Microbiological methods of water, soil, air

Microorganisms are the smallest, mostly single-celled creatures, widespread in nature. They are found in all environments (air, soil, water), in the human body and animals, in plants.

The qualitative diversity and the number of microorganisms depend primarily on nutritional compounds. However, the humidity, temperature regime, aeration, the action of sun rays and other factors are also of great importance.

природных сред позволяют выявить наличие патогенных микроорганизмов, определить их количество и, в соответствии с полученными результатами, выработать меры по устранению или предупреждению инфекционных заболеваний. Methods of sanitary-microbiological study of natural media 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 principles for managing natural processes. . Let's consider further, what are the methods of microbiological research .

The soil

It is considered by scientists as one of the possible ways of transmission of infectious pathologies. With the excretions of sick people or animals, pathogenic microorganisms penetrate the soil. Some of them, in particular, spore, can survive in the ground for a long time (sometimes several tens of years). позволяют определить "микробное число" (кол-во микроорганизмов в грамме грунта), а также коли-индекс (количество кишечных палочек). Pathogens of such dangerous infections, such as tetanus, anthrax, botulism, etc., enter the soil. Methods for the sanitary-microbiological study of the soil make it possible to determine the "microbial number" (number of microorganisms in a gram of soil), as well as the coli-index (the number of E. coli) .

Soil analysis: general information

следует в первую очередь отнести прямое микроскопирование и посев на плотную питательную среду. The methods of microbiological soil research should first of all include direct microscopy and seeding on a dense nutrient medium. Populations of microorganisms and their groups inhabiting the soil differ in their taxonomic position and ecological functions. In science they are united under the general term "soil biota". Ground is the habitat of a huge number of microorganisms. In a gram of soil there are from 1 to 10 billion of their cells. In this environment, the decomposition of organic substances with the participation of various saprophytic microorganisms proceeds.

Microscopic method of microbiological research: stages

The analysis of the environment begins with sampling. To do this, use a previously cleaned and rubbed alcohol knife (you can use a shovel). After this, the sample is prepared. The next step is the counting of 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, remove 2-3 cm from the top of the soil, since it may contain foreign microflora. After this, monoliths are taken from the studied site of the soil. The length of each of them should correspond to the thickness of the layer from which you need to take a sample.

On the site of 100-200 square meters. M, 7-10 samples are taken. The weight of each is about 0.5 kg. The samples must be thoroughly mixed in a bag. After this, take an average sample weighing approximately 1 kg. It should be placed in a parchment (sterile) bag, embedded in a tissue bag. Prior to direct analysis, the sample is stored in the refrigerator.

Preparation for research

The mixed soil is poured onto the dry glass. Preliminary it must be wiped with alcohol and burned over the burner. Using a spatula, the soil is thoroughly mixed and laid out evenly. Without fail it is necessary to remove roots, other extraneous elements. For this, tweezers are used. Before work, the tweezers and spatula are calcined over the burner and cooled.

From small parts of the soil distributed over the glass, small portions are taken. They are weighed in a porcelain cup on a technical scale. является специальная обработка образца. An obligatory stage of the microscopic method of microbiological investigation is the special processing of the sample. It is necessary to 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. From it take 0.4-0.8 ml of liquid and moisten the hanging of the soil 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 the rocker for 5 minutes. After that, it is left to settle for about 30 seconds. This is necessary in order for large particles to settle. After half a minute, the mass is used to prepare the preparation.

Cell counting on fixed smears

, разработанному Виноградским. 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 microorganism cells 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 volume of the suspension (usually 0.02-0.05 ml) is applied using a micropipette on a slide. A drop of agar-agar solution (agaropectin polysaccharide mixtures and agarose extracted from brown and red seaweed of the Black Sea) is added to it, mixed rapidly and distributed on 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 the carbolic erythrosin solution for 20-30 minutes.

After staining, it is washed and dried in air. The cells are counted with an immersion objective.

Sowing on dense media

позволяют выявить большое количество микроорганизмов. Microscopic methods of microbiological research make it possible to 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 preparation (soil suspension) in a Petri dish on a dense medium.

позволяет учитывать не только количество, но и групповой, а в ряде случаев и видовой состав микроскопической флоры. This method of microbiological research allows us to take into account not only the quantity, but also the group composition, and in some cases the species composition of the microscopic flora. The count of the number of colonies is usually made from the bottom of the Petri dish in transmitted light. On the calculated section, the dot is placed with a marker or ink.

Water analysis

The microflora of a water body, as a rule, reflects the microbial composition of the soil near it. имеют особое практическое значение при изучении состояния конкретной экосистемы. In this regard, the methods of sanitary and microbiological study of water and soil are of particular practical importance in studying the state of a particular ecosystem. Fresh ponds contain, as a rule, cocci, rod-shaped bacteria.

Anaerobes in water are found in small quantities. As a rule, they reproduce 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.

Virtually no artesian wells are found in microorganisms. This is due to the filtering ability of the soil layer.

считаются определение микробного числа и коли-титра либо коли-индекса. The generally accepted methods of microbiological water testing are the determination of the microbial number and the colitis or coliform index. The first indicator characterizes the number of bacteria in 1 ml of liquid. The coli-index is the number of E. coli present in a liter of water, and the colite-titer is the minimum quantity or maximum dilution of the liquid in which they can still be detected.

Determination of the microbial number

This method of sanitary microbiological water research is as follows. In 1 ml of water, the amount of facultative anaerobes and mesophilic (intermediate) aerobes that are capable of meat-agitated agar (the main nutrient medium) at 37 ° C is determined. During the day to form colonies, visible with an increase of 2-5 r. Or with the naked eye.

является посев. The key stage of this method of microbiological water research is sowing. From each sample, at least 2 different volumes are sown. When analyzing tap water, 1-0.1 ml of pure liquid is added to each cup and 0.01-0.001 ml of contaminated liquid. For sowing 0.1 ml or less, the liquid is diluted with distilled (sterile) water. Consistently prepare tenfold dilutions. 1 ml of each of them is added to two Petri dishes.

The dilutions are filled with nutrient agar. It must be pre-melted and cooled to 45 deg. After active mixing, the medium is left on a horizontal surface to solidify. At 37 degrees. Crops are grown throughout the day. позволяет учитывать результаты на тех чашках, где количество колоний находится в пределах от 30 до 300. The method of microbiological water research under consideration makes it possible to take into account the results on those plates where the number of colonies is in the range from 30 to 300.

Air

It is considered a transit medium for microorganisms. являются седиментация (оседание) и аспирация. The main methods of microbiological study of air are sedimentation (sedimentation) and aspiration.

The microflora of the air environment is conditionally divided into variable and constant. The first include yeast, pigment-forming cocci, sporiferous bacilli, rods and other microorganisms, resistant to drying, light exposure. Representatives of the variable microflora, penetrating into the air from their usual habitat, do not last long their viability.

In the air of large megacities microorganisms are much more than in the air environment of rural areas. Over the seas, the forests of bacteria are very few. Clearing of air is facilitated by precipitation: snow and rain. In enclosed premises of microbes much more than in open spaces. Their number increases in the winter period in the absence of regular airing.

Sedimentation

считается простейшим. 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 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 drops on an open cup. Primarily large particles are retained on the surface.

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 sophisticated instruments or a source of electricity.

Determination of the microbial number is carried out by the method of Omelyansky. In accordance with it, for 5 minutes on the surface of agar with an area of 100 square meters. Cm settles the number of bacteria that is present in 10 liters of air.

Order 535 "On the unification of microbiological methods of research"

Bacteriological analysis occupies an important place in a complex of clinical and laboratory activities aimed at the diagnosis, prevention and treatment of a variety of infectious diseases. However, they are not limited to environmental research.

Of particular importance is the bacteriological analysis of biological material in medical institutions. To the researches spent in medical institutions, raised requirements are made. The purpose of the Order "On the Unification of Microbiological Methods of Research" is to improve bacteriological analysis, improve the quality and effectiveness of microbiological diagnosis.

Microscopic examination of smears in women

It is a key method of analysis in the diagnosis of sexually transmitted infections and opportunistic diseases (caused by opportunistic bacteria).

Microscopic analysis allows assessing the qualitative and quantitative composition of microflora, verify the correctness of sampling. For example, the presence of vaginal epithelium in a smear taken from the cervical canal indicates a violation of the biological sampling rules.

It is worth saying that the microbiological examination in this case is generally accompanied by certain problems. They are associated with the fact that in the lower parts of the genital tract there is normally a diverse microflora that varies in different age periods. To improve the effectiveness 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 produced nucleic acids complementary to viral acids labeled with a radioactive label or biotin.

A special feature of the method is the multiple copying of a specific DNA fragment, which includes several hundred (or tens) of nucleotide pairs. The mechanism of replication (copying) is that the completion can begin only in certain blocks. To create them, primers (primers) are used. They are synthesized oligonucleotides.

PCR diagnostics (polymerase chain reaction) are easy to perform. This method allows you to quickly obtain a result 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 preferable. However, currently the test systems are not reliable enough, so PCR diagnostics can not completely replace the traditional methods.