Microbiological methods of research of water, soil, air

Micro-Organisms-tiny, mostly single-celled creatures, which are widespread in nature. They are found in all environments (air, soil, water), in humans and animals, in plants.

High-Quality variety and number of microorganisms depend primarily on nutritional compounds. However, equally important are humidity, temperature, aeration, sunlight, and other factors.

Methods of sanitary-microbiological research natural environments reveal the presence of pathogens, to determine their number and, in accordance with the received results, to develop measures for elimination or prevention of infectious diseases. In addition, quantitative records are necessary to model ecosystems and developing principles of management of natural processes. Consider next, what are the Methods in microbiological research.

Ground

It is regarded by scientists as one of the possible ways of transmission of infectious pathologies. With secretions of sick people or animals in the soil penetrate pathogenic microorganisms. Some of them, in particular spore which can survive in the soil for a long time (sometimes several decades). In the soil are activators of such dangerous infections, such as tetanus, anthrax, botulism, etc. Methods of sanitary-microbiological research of soil allow you to define "microbial count" (number of microorganisms per gram of soil), and coli-index (number of E. coli).

Soil Analysis: General information

Methods in microbiological studies of the soil should first include direct microscopy and inoculation of the dense nutrient medium. Populations of microorganisms and their groups inhabiting the soil, and different taxonomic position and ecological functions. In science they United under the generic term "soil biota". Soil-habitat of a vast number of microorganisms. In a gram of soil is present from 1 to 10 billion of their cells. In this environment, actively proceeds the decomposition of organic substances with the participation of diverse saprophytic microorganisms.

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Microscopic method microbiological examination: stages

Analysis of the environment begins with sampling. To do this, use pre-cleaned and rubbed with alcohol knife (you can use a shovel). This is followed by specimen preparation. The next stage – counting cells on stained smears. Consider each stage separately.

Samples

The analysis of agricultural soils, as a rule, samples are taken from the depth of the entire layer. First, remove the top 2-3 cm of soil, as it may contain extraneous microflora. After that, the studied area taking soil monoliths. The length of each of them should correspond to the thickness of the layer from which to sample.

On a plot of 100-200 square meters. m is selected 7-10 samples. The weight of each-of the order of 0.5 kg. Sample should be thoroughly mixed in the bag. Then take an average sample, weighing approximately 1 kg. It should be placed in the parchment (sterile) package, embedded in a fabric bag. Prior to analysis the sample is kept in the refrigerator.

Preparation

Mixed soil is poured on a dry glass. First it must be wiped with alcohol and burn above the burner. With the spatula the soil is thoroughly mixed and decomposed into an even layer. It is mandatory to remove the roots, and other extraneous elements. To do this, use the tweezers. Before work a pair of tweezers and a spatula calcined over a burner and cool.

From a variety of soils distributed on the glass, selected small portions. They weighed in a porcelain Cup on a technical scale. A mandatory step in the microscopic Method microbiological study Is a special treatment of the sample. It is necessary to prepare 2 sterile flasks. Their capacity shall not exceed 250 ml. In one of the flasks, pour 100 ml of tap water. From it we take 0.4-0.8 ml of liquid and moisten the soil suspension to a paste. The mixture must be rubbed with your finger or a rubber pestle for 5 min.

Water from the first flask soil mass transferred to the empty flask. Further, it is again rubbed. After this mass is transferred into the flask near the flame. The capacity of a soil suspension is shaken on a rocker for 5 min then leave it to settle for about 30 s. It is necessary to ensure that large particles have settled. Half a minute later, the mass used for the preparation of the drug.

Calculation of cells in fixed smears

The Direct microscopic study of soil is carried out on the Method microbiological study developed by Vinogradsky. In a certain volume of the suspension was counted the number of cells of microorganisms. The study of fixed smears allows you to save the drugs for long period and perform the calculations at any time.

The Preparation is carried outin the following way. A certain amount of suspension (usually 0.02-0.05 ml) is applied with a micropipette on a glass slide. To it add a drop of a solution of agar-agar (a mixture of polysaccharides of agaropectin and agarose extracted from brown and red algae of the Black sea), quickly mix and distribute on the area of 4-6 sq. cm Smear is dried in air and fixed for 20-30 min. alcohol (96 %). Further, the drug is moistened with distilled water, placed in a R-R carbolic erythrosine for 20-30 minutes.

After staining it is washed and dried in air. Cell counting is performed with immersion objective.

Planting on a dense medium

Microscopic Methods in microbiological research allow to identify a large number of microorganisms. But in spite of this, the method of sowing is considered the most common in practice. It consists in sowing the amount of the drug (soil suspension) in a Petri dish in the dense medium.

This Method microbiological study allows to consider not only the quantity but also the group, and in some cases the species composition of microscopic flora. Counting the number of colonies produced, as a rule, from the bottom of the Petri dish in transmitted light. Counted on the plot a dot with a marker or ink.

Water Analysis

The Microflora of a water body, usually reflects the microbial composition of the soil around it. In this connection, Methods of sanitary-microbiological research of water and soil Have a special practical importance in the study of the condition of specific ecosystems. In fresh water are generally cocci, rod-shaped bacteria.

Anaerobic bacteria in the water found in small quantities. Usually, they reproduce at the bottom of ponds, in the mud, taking part in the processes of purification. Microflora of the oceans and seas represented mainly salt (halophilic) bacteria.

In the water of artesian wells of the micro-organisms are almost there. This is due to the filtration capacity of the soil.

Common Methods microbiological examination of water Are considered to be the determination of microbial number and coli-titre or coli-index. The first indicator describes the number of bacteria in 1 ml of liquid. If-the index represents the number of E. coli present in a liter of water, and if the caption is – the minimum number or maximum dilution of the fluid in which they can still be found.

Determination of microbial number

This method sanitary-microbiological research of water consists in the following. In 1 ml of water, determine the number of facultative anaerobes and mesophilic (intermediate), aerobic, capable of mesopatamia agar (basic medium) at 37 deg. during the day to form colonies that are visible with magnification 2-5 p. or the naked eye.

Key stage of the considered Method microbiological examination of water Is seeding. Of each sample is seeding not less than 2 different volumes. In the analysis of tap water in each Cup making for a 1-0 lead.1 ml of the pure liquid and 0.01-0.001 ml contaminated. For sowing 0.1 ml or smaller volume of fluid is diluted with distilled (sterile) water. Consistently prepare tenfold dilutions. 1 ml from each of them bring in two Petri dishes.

Dilution poured nutrient agar. It must be pre-melt and cool to 45 deg. After an active mixing environment leave on a level surface for solidification. At 37 deg. crops grown during the day. Consider the Method microbiological examination of water allows you to post the results on those cups where the number of colonies is in the range from 30 to 300.

Air

It is a transit medium for microorganisms. The main Methods microbiological studies of the air are sedimentation (settling) and aspiration.

The Microflora of the air environment is conventionally divided into variable and constant. The first is the yeast, pigment-cocci, spore-bearing bacilli, coli and other microorganisms that are resistant to drying, exposure to light. Representatives of variable microflora penetrating the air from a familiar environment, long retain their viability.

In the air of large cities of the microorganisms is much greater than in air of the countryside. Over the seas and forests of the bacteria is very small. Cleanse air contributes to precipitation: snow and rain. In closed spaces germs a lot more than in open spaces. Their number increases in winter when the lack of regular ventilation.

Sedimentation

This Method microbiological research in Microbiology is considered to be the simplest. It is based on the deposition of droplets and particles on the surface of the agar in an open Petri dish under the influence of gravity. The method of sedimentation does not allow to accurately determine the number of bacteria in the air. The fact that the open Cup to catch fine fractions of dust particles and bacterial drops quite difficult. On the surface delayed mainly large particles.

This method is notused 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 external power source.

Determination of microbial count is done by the method Omelyanska. In accordance with him, 5 minutes on the surface of the agar with an area of 100 sq cm is deposited a number of bacteria, which is present in 10 l of air.

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

The Bacteriological analysis has an important role in clinical and laboratory activities aimed at the diagnosis, prevention and treatment of various infectious diseases. However, the study of the environment they are not limited.

Of Particular importance is bacteriological analysis of biological material in hospitals. The research conducted in health facilities, increased demands. The purpose of the Order "On the unification of microbiological methods of research" is to improve the bacteriological analysis, improving the quality and efficiency of microbiological diagnostics.

Microscopic examination of smears in women

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

Microscopic analysis allows to evaluate the qualitative and quantitative composition of microflora, to check the correctness of sampling. For example, the presence of vaginal epithelium in a smear taken from the cervix indicates a violation of the rules of collection of a biological sample.

It is Worth saying that the microbiological examination in this case is generally accompanied by certain problems. They are connected with the fact that in the lower genital tract normal microflora present diverse, varying in different ages. To improve the efficiency of research and developed the uniform rules.

Diagnosis of viral infections

It is the methods of identifying RNA and DNA pathogens. They are based mainly on the determination of the nucleotide sequences in the pathological material. For this purpose, molecular probes. They represent an artificially produced nucleic acids complementary to (complementary to) the viral acids marked with radioactive label or Biotin.

The Peculiarity of the method is multiple copying of a specific DNA fragment that includes a few hundred (or dozen) nucleotide pairs. Replication (copying) is that the building up can begin only in certain blocks. To create them using primers (starters). They represent the synthesized oligonucleotides.

PCR-diagnostics (polymerase chain reaction) is simple to implement. This method allows to quickly obtain results using a small amount of pathological material. Using PCR diagnostics revealed acute, chronic and latent (hidden) infection.

The sensitivity of this method is more preferred. However, currently the test system is not sufficiently reliable, so PCR diagnosis cannot completely replace conventional methods.