GB 5749-2022 Key Insights: E. coli Detection & Prevention in Drinking Water

The microbial safety of drinking water for daily use is the "first line of defense" for safeguarding public health. As the core criterion for drinking water safety in China, the National Standard for Drinking Water Quality (GB 5749-2022) explicitly designates Escherichia coli as a key microbial indicator for routine inspection. Compared with total coliforms and aerobic bacterial count, Escherichia coli is the most specific "indicator bacterium" for reflecting fecal contamination, and its detection results are directly related to the judgment of drinking water safety. This article will systematically analyze the core points of Escherichia coli detection in drinking water from three dimensions: standard requirements, practical detection operations, and safety significance.

I. Core of the Standard: Indicator Positioning and Significance of Escherichia coli

Among the 3 routine microbial indicators specified in GB 5749-2022, Escherichia coli has become the "gold standard" for drinking water safety assessment due to its high specificity for fecal contamination. To accurately understand its detection value, it is necessary to first clarify the differences and correlations among the three types of microbial indicators.

1. Positioning Differences of the Three Routine Microbial Indicators

The aerobic bacterial count is the "basic ruler" for water quality cleanliness, which only reflects the total number of microorganisms in water. It is used to evaluate the effect of purification processes but cannot directly indicate the source of pollution. Total coliforms include 4 genera such as Escherichia and Citrobacter. Although related to fecal contamination, some genera can survive in nutrient-rich natural water bodies, leading to the possibility of "detection from non-fecal contamination", which limits its hygienic significance. In contrast, Escherichia coli, as the core component of thermotolerant coliforms, is a characteristic flora of the human and animal intestinal tracts. Its detection can directly prove the presence of fecal contamination in water bodies, thereby indicating the risk of intestinal infectious disease transmission. Therefore, it has the highest early warning value among the three indicators.

2. Safety Significance of Escherichia coli: From "Indicator Bacterium" to "Risk Early Warning"

The detection significance of Escherichia coli (commonly known as E. coli) stems from its "symbiotic relationship" with intestinal pathogenic bacteria. When water bodies are contaminated by feces, Escherichia coli coexists with pathogenic bacteria such as Salmonella typhi and Vibrio cholerae, and its survival ability is similar to that of these pathogens. Therefore, the risk of pathogenic bacterial contamination can be indirectly assessed by detecting Escherichia coli. GB 5749-2022 explicitly stipulates that Escherichia coli "shall not be detected" in drinking water for daily use (Unit: MPN/100mL or CFU/100mL). This strict limit sets a "red line" for the microbial safety of drinking water.

It should be noted that not all strains of Escherichia coli are pathogenic. Most strains are normal flora of the human intestinal tract, which can synthesize vitamins B and K and have a symbiotic relationship with the host. Only diarrheagenic Escherichia coli (such as enterotoxigenic ETEC and enterohemorrhagic EHEC) can cause diseases such as diarrhea and urinary tract infections. Among them, EHEC may also lead to serious complications such as hemolytic uremic syndrome, which requires timely early warning through accurate detection.

II. Practical Detection Operations: Full-Process Specifications from Sampling to Determination

The accuracy of Escherichia coli detection results depends on the standardized operation of the entire "sampling-cultivation-judgment" process. GB/T 5750.2-2023 and GB/T 5750.12-2023 provide authoritative guidelines for practical operations, with core points focusing on three links: sampling preparation, aseptic operation, and membrane filtration method determination.

1. Sampling Preparation: "Preparatory Guarantee" of Containers and Sterilization

The cleanliness and sterility of sampling containers are crucial for avoiding detection errors, which must strictly follow the triple requirements of "cleaning-sterilization-timeliness". For container selection, clean ground-glass hard glass bottles or polyethylene bottles (barrels/bags) can be used. The former is suitable for routine laboratory sampling, while the latter is convenient for on-site portable sampling.

The cleaning process must be "two-step in place": first, clean with tap water and detergent, then rinse thoroughly with tap water, soak in 10% nitric acid (or hydrochloric acid) for more than 8 hours, and finally rinse sequentially with tap water and pure water to completely remove residual impurities and metal ions. Sterilization can be carried out by two methods: dry heat or high-pressure steam. Dry heat sterilization requires maintaining at 160℃ for 2 hours, while high-pressure steam sterilization requires maintaining at 121℃ for 15 minutes. If not used immediately after sterilization, the containers should be placed in an oven at 60℃ to dry the condensed water. In addition, the sterilized containers must be used within 2 weeks to avoid secondary contamination.

2. Sampling Operation: Aseptic Principle and Scenario Adaptation

The core of the sampling process is "aseptic operation", which must prevent the interference of external contamination on the water sample. There are clear differences in sampling methods for different water sources. For tap water samples, the faucet must first be sterilized by flaming for 3 minutes, then run water for 5 minutes (1-3 minutes for frequently used faucets) to drain the stagnant water in the pipeline before sampling. For source water samples, sampling should be carried out at a depth of 10-15cm below the water surface to ensure representativeness. After sampling, the lid should be tightly closed in the water before taking it out to avoid contamination by floating substances on the water surface.

In addition, the samples must be sent for inspection immediately after sampling, stored at 0-4℃ for refrigeration throughout the process, and the time from collection to inspection should not exceed 8 hours. Escherichia coli is prone to reproduction at room temperature, and long-term storage will lead to high detection results, losing their authenticity.

3. Core of Determination: "Principle and Steps" of the Membrane Filtration Method

GB/T 5750.12-2023 recommends the membrane filtration method for Escherichia coli detection. This method achieves accurate identification through fluorescence reaction, which mainly relies on the specificity of NA-MUG medium and standardized operation.

① Core Principle

Escherichia coli can produce β-glucuronidase, which can decompose the fluorescent substrate (4-methylumbelliferyl-β-D-glucuronide) in NA-MUG medium to release fluorescent products. Under the irradiation of a 366nm ultraviolet lamp, colonies containing Escherichia coli will produce blue fluorescence. Positive results can be determined through fluorescence observation. This method has both specificity and sensitivity, with a detection limit of up to 1 CFU/100mL.

② Medium Preparation

The formula and preparation process of NA-MUG medium directly affect the detection effect, and the component ratio and sterilization conditions must be strictly controlled. The formula is as follows: peptone 5.0g, beef extract 3.0g, agar 15.0g, MUG 0.1g, add pure water to 1000mL. During preparation, the components should first be fully mixed, heated to dissolve, then sterilized by high-pressure steam at 121℃ for 15 minutes. When cooled to about 50℃, pour the plates. The final pH must be controlled at 6.6-7.0. The prepared plates can be stored at 0-4℃ for 2 weeks. If exceeding the time limit, they must be prepared again.

③ Experimental Steps and Result Judgment

The experiment must be carried out in an aseptic laboratory, strictly following the biosafety requirements of GB 19489. The core steps are divided into three stages: inoculation, cultivation, and judgment. During inoculation, transfer the filter membrane positive for total coliforms detection (with the bacterial retention surface facing up) to the NA-MUG plate. Then place it in an incubator at 36℃±1℃ for 4 hours of cultivation. After cultivation, irradiate the plate with a 6W, 366nm ultraviolet lamp in the dark. If blue fluorescence appears on the edge or back of the colony, it is positive for Escherichia coli.

The result calculation must adopt the standard formula: Escherichia coli colony count (CFU/100mL) = (number of detected positive colonies × 100) / volume of filtered water sample (mL). If no positive colonies are detected, the result is reported as "not detected". If detected, the specific colony count must be recorded, and it must meet the standard limit requirement of "shall not be detected".

III. Risk Prevention and Control: Extension from Detection to Safety Guarantee

The detection of Escherichia coli is not the end. Its core purpose is to achieve risk prevention and control through pollution early warning. Combined with its transmission routes and survival characteristics, a safety defense line can be constructed from two aspects: source control and terminal disinfection.

1. Source Control: Blocking the Path of Fecal Contamination

The contamination of Escherichia coli mainly comes from domestic sewage, livestock and poultry manure, agricultural non-point sources, etc., which requires targeted prevention and control: protected areas must be designated for drinking water sources, and activities such as breeding and sewage discharge are prohibited; urban pipe networks must implement rainwater-sewage diversion to avoid source water contamination by sewage overflow during heavy rains (as mentioned earlier, the concentration of Escherichia coli in source water can surge 50 times after heavy rains); in rural areas, the treatment of decentralized sewage must be strengthened to avoid the direct discharge of fecal sewage into source water.

2. Terminal Disinfection: Key to Killing Pathogenic Strains

Pathogenic strains such as diarrheagenic Escherichia coli are sensitive to high temperatures. They can be killed by soaking in 80℃ hot water for 5-10 minutes or boiling water for 2-5 minutes. For drinking water, water plants must ensure sterilization effects through processes such as chlorination disinfection and ozone disinfection. For household drinking, if the water quality is suspected to be contaminated, thorough disinfection can be achieved by boiling. In addition, for scenarios such as catering and food processing, regular detection of Escherichia coli in water bodies is required to avoid cross-contamination.

3. Portable Detection: Technical Support for On-Site Prevention and Control

Although traditional laboratory detection is accurate, it is time-consuming and cannot meet the needs of on-site emergency response. The VN-M50 Portable Microbial Detection Kit developed by VVNA integrates a full set of equipment including aseptic sampling bottles, NA-MUG medium, and small incubators. It highly integrates the sampling, inoculation, and cultivation processes, enabling on-site detection and greatly shortening the early warning time. It is suitable for scenarios such as source water patrols and emergency response to sudden pollution, providing an efficient tool for microbial safety prevention and control.

IV. Conclusion: Safeguarding the Microbial Safety of Drinking Water Through Standardized Detection

The detection of Escherichia coli is a core link in the microbial safety control of GB 5749-2022. The full-process standardized operation from sampling preparation to result judgment is the key to ensuring data accuracy. Understanding the indicator significance of Escherichia coli, mastering standardized detection methods, and constructing a closed-loop system of "detection-early warning-prevention and control" are not only the implementation of standards but also the direct safeguard of public health. With the development of portable detection technology, the future microbial safety control of drinking water for daily use will be more efficient and accurate, building a solid "microbial defense line" for the drinking water safety of the whole people.

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