E. coli (Escherichia coli), GKB, OKB, TKB. Principles of drinking water rationing

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Drinking water

The mismatch of water, as well as chemical, makes it undrinkable. If your water source is not protected from direct environmental exposure or the utility systems are outdated or have not been cleaned for a long time, then microbiological testing is a must. Your health and safety depends on it! This is especially important for those who use the well. - ground, it directly contacts the soil, which means it threatens to "drink" you with nitrates, heavy metals, ammonia, and, of course, harmful organic substances that enter the soil as a result of the activities of agricultural farms or lands.

Table 1 shows the microbiological indicators of the current standard SanPiN 2.1.4.1074-01 for drinking water:

Table 1. Microbiological standards for drinking water

Standard microbiological analysis

The standard microbiological analysis of drinking water at Moscow State University includes the determination of three indicators: the total microbial number, the number of total coliform and thermotolerant coliform bacteria.

Advanced microbiological analysis

An extended microbiological analysis of water includes the analysis of five indicators: total microbial count, total coliform bacteria count, thermotolerant coliform bacteria count, coliphage titer and content of spores of sulfite-reducing bacteria.

Microbiological analysis of surface water bodies (ponds, rivers, pools)

Often there are bodies of water on our sites or nearby, where we and our children like to spend time with pleasure. Of course, the water in these reservoirs is not potable, but its safety for humans, as well as drinking, is regulated. Table 2 presents the microbiological indicators of the current standard for hygienic requirements for the protection of surface waters (SanPiN 2.1.5.980-00)

Table 2. Microbiological standards for recreational water use, as well as within the boundaries of populated areas

Standard microbiological analysis (surface waters)

Microbiological analysis of water not intended for drinking includes the determination of the number of two indicators: total coliform and coliform thermotolerant bacteria.

Advanced microbiological analysis (surface waters):

In addition to the two main indicators, we propose to conduct an additional analysis for the content of: coliphages, opportunistic yeasts and micromycetes (frequent companions of opportunistic diseases) and the self-purification index of the reservoir.

Determination of bacteria of the genus Salmonella and the genus Enterococcus

With a significant excess of the SanPiN 2.1.5.980-00 standards, as well as possible fecal contamination of the reservoir, we propose to analyze for the presence of pathogens of intestinal infections (genus Salmonella and Enterococcus).

Glossary

Total Microbial Abundance (TMC)

The method determines in drinking water the total number of mesophilic aerobic and facultative anaerobic microorganisms (FMC) capable of forming colonies on nutrient agar at a temperature of 37 ° C for 24 hours, visible with a 2-fold increase. This indicator identifies potential bacteria that can harm human health.

Common coliform bacteria (TCB)

Common coliform bacteria (CBC) are gram-negative, oxidase-negative, non-spore-forming rods capable of growing on differential lactose media, fermenting lactose to acid, aldehyde and gas at a temperature of (37 + 1) ° C for (24-48) hours. Many representatives of this group are microorganisms of the normal microflora of the stomach, so the excess of this group of microorganisms may indicate possible anthropogenic (including fecal) water pollution.

Thermotolerant coliform bacteria (TCB)

Thermotolerant coliform bacteria (TCB) are among the common coliform bacteria, have all their characteristics and, in addition, are able to ferment lactose to acid, aldehyde and gas at a temperature of (44 ± 0.5) ° C for 24 hours. As well as OKB, they are an indicator group, however, they are more stable in the environment: that is why the detection of this group of microorganisms in water can indicate unequivocal contamination of it with human waste products.

coliphages

Coliphages, determined by the standard method (MUK 4.2.1018-01), are Escherichia coli viruses and are considered by epidemiologists as an additional, and sometimes more sensitive, method in determining water pollution by microorganisms of the Escherichia coli group. Virus particles, and coliphages in particular, are more resistant to the environment than their host bacteria. In this regard, the presence of coliphages can serve as a reliable marker of more ancient fecal contamination of the water source. A direct correlation was shown between the content of coliphages in water and enteroviruses dangerous to humans, so the presence of coliphages in water may indicate a viral infection of the source. The current regulatory document (SanPiN 2.1.4.1074-01) implies the absence of coliphages in 100 ml of water.

Spores of sulfite-reducing clostridia

Sulphite-reducing clostridia are spore-forming anaerobic rod-shaped microorganisms, which are an additional microbiological indicator of fecal pollution of a reservoir. Unlike relatively unstable coliform and thermotolerant coliform bacteria, Clostridium spores can persist in water bodies for a long time. Clostridia are found in the intestines of humans and domestic animals, however, if ingested with water in large quantities, they can cause food poisoning. Sulfite-reducing clostridia include clostridium dangerous to humans (Clostridium botulinum, Clostridium perfringens, Clostridium tetani), which cause severe diseases. According to the current standard (SanPiN 2.1.4.1074-01), Clostridia spores should be absent in 20 ml of water.

Opportunistic yeasts and micromycetes

Conditionally pathogenic yeasts and micromycetes (molds) include a large heterogeneous group of fungal organisms that can grow saprotrophically at 37 °C. It includes representatives such as Candida albicans and Cryptococcus neoformans, which are a frequent factor in human opportunistic diseases, causing candidiasis (fungal skin diseases), thrush, and so on. Other micromycete organisms (Cladosporium cladosporioides, Aspergillusniger) can be active sensitizers of allergic reactions, and sometimes allergens themselves. In the Russian Federation, water is not standardized for molds and yeast organisms in water.

Determination of the self-cleaning index (from MUK 4.2.1884-04)

The total number of microorganisms is not standardized in the water of reservoirs in recreation areas, since the level of this group of microorganisms largely depends on the natural characteristics of each object, season, etc.

However, when choosing a new source of water supply or a place of recreation in the water of reservoirs, it is additionally necessary to determine the total microbial population, which grows:

at a temperature of 37 ° C for 24 hours;
at 22°C for 72 hours.

It is assumed that:

TMP at 37 °C is mostly represented by alochthonous microflora (introduced into the reservoir as a result of anthropogenic pollution, including fecal pollution);
MCH at 20-22 °C is represented, in addition to alochthonous, aboriginal microflora (natural, characteristic of this reservoir).

The ratio of the numbers of these groups of microorganisms makes it possible to judge the intensity of the self-purification process. At the end of the self-cleaning process, the OMC coefficient is 22 ° C / OMC 37 ° C. In places of pollution by household sewage, the numerical values of both groups are close.

The indicator provides additional information on the sanitary state of water bodies, sources of pollution, and self-purification processes.

With this method of water analysis, a certain amount of water is passed through a special membrane with a pore size of about 0.45 µm. As a result, all bacteria present in the water remain on the membrane surface. After that, the membrane with bacteria is placed for a certain time in a special nutrient medium at a temperature of 30-37 ° C. During this period, called the incubation period, the bacteria get the opportunity to multiply and form well-defined colonies that are already easy to count. As a result, you can observe this: Or even this picture:

Since this method of water analysis involves only determining the total number of colony-forming bacteria of different types, its results cannot unambiguously judge the presence of pathogenic microbes in the water. However, a high microbial count indicates a general bacteriological contamination of water and a high probability of the presence of pathogenic organisms.

When analyzing water, it is necessary to control not only the content of toxic chemicals, but also the number of microorganisms that characterize the bacteriological contamination of drinking water. TMF is the total microbial number. In the water of centralized water supply, this number should not exceed 50 CFU / ml, and in wells, wells - no more 100 cfu/ml

Sanitary and microbiological research of water is carried out in a planned
order for the purpose of current surveillance, as well as for special epidemiological
kim testimony. The main objects of such research are:

Drinking water of the central water supply (tap water);

Drinking water of non-centralized water supply;

Water from surface and underground water sources;

Wastewater;

Water of coastal zones of the seas;

Swimming pool water.

The main indicators for assessing the microbiological state of drinking water in accordance with the current regulatory documents are:

1. Total microbial count (TMC) - the number of mesophilic bacteria in 1 ml of water.

If titer- the smallest volume of water (in ml) in which at least one living
microbial cell related to BGKP.
BGKP index- the amount of BGKP in 1 liter of water.

3. The number of spores of sulfite-reducing clostridia in 20 ml of water.

4. Number of coliphages in 100 ml of water.

Determination of TMC makes it possible to assess the level of microbiological contamination of drinking water. This indicator is indispensable for the urgent detection of massive microbial contamination.

Total microbial count- this is the number of mesophilic aerobic and facultative anaerobic microorganisms capable of forming colonies on nutrient agar at a temperature of 37 ° C and within 24 hours, visible at a twofold increase.

When determining the total microbial number, 1 ml of the test water is added to a sterile Petri dish and 10-12 ml of warm (44 ° C) molten nutrient agar is poured. The medium is gently mixed with water, uniformly and
without air bubbles distributing along the bottom of the cup, then cover with a lid and leave to solidify. Crops are incubated in a thermostat at 37 °C for 24 hours. Count the total number of colonies grown in both dishes and determine the average value. The final result is expressed as the number of colony forming units (CFU) in 1 ml of the test water. 1 ml of drinking water should contain no more than 50 CFU

Definition of BGKP
At the same time, common coliform bacteria - OKB and thermotolerant coliform bacteria - TKB are determined.

GKB are gram-negative, non-spore-forming rods that ferment lactose to acid and gas at 37°C for 24-48 hours. TKB are among the OKB, they have their signs, but I ferment at 44 ° C. For the determination of enterobacteria - the method of membrane filters or titration.

Microbial number - the main criteria for assessing the microbiological state of drinking water, based on the current regulatory documents, is TMC (total microbial number), which characterizes the number of aerobic and anaerobic bacteria in one milliliter of water, formed per day at a temperature of 37 degrees, in a nutrient medium. This indicator is virtually indispensable for the rapid detection of massive microbial contamination.

For determination of the total microbial number one milliliter of the test water is added to a sterile Petri dish, then 10-15 ml of warm (about 44 ° C) nutrient agar is poured in a molten form. The medium is carefully mixed with water, distributed evenly and without air bubbles over the bottom of the dish, then closed with a lid and left in the Petri dish until solidified. The same is done in the other cup. Sowing in a thermostat is incubated at a temperature of 37 ° C during the day. Then, at double magnification under a microscope, the total number of colonies grown in two cups is counted, and the average value is determined. There should not be more than 50 CFU in 1 ml of drinking water.

With this method of water analysis, a certain amount of water is passed through a special membrane with a pore size of about 0.45 µm. As a result, all bacteria present in the water remain on the membrane surface. After that, the membrane with bacteria is placed for a certain time in a special nutrient medium at a temperature of 30-37 °C. During this period, called the incubation period, the bacteria have the opportunity to multiply and form well-defined colonies that are already easy to count. As a result, you can observe this: Or even this picture:

When analyzing water, it is necessary to control not only the content of toxic chemicals, but also the number of microorganisms characterizing the bacteriological contamination of drinking water. 100 cfu/ml

- drinking water of the central water supply (tap water);

- drinking water of non-centralized water supply;

— water from surface and underground water sources;

- wastewater;

- water of coastal zones of the seas;

- Swimming pool water.

The main indicators for assessing the microbiological state of drinking water in accordance with the current regulatory documents are:

1. Total microbial count (TMC) - the number of mesophilic bacteria in 1 ml of water.

If titer- the smallest volume of water (in ml) in which at least one living
microbial cell related to BGKP.
BGKP index- the amount of BGKP in 1 liter of water.

3. The number of spores of sulfite-reducing clostridia in 20 ml of water.

4. Number of coliphages in 100 ml of water.

Definition of BGKP
At the same time, common coliform bacteria - OKB and thermotolerant coliform bacteria - TKB are determined.

Quality indicators of drinking water of water supply systems.

This indicator is virtually indispensable for the rapid detection of massive microbial contamination.

(main method)

The method is based on filtering a certain volume of water (300 ml) through membrane filters, growing crops on a differential diagnostic medium with lactose (Endo) and subsequent identification of colonies by cultural and biochemical characteristics.

Membrane filters prepared for analysis (boiled or sterilized in another way) are placed with sterile tweezers into the funnel of the filter apparatus. A measured volume of water is poured into the funnel of the device and a vacuum is created. After filtration, the filter is removed and placed on the surface of the Endo nutrient medium without turning over.

One cup can fit 3 filters. In the study of drinking water, 3 volumes of 100 ml are filtered. when analyzing water of unknown quality, it is advisable to filter other volumes of water to obtain isolated colonies on the filter (10.40, 100 and 150 ml).

The filter dishes are incubated upside down in an incubator at t 37°C for 24 hours.

If there is no growth on the filters or atypical membranous, moldy, blurry colonies have grown, they give a negative result. OKB and TKB are absent in 100 ml of the studied water.

With the growth of typical isolated lactose-positive (dark red with prints on the reverse side of the filter) colonies on the filters, their number is counted and they begin to confirm their belonging to the OKB and TKB.

Spend microscopy of smears from 3-4 colonies stained by Gram (take into account gram-negative);

The presence of oxidase is determined (oxidase-negative ones are taken into account, since oxidase-positive gram-negative rods do not belong to enterobacteria, but can be, for example, pseudomonads);

The fermentation of lactose to acid and gas is determined at a temperature of 37 ° C, which is important for slightly colored colonies and their relationship to TKB, and at a temperature of 44 ± 0.5 ° C, in order to decide whether they belong to TKB.

Setting up an oxidase test

On paper moistened with 1% alcohol solution of α-naphthol and 1% aqueous solution of dimethylphenylenediamine, a part of the colored colony is applied with a platinum loop or a glass rod. the reaction is considered positive if within 1 minute, a maximum of 4 minutes, a blue or purple color appears. Oxidase-positive colonies are not taken into account and are not subjected to further research.

It is possible to transfer the filter with colonies to the paper moistened with the reagent. You can use ready-made paper systems (NIBs) moistened with distilled water.

Part of the colonies of gram-negative oxidase-negative bacteria are tested for the ability to ferment lactose. This uses a semi-liquid medium with lactose and a pH indicator. Sowing is done by injection to the bottom in 2 test tubes. One is incubated at a temperature of 37 ± 1 ° C for 24-48 hours to confirm the relationship to TKB, the other at a temperature of 44 ± 0.5 ° C for 24 hours, registration is possible after 4-6 hours to confirm the presence of TKB.

When superimposing colonies on the filter, they are sieved, then the resulting isolated colonies are identified. Colonies are counted as OKB - if they are red on Endo, they contain gram-negative oxidase-negative rods that decompose lactose at a temperature of 37 ° C to acid and gas. Colonies are counted as TKB if they contain gram-negative oxidase-negative rods that ferment lactose at a temperature of 44 °C to acid and gas (Scheme No. 2).

SCHEME № 2

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Total microbial count

With this method of water analysis, a certain amount of water is passed through a special membrane with a pore size of about 0.45 µm. As a result, all bacteria present in the water remain on the membrane surface. After that, the membrane with bacteria is placed for a certain time in a special nutrient medium at a temperature of 30-37 °C. During this period, called the incubation period, the bacteria have the opportunity to multiply and form well-defined colonies that are already easy to count. As a result, you can observe this: Or even this picture:

- drinking water of the central water supply (tap water);

- drinking water of non-centralized water supply;

— water from surface and underground water sources;

- wastewater;

- water of coastal zones of the seas;

- Swimming pool water.

The main indicators for assessing the microbiological state of drinking water in accordance with the current regulatory documents are:

1. Total microbial count (TMC) - the number of mesophilic bacteria in 1 ml of water.

If titer- the smallest volume of water (in ml) in which at least one living
microbial cell related to BGKP.
BGKP index- the amount of BGKP in 1 liter of water.

3. The number of spores of sulfite-reducing clostridia in 20 ml of water.

4. Number of coliphages in 100 ml of water.

Definition of BGKP
At the same time, common coliform bacteria - OKB and thermotolerant coliform bacteria - TKB are determined.

Principles of drinking water rationing

The same is done in the other cup. Sowing in a thermostat is incubated at a temperature of 37 ° C during the day. Then, at double magnification under a microscope, the total number of colonies grown in two cups is counted, and the average value is determined. There should not be more than 50 CFU in 1 ml of drinking water.

For the period from January 9 to October 10, 2014. The Department of Veterinary and Sanitary Expertise of the Federal State Budgetary Institution "Tula MVL" received - 302 water samples, conducted - 693 studies, of which, for the indicators of the Design Bureau, TKB-458 studies.

What are these indicators, and why exactly are they given attention when assessing drinking water?

Water - the main component of any organism, plays a huge role in its life. It is a habitat for various microorganisms, including pathogens. Pathogen detection is the most accurate indicator of water pollution. These microorganisms include bacteria of the Escherichia coli group - E. coli (bacteria of the Escherichia coli group, also called colimorphic and coliform bacteria) - a group of bacteria of the Enterobacteriaceae family, conditionally distinguished by morphological and cultural characteristics, used by sanitary microbiology as a marker of fecal contamination. Among coliform bacteria, the presence of common and thermotolerant coliform bacteria (TCB, TKB) in drinking water is often determined, which indicates poor-quality water supply and possible fecal contamination of the water source, which creates a potential threat to the development and spread of intestinal diseases.

In water supply systems with prepared water, coliform bacteria should not be detected (SanPiN). The presence of coliform organisms indicates insufficient water purification, its secondary pollution, and the presence of nutrients in the water. Accidental entry of coliform organisms into the system is allowed, but not more than 5% of samples taken during the year. If TKB, OKB) are detected in a sample of drinking water, they are immediately determined in repeated samples.

TKB (thermotolerant coliform bacteria). This is a group of coliform organisms capable of fermenting lactose at 44-45°C. They are quickly detected, therefore, they serve to evaluate the effectiveness of water purification from fecal bacteria.

OKB (Common Coliform Bacteria) - The OKB group includes a fairly large number of genera of the Enterobacteriacea family, whose representatives are able to ferment lactose: Citrobacter, Enterobacter, Klebsiella, Serratia, Pantoea, Rahnella, etc. Among these microorganisms there is also a large number of free-living saprophytes, therefore, the TKB indicator is an important technological (indicator) indicator.

Accordingly, if these bacteria are found in drinking water, then this means that there is a possibility of water pollution by sewage.

The results of determining the indicators of OKB, TKB are presented as CFU / 100 ml; coliform bacteria should not be detected in 100 ml of drinking water in a three-fold study of the normalized volume.

Be that as it may, any increased bacteria content in the water is a warning sign, and when it appears, something needs to be done with the water.

To conduct microbiological studies of drinking water, you can contact the Tula MVL Federal State Budgetary Institution.

8.1. Determination of the total number of microorganisms forming colonies on nutrient agar

8.1.1. Definition of the concept of an indicator

8.1.2. Performing analysis

From each sample, at least two volumes of 1 ml are inoculated.

After thorough mixing, water samples are added 1 ml into sterile Petri dishes, slightly opening the lids. After adding water, (8-12) ml (per a cup with a diameter of 90-100 mm) of molten and cooled to (45-49) ° C nutrient agar is poured into each cup after flaming the edge of the dish in which it is contained. Then quickly mix the contents of the cups, evenly distributing over the entire bottom, avoiding the formation of air bubbles, getting agar on the edges and lid of the cup. This procedure is carried out on a horizontal surface, where the plates are left until the agar solidifies.

The molten agar for the period of analysis is placed in a water bath or a thermostat that maintains a temperature of (45-49) °C.

After the agar solidifies, the plates with the cultures are placed upside down in a thermostat and incubated at a temperature of (37 ± 1)°C for (24 ± 2) hours.

8.1.3. Even results

All colonies grown on the plate, observed at 2-fold magnification, are counted. Only those dishes on which no more than 300 isolated colonies have grown are taken into account.

The number of colonies on both plates are summed up and divided by two. The result is expressed as the number of colony forming units (CFU) in 1 ml of the test water sample.

If counting is not possible on one of the 2 plates, the result is given based on the count of colonies on one plate. If two plates show diffuse colony growth that does not cover the entire surface of the plate, or more than 300 colonies have grown and the assay cannot be repeated, count the sector of the dish and then recalculate the entire surface. In these cases, the protocol notes “the number of CFU / ml - approximately”.

If colony counts on plates are not possible, record “continuous growth” on the protocol.

8.2. Determination of common and thermotolerant coliform bacteria by membrane filtration (main method)

8.2.1. Definition of the concept of an indicator

Common coliform bacteria (CBC) are Gram-negative, oxidase-negative, non-spore-forming rods capable of growing on differential lactose media, fermenting lactose to acid, aldehyde and gas at a temperature of (37 ± 1) °C for (24-48) h .

8.2.2. Method principle

The method is based on filtering a set volume of water through membrane filters, growing crops on a differential nutrient medium with lactose and subsequent identification of colonies by cultural and biochemical properties.

8.2.3. Performing analysis

8.2.3.1. Research order

In the study of drinking water, 3 volumes of 100 ml are analyzed.

If stable negative results are obtained, 300 ml of water can be filtered through one filter.

When filtering water of unknown quality, it is advisable to increase the number of filtered volumes to obtain isolated colonies on the filter (for example, 10, 40, 100, 150 ml of water).

The measured volume of water is filtered through membrane filters in compliance with the requirements set forth in paragraph 7.

The filters are placed on the Endo medium prepared according to paragraph 5.4. Cups with filters are placed in a thermostat upside down and incubated at a temperature of (37 ± 1) °C for (24 ± 2) hours.

If there is no growth on the filters or the colonies are membranous, spongy, moldy, transparent, vague, they give a negative answer: the absence of OKB and TKB in 100 ml of the test water. The analysis is completed after 24 hours.

If growth of isolated typical lactose-positive colonies is detected on the filters: dark red, red with or without a metallic sheen, or other similar type of colonies with an imprint on the back of the filter, count the number of colonies of each type separately and proceed to confirm their belonging to the OKB and TKB.

To confirm the presence of OKB, examine:

All colonies if less than 5 colonies grew on the filters;

At least 3-4 colonies of each type.

To confirm the presence of TKB, all typical colonies are examined, but no more than 10.

Each selected isolated colony is examined for:

The presence of oxidase activity;

Gram affiliation (microscopy of a Gram-stained preparation or Gregersen test);

Fermentation of lactose to acid and gas.

8.2.3.2. Setting up an oxidase test

A strip of filter paper is placed in a clean Petri dish and moistened with 2-3 drops of the oxidase test reagent according to paragraph 5.7. Finished paper systems are moistened with distilled water. Part of the isolated colony with a glass folder or a platinum loop (nichrome metal loop can give a false positive reaction) is streaked onto the prepared filter paper. The reaction is considered positive if a violet-brown (section 5.7.1 option 1) or blue (section 5.7.2 option 2 and NIB oxidase) staining of the stroke appears within 1 min. With a negative reaction, the color at the site of application of the culture does not change. With a positive result, this colony is excluded from further research.

If, when examining colonies stained in dark red, an insufficiently clear result is obtained, it is necessary to transfer the culture from Endo medium to nutrient agar. After incubation, the test is repeated.

8.2.3.3. Determination of belonging to Gram

A smear is taken from the oxidase-negative colony, Gram-stained, and microscopically examined.

On a glass slide defatted with alcohol, 1 drop of distilled water is applied in a loop, a small amount of culture from the analyzed colony is added and spread over the surface of the glass. The smear is dried at room temperature and fixed three times through the flame of the burner. A strip of filter paper is applied to the preparation and a carbolic solution of violet gentian is poured on it for (0.5-1) min, the paper is removed, Lugol's solution is poured for (0.5-1) min, Lugol's solution is drained and the glass is washed in ethyl alcohol in for (0.5-1) minutes, until the dye stops leaving. Then the glass is thoroughly washed with water and stained for (1-2) min with Ziel's fuchsin, diluted 1:10 with distilled water. After washing and drying the preparation, the smear is microscopically examined.

The preparation of reagents for Gram staining is described in Section 5.9.

Gram-negative microorganisms are pink, Gram-positive are blue. Coliform bacteria are gram-negative rods.

The Gram stain can be replaced by the Gregersen test, which does not require the use of optics.

Gregersen's test: in a drop of a 3% aqueous solution of KOH on a glass slide, a bacterial mass taken from a solid medium is emulsified. After a few seconds of stirring with the loop, the suspension becomes mucilaginous and mucous threads stretch behind the loop, which indicates that the test culture or colony belongs to a gram-negative species. In gram-positive bacteria, mucous threads are not formed - the reaction is negative.

8.2.3.4. Determination of lactose fermentation

The rest of the oxidase-negative gram-negative isolated colony is seeded in parallel in two test tubes with lactose medium (p. 5.6):

To confirm the presence of OKB, the inoculation is incubated at a temperature of (37 ± 1) °C for 48 hours;

To confirm the presence of TKB, inoculation is carried out in a medium preheated to a temperature of (43-44) °C and incubated at a temperature of (44 ± 0.5) °C for 24 hours.

Primary accounting for the formation of acid and gas on confirming semi-liquid media and NIB (section 5.6) is possible after (4-6) hours. If acid and gas are detected, a positive answer is given. In the absence of acid and gas or in the presence of only acid, tubes with cultures for the final count of TKB are left up to 24 hours. Tubes with crops to confirm the presence of TTB after viewing after 24 hours and receiving a negative result are left for final counting up to 48 hours.

If the colony to be examined is of small size, subculture it on nutrient agar slant and, after incubation for (18-24) hours, perform all necessary confirmatory tests.

8.2.3.5. Perform confirmatory tests in colony overlay or continuous growth

If colonies or continuous growth are observed on part or all of the filter surface, an oxidase test is performed by placing the membrane filter on a circle of filter paper of a larger diameter than the filter, richly moistened with the reagent, or on a NIB oxidase disc moistened with distilled water. When the first signs of a reaction appear, but not more than 5 minutes later, the membrane filter is transferred back to the Endo medium. After a clear manifestation of the reaction, the result is determined. If a violet-brown or blue color appears (depending on the reagent used), the oxidase test is considered positive.

If all colonies on the filters are oxidase-positive, they are not taken into account and give an answer about the absence of TKB and TKB and complete the analysis.

In case of a negative oxidase reaction, sieving is carried out until isolated colonies are obtained and their belonging to the OKB and TKB is confirmed according to paragraphs 8.2.3.3-8.2.3.4 (qualitative analysis).

8.2.4. Accounting for results

8.2.4.1. Gram-negative colonies are counted as TBCs for a negative oxidase test and lactose fermentation at 37°C to produce acid and gas.

Gram-negative colonies are counted as TKB in a negative oxidase test and lactose fermentation at 44°C with acid and gas production.

8.2.4.2. In the absence of common and thermotolerant coliform bacteria on all filters, the result is recorded as “no CFU of TCB in 100 ml” and “no CFU of TCB in 100 ml”.

8.2.4.3. In the case of identification of all grown suspicious colonies, the number of colony-forming units of TKB and TKB is counted on all filters and the result of the CFU analysis is expressed in 100 ml of water.

The calculation is carried out according to the formula:

X is the number of colonies in 100 ml;

the filtered volume of water through the filters on which the account was kept;

a is the total number of colonies counted on these filters.

1. When sowing 3 filters of 100 ml, two colonies grew in 100 ml, there was no growth on the other two filters. The number of total or thermotolerant coliforms would be:

CFU OKB (TKB) in 100 ml

2. When sowing 10, 40, 100 and 150 ml on filters with a filtered volume of 40 ml, 4 isolated colonies grew, with a filtered volume of 100-3 OKB. Filters with volumes of 10 ml and 150 ml are overgrown and are not subject to accounting. The total number of OKB (TKB) colonies on those filters where isolated colonies were obtained is summed up and recalculated for a volume of 100 ml.

CFU in 100 ml

8.2.4.4. If, during a selective check of colonies of the same type, unequal results are obtained, then the numbers of OKB or TKB among colonies of this type are calculated according to the formula:

, where

X is the number of confirmed bacteria of the same type;

a is the total number of colonies of this type;

- number of tested ones;

c is the number of colonies with a positive result.

The results of accounting for each type of colonies are summarized and then calculated according to clauses 8.2.4.3-8.2.4.4.

8.2.4.5. The final result is given: the number of CFU TCB in 100 ml, of which the number of CFU TCB in 100 ml.

An indicative result can be issued by detecting typical coliform colonies on Endo's medium, formed by gram-negative oxidase-negative bacteria. The final answer is confirmed by the results of lactose fermentation.

8.2.4.6. When superimposing colonies or continuous growth on all filters (clause 8.2.3.5), in case of confirmation of belonging to OKB and TKB, a qualitative result “detected OKB in 100 ml” is issued.

If all the colonies on the filter are oxidase-positive or their belonging to the OKB and TKB is not confirmed, the analysis is completed, the protocol says “filters bury”.

In both cases, the analysis is repeated.

8.3. Determination of common and thermotolethal coliform bacteria by titration method

8.3.1. Definition of the concept of an indicator

Definition of the concept of OKB and TKB indicators according to clause 8.2.1.

8.3.2. Application area

The titration method can be used:

In the absence of materials and equipment necessary to perform the analysis by membrane filtration;

When analyzing water with a high content of suspended solids;

In the case of the predominance of foreign microflora in the water, which prevents the production of isolated colonies of common coliform bacteria on the filters.

8.3.3. Method principle

The method is based on the accumulation of bacteria after inoculation of a fixed volume of water into a liquid nutrient medium, followed by subculture on a differential dense nutrient medium with lactose and identification of colonies by cultural and biochemical tests.

8.3.4. Performing analysis

In the study of drinking water qualitative method(current sanitary and epidemiological supervision, production control) inoculate 3 volumes of 100 ml.

When studying water for the purpose quantification OKB and TKB, when reanalyzed, inoculate: 3 volumes of 100 ml, 3 volumes of 10 ml, 3 volumes of 1 ml.

Each volume of the test water is inoculated into a lactose-peptone medium prepared according to clause 5.5. Inoculation of 100 ml and 10 ml of water is carried out in 10 and 1 ml of concentrated lactose-peptone medium, inoculation of 1 ml of the sample is carried out in 10 ml of medium of normal concentration.

Crops are incubated at (37 ± 1) °C for 48 hours. Not earlier than 24 hours. incubation, a preliminary assessment of crops is carried out. From the containers, where the presence of growth (turbidity) and the formation of gas are noted, a bacteriological loop is inoculated into sectors of the Endo medium (section 5.4.1) to obtain isolated colonies.

The containers without growth and gas formation are left in a thermostat and finally examined after 48 hours. Crops without signs of growth are considered negative and they are not subject to further research. From the containers where turbidity and the formation of gas or only turbidity are noted, seeding is done on the sectors of the Endo medium.

Inoculations on Endo medium are incubated at a temperature of (37 ± 1) °C for (18-20) hours.

With the formation of turbidity and gas in the accumulation medium and growth on Endo medium, colonies typical of lactose-positive bacteria: dark red or red, with or without a metallic sheen, convex with a red center and an imprint on the nutrient medium, give a positive response to the presence of common coliforms. bacteria in a given sample volume.

The presence of the OKB is required to be confirmed:

If only turbidity is noted in the accumulation medium;

If belonging to lactose-positive colonies is questionable by the researcher. In these cases:

Check for an imprint on Endo's medium after looping a suspicious colony;

Perform the oxidase test according to clause 8.2.3.2;

Confirm belonging to Gram according to clause 8.2.3.3;

The ability to gas formation is confirmed by inoculation of isolated 1-2 colonies of each type from each sector on a medium with lactose according to clause 5.6, followed by incubation of the inoculations at a temperature of (37 ± 1) ° C for (24-48) hours.

In the absence of isolated colonies, sieving is carried out on Endo medium by conventional bacteriological methods.

A negative answer is given if:

There are no signs of growth in the accumulation environment;

There is no growth on the sectors of the Endo environment;

Colonies not characteristic of coliform bacteria (transparent with jagged edges, blurry, etc.) grew on the sectors of the Endo medium;

All colonies were oxidase positive;

All colonies were Gram-positive;

If gas formation is not noted in the confirmatory test on the medium with carbohydrate.

For determining thermotolerant coliform bacteria work with sectors of the Endo medium where typical lactose-positive colonies have grown. Sowing 2-3 isolated colonies of each type from each sector in test tubes with any of the lactose media prepared according to paragraph 5.6.

Before sowing, the medium is heated in a water bath or in a thermostat to 44 °C. Immediately after inoculation, the tubes are placed in a thermostat and incubated at a temperature of (44 ± 0.5) °C for 24 hours. It is allowed to view the inoculations after (4-6) hours.

With the formation of gas in the accumulation medium, the growth of lactose-positive bacteria on the Endo medium, and the detection of the ability of these bacteria to ferment lactose to acid and gas for 24 hours at a temperature of 44 ° C, they give a positive response to the presence of a TKB water sample in this volume. In all other cases, they give a negative answer.

It is permissible to speed up the issuance of a response to the presence of TKB to inoculate 1 ml from the volumes of the accumulation medium, where turbidity and gas formation are noted in a test tube with a lactose-peptone medium with a float according to clause 5.6 and preheated to a temperature of 44 ° C. Crops are kept in a thermostat at a temperature of (44 ± 0.5) ° C for 24 hours. If acid and gas are detected, they give a positive answer.

8.3.5. Accounting for results

In the study of 3 volumes of 100 ml, the results are evaluated qualitatively and if OKB and TKB are detected in at least one of the 3 volumes, an entry is made in the protocol “found in 100 ml”.

In the study of the quantitative method, the most probable number (MPN) of the OKB and TKB is determined according to Table. 1.1 applications 1.

The result is reported without a confidence interval.

In case of a negative response to the presence of TKB and TKB in all investigated volumes, a conclusion is issued in the protocol “not found in 100 ml”.

8.4. Determination of spores of sulfite-reducing clostridia

8.4.1. Definition of the concept of an indicator

Sulfite-reducing clostridia are spore-forming anaerobic rod-shaped microorganisms that reduce sodium sulfite on iron-sulfite agar at a temperature of (44 ± 1) ° C for (16-18) hours.

8.4.2. Method principle

The method is based on growing crops in iron sulfite agar under conditions close to anaerobic and counting the number of black colonies.

8.4.3. Performing analysis

8.4.3.1. A 20 ml sample of water is heated in a water bath in test tubes at a temperature of (75 ± 5)°C for 15 minutes to exclude vegetative forms.

In the study of chlorinated water, heating of the sample can be omitted.

From each sample of drinking water, 20 ml are cultured or filtered. If necessary, select volumes so that no more than 10-15 colonies grow in crops (on filters). In this case, they are guided by the results of previous studies.

Water filtration is carried out in accordance with the requirements set forth in paragraph 7.

8.4.3.2. Determination by filtration in test tubes

Before inoculation, tubes with iron sulfite agar prepared according to 5.8 are melted in a water bath (do not boil!). During sowing, the medium is kept heated to (70-80) °C in a water bath.

After filtering the set volume of water, the membrane filter is taken with flambéed tweezers by two opposite edges and bent in the form of a tube is placed in a test tube with hot agar. The side of the filter with settled bacteria is facing inwards. In this case, the filter is straightened and located along the wall of the test tube.

Immediately after inoculation, the tube with agar and filter to create anaerobic conditions is rapidly cooled by placing in a container of cold water. Cultivate crops at (44 ± I) °C for (16-18) hours.

8.4.3.3. Determination by filtration in Petri dishes

Petri dishes with a diameter of (55-60) mm are filled with a thin layer of iron-sulfite agar. After filtration, place the filter with the filter surface down on the solidified nutrient medium so that there are no air bubbles under the filter. Then pour molten iron sulfite agar up to the top of the dish so that the lid fits snugly on the medium to create anaerobic conditions. Cultivate crops at (44 ± 1) ° C for (16 - 1 8) hours.

8.4.3.4. Determination by direct seeding

Iron sulfite agar vials and water sample are prepared as described in 8.4.3.1.

Add to sterile test tubes:

10 ml in 2 test tubes (at least 30 ml in volume) or

5 ml in 4 test tubes (15 ml each).

Crops are poured with hot iron-sulfite agar in an amount exceeding the volume of water by 2 times. Pour the medium along the wall of the test tube, avoiding the formation of air bubbles. After that, the tube is rapidly cooled by placing it in a container of cold water to create anaerobic conditions. The inoculations are incubated at (44 ± 1) °C for (16-18) hours.

8.4.4. Accounting for results

Only those crops where isolated colonies are obtained are subject to quantitative accounting. Black colonies are counted, grown both on filters and in the thickness of the nutrient medium.

The result of the analysis is expressed as the number of colony-forming units (CFU) of spores of sulfite-reducing clostridia in 20 ml of water.

If there is no growth of black colonies on all filters, the answer is “not found in 20 ml of water”.

If it is impossible to count colonies due to confluent growth, the result is assessed as qualitative, the protocol notes “found in 20 ml”. If necessary, to obtain a quantitative result, the analysis is repeated.

8.5. Definition of coliphages

8.5.1. Definition of the concept of an indicator

Coliphages are bacterial viruses capable of lysing E. coli and forming at a temperature of (37 ± 1)°C after (18 ± 2) h bacterial lawn lysis zones (plaques) on nutrient agar.

8.5.2. Titration method for the determination of coliphages

8.5.2.1. Method principle

The determination of coliphages in drinking water consists in the preliminary accumulation of coliphages in the enrichment medium on the culture of E. coli and the subsequent identification of zones of lysis (enlightenment) of the E. coli lawn on nutrient agar.

8.5.2.2. Application area

The method is intended for current monitoring of drinking water quality.

8.5.2.3. Preparation of test culture E. coli K12 StrR.

At all stages of the study, a bacterial suspension prepared as follows is used: the culture of E. coli is inoculated into a test tube with slanted nutrient agar with streptomycin (section 5.3.5). After (18 ± 2) hours of incubation at a temperature of (37 ± 1) ° C, wash the bacteria from the joint with 5 ml of sterile saline (0.85% NaCl solution) and, according to the turbidity standard, prepare a suspension of E. coli at a concentration of 109 bacterial cells in 1 ml.

It is allowed to use a 4-hour broth culture of E. coli obtained by growing in a thermostat at a temperature of 37°C. The concentration of 109 E. coli bacterial cells is contained in 2 ml.

8.5.2.4. Conducting a qualitative analysis

Add 10 ml of a 10-fold nutrient broth (prepared according to clause 5.2.2) and 1 ml of a prepared washing of the test culture or 2 ml of a 4-hour broth culture (clause 8.5.2.3) into the test water sample with a volume of 100 ml.

For culture control, 0.1 ml of an E. coli wash (or 0.2 ml of a 4-hour broth culture) is placed in a Petri dish and covered with nutrient agar.

The test water sample (100 ml) and the Petri dish with E. coli control are placed in a thermostat and incubated at a temperature of (37 ± 1) °C for (18 ± 2) hours.

After incubation, 10 ml of the test water sample is poured into a test tube and 1 ml of chloroform is added.

The test tube is closed with a sterile rubber or silicone stopper, shaken vigorously to evenly distribute chloroform over the sample volume, and leave at room temperature for at least 15 minutes until the chloroform is completely precipitated.

In pre-melted and cooled to (45-49) °C Nutrient agar, add the prepared washout of E. coli bacteria (clause 8.5.2.3) at the rate of 1.0 ml of washout (or 2 ml of a 4-hour broth culture) per 100 ml of agar.

Transfer 1 ml of the sample treated with chloroform (without touching chloroform) into a sterile Petri dish with a pipette from a test tube and fill it with a mixture of melted and cooled to (45-49) ° C nutrient agar with a volume of (12-15) ml, as well as one additional Petrid dish for control E. coli cultures and shake gently to evenly mix the water and agar samples. For complete solidification, the cups are left on the table at room temperature for 10 minutes. After solidification, the cups are turned over and placed in a thermostat for (18 ± 2) hours at 37 °C.

When performing a series of samples, a general control is placed for the entire series.

Accounting for results

View crops carried out in transmitted light.

The sample is considered positive in the presence of complete lysis, clarification of several plaques, one plaque on the dish with a water sample in the absence of lysis zones on the control dish.

The analysis protocol notes: coliphages were found or not found in 100 ml of water (qualitative result).

If there are lysis zones in the culture control, the result is considered invalid.

8.5.2.5. Performing Quantitative Analysis

Pour the investigated water sample in the amount of 100 ml into 6 volumes: 1 bottle of 50 ml and 5 test tubes of 10 ml. To 50 ml of the sample, add 5 ml of tenfold nutrient broth (according to 5.2.2) and 0.5 ml of a wash (or 1 ml of a 4-hour broth culture) of E. coli bacteria (clause 8.5.2.3). Into each 10 ml of the sample, add 1 ml of tenfold nutrient broth and 0.1 ml of a wash (or 0.2 ml of a 4-hour broth culture) of E. coli bacteria.

For culture control, OD ml of bacteria wash (or 0.2 ml of 4-hour broth culture) of E. coli is placed in a Petri dish and filled with nutrient agar.

Crops are incubated at a temperature of (37 ± 1) °C for 18 ± 2 hours.

After incubation, pour 10 ml from a volume of 50 ml into a test tube. Add 1 ml of chloroform to all 6 test volumes. Close the test tubes with sterile rubber or silicone stoppers, shake vigorously to evenly distribute chloroform over the sample volume, and leave at room temperature for at least 15 minutes to precipitate chloroform.

In the previously melted and cooled to (45-49) °C nutrient agar, add the prepared washing of E. coli bacteria (section 8.5.2.3) at the rate of 1.0 ml of washing (or 2 ml of a 4-hour broth culture) per 100 ml of agar . Pour the prepared mixture into Petri dishes: 1 cup to control the culture of E. coli for lysogenicity and one cup for each water sample under study. With the simultaneous analysis of several water samples, one control of the E. coli culture is placed.

After the agar solidifies, the dishes intended for inoculation of samples are divided into 6 sectors, labeled in accordance with the volumes under study. With a Pasteur pipette (micropipette or bacteriological loop with a longitudinal stroke), apply 1 drop of supernatant liquid (without chloroform) to each sector from the corresponding test tube.

After the drops have dried, place the cups with the test samples and the control cup in a thermostat at (37 ± 1) °C for (18 ± 2) hours.

Accounting for results

The results are viewed in transmitted light.

Accounting is carried out by the presence of zones of enlightenment (lysis) on the sectors of the E. coli lawn.

When using the drip seeding method with a pipette, a lysis zone is formed in the form of a rounded spot or individual plaques. When sowing a longitudinal stroke with a bacteriological loop, lysis is noted along the stroke.

The sample is considered positive if there is a lysis zone on at least one sector in the absence of lysis zones on the control plate.

The assessment is carried out according to the table of the most probable number (MPN) of plaque-forming units (PFU) (Table 1.2). The analysis protocol indicates the most probable number of coliphages in 100 ml of water and the range of possible fluctuations: LF PFU (lower limit - upper limit) of coliphages in 100 ml. The result is semi-quantitative.

If there are zones of lysis in the control dish, the result is considered invalid.

8.5.3. Direct method for determining coliphages

.one. Method principle

The determination of coliphages in drinking water consists in the study of a normalized volume of water (100 ml) by direct inoculation and subsequent registration of lysis zones (plaques) on the E. coli lawn in Petri dishes with nutrient agar.

8.5.3.2. Domain

The direct method of isolating coliphages from water is carried out in parallel with the titration method in studies according to epidemic indications.

8.5.3.3. Conducting an analysis

In nutrient agar of double concentration (p. 5.3.2), melted and cooled to (45-49) ° C, add E. coli wash (p. 8.5.2.3) at the rate of 2.0 ml of wash (or 4 ml of 4- hour broth culture) for every 100 ml of agar, mix. Pour the investigated 100 ml of water into 20 ml large test tubes, heat to (35-44) ° C and immediately (no more than 5 minutes after reaching the required temperature) pour into 5 Petri dishes and immediately add 20 ml to each dish agar mixtures with E. coli culture.

To control the culture of E. coli, add 20 ml of sterile tap water, preheated to (35-44) ° C, into one Petri dish, pour 20 ml of the prepared agar with E. coli and mix gently.

Stir the contents of the cups gently and leave at room temperature until solidified. Place the plates with frozen agar upside down in a thermostat and incubate at a temperature of (37 ± 1) °C for (18 ± 2) hours.

Accounting for results

Viewing crops is carried out in transmitted light.

Accounting for the results is carried out by counting and summing plaques grown on 5 Petri dishes. The results are expressed in plaque forming units (PFU) per 100 ml of water sample. There should be no plaques in the control dish.

Most often, the lysis zones look like transparent spots against the background of the nutrient agar test culture lawn in the form of round isolated plaques (from 1 to 5-7) mm in diameter with clearly defined or erased borders.

At high phage concentrations, a different pattern of lysis is observed.

The fusion of negative colonies gives an “openwork” lawn of E. coli, the growth of single colonies of E. coli against the background of continuous lysis, or a complete lack of growth on the dish.

With direct inoculation, lysis is possible, masked by inhomogeneously solidified agar, as well as closed by accompanying microflora. Droplets of condensate and inhomogeneously set agar from direct inoculation can lead to the formation of artefacts in the E. coli lawn that visually resemble lysis.

Preliminary accounting of the results can be carried out after (5-6) hours of incubation. At this stage, in the presence of clear zones of lysis, a preliminary answer about the presence of coliphages in the water can be issued.

The final quantitative record of direct inoculation is carried out after (18 ± 2) hours. The results are expressed as the number of plaque-forming units (PFU) per 100 ml of water sample.

If confluent plaque growth is noted and counting is difficult, then a qualitative result can be issued according to direct seeding: “found in 100 ml of water”.

If a negative result is obtained when working with the direct method, the final answer is issued according to the results of the titradion method.

If there are zones of lysis in the control dish, the result of the study is considered invalid.

8.5.4. Setting up controls

8.5.4.1. Negative control

The negative control confirms the absence of phage contamination of nutrient media, laboratory glassware, equipment at the stages of preparation and analysis, and also allows you to evaluate the ability of the test culture E . coli to give a uniform lawn.

The negative control is the study of sterile tap water, carried out similarly to the analyzed water sample. So, when analyzing water by the titration method, 10 ml of sterile tap water is added to an additional test tube. When analyzing water by direct inoculation, 20 ml of sterile tap water is added to an additional sixth Petri dish.

Additional crops are examined for coliphages in the same way as the main samples.

When analyzing a series of samples, there can be one negative control for each type of analysis: titration and direct. In this case, the negative control is staged after processing all samples of this series.

If plaques of coliphages are found in the negative control plates, the results of the study of the entire series of water samples are invalid.

It is necessary to check the sterility of laboratory equipment, utensils, nutrient media, and repeat the control inoculation for the purity of the E. coli K12 F + StrR test strain.

The frequency of negative control - 1 time per day.

8.5.4.2. Method for confirming the phage nature of lysis

In doubtful cases, when working with both titration and direct methods, it is necessary to conduct a control inoculation to confirm the phage nature of lysis.

For this purpose, a bacteriological loop is used to remove a section of agar that is suspicious of coliphages, place it in 5 ml of nutrient broth, where a drop of E. coli test culture is added and incubated at 37 ° C for (16-18) hours. The resulting culture is treated with chloroform and examined for the presence of phage. Seeding is carried out with a loop or pipette on nutrient agar sectors in the same way as described in paragraph 8.5.2.5. Lysis on any of the sectors is regarded as confirmation of the presence of the phage.