Air sampling methods for microbial contamination. Planning and creating secure products. Scheme for obtaining sterile air
From a sanitary and microbiological point of view, air is an environment in which microorganisms are not able to multiply, since it does not contain nutrients and moisture, and the sun's rays have a bactericidal effect. Nevertheless, pigment-forming cocci, spores of bacteria, molds and actinomycetes are constantly present in the air. Microbial air pollution is unstable and depends on many factors. So, pathogenic microbes get into the air with dust from the soil and with the secretions of sick people and animals. Indoor air is polluted during dry cleaning, sneezing and coughing. At the same time, aerosol droplets in the air serve as a source of aerogenic contamination of others. The droplet settling rate depends on the aerosol diameter.
Bacterial aerosols are divided into three phases:
1. Coarse droplet phase with an aerosol particle diameter of more than 0.1 mm; the residence time of such particles in the air is several seconds, the droplets settle quickly.
2. Drop nuclear phase having a particle diameter of 0.1 mm or less. Particles are in the air long time and dispersed over long distances with air currents, along with which various microorganisms, including pathogens, spread.
3. Bacterial Dust Phase has particles different diameter from 1 to 0.01 mm. This phase has the greatest epizootological and epidemiological significance, since it penetrates deep into the respiratory tract. Infectious diseases are transmitted by aerogenic method mainly in enclosed spaces.
The survival of pathogenic microorganisms in suspension depends on the biological properties of the pathogen, as well as temperature and humidity. For example, the causative agents of tuberculosis, anthrax, which tolerate desiccation well, remain in the environment for a long time.
A microbiological study of air is carried out to determine the amount of MAFAnM, i.e., the total microbial number and the number of sanitary-indicative microorganisms. The amount of MAFAnM in the air is determined by inoculation on the surface of the MPA; the number of sanitary-indicative microbes is determined by inoculation on blood agar, yolk-salt agar. To determine the presence of mold and yeast spores, wort_agar or Sabouraud and Czapek medium are used. There are many methods bacteriological research air, the most accessible are the methods of Koch and Krotov.
Koch sedimentation method(lat. sedimentum - sediment). The essence of the method is deposition of microbial particles and aerosol drops on the surface of a dense nutrient medium under the action of gravity.
Methodology. Petri dishes with MPA, Saburo's medium are left open for 5–20 minutes in the test room (classroom, in the workshops of a dairy plant, a meat processing plant, etc.). Then the cups are closed and placed in a thermostat at a temperature of +30_C, if it is MPA or blood agar, after which they are cultivated for 48 hours; if it is Sabouraud's medium, it is cultivated at a temperature of +25_С for 4–7 days. Then carry out the calculation of the grown colonies in the entire cup.
After counting the grown colonies in a Petri dish, the number of microorganisms in 1 m3 of air is determined according to the Omelyansky formula, according to which as many microbial cells settle in dishes with a nutrient medium of 100 cm2 within 5 minutes as they are contained in 10 liters of air:
X \u003d a * 100 * 1000 * 5 / b * 10 * T
where X- the number of microbes in 1 m3 (1000 l) of air; a- the number of grown colonies in the cups; b- cup area (80 cm2); 5 - exposure time according to the Omelyansky rule; T- the time during which the cup was open; 10 - 10 liters of air according to the Omelyansky rule; 1000 - 1 m3 of air; 100 -100 cm2 of nutrient medium.
Aspiration method of Krotov is more accurate, since the device is equipped with a micromanometer showing the number (volume) of liters of air sown. Krotov's apparatus is a cylindrical device, inside of which there is an electric motor with centrifugal fan. When the fan rotates, air is sucked from the test room through a narrow wedge-shaped slit in the lid of the device, under which there is a rotating platform with a Petri dish, the air stream hits the moist surface of the nutrient medium, microorganisms from the air settle. Cups with crops are placed in a thermostat for 24–48 hours at a temperature of +30_C. Colonies are counted in the same way as in the sedimentation method. In the future, the number of microbes in 1 m3 of air is determined by the formula
where X- the number of microbes in 1 m3 of air; a- the number of grown colonies; 1000 l - 1 m3 of air; b- amount of seeded air.
The requirements for microbiological parameters of air are presented in Table. 19 (examined once a month).
Each bacteriological laboratory has a box for carrying out crops and subcultures, the air in the box should be checked for bacterial contamination at least twice a week, special requirements are imposed on the quality of the air in the box. To conduct the study, Petri dishes with MPA and Sabouraud medium are left open in the box for 15 minutes, then the dishes with MPA medium are kept in a thermostat for 48 hours at a temperature of +37_C, the dishes with Sabouraud medium - 96 hours at a temperature of +25...+27_C. 5 mold colonies are allowed in the cups.
Questions for preparation on the topic.
Characteristics of air microflora.
Microbial number of air and its definition.
Sanitary-indicative air microorganisms and their detection.
Air is an unfavorable environment for the life of microorganisms; microbes enter the air from soil, water, human and animal organisms and, not finding nutrients in the air, gradually die off under the influence of solar radiation, drying, temperature changes and other factors.
The number of microorganisms in the air and their qualitative composition fluctuate significantly, depending on meteorological conditions, distance from the earth's surface, the presence settlements etc. The largest number microorganisms contains the air of large cities with heavy traffic, the least microbes in the air of forests, mountains, but as you rise up, the air becomes more and more clean even over large industrial cities. The indoor air contains a significant number of microorganisms, especially when there are large crowds of people.
Microbes in the air are on particles of dust or moisture in the form of aerosols. An aerosol is a colloidal system consisting of a gaseous medium, for example, air, which contains the smallest particles in the spray state. solid or droplets of liquid. On the surface of the particles there is a layer of adsorbed air, the presence of a gaseous medium protects the particles from wetting.
The stability of the dispersed phase of an aerosol depends on the size of the particles, their surface energy, and electric charge. In the kinetics of microbial aerosol, 3 phases are schematically distinguished:
Large-nuclear Phase. The particles of which have a diameter of more than 0.1 mm and settle relatively quickly, the duration of their stay in the air is several seconds;
Small nuclear phase, particle size less than 0.1mm; these small droplets, due to their large specific surface and low weight, are kept in the air for a long time, forming a fairly stable colloidal system, the microorganisms in it are protected by a layer of moisture;
Phase of bacterial dust. Drops of the large-nuclear and small-nuclear phases of the aerosol, gradually settling and drying, can turn into the so-called bacterial dust, the particle size of which is from 1 µm to 100 µm, bacterial dust particles stay in the air for a long time in a suspended state and can penetrate into the upper respiratory tract and lungs person.
In the air under natural conditions, up to 100 species of various microorganisms are found, most of which are represented by saprophytes.
The most commonly found microbes in the open air include various cocci, spore-forming rods in the form of spores (Bacilluc nesentericus, 3;.c.subtilis, Lao .mecateriui.i),
non-spore-forming pigment bacteria (serrc.tia narcescono), numerous fungal spores FROM the genera Fenicilliuia,..s;-)ergillus,;.D1C yeast and yeast-like fungi. The most resistant to adverse factors in the atmosphere are pigment microerganisms belonging to various systematic groups.
Conditionally pathogenic and pathogenic microorganisms can get into the air, especially indoors, from humans and animals: staphylococci, streptococci, pneumococci, diphtheria and tuberculosis bacilli, influenza viruses, chickenpox, measles, parotitis, etc. Especially many microorganisms enter air when coughing, sneezing, talking; even a perfectly healthy person, with each act of sneezing, secretes into environment approximately 10,000 - 20,000 microorganisms. In the distribution of many infectious diseases so-called aerogenic, for example, influenza, chickenpox and so on., great importance has an airborne route of transmission. In those cases when the drying of drops of saliva or sputum leads to the formation of drop nucleoli, in which microbes are protected by a protein film, the latter can remain viable for a long time. So, diphtheria bacilli persist for a day, hemolytic streptococci up to 2 days, tuberculous microbacteria up to 18 days. For the prevention of infections, for air control in the workshops of medical industry enterprises, health care institutions and other purposes, sanitary and bacteriological air testing is widely used. It includes determining the total number of microorganisms
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mov in T m (1000 l) of air, i.e. microbial number of air and sanitary-indicative microbes.
Methods of microbiological examination of air are divided into sedimentation and filtration.
A variation of the sedimentation method is the shock air jet method. The most simple is the Koch sedimentation method: sterile Petri dishes with MPA are opened in
air sampling sites and kept for a certain time (most often from 5 minutes to 30 minutes), after which they are closed and placed in a thermostat at 37 ° for 24 hours, and then left for a day at room temperature. Microorganisms contained on aerosol particles settle on a nutrient medium and form colonies on it. By the number of grown colonies, the microbial number of air is calculated using the Omelyansky rule, according to which it is believed that as many microorganisms settle on the surface of a nutrient medium with an area of 100 cm2 within 5 minutes as they are contained in 3 liters of air. Knowing the number of grown colonies and exposure time, calculate the number of microbes contained in I (1000 l) of air.
The Koch method, with its simplicity and convenience, has a number of disadvantages: first of all, only relatively large aerosol particles settle on the agar, particles of the bacterial dust phase can stay in the air for a long time in a suspended state without settling, in addition, the direction and force of sedimentation affect the process of sedimentation. air currents. Seeding by the Koch method is not
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gives an idea of the number of rickettsiae and viruses in the air.
Fig.92. Krotov's device (general view).
6811‘**8
Fig.91. Scheme of the Krotov device, ([-cylindrical case; 2-base of the case; 3-electric motor; 4-centrifugal fan; 5-eight-bladed impeller; 6-disk; 7-spring; 8-Petri dish; 9-lid of the device; 10-on - toggle locks; 11-plexiglass discs; T2-wedge-shaped slot; 13-split ring; 14-nipple with a diaphragm; 15-lead tube.
More advanced are the methods of instrumental study of air microflora using Krotov apparatus and impactors (Fig. 91,92,93). Krotov's device is a cylindrical body, closed from above with a removable lid, under which a Petri dish with MPA is installed on a rotating disk, an electric motor is placed inside the cylinder, the latter, rotating at a speed of 4-5 thousand revolutions per minute, provides air suction through the plexiglass cover, having a wedge-shaped slit or holes. As a result of the turbulent air flow inside the cylinder, a disk with a Petri dish rotates, which ensures a uniform distribution of microflora over the entire surface of the nutrient medium, and aerosol particles of all three phases are actively sucked in. Using a rotameter, which is designed to determine the amount of air sucked in, from 50 to 200 liters of air are passed through the device. After sampling, the dishes are closed and placed in a thermostat at 37° for 24 hours, and then for 24 hours at room temperature. By counting the number of grown colonies and knowing the volume of air passed, it is easy to calculate the microbial number.
Fig.93. Schematic of the four-stage May impactor (see description in the text).
Impactors - devices equipped with tubes with conical nozzles - cascades through which air is sucked. In front of the narrow end of each nozzle, receiving plates are placed, which are glass slides lubricated with glycerin and saline. Is air leaking through tubes with nozzles? hits the receiving plates, microorganisms settle on them. After sampling the air, the glass slides are removed from the impactors and the settled microbes are examined either by microscopy, or they are washed from the glass with saline solution, from which they are then seeded onto nutrient media.
Filtration methods for studying air are based on filtering or aspiration (sucking) it through special filters, liquids, powders, etc., adsorbing microflora.
Filters used for air analysis can be insoluble - cotton, paper, membrane, millipores, and soluble - glycerol-gelatin, from sodium alginate, sugar
і
powder
A filter plate of the appropriate material is placed in the Seitz apparatus and a certain amount of air is sucked through the filter using a vacuum pump. Then the filter plate is removed, immersed in physiological solution and shaken, microorganisms are desorbed into the solution and quantitative seedings are made from it on nutrient media. If soluble materials are used as filters, then after sucking out the air, they are dissolved in a sterile saline solution.
Air can be sucked through a sterile liquid (water, saline, meat-peptone broth, etc.) using the Dyakonov device (Fig. 94)
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t
VT
Fig.94. Dyakonov's device
This device consists of a glass cylinder with a capacity of 100-200 ml, in a hermetically sealed stopper of which two glass tubes are inserted; When examining air, 10-20 ml of water is poured into the device, glass beads are placed and sterilized. After sterilization, the outlet tube is connected to a vacuum pump, to which a rheometer is connected to measure the volume of air passing through the device, and air is sucked in in an amount of 100-200 liters. After turning off the device, take 1 ml of water through which the air was filtered, add it to an empty sterile Petri dish and pour 15 ml of molten MPA (+ 45 °). Cups with crops are incubated in a thermostat at 37 ° for 1-2 days, and then the number of grown colonies is counted and, knowing the volume of filtered air, the microbial number is calculated.
In indoor air, as well as in water and soil, the presence of sanitary-indicative microorganisms is determined.
They are recognized as Streptococcus viridans, (green streptococcus) (type L.), Str.haemolyticus (hemolytic streptococcus Staphylococcus aureus, which has hemolytic properties. The presence of these microbes in the air indicates that it is contaminated with the microflora of the human upper respiratory tract. The number of sanitary-indicative microorganisms , containing-
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in I m (1000 liters) of air is called streptococcalmindex.
To identify sanitary-indicative air microbes, all the methods described above are used, but sowing is carried out on elective and differential diagnostic media, which make it possible to quickly detect these bacteria and separate them from other representatives of the air microflora. These media include blood agar, on which hemolytic staphylococci and streptococci give a zone of hemolysis (erythrocyte destruction). Yolk-salt agar, etc.
The results of air assessment in residential premises in winter and summer are presented in Table 10.
Table 10
Criteria for sanitary assessment of indoor air
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(number of microorganisms in I m of air) Air assessment
micro-
orga
nisms of viridescent and hemolytic streptococci Pure 1500 16 4500 36 Contaminated .... 2500 36 7000 124 The purpose of the work: to determine the microbial number of air and the content of sanitary-indicative microorganisms.
Materials: Petri dishes with MPA, wort agar and blood agar, Krotov apparatus.
Working process. I. Determine the microbial count by the Koch method: leave Petri dishes with MPA, blood agar and wort agar open in different parts laboratory for 5 min. Close the dishes and place them in a thermostat at 37°C for 48 hours. Count the number of grown colonies, determine the microbial number of air (see above).
Determine the microbial number and the number of sanitary-indicative microorganisms using the Krotov apparatus. Use plates with MPA, blood agar and wort agar. For each
cups skip 200 liters of air at a speed of 20-30 l / min. After sampling, close the cups and place in a thermostat at 37°C for 48 hours. Count grown colonies, microscopically prepare smears and stain them according to Gram. Note the presence of areas of hemolysis on blood agar.
Compare the results obtained by the two methods. Assess the purity of the air. The results are presented in the form of a table.
Sanitary assessment of air
Medium Number of colonies per plate as determined
by the Koch method in the Krotov apparatus
MPA
blood agar wort agar
microbial count
The number of green and
hemolytic
streptococci
Literature
Labinskaya A.S. Microbiology with the technique of microbiological research. M., 1972.
Pereti L.G. The value of normal microflora for the human body. Medgiz., 1955.
P I t to and n K.D., Krivoshein B.S. Microbiology.M. ,1980. Sanitary microbiology. Ed. G. P. Kalina and G. K. Chis- tovich. M., 1969.
Tep V.I. Sanitary microbiology. 1958.
T i m a k o v V.D. Microbiology. M., 1973. .
Sanitary and microbiological examination of air can be divided into into 4 stages:
1) sampling;
2) processing, transportation, storage of samples, obtaining a concentrate of microorganisms (if necessary);
3) bacteriological seeding, cultivation of microorganisms;
4) identification of the isolated culture.
Sample selection:
Correct sampling ensures the accuracy of the study. In enclosed spaces, sampling points are set for every 20 m2 of area - one air sample, according to the type of envelope: 4 points at the corners of the room (at a distance of 0.5 m from the walls) and the 5th point - in the center. Air samples are taken at a height of 1.6-1.8 m from the floor - at the level of breathing in living quarters. Samples must be taken during the day (during the period of active human activity), after wet cleaning and ventilation of the room. Atmospheric air is examined in a residential area at a level of 0.5-2 m from the ground near pollution sources, as well as in green areas (parks, gardens, etc.) to assess their impact on air microflora.
It should be noted that during air sampling, in many cases, it is sown on a nutrient medium.
All methods of air sampling can be divided into sedimentation and aspiration.
sedimentation- most old method, is widely used due to its simplicity and availability, but is inaccurate. The method was proposed by R. Koch and consists in the ability of microorganisms under the influence of gravity and under the influence of air movement (together with dust particles and aerosol droplets) to settle on the surface of the nutrient medium in open Petri dishes. Cups are installed at sampling points on horizontal surface. When determining the total microbial contamination, the dishes with meat-peptone agar are left open for 5-10 minutes or longer, depending on the degree of expected bacterial contamination. To identify sanitary-indicative microbes, Garro or Turzhetsky medium (to detect streptococci), milk-salt or yolk-salt agar (to determine staphylococci), wort agar or Sabouraud medium (to detect yeast and fungi) are used. When determining sanitary-indicative microorganisms, the cups are left open for 40-60 minutes.
At the end of the exposure, all dishes are closed, placed in a thermostat for a day for cultivation at a temperature optimal for the development of the isolated microorganism, then (if required by research) they are left at room temperature for 48 hours for the formation of pigment by pigment-forming microorganisms.
The sedimentation method has a number of disadvantages: only coarsely dispersed aerosol fractions settle on the surface of the medium; often colonies are formed not from a single cell, but from an accumulation of microbes; only part of the air microflora grows on the nutrient media used. In addition, this method is completely unsuitable for the study of bacterial contamination. atmospheric air.
More advanced methods are aspiration based on the forced deposition of microorganisms from the air onto the surface of a dense nutrient medium or into a trapping liquid (meat-peptone broth, buffer solution, isotonic sodium chloride solution, etc.). In the practice of the sanitary service for aspiration sampling, the Krotov apparatus, the Rechmensky bacteriotrap, the air sampling device (POV-1), the aerosol bacteriological sampler (PAB-1), the bacterial-viral electroprecipitator (BVEP-1), the Kiktenko device, Andersen devices are used , Dyakonova, MB, etc. Membrane filters No. 4 can also be used to study the atmosphere, through which air is sucked using the Seitz apparatus. A wide variety of instruments testifies to the absence of a universal apparatus and to a greater or lesser degree of their imperfection.
Krotov's device. At present, this device is widely used in the study of indoor air and is available in laboratories.
Krotov's apparatus
The principle of operation of the Krotov apparatus (Fig. 22) is based on the fact that air sucked through a wedge-shaped slot in the lid of the apparatus hits the surface of the nutrient medium, while dust and aerosol particles adhere to the medium, and with them the microorganisms in the air .
Bacterial-viral electroprecipitator (BVEP-1). The device is based on the aspiration-ionization principle of operation. BVEP-1 consists of a settling chamber, in which electrodes are mounted: a negative one in the form of a supply tube through which air enters (and aerosol particles, respectively, are negatively charged), and a positive one, on which bacteria settle.
MB device. This device serves not only to determine the total microbial contamination, but also to take air samples with aerosol particles of various sizes. The MB device is built on the “sieve” principle and is a cylinder divided into 6 horizontal strips, on each of which Petri dishes with MPA are placed. The air is sucked in starting from the upper stage, in the plate of which the holes are the largest, and the lower the stage, the smaller the hole size (only fine fractions of air aerosol pass through the latter). The device is designed to capture aerosol particles larger than 1 µm at an air sampling rate of 30 l/min. Reducing the number of holes provides a more uniform distribution of the aerosol from the air throughout the nutrient medium. To capture even more small particles aerosol, you can add an additional filter of AFA filter material.
When using any of the listed devices, the results obtained are approximate, but they give a more correct assessment of air contamination in comparison with the sedimentation method. Since both sampling and sanitary and microbiological studies of air are not regulated by GOST, any device can be used to assess bacterial air pollution. In many cases, sampling is combined with the seeding stage.
To reduce the number of microorganisms in indoor air, the following means are used: a) chemical - treatment with ozone, nitrogen dioxide, spraying lactic acid, b) mechanical - passing air through special filters, c) physical - ultraviolet irradiation.
TOPIC 3. HYGIENIC ASSESSMENT OF MICROBIAL POLLUTION IN ROOM AIR
TOPIC 3. HYGIENIC ASSESSMENT OF MICROBIAL POLLUTION IN ROOM AIR
Purpose of the lesson:study of methods for determining and evaluating bacterial contamination of indoor air.
In preparation for class, students should work on the following: theory questions.
1. Epidemiological significance of the air environment. Sources of microbial air pollution.
2. Characteristics of the bacterial composition of atmospheric air and indoor air. Factors contributing to the reduction of microbial indoor air pollution.
3. Significance of bacterial air pollution in manufacturing medicines.
4. Methods of research and assessment of the degree of bacterial pollution of indoor air.
After mastering the topic the student must know:
Methodology for air sampling, analysis, determination of the degree of bacterial air pollution pharmacy premises;
Calculation of the required power and the number of bactericidal irradiators for the disinfection of air and surfaces of pharmacy premises;
be able to:
Evaluate the results of air studies for compliance with hygienic standards;
Assess the working conditions of pharmacy staff under the influence of biological factors according to the sanitary and hygienic examination and laboratory tests;
Use basic regulations and reference information sources for organizing control over the level of microbial pollution in the air of pharmacy premises and developing preventive measures to prevent and reduce the level of air pollution in pharmacy premises.
Training material for the assignment
The air can be polluted by aeroplankton, i.e. bacteria, viruses, mold spores, yeast fungi, protozoan cysts, moss spores, etc. Soil is the main source of air pollution. Microorganisms that enter the atmospheric air die relatively quickly due to drying, action ultraviolet rays Sun and lack of nutrient material. However, saprophytic and sometimes pathogenic microorganisms are always found in the surface layer of the atmosphere and in the air of poorly ventilated enclosed spaces.
In the production of biosynthetic drugs, workers may be exposed to aerosols of living cells of microbial producers, metabolic products of microorganisms, and dusty end products, often containing more than 50% protein (for example, in factories producing protein-vitamin concentrates). At the stages of obtaining and isolating antibiotics, as well as at the final stages (drying, packaging, packaging), workers can be exposed to antibiotic dust. Air content control harmful substances biological nature (antibiotics, enzymes, vitamins, etc.) are carried out in a similar way: as is customary for chemicals in accordance with the requirements Guidelines "Microbiological monitoring of the working environment" (MU 4.2.734-99) and Appendix 10 of Guidelines 2.2.755-99 "Methodology for monitoring the content of microorganisms in the air of the working area."
In the premises of pharmacies, bacterial air pollution, which occurs due to the discharge of visitors and pharmacy workers, is of great importance, as it is the cause of possible infection of personnel with pathogens of various infectious diseases, as well as the danger of microorganisms entering medicines. The microflora that got into drugs leads to a change in their physicochemical properties, a decrease in therapeutic activity, a decrease in shelf life, and can cause the development of diseases and complications in a patient. The most intense bacterial air pollution is observed in trading floor, washing and auxiliary premises.
Biological components of indoor dust are microflora (bacteria, viruses and fungi) of the upper respiratory tract, skin, microscopic mites, mold spores. Sanitary-indicative microorganisms in indoor air are staphylococci, viridescent streptococci, and hemolytic streptococci are indicators of direct epidemic danger. Despite the relatively short period of stay in the air, microbes create an epidemic danger. The sources of microbial air pollution in hospitals of all types are medical personnel and patients suffering from erased (asymptomatic) forms of infectious diseases, as well as carriers of multidrug-resistant strains of pathogenic and conditionally pathogenic microorganisms.
There are no standards for the content of microorganisms in the air of residential premises. Bacterial Purity Guidelines industrial premises(hospitals, pharmacies) are designed depending on their functional purpose taking into account the intensity of bacterial contamination and the risk of nosocomial infections. In accordance with regulatory documents (SanPiN 2.1.3.1375-03), bacterial air purity is assessed differentially by the total number of microorganisms in 1 m 3 of air, and in rooms of classes A, B, and C, it is necessary to control the presence of colonies Staphylococcus aureus, which should not be determined in 1 m 3 of air, and mold and yeast fungi, which should not be determined in 1 dm 3 of air.
One of effective methods air disinfection is the use of bactericidal action of ultraviolet rays with a wavelength of 254-257 nm. In order to rehabilitate pharmacy and medical premises, bactericidal uvio lamps BUV-15, BUV-30 are currently used, which are gas-discharge mercury lamps low pressure. The lamps are made in the form of uviol glass tubes of different lengths and are filled with a gas mixture consisting of mercury vapor and argon. Tungsten electrodes are soldered into the ends of the tubes. When a current is passed through the tube, a gas discharge occurs, as a result of which a glow occurs. The uvio glass of the lamp transmits UV rays that kill germs, while providing a high disinfecting effect.
In pharmacies, ceiling-mounted bactericidal irradiators (CBOs) and wall-mounted bactericidal irradiators (NBOs) are used. PSPs have
two shielded lamps BUV-15 and two open lamps BUV-30. When using PBO, especially when unshielded bactericidal lamps are turned on, the disinfecting effect occurs due to the action of a direct beam beam. The NBO has two germicidal lamps: one, a shielded lamp, irradiates the upper zone and the other, unshielded, the lower zone. Reliable bactericidal effect achieved with the operation of bactericidal irradiators within two hours at a lamp power of 3 W per 1 m 3.
During long-term operation of bactericidal lamps, ozone and nitric oxide can accumulate in the indoor air in quantities exceeding the MPC of these substances, so the use of ultraviolet irradiation requires compliance with safety regulations. In the presence of workers, it is recommended to use shielded bactericidal lamps with a power of 1 W per 1 m 3, and in the absence of people, bactericidal lamps are used open type(NE) with a power of 3 W per 1 m 3. PBO and NBO are stationary bactericidal installations. Currently, medical institutions and pharmacies use mobile bactericidal irradiators, which makes it possible to more effectively disinfect the air.
Determining the number of bacteria is carried out sedimentation or aspiration methods.
sedimentation method is based on the natural deposition of bacteria from the air onto a Petri dish with a nutrient medium and subsequent keeping in a thermostat for two days at a temperature of 37 ° C and counting the colonies that have grown during this time on the entire area of the dish.
Principle aspiration method- aspiration of a certain volume of air with the sowing of the bacteria contained in it on the surface of the nutrient medium using the Krotov slit device (Fig. 10) or using the Flora-100 microbiological air impactor.
Krotov's device is a cylinder with a removable cover, in which there is an electric motor with a centrifugal fan. The principle of operation of the device is based on the inertial deposition of aerosol particles on the surface of the nutrient medium. The test air is sucked in at a rate of 20-25 l / min through a wedge-shaped
the gap in the lid of the device hits the surface of a dense nutrient medium, and microbes linger on its wet surface. For uniform inoculation of microbes, a Petri dish with a nutrient medium is placed on a stand rotating at a speed of 1 revolution in 1 s. The rate of air aspiration is regulated by the micromanometer (rheometer) of the device. The total volume of the sample with significant air pollution should be 40-50 liters, with a slight one - more than 100 liters. The duration of aspiration is 2-5 minutes. After incubation of the selected samples at a temperature of 37 ° C for 1-2 days, depending on the isolated microorganisms, the grown colonies are counted. Given the volume of the air sample taken, the number of microbes in 1 m 3 of air is calculated.
Rice. ten.Krotov's device for bacteriological examination of air
Impactor "Flora-100", modern model device for capturing bacteria from the air, operates in automatic mode and surpasses Krotov's device in terms of technical characteristics.
Determining the number of microorganisms in the air is one of the hygienic criteria for its purity. The degree of bacterial air pollution is judged by the total number of bacteria contained in 1 m 3 of air. In addition, air can be assessed by the content of sanitary indicative microorganisms ( different types streptococci and staphylococci) are common inhabitants of the mucous membranes of the human respiratory tract. The content of microorganisms in the air is different in different seasons of the year. In the cold
During the winter period, the air has less microbial pollution, and in summer the air is more polluted with microbes that enter it in large quantities along with particles of soil dust. As indicative indicators for assessing bacterial air pollution in residential premises, the proposed by A.I. Shafir the following values (Table 9).
Table 9Evaluation of air purity by bacteriological indicators of air in pharmacy premises in different periods of the year
Air purity rating | ||||
Summer period (April-September) | Winter period (October-March) |
|||
Total Microorganisms | Hemolytic streptococcus | Total Microorganisms | Hemolytic streptococcus |
|
Clean | <3500 | <5000 | ||
moderately polluted | 3500-5000 | 24-52 | 5000-7000 | 52-124 |
Contaminated | >5000 | >7000 | >124 |
|
Laboratory work "Identification and assessment of microbial air pollution"
Student assignments
1. Perform a bacteriological air culture using the Krotov device.
2. Count the colonies in a Petri dish, inoculated with air on the nutrient medium of which was made using the Krotov apparatus a day ago at a rate of 20 l/min for 5 min and which was in a thermostat at a temperature of 37 °C for a day.
3. Determine the level of bacterial contamination in the pharmacy premises.
4. Give a hygienic assessment of the efficiency of germicidal lamps according to the conditions of the situational task.
Method of work
Determination of microbial air pollution
Having received one of the Petri dishes with grown microbial colonies, familiarize yourself with the time information contained in the task,
place and conditions of air sampling (speed and time of aspiration).
To count the number of colonies, it is necessary to divide the surface of the dish into 4 equal sides, drawing the dividing lines on the glass of the lid. Count the total number of colonies on the surface j of the dish and multiply by 4. Counting can be done with a simple eye or through a magnifying glass. The number of grown colonies can be taken approximately equal to the number of microbial bodies in the volume of air sown on a Petri dish. Then, taking into account the sampling conditions, calculate the total number of microorganisms in 1 m 3 of room air.
The degree of microbial air pollution should be assessed in accordance with the gradations given in Table. nine.
Calculation of the required power and the number of UV irradiators in the room
The required power (N) of germicidal lamps is determined by the formula:
N= E V,
where: E- normalized value of the specific power of the lamps:
3 W/m 3 - for open type lamps,
1 W/m 3 - for shielded lamps,
V- the volume of the room, m 3.
Required amount germicidal lamps (K) is determined by the formula:
To= N/ (power of the germicidal lamp).
Microorganisms are tiny, mostly single-celled creatures that are widely distributed in nature. They are found in all environments (air, soil, water), in the human body and animals, in plants.
The qualitative diversity and quantity of microorganisms depend primarily on nutrient compounds. However, humidity is also important. temperature regime, aeration, action sun rays and other factors.
Methods of sanitary and microbiological research natural environments allow to identify the presence of pathogenic microorganisms, determine their number and, in accordance with the results obtained, develop measures to eliminate or prevent infectious diseases. In addition, quantitative accounting is necessary for modeling ecosystems and developing management principles. natural processes. Let's consider further what they are.
The soil
It is regarded by scholars as one of the possible ways transmission of infectious diseases. With the secretions of sick people or animals, pathogenic microorganisms penetrate the soil. Some of them, in particular, spore ones, are able to remain in the soil for a long time (sometimes several decades). pathogens enter the soil dangerous infections like tetanus, anthrax, botulism, etc. Methods of sanitary and microbiological study of soil allow you to determine the "microbial number" (number of microorganisms per gram of soil), as well as the coli index (number of Escherichia coli).
Soil analysis: general information
To soil microbiological research methods First of all, direct microscopy and inoculation on a dense soil should be attributed. Populations of microorganisms and their groups inhabiting the soil differ in taxonomic position and ecological functions. In science, they are united under the general term "soil biota". Soil is a habitat for a huge number of microorganisms. In a gram of soil there are from 1 to 10 billion of their cells. Decomposition is active in this environment. organic matter with the participation of various saprophytic microorganisms.
Microscopic method of microbiological research: stages
Environmental analysis begins with sampling. To do this, use a knife that has been previously cleaned and rubbed with alcohol (you can use a shovel). This is followed by sample preparation. The next step is to count cells on stained smears. Let's consider each stage separately.
Sampling
When analyzing arable soil, as a rule, samples are taken from the depth of the entire layer. First, 2-3 cm is removed from the top of the soil, since foreign microflora may be present in it. After that, monoliths are taken from the studied area of the soil. The length of each of them should correspond to the thickness of the layer from which the sample is to be taken.
On a plot of 100-200 sq. m 7-10 samples are taken. The weight of each is about 0.5 kg. The samples must be thoroughly mixed in the bag. After that, an average sample is taken, weighing approximately 1 kg. It should be placed in a parchment (sterile) bag enclosed in fabric bag. The sample is stored in a refrigerator until the actual analysis.
Study preparation
The mixed soil is poured onto dry glass. First, it must be wiped with alcohol and burned over the burner. Using a spatula, the soil is thoroughly mixed and laid out in an even layer. It is imperative to remove the roots, other foreign elements. For this, tweezers are used. Before work, the tweezers and spatula are calcined over the burner and cooled.
From various sites soil distributed over the glass, small portions are taken. They are weighed in a porcelain cup on technical scales. Mandatory stage of microscopic microbiological research method is a special sample processing. It is necessary to prepare 2 sterile flasks in advance. Their capacity should not exceed 250 ml. Pour 100 ml of tap water into one of the flasks. 0.4-0.8 ml of liquid is taken from it and a sample of soil is moistened to a pasty state. The mixture must be rubbed with a finger or a rubber pestle for 5 minutes.
With water from the first flask, the soil mass is transferred to an empty flask. Then it is rubbed again. After that, the mass is transferred to the flask near the flame of the burner. The container with the soil suspension is shaken on a rocking chair for 5 minutes. After that, it is left to settle for about 30 s. This is necessary in order for large particles to settle. After half a minute, the mass is used to prepare the drug.
Cell count on fixed smears
Direct microscopic examination of the soil is carried out according to microbiological research method developed by Vinogradsky. In a certain volume of the prepared suspension, the number of microorganism cells is counted. The study of fixed smears allows you to save preparations for a long time and perform calculations at any convenient time.
Preparation of the drug is carried out as follows. A certain volume of suspension (usually 0.02-0.05 ml) is applied with a micropipette to a glass slide. A drop of agar-agar solution (a mixture of agaropectin and agarose polysaccharides extracted from brown and red algae of the Black Sea) is added to it, quickly mixed and distributed over an area of 4-6 square meters. see. The smear is dried in air and fixed for 20-30 minutes. alcohol (96%). Next, the preparation is moistened with distilled water, placed in a solution of carbolic erythrosin for 20-30 minutes.
After staining, it is washed and air dried. Cell counting is carried out with an immersion objective.
Seeding on solid media
microscopic microbiological research methods allow the detection of a large number of microorganisms. But despite sowing is considered the most common in practice. Its essence consists in sowing the volume of the drug (soil suspension) in a Petri dish on a dense medium.
This microbiological research method allows you to take into account not only the quantity, but also the group, and in some cases even species composition microscopic flora. The number of colonies is usually counted from the bottom of the Petri dish in transmitted light. A dot is placed on the calculated area with a marker or ink.
Water analysis
The microflora of a water body, as a rule, reflects the microbial composition of the soil around it. In this connection methods of sanitary and microbiological research of water and soil have a special practical value when studying the state of a particular ecosystem. Freshwater bodies contain, as a rule, cocci, rod-shaped bacteria.
Anaerobes in water are found in small quantities. As a rule, they breed at the bottom of reservoirs, in silt, taking part in purification processes. The microflora of the oceans and seas is represented mainly by salt-loving (halophilic) bacteria.
There are practically no microorganisms in the water of artesian wells. This is due to the filtering ability of the soil layer.
generally accepted methods of microbiological research of water determination of the microbial number and coli-titer or coli-index are considered. The first indicator characterizes the number of bacteria in 1 ml of liquid. The coli index is the number of Escherichia coli present in a liter of water, and the coli titer is the minimum amount or maximum dilution of the liquid in which they can still be detected.
Determination of microbial number
This method of sanitary microbiological research of water consists in the following. In 1 ml of water, the number of facultative anaerobes and mesophilic (intermediate) aerobes is determined, capable of on meat-peptone agar (the main nutrient medium) at 37 deg. during the day to form colonies, visible with an increase of 2-5 r. or with the naked eye.
The key stage of the considered method of microbiological research of water is the sowing. From each sample, at least 2 different volumes are inoculated. With 1-0.1 ml of pure liquid and 0.01-0.001 ml of contaminated liquid are added to each cup. For inoculation of 0.1 ml or less, the liquid is diluted with distilled (sterile) water. Tenfold dilutions are prepared sequentially. 1 ml from each of them is added to two Petri dishes.
Dilutions are filled with nutrient agar. It must first be melted and cooled to 45 degrees. After active mixing, the medium is left on a horizontal surface to solidify. At 37 deg. crops are grown throughout the day. under consideration method of microbiological research of water allows you to take into account the results on those dishes where the number of colonies is in the range from 30 to 300.
Air
It is considered a transit medium for microorganisms. Main methods of microbiological research of air are sedimentation (settlement) and aspiration.
The microflora of the air environment is conditionally divided into variable and constant. The first includes yeast, pigment-forming cocci, spore-bearing bacilli, bacilli and other microorganisms that are resistant to desiccation and exposure to light. Representatives of variable microflora, penetrating into the air from their usual habitat, do not retain their viability for long.
There are much more microorganisms in the air of large megacities than in the air countryside. There are very few bacteria over the seas and forests. Precipitation contributes to air purification: snow and rain. There are many more germs indoors than outdoors. Their number increases in winter period in the absence of regular ventilation.
Sedimentation
This method of microbiological research in microbiology considered to be the simplest. It is based on the settling of droplets and particles on the surface of agar in an open Petri dish under the action of gravity. The sedimentation method does not accurately determine the number of bacteria in the air. The fact is that it is rather difficult to catch small fractions of dust particles and bacterial droplets on an open cup. Mostly large particles are retained on the surface.
This method is not used in the analysis of atmospheric air. This medium is characterized by large fluctuations in the speed of air flow. Sedimentation, however, can be used in the absence of better instruments or a power source.
Determination of the microbial number is carried out according to the Omelyansky method. In accordance with it, in 5 minutes on the surface of a 100 sq. cm settles such a number of bacteria that is present in 10 liters of air.
Order 535 "On the unification of microbiological research methods"
It is worth saying that microbiological examination in this case is generally accompanied by certain problems. They are due to the fact that in the lower parts of the genital tract, there is normally a diverse microflora that changes in different age periods. To improve the efficiency of the study, unified rules were developed.
Diagnosis of viral infections
It is carried out by methods of detection of RNA and DNA pathogens. They are based mainly on the determination of nucleotide sequences in pathological material. For this, molecular probes are used. They are artificially obtained nucleic acids, complementary (supplementing) to viral acids, labeled with a radioactive label or biotin.
A feature of the method is the repeated copying of a specific DNA fragment, which includes several hundred (or tens) nucleotide pairs. The mechanism of replication (copying) is that completion can only start in certain blocks. Primers (seeds) are used to create them. They are synthesized oligonucleotides.
PCR diagnostics (polymerase chain reaction) is easy to perform. This method allows you to quickly get the result when using a small amount of pathological material. With the help of PCR diagnostics, acute, chronic and latent (hidden) infections are detected.
With sensitivity, this method is considered more preferable. However, at present, test systems are not sufficiently reliable, so PCR diagnostics cannot completely replace traditional methods.