Main indicators when studying water. Total microbial number for drinking water standardization

Comments on the table. When estimating the amount of OCB and TKB in 100 cm 3 of water, at least three volumes of water (100 cm 3 each) should be analyzed. When assessing OKB and OMC, exceeding the standard is not allowed in 95% of samples taken during the year. Coliphages are determined only in water supply systems from surface sources before supplying water to the distribution network, the same applies to the presence of Giardia cysts. The content of sulfite-reducing clostridia spores is determined only when assessing the effectiveness of water treatment technology. If TCB, OKB, coliphages or at least one of the indicated indicators is detected, a repeat emergency test of water for TKB, OKB and coliphages is carried out again. At the same time, water is tested for chlorides, ammonium nitrogen, nitrates and nitrites. If more than two TCB per 100 cm 3 and/or TCB and/or coliphages are detected in the repeated sample, then a study is carried out for pathogenic bacteria of the intestinal group and/or enteroviruses. The same study for pathogenic enterobacteria and enteroviruses is carried out for epidemiological indications by decision of the territorial centers of Rospotrebnadzor.

Thermotolerant coliform bacteria (TCB) are part of OCB and have all their characteristics, but, unlike them, are able to ferment lactose to acid, aldehyde and gas at a temperature of +44 ° C for 24 hours. Thus, TKB differ from OCB in their ability to ferment lactose to acid and gas at a higher temperature.

The indicators to be determined, the number and frequency of studies depend on the type of water supply source, the size of the population supplied with water from a given water supply system. These data are given in SanPiN 2.1.4.1074–01. In the guidelines for sanitary microbiological analysis drinking water (MUK 4.2.1018–01 of the Ministry of Health of the Russian Federation) methods of sanitary and microbiological control of drinking water quality are regulated.

Total number of microorganisms- this is the total number of mesophilic (having a temperature optimum of +37 °C) aerobic and facultative anaerobic microorganisms (MAFAnM) visible at a twofold increase, which are capable of forming colonies on nutrient agar at a temperature of +37 °C for 24 hours. To identify this indicator in Add 1 ml of water to a sterile Petri dish and fill it with molten (temperature no higher than +50 °C) meat peptone agar, and after 24 hours the number of grown colonies is counted.

DETERMINATION OF OBC AND TKB BY MEMBRANE FILTERS METHOD

The method is based on filtering certain volumes of water through membrane filters. For these purposes, filters with a pore diameter of 0.45 microns and a size of 35 or 47 mm in diameter are used (domestic Vladipor filters MFAS-S-1, MFAS-S-2, MFAS-MA (No. 4–6) or foreign ones - ISO 9000 or EN 29000). Membrane filters are prepared for analysis in accordance with the manufacturer's instructions.

DETERMINATION OF TCB AND TCB BY TITRATION METHOD

The method is based on the accumulation of bacteria after sowing certain volumes of water into liquid nutrient media, followed by reseeding on a differential solid medium with lactose and identifying colonies using cultural and biochemical tests. When studying drinking water using a qualitative method (current sanitary and epidemiological surveillance), three volumes of 100 cm 3 are inoculated. When studying water for the purpose of quantitative determination of TCB and TCB (repeated analysis), 100, 10 and 1 cm 3 are inoculated, respectively - three volumes of each series.

SANITARY AND MICROBIOLOGICAL STUDY OF SOIL

The soil provides shelter for a variety of microorganisms. Thus, the number of bacteria alone in the soil reaches 10 billion cells per 1 g. Microorganisms participate in soil formation and self-purification of the soil, in the circulation of nitrogen, carbon, and other elements in nature. In addition to bacteria, it contains fungi, protozoa and lichens, which are a symbiosis of fungi and cyanobacteria. There are relatively few microorganisms on the soil surface due to the destructive effects of UV rays, drying and other factors. The arable soil layer 10–15 cm thick contains the largest number of microorganisms. As you go deeper, the number of microorganisms decreases until they disappear at a depth of 3–4 m. The composition of the soil microflora depends on its type and condition, vegetation composition, temperature, humidity, etc. Most soil microorganisms are able to develop at neutral pH, high relative humidity, and temperatures from 25 to 45 ° C.

Spore-forming rods of genera live in the soil Bacillus And Closlridium. Non-pathogenic bacilli (Bac. megaterium, Bac. subtilis etc.). along with pseudomonads, proteus and some other bacteria, they are ammonifying, forming a group of putrefactive bacteria that carry out the mineralization of organic substances. Pathogenic spore-forming bacilli (causative agents of anthrax, botulism, tetanus, gas gangrene) can persist for a long time, and some even reproduce in the soil ( Clostridiumbotulinum). The soil is also a habitat for nitrogen-fixing bacteria that assimilate molecular nitrogen. (Azotobacter, Azomonas, Mycobacterium etc.). Nitrogen-fixing species of cyanobacteria, or blue-green algae, are used to increase the fertility of rice fields.

Representatives of the family of intestinal bacteria (family. Enterobacteriaceae) - E. coli, pathogens of typhoid fever, salmonellosis and dysentery, once in the soil with feces, die. In clean soils, E. coli and Proteus are rare; The detection of coliform bacteria (coliform bacteria) in significant quantities is an indicator of soil contamination with human and animal feces and indicates its sanitary and epidemiological problems due to the possibility of transmission of pathogens of intestinal infections. The number of protozoa in the soil ranges from 500 to 500,000 per 1 g of soil. By feeding on bacteria and organic debris, protozoa cause changes in the composition of soil organic matter. The soil also contains numerous fungi, the toxins of which, accumulating in human food, cause intoxication - mycotoxicosis and aflatoxicosis.

The results of soil research are taken into account when determining and predicting the degree of their danger to health and living conditions of the population in populated areas (according to epidemiological indications), the prevention of infectious and non-infectious morbidity (preventive sanitary surveillance), and current sanitary control of objects that directly or indirectly impact the environment .

When conducting ongoing sanitary surveillance of soil conditions, they are limited to a brief sanitary-microbiological analysis, indicating the presence and degree of fecal contamination. The indicators included in this group also characterize the processes of soil self-purification from organic pollutants and enterobacteria. A complete sanitary-microbiological analysis of the soil is carried out in the form of preventive sanitary surveillance. The impact of chemical pollutants on biogeocenosis involves studying their bactericidal effect on soil microbiota, as a consequence: a change in the community of soil microorganisms and soil enzymatic activity. According to epidemic indications, pathogenic microbiota is indicated.

In the laboratory, an average sample is prepared from 5 spot soil samples taken from one area by thoroughly mixing and rubbing in a sterile porcelain cup with a rubber pestle for 5 minutes. Foreign impurities (plant roots, stones, wood chips) are removed by sifting the soil through a sieve, which is first wiped with a cotton swab moistened with 96% ethyl alcohol. From the average sample, weighed portions are taken (from 1 to 50–55 g, depending on the list of indicators being determined) and a 1:10 suspension is prepared in sterile tap water (10 g of soil per 90 cm 3 of water). To desorption of microorganisms from the surface of soil particles, the prepared soil suspension is shaken for 3 minutes on a mechanical dispersant mixer. After settling the suspension for 30 s, successive 10-fold dilutions of the soil are prepared to a concentration of 10 -4 –10 -5 g/cm 3 .

The results of a sanitary-microbiological study of soils are assessed by comparing data obtained on experimental and control plots of soils of the same composition located in close territorial proximity. Schemes for assessing the sanitary condition of soil based on individual sanitary and microbiological criteria are presented in MU No. 14446–76(Table 2).

Table 2. Scheme for assessing the sanitary condition of the soil according to microbiological indicators (according to MU No. 1446-76)

	Titer (g)			Thermophilic microorganism index (number of cells/g)
	coliform	Nitrifying bacteria	Clostridium	Thermophilic microorganism index (number of cells/g)
			0.01 and above
Polluted
Heavily polluted	0.009 and below	0.0009 and below	0.00009 and below

IN MU 2.1.7.730–99 “Hygienic assessment of soil quality in populated areas” A scheme for assessing the epidemic danger of soils in populated areas is presented. In this document, indicators such as coliform bacteria and the enterococci index are used to assess the intensity of the biological load on the soil, and pathogenic enterobacteria and enteroviruses are used to assess the epidemic danger of the soil.

STUDY OF MICROBIAL CONTAMINATION OF THE AIR ENVIRONMENT

Microorganisms enter the air from soil, water, as well as from the surface of the body, from the respiratory tract and with drops of saliva of humans and animals. Coccoid and rod-shaped bacteria, bacilli, clostridia, actinomycetes, fungi and viruses are found here. Air is considered as a factor in the transmission of respiratory infections, in which the pathogen is transmitted by airborne droplets or airborne dust. Sun rays and other factors contribute to the death of air microflora. To reduce microbial contamination of the air, wet cleaning of the room is carried out in combination with ventilation and cleaning (filtration) of the incoming air. Aerosol disinfection and treatment of premises with ultraviolet radiation lamps are also used (for example, in microbiological laboratories and operating rooms).

Many microorganisms are found in the air of enclosed spaces, the microbial contamination of which depends on the cleaning conditions of the room, the level of illumination, the number of people in the room, the frequency of ventilation, etc. A larger number of microorganisms are present in the air of large cities, and a smaller number in the air of rural areas. There are especially few microorganisms in the air over forests, mountains and seas.

Microbiological examination of air involves determining the total content of microorganisms, as well as staphylococci in 1 m 3 of air. In some cases, the air is examined for gram-negative bacteria, mold and yeast-like fungi. According to epidemic indications, the range of pathogens detected in the air can be expanded.

Air samples are taken by aspiration using a Krotov apparatus.

The use of the Koch sedimentation method is quite acceptable. The following premises of medical institutions are subject to research: operating rooms, dressing and treatment rooms, aseptic wards (boxes), wards of the anesthesiology and intensive care department, wards and corridors of medical departments, pharmacy premises, sterilization and obstetric and gynecological departments and blood transfusion stations (departments).

Air testing using the Koch method is used in extremely rare cases for an approximate assessment of the degree of microbial air pollution. To determine the total number of microorganisms in the air of operating rooms, before starting work, open cups with nutrient agar and place them approximately at the height of the operating table - one cup in the center and four in the corners of the room (“envelope method”) for 10 minutes, and to detect Staphylococcus aureus ( plates with yolk-salt agar (YSA) are used - for 40 minutes. The crops are incubated in a thermostat at +37 °C and for 24 hours at. room temperature, then count the number of colonies. In this case, they proceed from the classical formula of V.L. Omelyansky - on 100 cm 2 of the surface of the nutrient medium, in 5 minutes of exposure, the same amount of bacteria is deposited as is contained in 10 liters of air (1 m 3 contains 1000 liters). At the same time, more than 5 colonies of microorganisms should not grow on plates with nutrient agar, and Staphylococcus aureus should not be detected on LSA.

SANITARY AND MICROBIOLOGICAL CONTROL OF FOOD OBJECTS

Food products can be contaminated with various microorganisms, which leads to their spoilage, the development of foodborne illnesses and intoxications, as well as infections such as anthrax, brucellosis, tuberculosis, etc. Animal illness, injury or unfavorable conditions its content contributes to the disruption of the body’s protective barriers and the translocation (transfer) of microorganisms into usually sterile tissues and organs (intravital seeding). As a result, the tissues of the slaughtered animal become contaminated with protozoa, clostridia and other microbes; In case of mastitis, staphylococci and streptococci get into the milk. Secondary contamination of food products with microorganisms is also possible. In this case, the source of pollution is objects environment(soil, water, transport, etc.) as well as sick people and bacteria carriers. At low temperatures for storing meat and meat products even in frozen meat, microbes capable of reproducing under psychrophilic conditions (pseudomonas, proteus, aspergillus, penicillium, etc.) can predominate. Microbes living in meat cause it to mucus; processes of fermentation and putrefaction, caused by clostridia, Proteus, pseudomonads and fungi, develop in it.

Cereals and nuts in conditions of high humidity can become contaminated with fungi (aspergillus, penicillium, fusarium, etc.), which causes the development of food mycotoxicoses.

Meat dishes (jelly, meat salads, minced meat dishes) can cause diseases associated with Salmonella, Shigella, diarrheagenic E. coli, Proteus, enterotoxigenic strains of staphylococci, enterococci, Closlridium perfringens And Bacillus cereus.

Milk and dairy products can be a factor in the transmission of pathogens of brucellosis, tuberculosis and shigellosis. It is also possible to develop food poisoning as a result of the proliferation of salmonella, shigella and staphylococci in dairy products. Eggs, egg powder and melange with endogenous primary infection of eggs, especially duck eggs, with Salmonella, are the cause of salmonellosis.

Fish and fish products are more likely to be contaminated with bacteria Closlridium botulinum And Vibrio parahaemolylicus- causative agents of food intoxication and toxic infections. These diseases are also observed when consuming fish products contaminated with large amounts of Salmonella, Proteus, Bacillus cereus, Closridium perfringens.

Vegetables and fruits can become contaminated and become contaminated with diarrheagenic E. coli, Shigella, Proteus, and enteropathogenic strains of staphylococci. Pickled cucumbers can cause toxic infection caused by Vibrio parahaemolyticus.

All results of microbiological analysis of food products can be obtained no earlier than 48–72 hours, i.e. when the product can already be sold. Therefore, control over these indicators is retrospective in nature and serves the purposes of sanitary and hygienic assessment of an enterprise producing or selling food products.

The detection of increased general microbial contamination, coliform bacteria, suggests a violation of the temperature regime during the preparation and/or storage of the finished product. The detection of pathogenic microorganisms is regarded as an indicator of epidemiological problems in a canteen or retail establishment.

Standardization of microbiological indicators of food safety is carried out for most groups of microorganisms according to an alternative principle, i.e. the mass of the product is normalized, in which bacteria of the E. coli group, most opportunistic microorganisms, as well as pathogenic microorganisms, incl. salmonella and Listeria monocytogenes. In other cases, the standard reflects the number of colony-forming units in 1 g (cm 3) of the product (CFU/g, cm 3).

In consumer products, for which the tables SanPiN 2.3.2.1078–01. Hygienic requirements for safety and nutritional value food products There are no microbiological standards, pathogenic microorganisms, incl. salmonella are not allowed in 25 g of product.

Facilities for the preparation and sale of food products must be subject to sanitary and bacteriological control.

Data from a sanitary and microbiological study make it possible to objectively assess the sanitary and hygienic condition of the objects being examined, identify violations of the sanitary regime and promptly carry out targeted measures to eliminate them.

There are several methods of sampling from various equipment and equipment for microbiological research: methods of swab washes, fingerprints, and agar pouring. Of these, the most commonly used method is tampon flushing.

Sanitary and microbiological control is based on the detection of coliform bacteria (coliforms) in swabs - indicators of fecal contamination of the items under study. Tests for staphylococcus, pathogenic bacteria of the intestinal family, and determination of general microbial contamination are carried out according to indications. For example, taking swabs to detect staphylococci is necessary during inspections of confectionery shops, dairy kitchens and catering units of medical institutions.

Objects of sanitary and microbiological control:

∨ washing off the hands and work clothes of food (water supply) workers;

∨ equipment, inventory, utensils and other objects;

∨ ready-made meals, culinary and perishable products;

∨ raw materials and semi-finished products during the technological process (according to epidemiological indications);

∨ drinking water from centralized and especially decentralized water supply sources.

Hand washes from personnel involved in processing raw foods are collected before work begins. The swabs are delivered to the bacteriological laboratory within 2 hours. They can be stored and transported for no more than 6 hours at a temperature of +1–10 °C.

In the laboratory, swabs are inoculated on Kessler media with lactose or KODA, while a swab is lowered into a test tube with the medium and the remaining swab is transferred. Crops on Kessler and KODA media are incubated at 37 °C.

After 18–24 hours, all tubes with Kessler medium are sown onto sectors of cups with Endo medium; KODA medium is sown only if the color of the medium changes (from the original purple to yellow or green) or becomes turbid. Crops are grown on Endo medium at 37 °C for 18–24 hours.

Smears are prepared from colonies characteristic of coliforms, stained with Gram, microscopically examined, and, if necessary, additionally identified using generally accepted tests for coliform bacteria. When assessing the results of a sanitary microbiological examination, we proceed from the standards that there should be no coliforms in swabs taken from food products. Detection of coliform bacteria in washouts from the surfaces of clean, prepared-for-work items, equipment, hands and sanitary clothing of personnel indicates a violation of the sanitary regime. In case of repeated detection of coliform bacteria in a significant percentage of swabs, it is recommended to study the swabs for the presence of pathogenic enterobacteria. In this case, the swab and rinsing fluid are inoculated onto enrichment media - selenite broth or magnesium medium (it is possible to use Muller and Kaufman media). Further research is carried out according to generally accepted methods.

RESEARCH OF MILK AND DAIRY PRODUCTS

MICROFLORA OF DAIRY PRODUCTS

Milk is a very favorable nutrient medium for the development of many microorganisms. After eating infected milk and dairy products, infections such as typhoid fever, dysentery, cholera, escherichiosis, brucellosis, tuberculosis, scarlet fever, tonsillitis, Q fever, foot and mouth disease, tick-borne encephalitis, salmonella toxic infections, poisoning with staphylococcal enterotoxin, etc. can occur.

There are specific and nonspecific microflora of milk and dairy products.

TO specific microflora of milk and dairy products include microbes - causative agents of lactic acid, alcoholic and propionic acid fermentation. Microbiological processes due to the vital activity of these microorganisms underlie the preparation of fermented milk products (cottage cheese, kefir, curdled milk, acidophilus, etc.).

Lactic acid fermentation bacteria are considered normal microflora of milk and dairy products . Lactic acid streptococci play the main role in the souring of milk and dairy products. S. lactis, S. cremaris and others. Less active races of lactic acid streptococci ( S. citrovorus, S. lactis subsp. diacetylactis) produce volatile acids and aromatic substances and are therefore widely used in the production of cheeses. The group of lactic acid bacteria also includes lactic acid rods: Lactobacterium bulgaricum, Lactobacterium casei, Lactobacterium acidophilus etc.

The main causative agents of alcoholic fermentation in milk and dairy products are yeasts ( Saccharomyces lactis etc.).

Nonspecific microflora of milk putrefactive bacteria ( Proteus), aerobic and anaerobic bacilli ( B. subtilis, B. megatherium, C. putrificum) and many others

Microbial contamination of milk begins already in the udder. During the milking process, additional contamination occurs from the surface of the skin of the udder, from the hands, from the vessel into which it comes, and from the air in the room. The intensity of this additional contamination generally depends on how basic sanitary and hygienic conditions are observed when receiving milk. Poor storage conditions for milk can also contribute to the further growth of microflora in it.

Bactericidal phase. Freshly milked milk, although it already contains hundreds of microbes per 1 cm3 (mainly staphylococci and streptococci), has bactericidal properties due to the presence of normal antibodies in it. Therefore, the development of bacteria in milk is delayed for a certain period. This period is called the bactericidal phase. The duration of the bactericidal phase ranges from 2–36 hours depending on the physiological characteristics of the animal (in the early period of lactation, the bactericidal value of milk is higher).

Storing milk at elevated temperatures (30–37 °C) sharply reduces the duration of the bactericidal phase. Intensive additional contamination of milk with microbes has the same effect.

After the bactericidal phase has ended, the development of microflora begins. Its species composition changes over time under the influence of changes in the biochemical properties of the environment and due to antagonistic and symbiotic relationships between microbial species.

Mixed microflora phase. It lasts about 12 hours. During this period, there is no predominance of any species groups of microbes, since the abundance of nutrient substrate and spatial opportunities allow many types of microorganisms to develop quite freely.

Lactic acid streptococcus phase. In this phase, microorganisms of the named group become predominant ( S. lactis, S. termofilus, S. cremoris etc.). Lactose is intensively converted into lactic acid, the reaction changes in the acidic direction. The accumulation of lactic acid subsequently leads to the death of lactic acid streptococci and their replacement by more acid-resistant lactic acid bacteria. This occurs after 48 hours, marking the beginning of the third phase.

Lactic acid rod phase. In it, the rod-shaped forms of lactic acid bacteria acquire a dominant position. ( L. lactis, L. crusei, L. bulgaricum etc.). The resulting acidic reaction environment leads to inhibition of growth and gradual death of other types of bacteria.

By the end of the third phase, further possibilities for the development of lactic acid microflora are exhausted, and they are replaced by fungi, for which lactic acid serves as a nutrient substrate.

Fungal microflora phase. During this period, molds and yeasts develop, the vital activity of which leads to the loss of the product’s nutritional value. Yeasts are represented mainly by species from the genus Torula, some species of Saccharomycetes are found less frequently.

Among the molds found: milk mold ( Oidium lactis), covering the surface of curdled milk and sour cream in the form of a fluff, as well as Aspergillus, Penicillium and Mucoraceae.

The action of fungal flora leads to neutralization of the environment, and this makes it again suitable for bacteria. The development of putrefactive bacteria begins, causing proteolysis of casein, and, finally, a group of anaerobic spore-forming butyric acid bacteria.

The activity of the changing microflora ceases only with the onset of complete mineralization of all organic substances of milk.

Under some conditions, the process of changing microbial biocenoses may deviate from the above scheme. Thus, lactic acid bacteria can be inhibited from the very beginning by microbes of the E. coli group, if the latter are present in large quantities. Yeast can produce noticeable concentrations of alcohol, which is the case in products such as kefir (0.2–0.6%) and, especially, koumiss (0.9–2.5%). The presence of alcohol creates conditions for the subsequent development of acetic acid bacteria, which ferment alcohol into acetic acid. The presence of antibiotics and other substances that inhibit and neutralize microflora in milk can also slow down lactic acid 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 microns. As a result, all bacteria in the water remain on the surface of the membrane. After which the membrane with bacteria is placed on certain time into a special nutrient medium at a temperature of 30-37 oC. During this period, called incubation, bacteria are able to multiply and form clearly visible colonies that can be easily counted. As a result, you can see something like this: Or even this picture:

Since this method of water analysis only involves determining the total number of colony-forming bacteria different types, then based on its results it is impossible to unambiguously judge the presence of pathogenic microbes in water. However, a high microbial count indicates general bacteriological contamination of the 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 TMC - total microbial number. In centralized water supply water, this number should not exceed 50 CFU/ml, and in wells and wells - no more 100 CFU/ml

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

— drinking water from central water supply (tap water);

— drinking water from 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 according to current regulatory documents are:

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

If the titer- the smallest volume of water (in ml) in which at least one living organism was found
microbial cell related to coliforms.
Coliform Index- the amount of coliforms 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 allows one 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 is the number of mesophilic aerobic and facultative anaerobic microorganisms capable of forming colonies on nutrient agar at a temperature of 37 °C for 24 hours, visible at double magnification.

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 carefully mixed with water, evenly and
without air bubbles, spread over the bottom of the cup, then cover with a lid and leave until hardened. The crops are incubated in a thermostat at 37 °C for 24 hours. The total number of colonies grown in both dishes is counted and the average is determined. The final result is expressed by the number of colony-forming units (CFU) in 1 ml of test water. 1 ml of drinking water should contain no more than 50 CFU

Definition of coliforms
In this case, common coliform bacteria are determined - TCB and thermotolerant coliform bacteria - TCB.

OKB are gram-negative, non-spore forming rods that ferment lactose to acid and gas at a temperature of 37°C for 24-48 hours. TKB are included in the group of OKB, they have their symptoms, but I ferment at 44°C. To determine enterobacteria, use the membrane filter method or titration.

Microbial number is the main criteria for assessing the microbiological state of drinking water, based on current regulatory documents, is the 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.

Quality indicators of drinking water in water supply systems.

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) molten nutrient agar is poured. 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 it hardens. The same thing is done in another cup. The inoculations are incubated in a thermostat at a temperature of 37 °C for 24 hours. The total number of colonies grown in the two dishes is then counted under a microscope at 2x magnification and the average is determined. There should not be more than 50 CFU in 1 ml of drinking water.

(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 filtering, the filter is removed and, without turning over, placed on the surface of the Endo nutrient medium.

You can fit 3 filters on one cup. When studying 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 dishes with filters are incubated upside down in a thermostat at 37°C for 24 hours.

If there is no growth on the filters or if atypical filmy, moldy, or diffuse colonies have grown, a negative result is given. TCB and TCB are absent in 100 ml of the tested water.

When typical isolated lactose-positive (dark red with imprints on the back of the filter) colonies grow on the filters, their number is counted and they begin to confirm their belonging to OKB and TKB.

Microscopy of smears from 3-4 colonies stained by Gram is carried out (gram-negative ones are taken into account);

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 weakly colored colonies and their relationship to TKB, and a temperature of 44 ± 0.5°C, in order to decide whether they belong to TKB.

Performing an oxidase test

A part of the colored colony is applied to paper moistened with a 1% alcohol solution of α-naphthol and a 1% aqueous solution of dimethylphenylenediamine. the reaction is considered positive if a blue or violet color appears within 1 minute, maximum 4 minutes. Oxidase-positive colonies are not counted and are not subjected to further examination.

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

Parts of 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 injecting 2 test tubes to the bottom. One is incubated at a temperature of 37±1oC for 24-48 hours to confirm the relation to TKB, the other at a temperature of 44±0.5oC for 24 hours, counting is possible after 4-6 hours to confirm the presence of TKB.

When colonies are placed 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 oC 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 oC to acid and gas (scheme No. 2).

SCHEME No. 2

Date of publication: 2014-11-02; Read: 1811 | Page copyright infringement

studopedia.org - Studopedia.Org - 2014-2018 (0.001 s)…

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 microns. As a result, all bacteria in the water remain on the surface of the membrane. After which the membrane with bacteria is placed for a certain time in a special nutrient medium at a temperature of 30-37 oC. During this period, called incubation, bacteria are able to multiply and form clearly visible colonies that can be easily counted. As a result, you can see something like this: Or even this picture:

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

— drinking water from central water supply (tap water);

— drinking water from 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 according to current regulatory documents are:

1. Total microbial number (TMC) - the number of mesophilic bacteria in 1 ml 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 allows one to assess the level of microbiological contamination of drinking water. This indicator is indispensable for the urgent detection of massive microbial contamination.

Definition of coliforms
In this case, common coliform bacteria are determined - TCB and thermotolerant coliform bacteria - TCB.

Microbial number is the main criteria for assessing the microbiological state of drinking water, based on 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 water being tested is added to a sterile Petri dish, then 10-15 ml of warm (about 44 ° C) molten nutrient agar is poured. 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 it hardens.

Principles of drinking water rationing

The same thing is done in another cup. The inoculations are incubated in a thermostat at a temperature of 37 °C for 24 hours. The total number of colonies grown in the two dishes is then counted under a microscope at 2x magnification and the average is determined. There should not be more than 50 CFU in 1 ml of drinking water.

OKB is an international qualification and they are included in large group Coliform bacteria (coliform bacteria). The content of OCB in water can be determined by two methods: the membrane filter method and the titration (fermentation) method.

Study of water using the membrane filter method. The method is based on filtration of a specified volume of water through membrane filters, growing crops on a differential diagnostic medium and subsequent identification of colonies based on cultural and biochemical characteristics.

Titration method for water testing. The method is based on the accumulation of bacteria after inoculating a set volume of water into a liquid nutrient medium, followed by reseeding into a differential diagnostic medium and identifying colonies using cultural and biochemical tests.
“Coliform organisms” belong to a class of gram-negative rod-shaped bacteria that live and reproduce in the lower digestive tract of humans and many warm-blooded animals such as livestock and waterfowl, capable of fermenting lactose at 35-37 0C to form acid , gas and aldehyde. When they get into water with fecal waste, they are able to survive for several weeks, although the vast majority of them are unable to reproduce.

According to research recent years Along with the bacteria Escherichia (E.Coli), Citrobacter, Enterobacter and Klebsiela usually classified in this class, it also includes the lactose-fermenting bacteria Enterobacter cloasae and Citrobadter freundii. These bacteria can be found not only in feces, but also in the environment, and even in drinking water with relatively high concentrations nutrients. In addition, this includes species that are rarely or not at all found in feces and can reproduce in water of sufficiently good quality.

TCB - thermotolerant coliform bacteria. The TCB number characterizes the degree of fecal contamination of water in water bodies and indirectly determines the epidemic danger in relation to pathogens of intestinal infections. TCB is determined by the same methods as coliforms (OCB).
Sampling for sanitary microbiological studies must be carried out in compliance with the rules of sterility and all necessary conditions regulated for each object under study by the relevant regulatory documents.

Errors made when taking samples lead to incorrect results. When packaging and transporting samples, it is necessary to create conditions that exclude the death or proliferation of the original microbiota in the object being studied. Therefore, the collected samples should be delivered to the laboratory for testing as quickly as possible.

The basis of hygienic requirements for the quality of water for drinking and domestic needs is the principle that places the focus on the quality of water, on which human health and living conditions depend. In accordance with modern sanitary legislation, drinking water must be safe in terms of epidemics and radiation, harmless in terms of chemical composition and have favorable organoleptic properties.

The safety of drinking water in epidemic terms is determined by its compliance with standards for microbiological indicators. The microbiological composition of drinking water is the main indicator of its quality and suitability for consumption. This takes into account both bacterial and viral contamination.

The epidemiological safety of drinking water in SanPiN is assessed according to several indicators. A major role among them is given to thermotolerate coliforms as true indicators of fecal contamination and general coliforms.

Common coliform bacteria (TCB) are gram-negative, oxidase-negative, non-spore forming rods, capable of growing on differential lactose media, fermenting lactose to acid and gas at a temperature of +37 for 24-48 hours.

Thermotolerant coliform bacteria (TCB) are part of the TCB and have all their characteristics, but unlike them, they are able to ferment lactose to acid, aldehyde and gas at a temperature of +44 within 24 hours. Thus, TKB differs from OCB in its ability to ferment lactose to acid and gas at a higher temperature. Thermotolerant and common coliforms should be absent in 100 ml of drinking water (in any sample with three repetitions of the analysis).

In the distribution network of large centralized systems drinking water supply(if the number of samples studied is at least 100 per year), 5% of non-standard samples for common coliforms are allowed, but not in two consecutive samples taken at one point.

The total number of microorganisms (total microbial number - TMC) is determined by growth on meat peptone agar at an incubation temperature of 37. This indicator is used to characterize the effectiveness of drinking water purification; it must be considered when monitoring water quality over time. A sharp deviation of the TMC even within the standard value (but not more than 50 in 1 ml) serves as a signal of a violation in the water treatment technology. The growth of TMC in the water of the distribution network may indicate its unfavorable sanitary condition, which promotes the proliferation of microorganisms due to the accumulation of organic substances or leaks, leading to the infiltration of contaminated groundwater.

Aerobic saprophytes make up only part of the total number of microbes in water, but are an important sanitary indicator of water quality, since there is a direct relationship between the degree of contamination with organic substances and the microbial number. In addition, it is believed that the higher the total microbial count, the greater the likelihood of pathogenic microorganisms being present in the water. Microbial number in tap water should not exceed 100.

The safety of drinking water in epidemic terms is determined by its compliance with standards for microbiological indicators (Table 1).

Table 1. Microbiological indicators drinking water

The concept of sanitary indicator microorganisms

Basic requirements for sanitary-indicative microorganisms: 1. they must have a common natural habitat with pathogenic microorganisms and be released into the external environment in large quantities; 2. in the external habitat, sanitary-indicative microorganisms should be distributed as evenly as possible and be more stable than pathogenic ones. They should remain in water longer, practically without reproducing, have greater resistance to various unfavorable factors, and they should exhibit less variability in properties and characteristics; 3. methods for determining sanitary indicative microorganisms must be simple and have a sufficient degree of reliability.

From the point of view of sanitary microbiology, water quality assessment is carried out in order to determine its sanitary and epidemiological danger or safety. For human health. Water plays an important role in the transmission of pathogens of many infections, mainly intestinal.

Direct quantitative determination of all infections for water quality control is not feasible due to the diversity of their types and the complexity of analysis.

Analysis of only one water sample for the possible presence of pathogens of typhoid fever, paratyphoid A, paratyphoid B, dysentery, infectious jaundice, water fever and tularemia would completely load the entire staff of even a large bacteriological laboratory. In addition, the answer in this case would be given only after 2-3 weeks, i.e. when the population had long since drunk the water being tested.

In view of the obvious inexpediency of a detailed determination of the harmlessness of water, even in late XIX centuries, attempts have been made to replace the search for all aquatic pathogenic microbes with one microbe, albeit non-pathogenic, but constantly present in human feces. Then one could assume that if the water being tested is indeed contaminated with feces, then it may be dangerous to drink, since both sick people and carriers of the bacilli can be found among the healthy population. The search for such bacteriological indicators of fecal pollution has been successful. It turned out that the following three microbes are constantly present in human feces: 1) E. coli; 2) enterococci; 3) anaerobic spore-forming bacteria, mainly Bac. perfingens.

Thus, in household wastewater Escherichia coli predominates. But it's not just about its greater content. The main value of a bacterial indicator of fecal contamination is its rate of death of most pathogenic microbes. Only if this condition is met will a microbe that is constantly present in human feces be an indicator of fecal contamination.

If from this point of view we approach the discovered permanent inhabitants of the intestine, we will find the following: microbes of the Bac group. perfingens persists in water much longer than pathogenic microbes; enterococci, on the contrary, die much faster; As for E. coli, the time of its preservation in water approximately corresponds to the survival time of pathogenic microbes.

Therefore, the main sanitary and bacteriological indicator of water is E. coli. Only in Russia, the only country in the world, water quality is controlled by bacteria of the Escherichia coli group (coliform index). This group includes all representatives of the group of intestinal bacteria and opportunistic representatives.

In accordance with GOST 2874-73 and GOST 18963-73, coliform bacteria (coliforms) include gram-negative, non-spore-forming bacilli that ferment lactose or glucose to acid and gas at 37° in 24 hours and do not have oxidase activity. Coliform bacteria include representatives of various genera - Escherichia, Citrobacter, Enterobacter, Klebsiella, but all of them are released into the environment from the intestines of humans and animals. In this regard, their detection in the environment should be considered as an indicator of fecal contamination.

Of the genera included in the composition of coliforms, the genus Escherichia has the most sanitary and indicative importance. The presence of all these bacteria in the environment is considered fresh fecal contamination.

Escherichia is one of the background species of the intestines of humans and animals. The genus Escherichia, including the type species E. coli, an indicator of fresh fecal contamination, possible reason toxic infections. Representatives of the genus found in water are interpreted as thermotolerant coliform bacteria.

Citrobacter - live in wastewater, soil and other environmental objects, as well as in the feces of healthy and patients with acute intestinal infections. They belong to the group of opportunistic bacteria. (Microbiological dictionary-reference book, 1999)

The disadvantages of Citrobacter as a SPMO include the following:

1. abundance of analogues in the external environment.

2. variability in the external environment.

3. insufficient resistance to adverse effects.

4. ability to reproduce in water.

5. unclear indicator even for the presence of salmonella.

Research in recent years has revealed that there is no direct correlation between the presence of pathogenic bacteria and indicators in water. In regions with intense anthropogenic pressure on water bodies a decrease in the content of indicator microorganisms was noted with a change in their biological and cultural properties against the background of a quantitative predominance of potential pathogenic and pathogenic bacteria.

Enterobacter - live in the intestines of humans and other animals, found in soil, water, food products, cause intestinal, urogenital, respiratory, purulent-inflammatory diseases of humans.

Klebsiella - lives in water, soil, food, intestines and respiratory tracts of humans, mammals, and birds.

In 1910 Enterococci (Enterococcus faecalis, Enterococcus faecium) have been proposed for the role of SPMO.

Enterococci are a genus of facultative anaerobic asporogenic chemoorganotrophic gram+ bacteria. Cells are polymorphic. Widely distributed in nature. They are one of the background species of the intestines of humans, mammals, and birds. They are often found in the flora of the skin of the perineum and genital tract, nasal cavities, pharynx, and nose. They survive for a long time in soil and food products.

Advantages of enterococcus as a SPMO:

1. is constantly located in the human intestine and is constantly released into the external environment. At the same time, Enterococcus faecalis mainly lives in the human intestines, so its detection indicates contamination with human feces. To a lesser extent, Enterococcus faecium occurs in humans. The latter is mainly found in the intestines of animals, although Enterococcus faecalis is also observed relatively rarely.

2. is not able to reproduce in the external environment; Enterococcus faecium mainly reproduces, but it has less epidemiological significance.

3. does not change its properties in the external environment.

4. has no analogues in the external environment.

5. resistant to adverse environmental influences. Enterococcus is 4 times more resistant to chlorine than E. coli. This is his main advantage. Thanks to this feature, enterococcus is used when checking the quality of water chlorination, as well as as an indicator of the quality of disinfection. Withstands temperatures of 60°C, which allows it to be used as an indicator of the quality of pasteurization. resistant to table salt concentrations of 6.5-17%. Tolerant to pH in the range 3-12.

6. Highly selective media have been developed for the indication of enterococci. The survival rate of enterococcus in water approaches the survival rate of pathogenic enterobacteria. Enterococcus is rightfully second only to E. coli as a sanitary indicator test when testing drinking water.

Enterococcometry is now legalized in the international water standard as an indicator of fresh fecal contamination. When atypical E. coli are detected in water, the presence of enterococci becomes the main indicator of fresh fecal contamination. Unfortunately, SanPiN 2.1.4.1074-01 for drinking water does not provide for the definition of enterococcus.

The Protea group is considered as the culprits of putrefactive processes in nature, and therefore as indicators of the presence of organic substances in the water of reservoirs. This applies mainly to one species – Pr.vulgaris; the second species – Pr.mirabilis – inhabits the intestines of humans and animals. This environmental difference made it possible to judge the nature of water pollution and the degree of its epidemic safety. Pr.vulgaris may be an indicator of fecal pollution, Pr.vulgaris may be an indicator of an increase in the concentration of organic matter in general. Weaknesses This indicator is the inconsistent presence of Pr.mirabilis in the human intestine and the ability of both species to reproduce quite intensively in water. There is also no research method that would allow differentiated consideration of both species when they are simultaneously present in the test sample. The proposed method does not perform this task.

Currently, it has been shown that bacteria of the genus Proteus are found in 98% of cases in the intestinal secretions of humans and animals, of which 82% of cases are Pr.mirabilis. detection of proteus in water indicates contamination of the object with decomposing substrates and indicates extreme sanitary problems. Proteometry is officially recognized in the USA.

Spores of sulfide-reducing clostridia are detected in water pipelines from surface sources to assess the effectiveness of technological water treatment. Spores of sulfide-reducing bacteria should not be present in 20 ml of drinking water after completion of water treatment.

As an indicator of viral contamination of drinking water, SanPiN includes coliphages, which in their biological origin, size, properties, and resistance to environmental factors are closest to intestinal viruses. Coliphages should not be detected in 100 ml of treated drinking water.

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

8.1.1. Definition of the indicator concept

The method determines in drinking water total number mesophilic aerobic and facultative anaerobic microorganisms (FAM), capable of forming colonies on nutrient agar at a temperature of 37 ° C for 24 hours, visible with a 2-fold magnification.

8.1.2. Performing analysis

At least two volumes of 1 ml are inoculated from each sample.

After thorough mixing, add 1 ml of water samples into sterile Petri dishes, opening the lids slightly. After adding water, pour (8-12) ml (per cup with a diameter of 90-100 mm) of melted and cooled to (45-49) °C nutrient agar into each cup after flaming the edge of the dish in which it is contained. Then quickly mix the contents of the cups, distributing them evenly over the entire bottom, avoiding the formation of air bubbles and 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 hardens.

For the period of analysis, molten agar is placed in a water bath or thermostat maintaining a temperature of (45-49) °C.

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

8.1.3. Even the results

All colonies grown on the plate, observed at 2x magnification, are counted. Only those plates on which no more than 300 isolated colonies grew were taken into account.

The number of colonies on both plates is summed and divided by two. The result is expressed by 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 on two plates there is a growth of diffuse colonies that does not extend to the entire surface of the plate, or more than 300 colonies have grown and the analysis cannot be repeated, count the sector of the plate and then recalculate the entire surface. In these cases, the protocol notes “number of CFU/ml - approximate.”

If counting colonies on plates is not possible, then “continuous growth” is noted in the protocol.

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

8.2.1. Definition of the indicator concept

Common coliform bacteria (TCB) are gram-negative, oxide-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 .

Thermotolerant coliform bacteria (TCB) are among the common coliform bacteria, have all their characteristics and, in addition, are capable of fermenting lactose to acid, aldehyde and gas at a temperature of (44 ± 0.5) ° C for 24 hours.

8.2.2. Principle of the method

The method is based on filtration of a specified 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 procedure

When testing drinking water, 3 volumes of 100 ml are analyzed.

If stable negative results are obtained, it is permissible to filter 300 ml of water 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 out in paragraph 7.

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

If there is no growth on the filters or filmy, spongy, moldy, transparent, blurry colonies have grown, they give a negative answer: the absence of TCB and TCB in 100 ml of the water being tested. The analysis is completed after 24 hours.

If the 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 OKB and TKB.

To confirm the presence of OKB, the following is examined:

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

At least 3-4 colonies of each type.

To confirm the presence of TSD, all typical colonies are examined, but not more than 10.

Each selected isolated colony is examined for:

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. Performing 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 clause 5.7. Finished paper systems are moistened with distilled water. A part of the isolated colony is streaked onto the prepared filter paper using a glass folder or a platinum loop (a metal loop made of nichrome can give a false positive reaction). The reaction is considered positive if, within 1 minute, a violet-brown (section 5.7.1 option 1) or blue (section 5.7.2 option 2 and SIB oxidase) streak color appears. In case of a negative reaction, the color at the site of application of the culture does not change. If the result is positive, this colony is excluded from further research.

If, when examining colonies colored dark red, the result is not clear enough, 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 Gram affiliation

A smear is made from an oxidase-negative colony, stained with Gram and examined under a microscope.

Apply 1 drop of distilled water in a loop onto a glass slide degreased with alcohol, add a small amount of culture from the analyzed colony and distribute it over the surface of the glass. The smear is dried at room temperature and fixed by passing it through the burner flame three times. A strip of filter paper is placed on the preparation and a carbolic solution of gentian violet is poured onto it for (0.5-1) min, the paper is removed, Lugol's solution is poured for (0.5-1) min, the Lugol's solution is drained and the glass is washed in ethyl alcohol in for (0.5-1) minutes until the dye stops coming off. Then the glass is thoroughly washed with water and repainted for 1-2 minutes with Ziehl fuchsin, diluted 1:10 with distilled water. After washing and drying the preparation, the smear is examined under a microscope.

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

Gram-negative microorganisms are pink in color, while gram-positive microorganisms are blue in color. 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 3% aqueous KOH solution on a glass slide, a bacterial mass taken from a solid medium is emulsified. After several seconds of mixing with a loop, the suspension becomes slimy 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 remaining part of the oxidase-negative gram-negative isolated colony is inoculated in parallel into two tubes with lactose medium (section 5.6):

To confirm the presence of OCD, the crop 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 of the formation of acid and gas on confirming semi-liquid media and NIB (clause 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, test tubes with cultures for final recording of TCB are left for up to 24 hours. Test tubes with cultures to confirm the presence of TCB after viewing after 24 hours and receiving a negative result are left for final counting for up to 48 hours.

If the colony to be examined is small in size, it is subcultured onto nutrient agar slants and, after incubation for 18-24 hours, all necessary confirmatory tests are performed.

8.2.3.5. Performing confirmatory tests for overlapping colonies or continuous growth

If colony overlay or continuous growth is observed on part or all of the filter surface, perform an oxidase test by placing the membrane filter on a circle of filter paper larger in diameter than the filter, generously moistened with reagent, or on a NIB-oxidase disk moistened with distilled water. When the first signs of a reaction appear, but no 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. When 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 a response about the absence of OCB and TCB and complete the analysis.

In case of a negative oxidase reaction, sieving is carried out until isolated colonies are obtained and their belonging to OKB and TKB is confirmed according to clauses 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 OCB if the oxidase test is negative and lactose is fermented at 37 °C with the formation of acid and gas.

Gram-negative colonies are counted as TCB if the oxidase test is negative and lactose is fermented at 44°C to produce acid and gas.

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

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

The calculation is carried out using the formula:

X is the number of colonies per 100 ml;

the volume of water filtered through the filters on which records were kept;

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

1. When 3 filters of 100 ml were inoculated, two colonies of 100 ml grew; there was no growth on the remaining two filters. The number of total or thermotolerant coliform bacteria will be:

CFU OKB (TCB) 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 TCB colonies on those filters where isolated colonies were obtained is summed up and recalculated to a volume of 100 ml.

CFU in 100 ml

8.2.4.4. If, during a random 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 using 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 verified ones;

c is the number of colonies with a positive result.

The obtained census results for each type of colony 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 TKB in 100 ml, of which the number of CFU TKB in 100 ml.

An approximate result can be given when typical coliform colonies are detected on Endo medium, formed by gram-negative oxidase-negative bacteria. The final answer is confirmed by the results of lactose fermentation.

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

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

In both cases, the analysis is repeated.

8.3. Determination of total and thermotoletarian coliform bacteria by titration method

8.3.1. Definition of the concept of indicator

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

8.3.2. Scope of application

The titration method can be used:

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

When analyzing water with a high content of suspended solids;

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

8.3.3. Principle of the method

The method is based on the accumulation of bacteria after inoculating a set volume of water into a liquid nutrient medium, followed by reseeding into a differential solid nutrient medium with lactose and identifying colonies using cultural and biochemical tests.

8.3.4. Performing analysis

When testing drinking water qualitative method(current sanitary and epidemiological supervision, production control) inoculate 3 volumes of 100 ml each.

When researching water for the purpose quantification During repeated analysis, OKB and TKB perform inoculation: 3 volumes of 100 ml, 3 volumes of 10 ml, 3 volumes of 1 ml.

Each volume of 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 sample is carried out in 10 ml of medium of normal concentration.

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

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

Crops 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 the 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 OKB must be confirmed:

If only turbidity is noted in the accumulation medium;

If belonging to lactose-positive colonies raises doubts among the researcher. In these cases:

Check for the presence of an imprint on the Endo medium after removing a suspicious colony with a loop;

Perform the oxidase test according to clause 8.2.3.2;

Confirm affiliation with Gram according to clause 8.2.3.3;

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

In the absence of isolated colonies, seeding is carried out on Endo medium using generally accepted bacteriological methods.

A negative answer is given if:

There are no signs of growth in the accumulation environment;

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

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

All colonies were oxidase positive;

All colonies were gram-positive;

If the confirmatory test on a medium with carbohydrate does not indicate gas formation.

To determine thermotolerant coliform bacteria work with sectors of the Endo environment where typical lactose-positive colonies have grown. Inoculate 2-3 isolated colonies of each type from each sector into test tubes with any of the lactose media prepared according to clause 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. Inoculations can be viewed after (4-6) hours.

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

To speed up the issuance of a response to the presence of TCB, it is permissible 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. The crops are kept in a thermostat at a temperature of (44±0.5)°C for 24 hours. If acid and gas are detected, a positive answer is given.

8.3.5. Accounting for results

When examining 3 volumes of 100 ml, the results are assessed qualitatively and if OKB and TCB are detected in at least one of the 3 volumes, an entry is made in the protocol “detected in 100 ml”.

When studying using a quantitative method, the most probable number (MPN) of OKB and TKB is determined according to the table. 1.1 appendices 1.

The result is reported without a confidence interval.

If the answer is negative for the presence of OCB and TCB in all tested volumes, a conclusion is issued in the protocol “not detected in 100 ml”.

8.4. Determination of sulfite-reducing clostridia spores

8.4.1. Definition of the concept of 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. Principle of the method

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

When studying chlorinated water, heating the sample does not need to be done.

From each drinking water sample, 20 ml are cultured or filtered. If necessary, volumes are selected so that no more than 10-15 colonies grow in the 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 out in paragraph 7.

8.4.3.2. Determination by filtration in test tubes

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

After filtering a set volume of water, the membrane filter is taken by two opposite edges with flaming tweezers and bent into a tube and placed in a test tube with hot agar. The side of the filter with settled bacteria faces inward. In this case, the filter straightens and is located along the wall of the test tube.

Immediately after inoculation, the test tube with agar and a filter to create anaerobic conditions is quickly cooled by placing it in a container with cold water. The crops are cultivated 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 thin layer iron sulfite agar. After filtration, place the filter with the filter surface down on the frozen nutrient medium so that there are no air bubbles under the filter. Then pour molten iron sulfite agar to the top edge of the dish so that the lid fits tightly to the medium to create anaerobic conditions. The crops are cultivated at (44 ± 1) °C for (16 - 1 8) hours.

8.4.3.4. Determination by direct seeding

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

Add into sterile tubes:

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

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

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

8.4.4. Accounting for results

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

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

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

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, obtain a quantitative result, the analysis is repeated.

8.5. Definition of coliphages

8.5.1. Definition of the indicator concept

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

8.5.2. Titration method for determining coliphages

8.5.2.1. Principle of the method

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

8.5.2.2. Scope of application

The method is intended for routine 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 is used, prepared as follows: the E. coli culture 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 off the bacteria from the school with 5 ml of sterile physiological solution (0.85% NaCl solution) and prepare a suspension of E. coli at a concentration of 109 bacterial according to the turbidity standard 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. A concentration of 109 E. coli bacterial cells is contained in 2 ml.

8.5.2.4. Conducting qualitative analysis

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

To control the culture, 0.1 ml of E. coli bacteria washout (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 a 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 water from the test 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 distribute chloroform evenly throughout the sample volume and left at room temperature for at least 15 minutes until the chloroform completely precipitates.

Add a prepared wash of E. coli bacteria (section 8.5.2.3) to the previously melted and cooled to (45-49) °Nutritional agar at the rate of 1.0 ml of wash (or 2 ml of a 4-hour broth culture) per 100 ml of agar.

Transfer 1 ml of the chloroform-treated sample (without touching the chloroform) into a sterile Petri dish using 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 Petri dish for control. E. coli culture and gently shake to ensure uniform mixing of the water sample and agar. To completely harden, leave the cups 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 set for the entire series.

Accounting for results

Viewing of crops is carried out in transmitted light.

The sample is considered positive if there is complete lysis, clearing of several plaques, one plaque on a plate with a water sample in the absence of lysis zones on the control plate.

The analysis protocol notes: coliphages are detected or not detected 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. Conducting quantitative analysis

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

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

The 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 volumes under study. Close the test tubes with sterile rubber or silicone stoppers, shake vigorously to distribute chloroform evenly throughout the sample volume, and leave at room temperature for at least 15 minutes to allow the chloroform to precipitate.

Add the prepared wash of E. coli bacteria (section 8.5.2.3) to the nutrient agar, previously melted and cooled to (45-49) °C, at the rate of 1.0 ml of wash (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 E. coli culture for lysogenicity and one cup for each water sample being tested. When simultaneously analyzing several water samples, one E. coli culture control is used.

After the agar has hardened, divide the dishes intended for inoculating samples into 6 sectors and label them in accordance with the volumes being tested. Apply 1 drop of supernatant liquid (without chloroform) to each sector from the corresponding test tube using a Pasteur pipette (micropipette or bacteriological loop with a longitudinal stroke).

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.

The count is carried out based on the presence of zones of clearing (lysis) in sectors of the E. coli lawn.

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

The sample is considered positive if there is a lysis zone in at least one sector and there are no 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: LHF PFU (lower limit - upper limit) of coliphages in 100 ml. The result is semi-quantitative.

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

8.5.3. Direct method for determining coliphages

.1. Principle of the method

Determination of coliphages in drinking water consists of examining a standardized volume of water (100 ml) by direct inoculation and subsequent recording of lysis zones (plaques) on the E. coli lawn in Petri dishes with nutrient agar.

8.5.3.2. Domain of definition

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

8.5.3.3. Carrying out analysis

In nutrient agar of double concentration (clause 5.3.2), melted and cooled to (45-49) °C, add E. coli wash (clause 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 test 100 ml of water into 20 ml into 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 mixture with E. coli culture.

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

Mix the contents of the cups carefully and leave at room temperature until solidified. Place the plates with frozen agar, bottom up, in a thermostat and incubate at a temperature of (37 ± 1) °C for (18 ± 2) hours.

Accounting for results

Viewing of crops is carried out in transmitted light.

The results are recorded by counting and summing plaques grown on 5 Petri dishes. Results are expressed in plaque forming units (PFU) per 100 ml water sample. There should be no plaques in the control plate.

Most often, lysis zones appear as 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 boundaries.

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

The fusion of negative colonies results in an “openwork” lawn of E. coli, the growth of single E. coli colonies against the background of continuous lysis, or a complete absence of growth on the plate.

With direct inoculation, lysis is possible, masked by inhomogeneously solidified agar, and also hidden by the accompanying microflora. Drops of condensation and inhomogeneously solidified agar during direct inoculation can lead to the formation of artifacts in the E. coli lawn, visually reminiscent of lysis.

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

The final quantitative count 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 growth of plaques is noted and counting is difficult, then according to direct culture data a qualitative result can be given: “found in 100 ml of water.”

If a negative result is obtained when working with the direct method, the final answer is given based on the results of the titration method.

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

8.5.4. Setting up controls

8.5.4.1. Negative control

Negative control confirms the absence of phage contamination of culture media, laboratory glassware, equipment at the stages of preparation and analysis, and also allows you to assess the ability of the test culture E. coli to produce a uniform lawn.

A negative control is a test of sterile tap water, carried out in the same way as the water sample being analyzed. Thus, when analyzing water by titration method, 10 ml of sterile tap water is added to an additional test tube. When analyzing water by direct seeding, add 20 ml of sterile tap water to an additional sixth Petri dish.

Additional cultures 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, setting up a negative control is carried out step by step after processing all samples of this series.

If coliphage plaques are detected in negative control plates, the results of the entire series of water samples are invalid.

The sterility of laboratory equipment, glassware, and culture media should be checked, and the control culture should be repeated for the purity of the test strain E. coli K12 F+ StrR.

The frequency of the negative control is 1 time per day.

8.5.4.2. Methodology for confirming the phage nature of lysis

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

For this purpose, a section of agar suspected of coliphages is removed using a bacteriological loop, placed 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 tested for the presence of phage. Sowing is carried out with a loop or pipette onto sectors of nutrient agar similarly to the method described in paragraph 8.5.2.5. Lysis on any of the sectors is regarded as confirmation of the presence of the phage.