MICROFLORA AND SANITARY-INDICATIVE BACTERIA OF SOIL, WATER, AIR.

THE METHODS OF STUDYING.

Microorganisms are widespread. Microbes are distributed everywhere in the environment surrounding us. They are found in the soil, water, air, in plants, animals, food products, various utensils, in the human body, and on the surface of the human body.

Potentially pathogenic bacteria can get to environment, for example, from patients, carriers and survive there for some time. From soil, water and air, microorganisms can enter the human body and cause diseases. So, the environment is a transmission factor of infectious diseases. Potentially pathogenic and pathogenic microorganisms get to environment mainly, in 2 ways:

1) fecal (with excrement from the intestine);

2) airborne (with droplets of mucus from the respiratory tract).

Thus sanitary-microbiological investigations are performed for study and evaluation of different objects for determination of their epidemic potential.

Sanitary microbiology is a science that studies the microflora of the environment and its harmful effect on the human body.

Methods for sanitary-microbiological investigation include:

1) determination of a total microbial contamination;

2)detection and titration of sanitary-indicative microorganisms;

3)detection of pathogenic microorganisms and/or their metabolites.

Direct detection of pathogenic microorganisms in the different objects of environment, in general, is complicated because of their small quantity, their temporarily staying in the environment and the duration and laboriousness of methods for their determination.

Thus indirect methods of detection of microbial contamination are used:

1) total microbial contamination as indicator of intensity of contamination by organic substances;

2) contamination by sanitary- indicative microorganisms.

А total microbial number (TMN) is used for evaluation of total microbial contamination.

TMN - the number of microbes in 1 ml of water, 1 g of soil , in 1 m3 of air.

Sanitary-indicative microorganisms - SIMs (or sanitary-indicative bacteria) are those, which are used for indirect evaluation of possible presence of pathogens in the environment.

SIMs are representatives of normal human microflora and homoiothermal animals.

- get to environment the same ways (fecal and airdrop ways), as pathogenic m/o;

- the same terms are maintained, as pathogenic m/o;

- do not have other habitats;

- the number is constant (they do not multiply in the environment);

- methods for determining them are easy and affordable;

- have stable and typical properties, so they are easily identified and are quantifiable.

For example, presence of Escherichia coli and Enterococcus faecalis on environmental objects is indicative of fecal contamination. Simultaneous isolation of Staphylococcus aureus and hemolytic streptococci indicates possible contamination by oral droplets.

If the amount of siMs increases in environmental objects, the probability of the presence of pathogenic and opportunistic microbes in them increases. For different objects there are specific siMs.

Presence of sanitary-indicative microorganisms is measured by titer and index.

The titer - min mass (in g) or volume (in ml), where else are detected SIMs.

The index is the amount of SIMs contained in a 1Lof water, 1g of soil, 1 m3 of air.

Microflora of the Water

Pseudomonasfluorescens, Micrococcus roseus, etc., are among the specific aquatic aerobic microorganisms. Anaerobic bacteria are very rarely found in water.

The microflora of rivers depends on the degree of pollution and the quality of purification of sewage waters flowing into river beds. Microorganisms are widespread in the waters of the seas and oceans. They have been found at different depths (3700-9000 m).

Water is an important factor for the transmission of a number of infectious diseases (enteric fever, paratyphoids, cholera, dysentery, leptospiroses, etc.).

How do we monitor the sanitary quality of water?

Explore:

1) tap (drinking) water;

2) swimming pool water;

 3) the water of open reservoirs;

4) sewage waters;

5) purified water for preparation of medicines;

6) distilled water for the preparation of sterile solutions (injections, eye drops)

Microbiological investigation of water. There are many kinds of pathogens that might be transmitted in water. These include bacteria, viruses and protozoa. Each type of bacterium, virus or protozoan requires a different test. Many of these tests are expensive because they require special materials or equipment or are time-consuming. It is impractical to monitor water quality for every pathogen on a routine basis.

The sanitary - bacteriological investigation of water includes:

1) determination of total number of microbes in 1 ml of water (TMN);

2) determination of coliform bacteria, as indicator of fecal pollution (they live in the intestine, representatives of normal human intestine microflora);

3) presence of spores of sulphite-reducing bacteria and cysts of Giardia lamblia;

4) presence of bacteriophages of E. coli;

5)detection of pathogenic microbes in case of epidemiological necessity

Due to the enormous sanitary-epidemiological role of water in relation to the intestinal group of diseases, it became necessary to work out rapid indicator methods for revealing coliform and pathogenic bacteria in water.

Determination of water TMN.

1. Sampling: 500 ml (tap water and purified water), 20 ml (water for injection), 100 ml (river water).

2. 1 ml of water is seeded in at least 2 Petri dishes according to Koch's deep method on MPA.

3. Incubation: 37 ° C, 24 hours.

4. Calculation: count the number of colonies on both plates, add up and divide by

The result is expressed in CFU(colony forming units) / ml.

Take into account only those Petri dishes, where no more than 300 colonies have grown.

If more than 300 - do 10-fold dilutions (1: 10; 1: 100, etc.). When calculating, multiply by the dilution rate.

Standards.

The drinking water should not have more than 50 microbes in 1 ml. The microbial number in

water of open reservoirs can be up 1000.

Coliform bacteria are Gram "-" asporogenous small rods that do not have oxidase activity and ferment

lactose to acid and gas at 37 ° C for 24-48 hours (or glucose to acid and gas at 37 ° C for 24 hours).

Detection of coliform bacteria is an indicator of fecal pollution of water.

Membrane filtration method.

The investigated water (3 x 100 ml) are filtered through the three (3) bacterial nitrocellulose filters. The filters are placed on Endo medium and incubated at 37 ° C for 24 hours.

After 24 h incubation at +37°C, number of colonies typical of Enterobacteriaceae is counted.

From 2 to 3 red-colored colonies are used for preparation of smear and Gram stain, followed by oxidase test allowing to distinguish Escherichia spp. Citrobacter spp, Enterobacter spp. and other Enterobacteriaceae from Pseudomonas spp. and other oxidase-positive non-fermenters which might be present in water.

For that purpose, filter with grown colonies (do not turn over! ) is transferred with forceps to filter paper disk wetted with dimethyl—n-phenyldiamine. Presence of oxidase will lead to development of blue coloration of colony.

After that, 2 or 3 colonies, which did not change color, are inoculated into semi-solid medium with 0.5% of glucose (lactose), followed by 24 h incubation at +37°C. In case of presence of formation of gas, you make a conclusion about the detection of coliform bacteria. Then number of red colonies is counted and coliform index is determined.

The index of CFU (colony forming units) of coliforms in 100 ml water is calculated according to the following formula: Х = , where X - coliforms CFU in 100 ml; V - total volume (300 ml) of water filtered through the 3 filters; a - the total number of colonies of coliforms grown on 3 filters.

If coliform bacteria are absent in all three samples of water of 100 ml, then the water accords to the requirements of microbial purity.

If coliform bacteria are detected in at least in one sample in a 100 ml the water does not accord to the requirements of microbial purity

In large settlements drinking water is being tested daily.

In the case of repeated detection of coliform bacteria, pathogenic microbes are determined.

What are the standards for drinking water? The USEPA issued revised Primary Drinking Water Standards in mid-1994. These standards address the source of water quality. The Primary Standards. If this test is used, and the sampling agency tests more than 40 samples, no more than 5% of those samples may test positive for total coliforms. If fewer than 40 samples are used, no more than 1 sample may test positive. In addition, the maximum contaminant levels, which vary with treatment technique, are specified for Giardia lamblia, Legionella (the bacterium which causes Legionnaire's disease) and viruses. The USEPA Safe Drinking Water Hotline provides more information. That number is 1-800-426- 4791.

The best way to ensure water safety - protection of water sources from microbial contamination!

Soil Microflora

Soil fertility depends not only on the presence of inorganic and organic substances, but also on the presence of various species of microorganisms which influence the qualitative composition of the soil. Due to nutrients and moisture in the soil the number of microbes in 1 g of soil reaches a colossal number — from 200 million bacteria in clayey soil to 5 thousand million in black soil.

Soil microflora consists bacteria (nitrifying, nitrogen-fixing, denitrifying), cellulose-splitting and sulfur bacteria, pigmented microbes fungi, protozoa, etc.

The greatest amount of microbes (1 000000 per cu cm) is found in the top layer of soil at a depth of 5-15 cm. In deeper layers (1.5-5 m) individual microbes are found. However, they have been discovered at a depth of 17.5 m in artesian water.

The number of microorganisms in the soil depends on the extent of contamination with faeces and urine, and also on the nature of treating and fertilizing the soil. Saprophytic spores (B. cereus. B, meguterium, etc.) survive for long periods in the soil. Pathogenic bacteria which do not produce spores due to lack of essential nutrients, and also as a result of the lethal activity of light, drying, antagonistic microbes, and phages do not live long in the soil (from a few days to a few months).

Usually the soil is an unfavourable habitat for most pathogenic species of bacteria, rickettsiae, viruses, fungi, and protozoa. However, the soil can act as a factor in the transmission of a number of pathogens of infectious diseases. Thus, for example, anthrax bacilli after falling on the soil produce spores which can remain viable for many years. As is known, the spores of Clostridia causing tetanus, anaerobic infections, and botulism, and of many soil microbes survive for long periods in the soil. The cysts of intestinal protozoa (amoeba, balantidium, etc.) spend a certain stage in the soil. The soil plays an important role in transmitting worm invasions (ascarids, hook-worms, nematode worms, etc.). Some fungi live in the soil. Entering the body they cause fusariotoxicosis, ergotism, aspergillosis, penicilliosis mucormycosis, etc.

Taking into consideration the definite epidemiological role played by the soil in spreading some infectious diseases of animals and man, sanitary-microbiological evaluation of soil is performed.

Microbiological investigation of soil. The sanitary - bacteriological investigation of soil includes:

1) a total quantity of saprophytes bacteria in 1 g of soil - a total microbial number (TMN);

2) contents of sanitary-indicative bacteria as indicator of fecal contamination.

The sanitary-indicative bacteria of the soil are

1) E. coli/Enterococcus faecalis;

2) Citrobacter spp. /Enterobacter spp.;

3) Clostridium perfringens.

Presence of E. coli/E. faecalis, Citrobacter spp. /Enterobacter spp. and Clostridium perfringens in the soil indicates the presence of recent, non- recent and bygone (old) fecal contamination, respectively.

More accurate evaluation is performed using coli-index — number of Enterobacteriaceae (so called coliform bacteria) found in 1 g of soil; perfringens-titer - mass of soil in which 1 cell C. perfringens is found.

Determination of soil TMN. For this purpose it is necessary to select most typical area not more then 25 m². The samples are taken from different places of the field along the diagonal, the angles and the center 10 — 20 cm deep. The weight of each sample must be 100 - 200 g. The total weight of the soil 0,5 - 1 kg.

After careful mixing take an average sample of weight 100 - 200 g. Put the samples of soil in the sterile banks, mark and deliver to the laboratory.

1. Prepare 10-fold dilutions (1:10, 1: 100, etc.) in an isotonic sterile solution of sodium chloride.

2. Make seeding of the soil dilutions on MPA (for bacteria) and on Saburo medium (for fungi): 1 ml in the depth of agar or 0.1 ml on the surface of agar.

3. Incubation: at 24 ° C (for fungi) and 37 ° C (for bacteria).

After incubation at optimal temperature count the colonies on the plates (1 colony=1 cell). The number of cells in 1 g of soil is calculated, taking into account:

- the weight of each sample;

- the rate of dilution;

- the volume of seeding.

Determination of perfringens-titer: seeding onto the Wilson-Blair medium: black colonies are formed and the gas breaks up the medium; Calculation: maximal dilution, where there are signs of growth of Clostridium perfringens.