The bacteriological laboratory is studying. Bacteriological laboratory - laboratories, paraclinics

Introduction

Like the general part of any other science, general bacteriology deals not with specific issues (say, the identification of individual species of bacteria), but with problems in general; its methodology covers basic procedures that are widely used in a wide variety of laboratory studies. This teaching aid is not aimed at identifying any group of microorganisms. This is the task of the following publications - on private and sanitary microbiology. However, the methods presented herein can be useful in any field where bacteria are involved and can be applied to practical problems involving the isolation and typification of bacteria.

Bacteriology became a science only after unique techniques were developed through which it continues to influence and penetrate later scientific fields such as virology, immunology and molecular biology. The technique of using pure cultures developed by R. Koch and the immunological reactions and chemical analysis first used by L. Pasteur have not lost their significance.

The methodology of general bacteriology is reflected in this publication using a structure that is typical of standard textbooks in this discipline. However, unlike laboratory workshops on the microbiology course for universities, it is presented in more detail in some sections and is for reference only. This structure takes into account the specific training and specialization of bacteriologists and veterinary experts. Often the material is presented arbitrarily, so some methods are mentioned several times, due to the desire to demonstrate their interrelation.

Bacteriological laboratory

Bacteriological laboratories as structural units are organized as part of regional, district, inter-district veterinary laboratories, as well as in the structure of zonal veterinary laboratories. They are also organized at sanitary and epidemiological surveillance centers, in infectious diseases hospitals, general hospitals, some specialized hospitals (for example, in tuberculosis, rheumatology, dermatovenerology), and in polyclinics. Bacteriological laboratories are part of specialized research institutions. Bacteriological laboratories are constantly used to confirm or establish an assessment of the suitability of meat for food purposes according to VSE.

The objects of research in bacteriological laboratories are:

1. Discharges from the body: urine, feces, sputum, pus, as well as blood, pathological and cadaveric material.

2. Objects of the external environment: water, air, soil, washouts from equipment, feed, technological raw materials obtained from the slaughter of farm animals.

3. Food products, samples of meat and meat products, milk and dairy products that need to be assessed for suitability for food purposes.

Bacteriological laboratory premises and workplace equipment

The specifics of microbiological work require that the room allocated for the laboratory be isolated from living rooms, food units and other non-core production premises.

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; bacteriological kitchen – for preparing, bottling, 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 bacteriological kitchen and the sterilization kitchen are combined in one room; There is no special room for keeping experimental animals.

The premises of microbiological laboratories are divided into 2 zones according to the degree of danger for personnel:

I. “Infectious” zone - a room or group of laboratory rooms where pathogenic biological agents are manipulated and stored; personnel are dressed in the appropriate type of protective clothing.

II. “Clean” zone - premises where no work is carried out with biological material, personnel are dressed in personal clothing.

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 or covered with tiles. 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 - an isolated room with a vestibule (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. Windows and doors of rooms in the “contagious” zone must be sealed. Existing exhaust ventilation from the “infectious” zone must be isolated from other ventilation systems and equipped with fine air filters.

The laboratory room is equipped with laboratory-type tables, cabinets and shelves for storing equipment, utensils, paints and reagents necessary for work.

The correct organization of the workplace for the bacteriologist and laboratory assistant is very important for work. 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 north or northwest, since work requires even, diffused light. The surface illumination of tables for work should be 500 lux. For ease of disinfection, the surface of laboratory tables is covered with plastic or upholstered with iron. Each laboratory employee is assigned a separate workplace measuring 150 x 60 cm.

All workplaces are equipped with items necessary for everyday bacteriological work, a list of which is given in Table 1.

Table 1.

Necessary items for bacteriological work

Item name	Approximate quantity
1. Set of paints and reagents for painting
2. Glass slides	25-50
3. Cover glasses	25-50
4. Glasses with holes	5-10
5. Test tube rack
6. Bacterial loop
7. Glass spatulas
8. Metal spatulas
9. Jar of cotton wool
10. Pipettes graduated to 1, 2, 5, 10 ml	25 of each volume
11. Pasteur pipettes	25-50
12. Tweezers, scissors, scalpel	By 1
13. Containers with disinfectant solutions
14. Microscope with illuminator
15. Magnifying glass 5´
16. Oiler with immersion oil
17. Filter paper	3-5 sheets
18. Jar with disinfectant solution for pipettes
19. Alcohol or gas burner
20. Installation for coloring preparations
21. Hourglass for 1 or 2 minutes	By 1
22. Pear with a rubber tube
23. Pencil on glass
24. Jar of alcohol cotton wool
25. Necessary sterile utensils	-

Disinfection

Disinfection is the destruction of microorganisms in environmental objects.

In microbiological laboratories, disinfection measures are used very widely. When finishing work with infectious material, employees of bacteriological laboratories carry out preventive disinfection of their hands and workplace.

Spent pathological material (feces, urine, sputum, various types of liquid, blood) is disinfected before throwing it into the sewer.

Graduated and Pasteur pipettes, glass spatulas and metal instruments contaminated with pathological material or a culture of microbes, immediately after their use, are lowered into glass jars with a disinfectant solution located on the table at each workplace.

Slides and cover glasses used in work are also subject to mandatory disinfection, since even a fixed and stained smear sometimes retains viable microorganisms that can be a source of intra-laboratory contamination. Only those dishes in which microorganisms were grown are not treated with disinfectants. It is placed in metal tanks or bins, sealed and submitted for autoclaving.

The choice of disinfectant, the concentration of its solution, the ratio between the amount of disinfectant and the material being disinfected, as well as the duration of the disinfection period are established depending on specific conditions, taking into account, first of all, the stability of the disinfected microbes, the degree of expected contamination, the composition and consistency of the material in which they are located .

Disinfection of hands after working with infectious material and when it comes into contact with the skin. Upon completion of work with infectious material, hands are disinfected prophylactically. A cotton ball or gauze napkin is moistened with a 1% chloramine solution, first the left, then the right hand is wiped in the following sequence: the back of the hand, the palmar surface, the interdigital spaces, the nail beds. Thus, the least contaminated areas are treated first, then move on to the most heavily contaminated areas of the skin. Wipe your hands for 2 minutes with two tampons in succession. When hands become contaminated with a culture of a pathogenic microbe or pathological material, the contaminated areas of the skin are first disinfected. For this purpose, they are covered for 3-5 minutes with cotton wool moistened with a 1% chloramine solution, then the cotton wool is dumped into a tank or bucket with waste material, and the hands are treated with a second swab in the same way as during preventive disinfection. After treatment with chloramine, wash your hands with warm water and soap. When working with bacteria that form spores, hand treatment is carried out with 1% activated chloramine. If infectious material comes into contact with hands, the exposure to the disinfectant is increased to 5 minutes.

Sterilization

Sterilization, in contrast to disinfection, involves the destruction of all vegetative and spore-bearing, pathogenic and non-pathogenic microorganisms in the object being sterilized. Sterilization is carried out in various ways: steam, dry hot air, boiling, filtration, etc. The choice of one or another sterilization method is determined by the quality and properties of the microflora of the object being sterilized.

Preparation and sterilization of laboratory equipment

Before sterilization, laboratory glassware is washed and dried. Test tubes, vials, bottles, mattresses and flasks are closed with cotton-gauze stoppers. Place paper caps over the stoppers on each vessel (except test tubes).

Rubber, cork and glass stoppers are sterilized in a separate bag tied to the neck of the dish. Petri dishes are sterilized wrapped in paper, 1-10 pieces each. Pasteur pipettes 3-15 pcs. wrapped in wrapping paper. A piece of cotton wool is placed in the top of each pipette to prevent the material from entering the environment. When wrapping the pipettes, great care must be taken so as not to break off the sealed ends of the capillaries. During operation, the pipettes are removed from the bag by the upper end.

Safety cotton is inserted into the upper part of graduated pipettes, as in Pasteur pipettes, and then wrapped in thick paper, pre-cut into strips 2-2.5 cm wide and 50-70 cm long. The strip is placed on the table, its left end is folded and wrapped the tip of the pipette, then, rotating the pipette, wrap a strip of paper onto it. To prevent the paper from unfolding, the opposite end is twisted or glued. The volume of the wrapped pipette is written on the paper. If there are pencil cases, graduated pipettes are sterilized in them.

Laboratory glassware is sterilized:

a) dry heat at a temperature of 180°C and 160°C, respectively, for 1 hour and 150 minutes.

b) in an autoclave at a pressure of 1.5 atm. for 60 minutes, to destroy spore microflora - 90 minutes at 2 atm.

Sterilization of syringes. Syringes are sterilized in disassembled form: separately the cylinder and piston in a 2% solution of sodium bicarbonate for 30 minutes. When working with spore-bearing microflora, sterilization is carried out in an autoclave at 132±2°C (2 atm.) for 20 minutes, at 126±2°C (1.5 atm.) - 30 minutes. The sterilized syringe is assembled after it has cooled, a piston is inserted into the cylinder, a needle is put on, having first removed the mandrel from it. The needle, cylinder and piston are taken with tweezers, which are sterilized along with the syringe.

Sterilization of metal instruments. Metal instruments (scissors, scalpels, tweezers, etc.) are sterilized in a 2% sodium bicarbonate solution, which prevents rust and loss of sharpness. It is recommended to wrap the blades of scalpels and scissors with cotton wool before immersing them in the solution.

Sterilization of bacterial loops. Bacterial loops made of platinum or nichrome wire are sterilized in the flame of an alcohol or gas burner. This method of sterilization is called calcination or flambéing.

The loop is placed in a horizontal position in the lower, coldest part of the burner flame to prevent splashing of the burned pathogenic material. After it burns, the loop is moved to a vertical position, first the lower, then the upper part of the wire is heated red-hot and the loop holder is burned through. Calcination generally takes 5-7 seconds.

Preparation for sterilization and sterilization of paper, gauze and cotton wool. Cotton wool, gauze, filter paper are sterilized in a dry-heat oven at a temperature of 160°C within an hour from the moment the thermometer reads this temperature or in an autoclave at a pressure of 1 atm. within 30 minutes.

Before sterilization, paper and gauze are cut into pieces, and cotton wool is rolled into balls or tampons of the required size. After this, each type of material separately, one or several pieces, is wrapped in thick paper. If the package breaks, the sterilized material should be sterilized again, since its sterility is impaired.

Sterilization of gloves and other rubber products. Rubber products (gloves, tubes, etc.) contaminated with the vegetative form of microbes are sterilized by boiling in a 2% sodium bicarbonate solution or running steam for 30 minutes; when contaminated with spore-bearing microflora, in an autoclave at a pressure of 1.5-2 atm. for 30 or 20 minutes. Before sterilization, rubber gloves are sprinkled with talc inside and outside to prevent them from sticking. Gauze is placed between the gloves. Each pair of gloves is wrapped separately in gauze and in this form is placed in boxes.

Sterilization of pathogenic microbial cultures. Test tubes and cups containing microbial cultures that are not needed for further work are placed in a metal tank, the lid is sealed and submitted for sterilization. Cultures of pathogenic microbes, vegetative forms, are killed in an autoclave for 30 minutes at a pressure of 1 atm. The delivery of tanks for sterilization to the autoclave is carried out by a specially designated person against a signature. The sterilization regime is recorded in a special journal. When destroying cultures of microbes of pathogenicity groups I and II, as well as material contaminated or suspected of being contaminated with pathogens assigned to these groups, tanks with waste material are transferred on metal trays with high sides in the presence of an accompanying person authorized to work with infectious material.

Types of sterilization

Sterilization by boiling. Sterilization by boiling is carried out in a sterilizer. Distilled water is poured into the sterilizer, since tap water forms scale. (Glass objects are immersed in cold water, metal objects in hot water with the addition of sodium bicarbonate). The items to be sterilized are boiled over low heat for 30-60 minutes. The beginning of sterilization is considered to be the moment the water boils in the sterilizer. At the end of boiling, the instruments are taken with sterile tweezers, which are boiled along with other items.

Dry heat sterilization. Dry heat sterilization is carried out in a Pasteur oven. The material prepared for sterilization is placed on the shelves so that it does not come into contact with the walls. The cabinet is closed and then the heating is turned on. The duration of sterilization at a temperature of 150°C is 2 hours, at 165°C - 1 hour, at 180°C - 40 minutes, at 200°C - 10-15 minutes (at 170°C paper and cotton wool turn yellow, and at higher temperatures charred). The beginning of sterilization is considered to be the moment when the temperature in the oven reaches the desired height. At the end of the sterilization period, the oven is turned off, but the cabinet doors are not opened until completely cooled, since cold air entering the cabinet can cause cracks to form on hot dishes.

Steam sterilization under pressure. Steam sterilization under pressure is carried out in an autoclave. The autoclave consists of two boilers inserted one into the other, a casing and a lid. The outer boiler is called a water-steam chamber, the inner boiler is called a sterilization chamber. Steam is generated in a water-steam boiler. The material to be sterilized is placed in the inner cauldron. There are small holes in the top of the sterilization boiler through which steam from the water-steam chamber passes. The autoclave lid is hermetically screwed to the casing. In addition to the main parts listed, the autoclave has a number of parts that regulate its operation: pressure gauge, water meter glass, safety valve, outlet, air and condensation valves. The pressure gauge is used to determine the pressure created in the sterilization chamber. Normal atmospheric pressure (760 mm Hg) is taken as zero, therefore, in an idle autoclave, the pressure gauge needle is at zero. There is a certain relationship between the pressure gauge readings and temperature (Table 2).

Table 2.

Relationship between pressure gauge readings and water boiling temperature

The red line on the pressure gauge scale determines the maximum operating pressure that is allowed in the autoclave. The safety valve serves to protect against excessive pressure build-up. It is set to a given pressure, that is, the pressure at which sterilization must be carried out; when the pressure gauge needle crosses the line, the autoclave valve automatically opens and releases excess steam, thereby slowing down the further rise in pressure.

On the side wall of the autoclave there is a water gauge glass showing the water level in the water-steam boiler. There are two horizontal lines on the water meter glass tube - lower and upper, indicating the permissible lower and upper water levels in the water-steam chamber, respectively. The air valve is designed to remove air from the sterilization and water-steam chambers at the beginning of sterilization, since air, being a poor heat conductor, disrupts the sterilization regime. At the bottom of the autoclave there is a condensation tap to free the sterilization chamber from condensate formed during the heating period of the sterilized material.

Rules for working with an autoclave. Before starting work, inspect the autoclave and control equipment. In autoclaves with automatic steam control, the arrows on the electric vacuum pressure gauge of the water-steam chamber are set in accordance with the sterilization mode: the lower arrow is set to 0.1 atm. lower, upper - by 0.1 atm. above the operating pressure, the water-steam chamber is filled with water to the upper mark of the measuring glass. During the period of filling with water, the valve on the pipe through which steam enters the chamber is kept open to allow free air exit from the boiler. The sterilization chamber of the autoclave is loaded with the material to be sterilized. After this, the lid (or door) of the autoclave is closed, tightly secured with a central lock or bolts; to avoid distortion, screw the bolts crosswise (along the diameter). Then turn on the heating source (electric current, steam), closing the valve on the pipe connecting the steam source to the sterilization chamber. With the beginning of steam formation and creation of pressure in the water-steam chamber, purging is carried out (removing air from the sterilization boiler). The method of air removal is determined by the design of the autoclave. At first, the air comes out in separate portions, then a smooth, continuous stream of steam appears, indicating that the air has been completely displaced from the sterilization chamber. After removing the air, the tap is closed, and a gradual increase in pressure begins in the sterilization chamber.

The beginning of sterilization is considered to be the moment when the pressure gauge needle shows the set pressure. After this, the heating intensity is reduced so that the pressure in the autoclave remains at the same level for the required time. At the end of the sterilization time, heating is stopped. Close the valve in the pipeline supplying steam to the sterilization chamber, and open the valve on the condensation (down) pipe to reduce the steam pressure in the chamber. After the pressure gauge needle drops to zero, slowly loosen the clamping devices and open the lid of the autoclave.

The temperature and duration of sterilization are determined by the quality of the material being sterilized and the properties of the microorganisms with which it is infected.

Temperature control in the sterilization chamber is carried out periodically using bacteriological tests. Biotests are produced by the bacteriological laboratories of the Center for Sensitive Sciences. If these tests are not passed, the technical condition of the autoclave is checked.

Sterilization with flowing steam. Sterilization with flowing steam is carried out in a Koch flowing steam apparatus or in an autoclave with the lid unscrewed and the outlet valve open. The Koch apparatus is a metal hollow cylinder with a double bottom. The space between the upper and lower bottom plates is filled 2/3 with water (there is a tap to drain the remaining water after sterilization). The lid of the device has a hole in the center for a thermometer and several small holes for steam to escape. The material to be sterilized is loaded loosely into the chamber of the device to ensure maximum contact with the steam. The beginning of sterilization is considered to be the time from the moment water boils and steam enters the sterilization chamber. In a fluid steam apparatus, nutrient media are mainly sterilized, the properties of which change at temperatures above 100°C. Sterilization with flowing steam should be repeated, since a single heating at a temperature of 100°C does not ensure complete disinfection. This method is called fractional sterilization: the sterilized material is treated with flowing steam for 30 minutes daily for 3 days. In the intervals between sterilizations, the material is kept at room temperature for the spores to germinate into vegetative forms, which die during subsequent heating.

Tyndalization. Tyndalization is fractional sterilization using temperatures below 100°C, proposed by Tyndall. The material to be sterilized is heated in a water bath equipped with a thermostat for an hour at a temperature of 60-65°C for 5 days or at 70-80°C for 3 days. In the intervals between heating, the treated material is kept at a temperature of 25°C for the spores to germinate into vegetative forms, which die during subsequent heating. Tindalization is used to defertilize nutrient media containing protein.

Mechanical sterilization using bacterial ultrafilters. Bacterial filters are used to free liquid from bacteria contained in it, as well as to separate bacteria from viruses, phages and exotoxins. Viruses are not retained by bacterial filters, and therefore ultrafiltration cannot be considered as sterilization in the accepted meaning of the word. For the manufacture of ultrafilters, finely porous materials (kaolin, asbestos, nitrocellulose, etc.) are used that can trap bacteria.

Asbestos filters (Seitz filters) are asbestos plates with a thickness of 3-5 mm and a diameter of 35 and 140 mm for filtering small and large volumes of liquid. In our country, asbestos filters are made in two brands: “F” (filtering), which retain suspended particles but allow bacteria to pass through, and “SF” (sterilizing), denser, retaining bacteria. Before use, asbestos filters are mounted in filter devices and together with them are sterilized in an autoclave. Asbestos filters are used once. Membrane ultrafilters are made of nitrocellulose and are white discs with a diameter of 35 mm and a thickness of 0.1 mm.

Bacterial filters differ in pore size and are designated by serial numbers (Table 3).

Table 3.

Bacterial filters

Immediately before use, membrane filters are sterilized by boiling. The filters are placed in distilled water, heated to a temperature of 50-60°C, to prevent them from twisting, and boiled over low heat for 30 minutes, changing the water 2-3 times. To avoid damage, sterilized filters are removed from the sterilizer with flambéed and cooled tweezers with smooth tips.

To filter liquids, bacterial filters are mounted in special filter devices, in particular, a Seitz filter.

It consists of 2 parts: an upper one, shaped like a cylinder or funnel, and a lower supporting part of the apparatus, with a so-called filter table made of a metal mesh or a clean ceramic plate, on which a membrane or asbestos filter is placed. The supporting part of the apparatus has the shape of a funnel, the tapering part of which is located in the rubber stopper of the neck of the Bunsen flask. In working condition, the upper part of the device is fixed to the lower part with screws. Before filtration begins, the junctions of various parts of the installation are filled with paraffin to create a seal. The outlet tube of the flask is connected with a thick-walled rubber tube to a water-jet, oil or bicycle pump. After this, the filtered liquid is poured into the cylinder or funnel of the apparatus and the pump is turned on, creating a vacuum in the receiving vessel. As a result of the resulting pressure difference, the filtered liquid passes through the pores of the filter into the receiver. Microorganisms remain on the surface of the filter.

Preparation of smears

To study microorganisms in stained form, a smear is made on a glass slide, dried, fixed, and then stained.

The test material is distributed in a thin layer over the surface of a well-degreased glass slide.

Smears are prepared from microbial cultures, pathological material (sputum, pus, urine, blood, etc.) and from the organs of corpses.

The technique for preparing smears is determined by the nature of the material being studied.

Preparation of smears from microbial cultures with a liquid nutrient medium and from liquid pathological material (urine, cerebrospinal fluid, etc.). A small drop of the test liquid is applied with a bacterial loop onto a glass slide and, using circular movements, the loops are distributed in an even layer in the form of a circle with the diameter of a penny coin.

Preparation of blood smears. A drop of blood is applied to the glass slide, closer to one of its ends. The second - ground - glass, which should be narrower than the object glass, is placed on the first at an angle of 45° and brought to a drop of blood until it comes into contact with it. After the blood has spread along the polished edge, a sliding movement is made with the glass from right to left, evenly distributing the blood in a thin layer over the entire surface of the glass. The thickness of the stroke depends on the angle between the glasses: the sharper the angle, the thinner the stroke. A properly prepared smear has a light pink color and the same thickness throughout.

Preparing a thick drop. A drop of blood is applied to the middle of the slide using a Pasteur pipette, or the glass is applied directly to the protruding drop of blood. The blood applied to the glass is smeared with a bacterial loop so that the diameter of the resulting smear corresponds to the size of a penny coin. The glass is left in a horizontal position until the blood dries. The blood in the “thick drop” is distributed unevenly, forming an uneven edge.

Preparation of a smear from a viscous material (sputum, pus). Sputum or pus applied to the slide closer to the narrow edge is covered with another slide. The glasses are slightly pressed against each other.

After this, the free ends of the glass are grabbed with fingers 1 and 2 of both hands and moved in opposite directions so that when moving, both glasses fit tightly to each other. The result is smears with evenly distributed material occupying a large area.

Preparation of smears from cultures from solid nutrient media. A drop of water is placed in the middle of a clean, well-greased glass slide, and a bacterial loop with a small amount of the microbial culture being studied is inserted into it, so that the drop of liquid becomes slightly cloudy. After this, the excess microbial material on the loop is burned in a flame and the smear is prepared according to the method described above.

Preparation of smears from organs and tissues. For the purpose of disinfection, the surface of the organ is cauterized with the heated jaws of tweezers, an incision is made in this place and a small piece of tissue is cut out from the depths with pointed scissors, which is placed between two glass slides. Next proceed in the same way as when preparing a smear from pus and sputum. If the tissue of the organ is dense, then a scraping is made from the depth of the incision with a scalpel. The material obtained by scraping is distributed in a thin layer over the surface of the glass with a scalpel or bacterial loop.

To study the relative position of tissue elements and the microorganisms in it, fingerprint smears are made. To do this, a small piece of tissue cut out from the middle of the organ is grabbed with tweezers and applied successively several times with the cut surface to a glass slide, thus obtaining a series of smears-imprints.

Drying and fixing smears. The smear prepared on a glass slide is dried in air and fixed after complete drying. When fixed, the smear is fixed on the surface of the glass slide, and therefore, during subsequent staining of the preparation, microbial cells are not washed off. In addition, killed microbial cells stain better than living ones.

There is a physical method of fixation, which is based on the effect of high temperature on the microbial cell, and chemical methods, which involve the use of agents that cause coagulation of proteins. Do not record smears containing pathogens of pathogenicity groups I–II over a flame.

Physical method of fixation. The slide with the preparation is taken with tweezers or fingers I and II of the right hand by the ribs with a stroke upward and smoothly moved 2-3 times over the upper part of the burner flame. The entire fixation process should take no more than 2 seconds. The reliability of fixation is checked by the following simple technique: the smear-free surface of a glass slide is applied to the back surface of the left hand. When the smear is correctly fixed, the glass should be hot, but not cause a burning sensation.

Chemical fixation method. To fix smears, the chemicals and compounds listed in Table 4 are also used.

Table 4.

Substances for chemical fixation

The slide with the dried smear is immersed in a bottle with a fixing agent and then dried in air.

Coloring strokes

Smear painting technique. To color smears, paint solutions or coloring paper are used, as proposed by A.I. Sinev. Simplicity of preparation, ease of use, as well as the ability to store ink paper for an indefinitely long time were the basis for their widespread use in various coloring methods.

Coloring strokes with ink paper. A few drops of water are applied to the dried and fixed preparation, and colored pieces of paper measuring 2´2 cm are placed. During the entire time of staining, the paper should remain moist and fit tightly to the surface of the glass. When drying, the paper is additionally moistened with water. The duration of staining of the smear is determined by the staining method. At the end of staining, the paper is carefully removed with tweezers, and the smear is washed with tap water and dried in air or with filter paper.

Staining smears with dye solutions. The dye is applied to the dried and fixed preparation with a pipette in such an amount that it covers the entire smear. When staining smears with concentrated solutions of dyes (Ziehl carbol fuchsin, carbolic gentian or crystal violet), staining is done through filter paper that retains dye particles: a strip of filter paper is placed on a fixed smear and a dye solution is poured onto it.

For microscopic examination, prepared smears, dried and fixed, are stained. Coloring can be simple or complex. Simple painting involves applying one paint to a smear for a certain period of time. Most often, for simple staining, alcohol-water (1:10) Pfeiffer fuchsin, Leffler's methylene blue and safranin are used. Eosin, like an acidic paint, is used only for staining the cytoplasm of cells and tinting the background. Acid fuchsin is completely unsuitable for staining bacteria.

photo from the site lentachel.ru

Just a hundred years ago, contamination during scientific research was considered virtually inevitable. Many scientists put their bodies at mortal risk by studying microbes and bacteria about the nature of which they knew little. Today, most of the dangerous microorganisms that surround us have been described and studied; moreover, special medical products are provided for bacteriological laboratories, the use of which with 99% probability protects researchers from any professional risks.

All objects that the bacteriological laboratory employees work with are saturated with pathogenic microflora. To maintain a healthy indoor environment and avoid direct contact with contaminated material, furniture, clothing and utensils with enhanced barrier and antimicrobial properties are used.

Hermetically sealed glass and metal cabinets and boxes, laboratory tables convenient for disinfection, sterilization and autoclave equipment, and a locked refrigerator are items that ensure the safety of people conducting research on infected samples.

All containers used for storing samples: flasks, graduated beakers are hermetically sealed to avoid the spread of microbes in the laboratory air.

To make containers, special unbreakable glass or high-strength plastic is used. Double walls, a special stable bottom shape, rubber elements on the lids, trays and cuvettes create the best conditions for isolating such dangerous neighbors as meningococci, streptococci, staphylococci, bacilli and clostridia.

Before starting research, the staff puts on special clothing: a protective gown, mask, goggles. To work with very dangerous substances, rubberized aprons or special gowns with moisture-repellent impregnation are used.

Correct timely treatment of air with ultraviolet irradiators and bactericidal lamps, the use of proven modifications of the washing room, supplying all employees with a full set of protective clothing is a generally accepted standard, deviation from which is administrative, and in case of severe consequences, criminally punishable.

Comprehensive equipment and implementation of all precautionary measures help preserve the health of employees, reduce occupational morbidity, and ensure high research productivity: it has been noted that the use of reliable, proven protective equipment reduces anxiety and promotes faster and more effective actions.

Bacteriological laboratory

The bacteriological laboratory was separated as an independent division in 1996.

Head - Candidate of Medical Sciences, bacteriologist of the highest category Polikarpova Svetlana Veniaminovna

PRIORITY AREAS OF ACTIVITY

The following types of analyzes are performed in the bacteriological laboratory:

Bacteriological examination of blood (hemoculture) and cerebrospinal fluid;
- bacteriological examination of sputum, tracheal aspirate, bronchial lavage water;
- bacteriological examination of discharge from various foci of inflammation: tonsillitis, pharyngitis, otitis, sinusitis, etc.;
- bacteriological examination of punctates, effusions, exudates;
- bacteriological examination of discharge from the mucous membrane of the pharynx and nose for conditionally pathogenic microflora;
- bacteriological examination of urine to determine the degree of bacteriuria;
- bacteriological examination of genital secretions for opportunistic microflora;
- bacteriological examination of conjunctival discharge;
- examination of feces for pathogenic intestinal flora;
- bacteriological examination of stool for dysbacteriosis;
- bacteriological examination of breast milk;
- qualitative determination of group B streptococcal antigen;
- qualitative determination of antigen Helicobacter pylori in human feces;
- qualitative determination of the antigen of toxins A and B Clostridium difficile in human feces;
- assessment of sensitivity/resistance of isolated microorganisms to antibiotics.

ACHIEVEMENTS

In the bacteriological laboratory, research is carried out to isolate, identify and determine the sensitivity to antibiotics of pathogenic and conditionally pathogenic microorganisms isolated from various biomaterials.

The work of the laboratory, based on the principles of classical clinical microbiology, has introduced new research methods that are based on the latest achievements of molecular genetic technologies.

The bacteriological laboratory carries out microbiological studies that meet modern Russian and international standards.

Every year, laboratory staff carry out bacteriological examinations of more than 10,000 patients hospitalized in hospital departments, 6,000 patients and newborns in the maternity hospital, more than 5,000 patients of the CDC and clinics of the Eastern Administrative District, performing more than 45,000 microbiological tests per year.

On the basis of the laboratory, an “Automated workstation for a microbiologist, epidemiologist and chemotherapist” has been developed and is constantly being modernized, which includes two programs: MICROB-AUTOMAT and MICROBIOLOGICAL MONITORING SYSTEM “MICROB” (SMM). The programs allow us to solve the main tasks of the hospital microbiological service: continuous monitoring of the microbial landscape and the level of its antibiotic resistance to prescribe adequate antibiotic therapy and timely detection of cases of healthcare-associated infections (HAI).

To ensure the proper quality of microbiological research, the following are being introduced into the laboratory:

• Quality management system according to standard GOST R ISO 15189 -15

“Medical laboratories. Particular requirements for quality and competence";

• Basics LEAN technologies (Lean manufacturing) - approaches to laboratory management aimed at improving the quality of work by reducing losses: material, financial, time;
• System 5S- a lean manufacturing tool - organizing a workspace in order to create optimal conditions for performing operations, maintaining order, cleanliness, neatness, saving time and energy.

Since 2014, the laboratory has been participating in the international program for monitoring antibiotic resistance in the countries of Central Asia and Eastern Europe, organized by WHO(UK NEQAS). The laboratory participates in external assessment of the quality of laboratory research as an expert laboratory in the section “Clinical Microbiology” (FSVOK).

For many years, bacteriologists have been conducting certification improvement cycles “Modern research methods in clinical microbiology” at the hospital for workers with a secondary medical education from medical institutions in Moscow.

HIGH TECH

• Introduction of molecular genetic methods in the diagnosis of infectious diseases - the real-time polymerase chain reaction (PCR) method;

• determination of the main mechanisms of development and spread of bacterial resistance to antibacterial drugs using phenotypic and genotypic methods;

• the use of immunochromatographic express methods (IMCT) for the etiological diagnosis of pathogens of infectious and inflammatory diseases;

• creation and implementation of an electronic module of expert opinions for bacteriologists, attending physicians, clinical pharmacologists to interpret the results of determining the sensitivity of microorganisms to antimicrobial drugs and identifying mechanisms of resistance.

SCIENTIFIC ACTIVITY

During the existence of the department, 2 candidate dissertations were completed in the laboratory. Employees of the department regularly make presentations at international, All-Russian, city congresses, conferences, symposiums and seminars, and regularly publish in scientific and practical publications.

The bacteriological laboratory cooperates:

• FSBI "Federal Research Center for Epidemiology and Microbiology"

them. Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation";

• FGAU "Scientific Center for Children's Health" of the Ministry of Health of the Russian Federation;
• Federal State Budgetary Institution "Medical Genetic Research Center" of the Russian Academy of Medical Sciences;
• Federal Budgetary Institution Central Research Institute of Epidemiology of Rospotrebnadzor;
• FBUN Moscow Research Institute of Epidemiology and Microbiology named after. G.N. Gabrichevsky Rospotrebnadzor;
• Department of Reproductive Medicine and Surgery of the Moscow State Medical and Dental University named after A.I. Evdokimov (MGMSU);

Scientific work is carried out in the following areas:

• microbiological diagnosis and prevention of healthcare-associated infections (HAI). Microbial ecology in health care facilities.
• features of microbiological diagnosis of lower respiratory tract infections in patients with cystic fibrosis;
• studying the mechanisms of resistance of enterobacteria to antimicrobial drugs;
• development of algorithms for rapid diagnosis of carriage of group B streptococci in pregnant women and newborns.

ARTICLES, CONFERENCES

2016

LABORATORY EQUIPMENT

Currently, the bacteriological laboratory is equipped with modern equipment:

• Automatic blood culture analyzer VersaTREK(TREK Diagnostic Systems) - the ability to diagnose sepsis in the shortest possible time - 90% of positive results are detected within the first 24 hours. The maximum time for a research protocol is 5
• Bacteriological analyzer for identifying and determining the sensitivity of microorganisms Phoenix 100(BD) - average time to obtain identification results is 6-8 hours, average time to obtain antimicrobial sensitivity results is 12-16 hours.
• Semi-automatic analyzer iEMSReader(ThermoLabsystems), which allows not only to identify and determine the sensitivity of microorganisms, but also to solve many practical and scientific-practical problems: determining the microbial contamination of urine and other biomaterials, carrying out laboratory monitoring of the effectiveness of antibacterial therapy, assessing the bactericidal activity of blood serum, and also researching kinetic models of microbial growth.

The ALISA laboratory information system has been introduced into the work of the laboratory; each workstation of a bacteriologist is computerized.

TEAM

Pivkina Nadezhda Vasilievna

Doctor-bacteriologist of the highest category, has professional retraining in the specialty “Bacteriology”. Member of the International Association of Clinical Microbiologists and Antimicrobial Chemotherapists (IACMAC), member of the Federation of Laboratory Medicine (FLM). He is a co-author of many scientific articles on the problems of clinical microbiology. Work experience in the specialty since 1992.

Timofeeva Olga Gennadievna

A bacteriologist of the highest category, she completed an internship in the specialty “Bacteriology” and underwent advanced training in the topic “PCR analysis in clinical diagnostic laboratories.” Member of the Federation of Laboratory Medicine (FLM). Currently working on writing a Ph.D. dissertation. Has publications on problems of clinical microbiology. Work experience in the specialty since 1996.

Bondarenko Natalia Alexandrovna

Bacteriologist of the first category, member of the Federation of Laboratory Medicine (FLM). He is a co-author of many scientific articles on the problems of clinical microbiology. Work experience in the specialty since 1988.

Balina Valeria Vladimirovna

Bacteriologist, clinical laboratory diagnostics doctor. She completed her internship in the specialty “Clinical Laboratory Diagnostics” and has professional retraining in the specialty “Bacteriology”. Passed qualification improvement on the topic “Molecular genetic methods in the diagnosis of infectious diseases.” Member of the Federation of Laboratory Medicine (FLM). Work experience in the specialty since 2013.

Nursing staff

The laboratory employs:

• 1 medical technologist
• 1 medical laboratory technician
• 5 medical laboratory assistants
• 1 laboratory assistant
• All employees have the highest qualification category.

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To conduct microbiological studies, there are bacteriological laboratories at hospitals and clinics or independently from them. They receive for research various material obtained from sick people (sputum, urine, pus, feces, blood, cerebrospinal fluid, etc.). In addition, there are also sanitary and bacteriological laboratories in which water, air and food products are subjected to bacteriological control.
The role of bacteriological laboratories is also great in the prevention of infectious diseases. Some people, after suffering an infectious disease (typhoid fever, dysentery, diphtheria, etc.), continue to release pathogenic microbes into the environment. These are the so-called bacteria carriers. Bacteria carriers are also found among healthy people. By identifying such bacteria carriers, bacteriological laboratories help health authorities carry out a number of preventive measures.
Water and food products contaminated with pathogenic microorganisms can cause epidemics (mass diseases) of typhoid fever, cholera, etc., which is why everyday sanitary and bacteriological control over the quality of drinking water, milk and other products is so important.
A bacteriological laboratory must have at its disposal at least three rooms: 1) a small room - a registration room for receiving and issuing tests; 2) medium cooking and washing - for preparing nutrient media and washing dishes; 3) a laboratory for bacteriological research. It is advisable to have a room for keeping experimental animals (vivarium). Each room should have appropriate furniture (kitchen and laboratory tables, various cabinets, stools, etc.).
The following is a list of the most important items needed in daily laboratory work. The purpose of their use, the method of handling them, as well as the principle of their design are described in the relevant sections of the course.
Optical devices. Biological microscope with immersion system, magnifiers, agglutinoscope.
Devices for sterilization and heating. Autoclave, fluid steam apparatus, drying cabinet, Seitz filters, thermostats, sterilizers for instruments.
Equipment for cooking media. A funnel for hot filtering, a funnel for pouring media, a water bath, pots of different sizes, tared scales with weights, a meat grinder, metal and wooden stands for filtering.
Tools. Scalpels of various shapes and types: scissors, straight, curved, blunt-tipped, intestinal scissors, anatomical and surgical tweezers, syringes.
Glass objects. Slide glasses, slide slides with a well, coverslips, bacteriological tubes, short tubes for serological reactions (agglutination), centrifuges, Heidepreich dishes*, glass tubes and rods, graduated pipettes of 1, 2, 5, 10 ml, Pasteur pipettes, glass paint bottles with pipettes, glass beakers and flasks of different sizes, cylinders of different sizes, filter funnels, etc.

*Until now, among most microbiologists and in textbooks, dishes for obtaining isolated colonies of microbes are called Petri dishes, not Heidenreich dishes, which does not correspond to the true state of affairs. Cups were first introduced into laboratory practice by the Russian microbiologist Heidenreich.

Various items. Bacterial loop, platinum wire, rubber tubes, manual horn scales with weights, racks (wooden, metal) for test tubes, thermometers, animal cages, devices for fixing animals, centrifuge.
Chemicals, paints, materials for media, etc. Agar-agar, gelatin, white in sheets, immersion oil, filter paper, absorbent and plain cotton wool, gauze, ethyl alcohol, aniline dyes (magenta, gentian and crystal violet, vesuvin, methylene blue, neutralrot, safranin, etc.), Giemsa paint, acids (nitric, hydrochloric, sulfuric, carbolic, phosphoric, picric, oxalic, etc.), alkalis (caustic potassium, caustic soda, ammonia, soda), salts (potassium nitrate, permanganate potassium, sodium sulfide, sodium chloride, etc.).

Laboratory table

To conduct microbiological research, the laboratory assistant must have an appropriately equipped workplace. The laboratory table must have a certain height so that it is easy to microscope while sitting at it (Fig. 9). If possible, the table should be covered with linoleum, and each workplace should be covered with a galvanized tray or mirror glass. The workplace should be equipped with a microscope, stands for test tubes and paints, a platinum loop and needle for inoculation, a cup with a bridge for preparations, a washer, an hourglass, slides and cover glasses, pipettes, a set of paints, filter paper, an alcohol or gas burner and a jar with a disinfectant solution (Lysol, carbolic acid, sublimate, chloramine or lysoform), into which used glass slides and coverslips, pipettes, glass rods, etc. are placed for disinfection. Dishes in which microbes are grown cannot be disinfected with chemicals. Traces of disinfectants on such dishes make them subsequently unsuitable for the growth and reproduction of microorganisms. After use, the dishes are placed in metal tanks or buckets with a lid, sealed and sterilized in an autoclave. After use, small instruments (tweezers, scalpels, scissors) are placed in a sterilizer and boiled for 30-60 minutes or immersed in a 3-5% soap-carbolic chloramine solution for 30-60 minutes.

Rice. 9. Technique for microscopy of bacteriological objects.

The workplace must be kept absolutely clean. It is unacceptable for the table to be contaminated with the infectious material being examined (urine, feces, pus, etc.). In the latter case, infectious material from the table can spread to other surrounding objects and then intra-laboratory infection is possible. After finishing work, the laboratory assistant must tidy up the workplace for which he is responsible, and for the purpose of prevention, wipe the glass at the workplace with a piece of cotton wool moistened with a 5% solution of carbolic acid or chloramine.

Rules of work and behavior in the laboratory

When working with infectious material, laboratory workers must remember the possibility of becoming infected themselves and transferring the infectious disease to their family, apartment, etc. Therefore, they must be attentive, careful, neat and pedantic in their work.
When working in laboratories, the following rules must be observed:

1. It is mandatory to be in the laboratory, and even more so to work in it, in a robe and a headscarf or cap.
2. Do not move from one laboratory room to another unnecessarily and use only the designated workplace and equipment.
3. Do not eat or smoke in the laboratory.
4. When working with infectious material and living cultures, use appropriate tools: tweezers, hooks, spatulas and other objects that must be destroyed or rendered harmless after use (burning on a burner flame, boiling, etc.). Suck out liquid infectious material into pipettes not with your mouth, but with the help of balloons, bulbs, pour liquid with infectious material from one vessel to another only over some receiver (tray, basin) into which a disinfectant liquid (carbolic acid solution, Lysol) is poured. . Sowing and re-seeding is carried out by burning test tubes, spatulas, platinum loops, pipettes, etc. on a burner flame.
5. If dishes break or liquid containing infectious material or live cultures spills, immediately thoroughly disinfect the contaminated work area, dress, and hands. All this should be done in the presence or under the supervision of the laboratory manager, who must be immediately informed of the accident.
6. If possible, destroy all used and unnecessary items and material (it is best to burn them or thoroughly neutralize them in sterilization machines or disinfectant liquids).

All items to be disinfected should be collected inside the laboratory in special receivers, tanks, buckets with lids, etc., and transferred closed to an autoclave, where they will be disinfected on the same day. The delivery of infectious material to the autoclave and its sterilization must be supervised by specially designated responsible laboratory workers.

1. Maintain strict cleanliness and neatness. Disinfect and wash your hands as often as possible during the working day and after finishing work.
2. Laboratory workers are subject to mandatory vaccination against major infectious diseases (primarily against intestinal diseases).
3. It is mandatory to conduct daily quantitative records of all living crops and infected animals with entries in special journals and accounting books.
4. After work, leave all material and cultures necessary for further work in a locked refrigerator or safe, and put the workplace in complete order.
5. Daily thorough cleaning of the laboratory premises must be done wet using a disinfectant liquid.

The presence of various bacteria in the intestines is considered normal. These bacteria take part in the processes of processing and assimilation of food. Proper digestion and functioning of the intestines is evidenced by feces consisting of small structureless particles called detritus.

To study the microbial composition of feces, a tank analysis is performed. If the number of bacteria is increased, then a person experiences intestinal pathologies, abdominal pain of various types, and pieces of undigested food appear in the feces. This study allows us to identify the causative agents of many ailments.

Classification of intestinal bacteria

However, upon detailed study they are classified into the following groups:

1. Healthy bacteria: lacto- and bifidobacteria, Eschecheria. These microorganisms activate the functioning of the intestines.
2. Opportunistic: enterococci, candida, clostridia, staphylococci. These microorganisms become pathogenic as a result of certain circumstances and are capable of provoking the development of various pathologies.
3. Pathogenic: bacilli, Klebsiella, Proteus, Salmonella, Shingella, Sarcinus. This group of bacteria provokes the development of serious diseases.

There are various methods for examining stool. One of the common methods is bacterial analysis.

What is a stool test?

Bacteriological examination of stool allows you to study its microbial composition, as well as determine the presence of pathogens of subsequent ailments:

• shigellosis;
• dysentery;
• salmonellosis;
• typhoid fever;
• cholera and other diseases.

Stool analysis takes quite a long time. The study is carried out before prescribing antibiotic therapy.

Indications for the study

The main reasons to give a stool test should be highlighted:

Scatological studies allow us to identify pathologies occurring in the intestinal cavity:

A tank test is also prescribed to diagnose pathologies of the digestive organs.

How is a stool sample taken?

Before undergoing the study, the patient should undergo special preparation for several days.

• greenery;
• beets;
• red fish;
• tomatoes.

In addition, meat products may influence the results of the study.

During the period of preparation for taking tests, it is necessary to stop taking antibiotics, anti-inflammatory drugs and medications containing enzymes and iron.

Collection of material for research must be carried out in the morning. To collect feces, use a sterile container, which can be purchased at a pharmacy. The duration of storage of biomaterial in the refrigerator is no more than 10 hours.

How is the research conducted?

Bacteriological examination of feces makes it possible to determine the physical and chemical composition of the material, its properties, and the presence of pathologies. This study helps to detect bacteria in the body and changes in biobalance.

A scatological analysis of stool is considered a complement to bacterial analysis. This study allows us to assess the presence of a specific odor of feces, its consistency and density, general appearance, and the presence or absence of microorganisms.

The study includes 2 stages:

1. Macroscopic analysis.
2. Microscopic.

Microscopic examination reveals mucus, protein, increased levels of bilirubin, blood clots, and iodophilic flora in the stool. The latter is formed thanks to active substances that convert starch into glucose. The detection of iodophilic flora does not indicate infection in all cases. The development of the disease is indicated by the accumulation of iodine bacteria caused by fermentation.

Since the child’s body does not fight pathogenic flora well, such bacteria are very often diagnosed in children’s feces.

Today, the method of sowing the biomaterial under study in a special environment with certain conditions is used. Experts determine the ability of bacteria to reproduce and form colonies. To obtain accurate results, all instruments used, as well as containers with collected biomaterial, must be sterile.

Pathogenic microorganisms are studied for sensitivity to various antibacterial medications. The study is characterized by highly accurate results, according to which the doctor can prescribe medication.

Only 10% of the total amount of the studied material can be pathogenic microflora.

Decoding the results

Examination of feces allows you to identify and determine the number of any bacteria. Based on the results obtained, the doctor makes a diagnosis and prescribes treatment.

Types of pathogenic microflora that can be found in feces:

1. E. coli. They interfere with the body's absorption of calcium and iron and usually indicate the presence of worms.
2. Enterobacteriaceae. Most often, these bacteria cause the development of dysentery and intestinal infections.
3. E. coli with reduced enzymatic activity indicate the formation of dysbacteriosis.
4. Lactose-negative bacteria. They cause disturbances in the digestive process and cause flatulence, heartburn, frequent belching, and a feeling of heaviness.
5. Hemolytic bacteria. They form toxins that negatively affect the nervous system and the intestines. Causes the formation of allergies.
6. Yeast-like fungi provoke the development of thrush.
7. Klebsiella, provokes the formation of gastroenterological pathologies.
8. Enterococci, provoke the occurrence of infectious pathologies of the genital organs, excretory tract and genitourinary system.

The interpretation of the tank analysis is indicated on the forms, which also indicate the normal indicators of bacteria.

Intestinal dysbiosis is a very dangerous pathology that destroys healthy microflora. This condition leads to the development of dysentery and staphylococcus. To avoid this, it is recommended to take a stool test to monitor intestinal biobalance at least once a year.

Tank analysis is considered a reliable study that provides information about the functioning of its important internal organs: the intestines and stomach. The study allows timely identification of pathogenic microorganisms that affect normal microflora. Prescribed for both adults and children.