Microorganisms are the smallest, mainly unicellular creatures, widespread in nature. They are found in all environments (air, soil, water), in humans and animals, in plants.
The qualitative diversity and quantity of microorganisms primarily depend on nutrient compounds. However, humidity, temperature, aeration, the action of sunlight and other factors are also important.
Methods of sanitary-microbiological research of natural environments make it possible 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 principles for managing natural processes. Let us further consider what are the methods of microbiological research .
The soil
It is considered by scientists as one of the possible ways of transmitting infectious pathologies. With the secretions of sick people or animals, pathogenic microorganisms penetrate the soil. Some of them, in particular, spore ones, are able to persist in the soil for a long time (sometimes several tens of years). Pathogens of such dangerous infections as tetanus, anthrax, botulism, etc. get into the soil. Methods of sanitary-microbiological investigation of the soil allow determining the "microbial number" (the number of microorganisms in a gram of soil), as well as the number of bacteria (number of Escherichia coli) .
Soil Analysis: General Information
The methods of microbiological soil research should include, first of all, direct microscopy and sowing on a solid nutrient medium. Populations of microorganisms and their groups inhabiting the soil differ in taxonomic position and ecological functions. In science, they are combined under the general term "soil biota". Soil - the habitat of a huge number of microorganisms. 1 to 10 billion of their cells are present in a gram of soil. In this environment, the decomposition of organic substances with the participation of various saprophytic microorganisms actively proceeds.
Microscopic method of microbiological research: stages
Environmental analysis begins with sampling. To do this, use a previously cleaned and rubbed with alcohol knife (you can use a shovel). After this, sample preparation is carried out. The next step is cell counting on stained smears. Let's consider each stage separately.
Sampling
In the analysis of 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, as extraneous microflora may be present in it. After that, monoliths are taken from the studied site of soil. The length of each of them should correspond to the thickness of the layer from which you want to take a sample.
On a plot of 100-200 square meters. m 7-10 samples are taken. Each weight is about 0.5 kg. Samples must be mixed thoroughly in a bag. After that take an average sample, weighing approximately 1 kg. It should be placed in a parchment (sterile) bag enclosed in a fabric bag. Before direct analysis, the sample is stored in the refrigerator.
Study preparation
Mixed soil spills out onto dry glass. It must first 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 and other extraneous elements. For this, tweezers are used. Before work, the tweezers and spatula are baked over the burner and cooled.
Small portions are taken from various sections of soil distributed over the glass. They are weighed in a porcelain cup on a technical scale. A mandatory step in the microscopic method of microbiological research is the special processing of the sample. Prepare 2 sterile flasks in advance. Their capacity should not exceed 250 ml. 100 ml of tap water is poured into one of the flasks. 0.4-0.8 ml of liquid is taken from it and moisten the soil sample to a pasty state. The mixture must be rubbed with a finger or a rubber pestle for 5 minutes.
The soil mass is transferred from the first flask to an empty flask. Then it is rubbed again. After this, the mass is transferred to the flask near the flame of the burner. A container with 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 so that large particles settle. After half a minute, the mass is used to prepare the drug.
Fixed Smear Cell Counting
Direct microscopic study of the soil is carried out according to the method of microbiological research developed by Vinogradsky. In a certain volume of the prepared suspension, the number of cells of microorganisms is counted. The study of fixed smears allows you to save drugs for a long time and perform calculations at any convenient time.
Preparation of the drug is as follows. A certain amount of suspension (usually 0.02-0.05 ml) is applied using a micropipette to a glass slide. A drop of agar-agar solution (a mixture of polysaccharides of agaropectin and agarose 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 drug is moistened with distilled water, placed in a carbol erythrosine solution for 20-30 minutes.
After staining, it is washed and dried in air. Cell counting is carried out with an immersion lens.
Seeding on solid media
Microscopic methods of microbiological research can identify a large number of microorganisms. But, despite this, the method of 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 solid medium.
This method of microbiological research allows you to take into account not only the quantity, but also the group, and in some cases the species composition of the microscopic flora. The number of colonies is usually calculated from the bottom of the Petri dish in transmitted light. In the calculated area, a dot is set 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 regard, the methods of sanitary-microbiological research of water and soil are of particular practical importance in studying the state of a particular ecosystem. Fresh water contains, 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 the processes of purification. 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.
The generally accepted methods of microbiological research of water are the determination of the microbial number and coli titer or coli index. 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 found.
Microbial number determination
This method of sanitary microbiological research of water is as follows. In 1 ml of water, the number of facultative anaerobes and mesophilic (intermediate) aerobes capable of meat-peptone agar (main nutrient medium) at 37 degrees is determined. throughout the day to form colonies visible with an increase of 2-5 p. or with the naked eye.
The key stage of the considered method of microbiological research of water is sowing. From each sample, at least 2 different volumes are sown. When analyzing tap water , 1-0.1 ml of clean liquid and 0.01-0.001 ml of contaminated water are added to each cup. For sowing 0.1 ml or less, the liquid is diluted with distilled (sterile) water. Tenfold dilutions are sequentially prepared. 1 ml from each of them is introduced into two Petri dishes.
Dilutions are poured with nutrient agar. It must first be melted and cooled to 45 degrees. After vigorous stirring, the medium is left on a horizontal surface for solidification. At 37 degrees crops are grown throughout the day. The considered method of microbiological research of water allows 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. The main methods of microbiological research of air are sedimentation (sedimentation) and aspiration.
The microflora of the air is conditionally divided into a variable and a constant. The first includes yeast, pigment-forming cocci, spore-bearing bacilli, bacilli and other microorganisms that are resistant to drying, exposure to light. Representatives of variable microflora, penetrating into the air from their habitual environment, do not retain their viability for long.
There are much more microorganisms in the air of large megacities than in the air of rural areas. Above the seas, forests of bacteria are very few. Precipitation is facilitated by precipitation: snow and rain. Indoor microbes are much more than in open spaces. Their number increases in the winter in the absence of regular ventilation.
Sedimentation
This method of microbiological research in microbiology is considered the simplest. It is based on the settling of droplets and particles on the surface of the agar in an open Petri dish under the influence of gravity. The sedimentation method does not accurately determine the number of bacteria in the air. The fact is that on an open cup it is rather difficult to catch small fractions of dust particles and bacterial drops. On the surface, mainly large particles are retained.
This method is not used in the analysis of atmospheric air. This environment is characterized by large fluctuations in the speed of air flow. Sedimentation, however, can be used in the absence of more advanced appliances or a source of electricity.
The determination of the microbial number is carried out according to the Omelyansky method. In accordance with it, for 5 minutes on the surface of the agar area of 100 square meters. see the number of bacteria that is present in 10 liters of air settles.
Order 535 "On the unification of microbiological research methods"
Bacteriological analysis occupies an important place in the complex of clinical and laboratory measures aimed at the diagnosis, prevention and treatment of a variety of infectious diseases. However, environmental studies are not limited to them.
Of particular importance is the bacteriological analysis of biological material in medical institutions. For research conducted in medical institutions, increased demands are made. The purpose of the Order "On the unification of microbiological research methods" is to improve bacteriological analysis, improve the quality and effectiveness of microbiological diagnostics.
Microscopic examination of smears in women
It is a key analysis method in the diagnosis of sexually transmitted infections and opportunistic diseases (caused by opportunistic bacteria).
Microscopic analysis allows you to evaluate the qualitative and quantitative composition of microflora, to verify the correctness of sampling. For example, the presence of a vaginal epithelium in a smear taken from the cervical canal indicates a violation of the rules for selecting a biological sample.
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 normally there is a diverse microflora that changes at different age periods. To increase the effectiveness of the study, unified rules were developed.
Diagnosis of viral infections
It is carried out by methods for detecting RNA and DNA pathogens. They are based primarily on the determination of nucleotide sequences in pathological material. For this, molecular probes are used. They are artificially obtained nucleic acids complementary to the viral acids labeled with a radioactive label or biotin.
A feature of the method is the multiple copying of a specific DNA fragment, which includes several hundred (or tens) nucleotide pairs. The mechanism of replication (copying) is that the completion can begin exclusively in certain blocks. To create them, primers (primers) are used. They are synthesized oligonucleotides.
PCR diagnostics (polymerase chain reaction) is simple to perform. This method allows you to quickly get the result when using a small amount of pathological material. Using PCR diagnostics, acute, chronic and latent (latent) infections are detected.
With sensitivity, this method is considered more preferable. However, at present, test systems are not reliable enough, therefore, PCR diagnostics cannot completely replace traditional methods.