Omch okb tkb in drinking water. Coliform bacteria in water
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, three volumes of 100 cm3 are inoculated. When studying water from the whole | for quantitative determination of OCB and TCB (repeated analysis), 1.10 and 100 cm3 are inoculated, respectively - three volumes of each series.
Inoculations of 10 and 100 cm3 of water are carried out, respectively, in 1 and 10 cm3 of accumulation medium - concentrated LPS without an indicator. Inoculation of 1 cm3 of sample is carried out in 10 cm3 of LPS of normal concentration. The crops are incubated at a temperature of 37 °C for 48 hours. After 24 hours, a preliminary assessment of the crops is carried out in the accumulation medium. From containers where growth (turbidity) and gas formation are noted, the material is sown with a bacteriological loop onto sectors of the Endo medium to obtain isolated colonies. Containers without visible signs of growth and gas formation are left in the thermostat for up to 48 hours and examined again for final assessment.
Culture results without signs of growth are considered negative and are not subject to further study. From containers where turbidity is noted and seeding is done on sectors of the Endo medium. Inoculations on Endo medium are incubated at a temperature of 37 °C for 18-20 hours. When turbidity appears, gas formation 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 conclusion about the presence of OCB in a given sample volume.
The presence of OKB must be confirmed in the following cases:
ü only turbidity was noted in the accumulation medium;
ü belonging to lactose-positive colonies is doubtful.
To confirm the presence of OKB, perform the following steps:
1. check for the presence of an imprint on the Endo medium after removing a suspicious colony with a loop;
2. perform an oxidase test;
3. check membership in the Gram group;
4. confirm the ability to form gases by inoculating 1-2 isolated colonies of all types from each sector into a confirmation medium (LPS with an indicator), followed by incubating the crops at a temperature of 37 °C for 24-48 hours.
In the absence of isolated colonies, seeding is carried out on Endo medium using generally accepted methods. A negative conclusion is given if:
ü there are no signs of growth in the accumulation environment;
ü there is no growth in the sectors of the Endo environment;
ü on sectors of the Endo medium, colonies uncharacteristic for coliform bacteria grew (transparent, with uneven edges, vague);
ü all colonies turned out to be oxidase-positive;
ü all colonies turned out to be gram-positive;
ü in the confirmatory test on LPS medium with an indicator, no gas formation was noted.
To determine TKB, they work with sectors of the Endo medium where typical lactose + colonies have grown. Two or three isolated colonies of each type from each sector are inoculated into test tubes with any of the lactose accumulation media and incubated at 44 °C for 24 hours. When gas is formed in a lactose accumulation medium, the growth of lactose-positive bacteria on Endo medium and the ability to ferment lactose to acid and gas in confirmatory lactose media at a temperature of 44 ° C for 24 hours is detected, a positive conclusion is given about the presence of TKB water in this volume. During a qualitative study (when examining three volumes of 100 cm3, if OKB and TBC are detected in at least one of the three volumes, a note is made: “OKB and TBC were detected in 100 cm3.”
When researching using a quantitative method, NHF, OKB and TKB are determined using special tables. If the results of the study for the presence of OKB and TCB in all examined volumes are negative, a conclusion is issued: “No OKB and TKB were detected in 100 cm3.”
Escherichia coli is the very first sanitary-indicative microorganism that has retained its importance to this day. Back in 1888, the French doctor E. Mase proposed using this bacterium as an indicator of fecal contamination of water. The third edition of the WHO Guidelines for Drinking-Water Quality recommends E. coli (index) as the indicator of choice for assessing fresh faecal contamination. Thermotolerant Coliform Bacteria (TCB) (index) is recommended as an alternative indicator of faecal contamination (in certain circumstances). The Coliform Bacteria (CB) indicator is recommended as a technological indicator for assessing the quality of water treatment (indicative). According to the domestic regulatory framework, Coliform Bacteria (CB) in WHO terminology correspond to the indicator Total Coliform Bacteria (TCB).
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To determine coliform indicators, the membrane culture method is widely used, although the titration method is no less important. Methods for determining these indicators vary greatly depending on the object under study and regulatory and methodological documents. The main dense differential medium for determining coliform indicators in domestic methods is Endo medium, however, in the latest edition of the ISO 9308-1:2000 standard, Endo medium is replaced by another lactose medium - Tergitol 7. The reason for this replacement was the potential carcinogenicity of fuchsin, an aniline dye that is part of the Endo medium. For the NHF method, liquid enrichment media are used. For potentially clean objects, lactose-peptone water is used; for potentially contaminated objects, Kessler's medium or its analogues are used.
It is necessary to note the new generation of nutrient media, which are often called “chromogenic”. Unlike traditional media, they make it possible to determine not a trait, for example, lactose utilization, but directly individual enzymes, the presence of which is characteristic of the desired microorganisms. Chromogenic media for identification of E. coli, e.g. Chromocult® or Coli ID, allow the determination of the β-glucuronidase enzyme, which is highly specific for Escherichia. The presence of this enzyme and the ability to form indole with 95% probability indicates that enterobacteria belong to the species E. coli. The same media also makes it possible to determine the β-galactosidase enzyme, characteristic of OCD, but the value of this diagnostic test is questionable: this enzyme is also possessed by aeromonads, free-living oxidase-positive rods that are not related to OCD. Merck tried to improve chromogenic media Chromocult EC and introduced into it a selective additive that inhibits the growth of aeromonads.
Among the innovative technologies in the field of sanitary bacteriology of water, noteworthy are test systems that use dry media on special plastic substrates. An example of such test systems are substrates Petrifilm™ rulers Aqua and in particular the product "Aqua Coliform Count Plate" (AQCC, 3M™Petrifilm™), which is intended for determining TCB and TCB in water. The uniqueness of petrifilms (media on substrates) lies in their ease of use. The labor-intensive stage of preparing culture media is eliminated, making their storage and disposal easier. However, the main advantage over traditional media and media on substrates from other manufacturers is that already at the stage of primary seeding when obtaining isolated colonies, petrifilms make it possible to determine not only the ability of bacteria to utilize lactose into acid, but also to detect gas formation. This allows in most cases to reduce the analysis to 1-2 days. In addition, AQCC petrifilms (unlike Endo medium) can be incubated at 44°C, which allows full use of the selective factor of high temperature already at the stage of primary inoculation, thereby significantly reducing the time and labor intensity of TCB analysis.
On selective petrifilms "Aqua Coliform Count Plate" (AQCC,3M™ Petrifilm™) OCB and TCB colonies turn intense red with the formation of gas bubbles around the colony.
OKB is an international qualification and they are part of the large group of 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 recent research, 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 a relatively high concentration of 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 and 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.
Coliform bacteria are always present in the digestive tract of animals and humans, as well as in their waste products. They can also be found on plants, soil and water, where contamination is a serious problem due to the possibility of disease transmission caused by various pathogens.
Harm to the body
Are coliform bacteria harmful? Most do not cause illness, however, some rare strains of E. coli can cause serious illness. In addition to people, sheep and cattle can be infected. It is of concern that contaminated water, in its external characteristics, is no different from ordinary drinking water in taste, smell and appearance. Coliform bacteria are found even in environments that are considered to be impeccable in every sense. Testing is the only reliable way to find out about the presence of pathogenic bacteria.
What happens when detected?
What to do if coliform or any other bacteria are found in drinking water? In this case, repairs or modifications to the water supply system will be necessary. When consumed, disinfection requires mandatory boiling, as well as repeated testing, which can confirm that the contamination has not been eliminated if it was thermotolerant coliform bacteria.
Indicator organisms
Common coliforms are often called indicator organisms because they indicate the potential presence of disease-causing bacteria in water, such as E. coli. Although most strains are harmless and live in the intestines of healthy people and animals, some can produce toxins, cause serious illness, and even be fatal. If pathogenic bacteria are present in the body, the most common symptoms are gastrointestinal upset, fever, abdominal pain and diarrhea. Symptoms are more pronounced in children or older family members.
Safe water
If there are no common coliform bacteria in the water, then it can be assumed with almost complete certainty that it is microbiologically safe for drinking.
If they were detected, then additional tests would be justified.
Bacteria love warmth and moisture
Temperature and weather conditions also play an important role. For example, E. coli prefers to live on the surface of the earth and likes warmth, so coliform bacteria in drinking water appear as a result of movement in underground streams during warm and humid weather conditions, while the least amount of bacteria will be found in the winter season.
Shock chlorination
To effectively kill bacteria, chlorine is used, which oxidizes all impurities. Its amount will be affected by water characteristics such as pH level and temperature. On average, the weight per liter is approximately 0.3-0.5 milligrams. It takes approximately 30 minutes to kill common coliform bacteria in drinking water. Contact time can be reduced by increasing the chlorine dose, but this may require additional filters to remove specific tastes and odors.
Harmful ultraviolet light
Ultraviolet rays are considered a popular disinfection option. This method does not involve the use of any chemical compounds. However, this remedy is not used where total coliform bacteria exceed one thousand colonies per 100 ml of water. The device itself consists of a UV lamp surrounded by a quartz glass sleeve through which a liquid flows, irradiated with ultraviolet light. The untreated water inside the apparatus must be completely clean and free from any visible contamination, blockages or turbidity to allow exposure to all harmful organisms.
Other cleaning options
There are many other treatments used to disinfect water. However, they are not recommended for long-term use for various reasons.
- Boiling. At 100 degrees Celsius for one minute, bacteria are effectively killed. This method is often used to disinfect water during emergencies or when necessary. This is a time-consuming and energy-intensive process and is typically only used in small quantities of water. This is not a long-term or permanent option for water disinfection.
- Ozonation. In recent years, this method has been used as a way to improve water quality and eliminate various problems, including bacterial contamination. Like chlorine, ozone is a strong oxidizing agent that kills bacteria. But at the same time, this gas is unstable and can only be obtained using electricity. Ozonation units are not generally recommended for disinfection because they are much more expensive than chlorination or ultraviolet systems.
- Iodization. The once popular method of disinfection has recently been recommended only for short-term or emergency water disinfection.
Thermotolerant coliform bacteria
This is a special group of living organisms that are capable of fermenting lactose at 44-45 degrees Celsius. These include the genus Escherichia and some species of Klebsiella, Enterobacter and Citrobacter. If foreign organisms are present in the water, this indicates that it has not been sufficiently purified, has been re-contaminated, or contains too many nutrients. If they are detected, it is necessary to check for the presence of coliform bacteria that are resistant to elevated temperatures.
Microbiological analysis
If coliforms were detected, this may indicate that they got into the water. Thus, various diseases begin to spread. In contaminated drinking water you can find strains of salmonella, shigella, E. coli and many other pathogens that range from mild digestive tract disorders to severe forms of dysentery, cholera, typhoid fever and many others.
Household sources of infection
The quality of drinking water is monitored and regularly checked by specialized sanitary services. What can an ordinary person do to protect themselves and protect themselves from unwanted infection? What are the sources of water pollution in domestic conditions?
- Water from the cooler. The more people touch this device, the greater the likelihood of harmful bacteria entering. Research shows that the water in every third cooler is simply teeming with living organisms.
- Rainwater. Surprisingly, moisture collected after rain is a favorable environment for the development of coliform bacteria. Advanced gardeners do not use such water even for watering plants.
- Lakes and reservoirs are also considered a risk group, since all living organisms, not just bacteria, multiply faster in stagnant water. An exception is the oceans, where the development and spread of harmful forms there is minimal.
- Pipeline condition. If the drain pipes have not been changed or cleaned for a long time, this can also lead to problems.
(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).
Dishes with filters are incubated upside down in a thermostat at 37 o 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 o C, which is important for weakly colored colonies and their relationship to TKB, and a temperature of 44 ± 0.5 o C, in order to resolve the issue of their belonging to TKB.
Performing an oxidase test
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±1 o C for 24-48 hours to confirm the relation to TKB, the other at a temperature of 44±0.5 o C for 24 hours, it is possible to count 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 o 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 o C to acid and gas (scheme No. 2).