects with conditionally pathogenic and pathogenic microflora: air, water, soil, food; conduct a survey of organized teams and individuals for carriage of pathogenic bacteria of the intestinal group, corynebacteria diphtheria, whooping cough, parapertussis, meningococcus. The work of the microbiological laboratory in conjunction with other departments of the SES has a specific task - health improvement environment and reducing the incidence of the population.

Bacteriological laboratories at medical institutions perform tests necessary to establish and clarify the diagnosis infectious disease, contributing the right choice specific treatment and determining the timing of the patient’s discharge from the infectious diseases hospital. Subjects for research in bacteriological laboratories are:

• secretions from the human body: urine, feces, sputum, pus, as well as blood, cerebrospinal fluid and cadaveric material;
• environmental objects: water, air, soil, food, washouts from equipment, hands, etc.

Bacteriological laboratory premises and workplace equipment

The specifics of microbiological work require that the room allocated for the laboratory be isolated from hospital wards, living rooms, and food units. The bacteriological laboratory includes: laboratory rooms for bacteriological research and utility rooms; autoclave or sterilization for disinfection of waste material and contaminated utensils; washing room equipped for washing dishes; media cooker for preparing, filling, sterilizing and storing nutrient media; vivarium for keeping experimental animals; material for storing spare reagents, dishes, equipment and household equipment.

The listed utility rooms, as independent structural units, are part of large bacteriological laboratories. In small laboratories, the preparation and sterilization rooms are combined in one room; There is no special room for keeping experimental animals.

The brightest, most spacious rooms are allocated for the laboratory rooms in which all bacteriological studies are carried out. The walls in these rooms, to a height of 170 cm from the floor, are painted in light colors with oil paint. The floor is covered with relin or linoleum. This type of finish allows you to use disinfectant solutions when cleaning the room.

Each room should have a sink with running water and a shelf for a bottle of disinfectant solution.

One of the rooms is equipped with a glass box with a pre-box for performing work under aseptic conditions. A table for sowing and a stool are placed in the box, and bactericidal lamps are mounted above the workplace. A cabinet for storing sterile material is placed in the antechamber. The laboratory room is equipped with laboratory tables, cabinets and shelves for storing equipment, utensils, paints, and reagents necessary for work.

Very great importance for work there is a proper organization of the workplace of a doctor - bacteriologist and laboratory assistant. Laboratory tables are installed near the windows. When placing them, you should strive to ensure that the light falls in front or from the side of the person working, preferably on the left side, but in no case from behind. It is advisable that rooms for analysis, especially for microscopy, have windows oriented to the north or northwest, since equal diffuse light is necessary for work. The surface illumination of tables for work should be 500 lux. For ease of disinfection, the surface of laboratory tables is covered with plastic, and each workplace on it is covered with mirror glass.

Each laboratory employee is assigned a separate workplace with an area of ​​150x60 cm. All workplaces are equipped with items necessary for daily work.

Rules of work and behavior in the laboratory

A feature of bacteriological work is the constant contact of laboratory staff with infectious material, cultures of pathogenic microbes, infected animals, blood and secretions of the patient. Therefore, all employees of the bacteriological laboratory are required to comply with the following work rules, which ensure sterility in work and prevent the possibility of intra-laboratory infections:

1. You cannot enter the premises of the bacteriological laboratory without special clothing - a robe and a white cap or headscarf.
2. You must not bring foreign objects into the laboratory.
3. It is prohibited to leave the laboratory in gowns or put an outer gown over a gown.
4. Smoking, eating, and storing food products are strictly prohibited in the premises of the bacteriological laboratory.
5. All material entering the laboratory must be considered contaminated.
6. When unpacking sent infectious material, care must be taken: upon receipt, jars containing material for research are wiped outside with a disinfectant solution and placed not directly on the table, but on trays or in cuvettes.
7. Transfusion of liquids containing pathogenic microbes is carried out over a vessel filled with a disinfectant solution.
8. Any accident involving glassware containing infectious material or spillage of liquid infectious material must be reported immediately to the head of the laboratory or his deputy. Measures to disinfect body parts, workplace objects and surfaces contaminated with pathogenic dress material are carried out immediately.
9. When studying infectious material and working with pathogenic microbial cultures, it is necessary to strictly adhere to the generally accepted technical techniques in bacteriological practice, which exclude the possibility of hand contact with infectious material.
10. Infected material and unnecessary crops must be destroyed, if possible on the same day. Tools used in working with infectious material are immediately disinfected after use, as is the surface of the workplace.
11. When performing bacteriological work, you must strictly monitor the cleanliness of your hands: after finishing work with infectious material, they are disinfected. At the end of the day, the workplace is tidied up and thoroughly disinfected, and the infectious material and microbial cultures necessary for further work are stored in a locked refrigerator or safe.
12. Bacteriological laboratory workers are subject to mandatory vaccination against those infectious diseases whose pathogens may be found in the objects under study.

Laboratory room cleaning

The microbiology laboratory must be kept clean. Hygienic cleaning of laboratory premises should be carried out regularly. It is very difficult to ensure complete sterility of the laboratory and it is not always necessary, but it is possible to significantly reduce the number of microorganisms in the air and on various surfaces in laboratory premises. This is achieved through the practical application of disinfection methods, that is, the destruction of pathogens of infectious diseases in environmental objects.

Floor, walls and furniture in a microbiological laboratory, they are vacuumed and wiped with various disinfectant solutions. Vacuum cleaning ensures that objects are freed from dust and a significant number of microorganisms are removed from them. It has been established that when a vacuum cleaner brush is passed 4 times over the surface of an object, approximately 47% of microorganisms are removed from it, and when it is passed 12 times, up to 97%. The most commonly used disinfectant solutions are a 2-3% solution of soda (sodium bicarbonate) or Lysol (a phenol preparation with the addition of green soap), a 0.5-3% aqueous solution of chloramine and some other disinfectants.

Air In the laboratory, the easiest way to disinfect is by airing. Prolonged ventilation of the room through the window (at least 30-60 minutes) leads to sharp decline the number of microorganisms in the air, especially when there is a significant difference in temperature between the outside air and the indoor air. A more effective and most commonly used method of air disinfection is irradiation with UV rays with a wavelength from 200 to 400 nm. These rays have a high antimicrobial activity and can cause the death of not only vegetative cells, but also microbial spores.

Literature

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