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HJ 1001-2018: Water quality - Determination of total coliforms, fecal coliforms and Escherichia coli - Enzyme substrate method
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Water quality - Determination of total coliforms, fecal coliforms and Escherichia coli - Enzyme substrate method
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HJ 1001-2018
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Basic data | Standard ID | HJ 1001-2018 (HJ1001-2018) | | Description (Translated English) | Water quality - Determination of total coliforms, fecal coliforms and Escherichia coli - Enzyme substrate method | | Sector / Industry | Environmental Protection Industry Standard | | Classification of Chinese Standard | Z16 | | Word Count Estimation | 38,320 | | Date of Issue | 2018-12-26 | | Date of Implementation | 2019-06-01 | | Regulation (derived from) | Ministry of Ecology and Environment Announcement No. 73 of 2018 | | Issuing agency(ies) | Ministry of Ecology and Environment | HJ 1001-2018: Water quality - Determination of total coliforms, fecal coliforms and Escherichia coli - Enzyme substrate method
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Water quality-Determination of total coliforms, fecal coliforms and Escherichia coli-Enzyme substrate method
National Environmental Protection Standard of the People's Republic
Determination of total coliforms, fecal coliforms and Escherichia coli in water quality
Published on.2018-12-26
2019-06-01 Implementation
Ministry of Ecology and Environment released
Content
Foreword...ii
1 Scope...1
2 Normative references...1
3 Terms and Definitions...1
4 Principles of the method... 2
5 Interference and elimination... 2
6 Reagents and materials...2
7 Instruments and Equipment...3
8 samples...3
9 Analysis steps...4
10 Calculation and representation of results...6
11 Precision and Accuracy...6
12 Quality Assurance and Quality Control...8
13 Waste treatment...8
Appendix A (informative) Result determination reference picture...9
Appendix B (Normative) 97 Hole Quantitative Disk Method MPN Table...12
Appendix C (Informative Appendix) 97 Hole Quantitative Disk Method MPN Table Corresponds to 95% Confidence Interval...20
Foreword
To protect the ecology of the People's Republic of China Environmental Protection Law and the Law of the People's Republic of China on Water Pollution Prevention and Control
This standard is established for the environment, to ensure human health, and to regulate the total coliform, fecal coliform and Escherichia coli in water.
This standard specifies the determination of total coliforms, fecal coliforms in surface water, groundwater, domestic sewage and industrial wastewater.
Enzyme substrate method for Escherichia coli.
Appendix A of this standard is a normative appendix, and Appendix B and Appendix C are informative appendices.
This standard is the first release.
This standard is formulated by the Department of Eco-Environmental Monitoring and the Department of Regulations and Standards of the Ministry of Ecology and Environment.
This standard was drafted. Shanghai Environmental Monitoring Center.
This standard is verified by the Jiangsu Provincial Environmental Monitoring Center, the Zhejiang Environmental Monitoring Center, and the Changzhou Environmental Monitoring Center.
Haisong District Environmental Monitoring Station, Shanghai Changning District Environmental Monitoring Station and Shanghai Qingpu District Environmental Monitoring Station.
This standard is approved by the Ministry of Ecology and Environment on December 26,.2018.
This standard has been implemented since June 1,.2019.
This standard is explained by the Ministry of Ecology and Environment. 1
Determination of total coliforms, fecal coliforms and Escherichia coli in water quality
1 Scope of application
This standard specifies the enzyme substrate method for the determination of total coliforms, fecal coliforms and Escherichia coli in water.
This standard applies to the determination of total coliforms, fecal coliforms and Escherichia coli in surface water, groundwater, domestic sewage and industrial wastewater.
The detection limit of this method is 10 MPN/L.
2 Normative references
This standard refers to the following documents or their terms. For undated references, the valid version applies to this standard.
GB/T 6682 analysis of experimental water specifications and experimental methods
GB/T 14581 Water quality lake and reservoir sampling technical guidance
HJ 494 Water Quality Sampling Technical Guidance
HJ/T 91 Surface Water and Wastewater Monitoring Technical Specifications
3 Terms and definitions
The following terms and definitions apply to this standard.
3.1
Total coliforms total coliforms
Cultured at 37 ° C for 24 h, can produce β-galactosidase (β-D-galactosidase), decomposes the adjacent nitrate in selective medium
Alkaloid-β-D-galactopyranoside (ONPG) Enterobacteriaceae bacteria producing yellow o-nitrophenol.
3.2
Fecal coliforms fecal coliforms
Also known as thermotolerant coliforms. Cultured at 44.5 ° C for 24 h, can produce β-galactosidase
(β-D-galactosidase), decomposition of o-nitrophenyl-β-D-galactopyranoside (ONPG) in selective medium
An Enterobacteriaceae bacterium producing a yellow o-nitrophenol.
3.3
Escherichia coli
Commonly known as E. coli. Cultured at 37 ° C for 24 h, can produce β-galactosidase (β-D-galactosidase), decomposition selectivity
o-Nitrobenzene-β-D-galactopyranoside (ONPG) in the medium produces yellow o-nitrophenol while producing β-grape
Uraculonic enzyme (β-Glucuronidase), decomposes 4-methylumbelliferyl-β-D-glucuronide 2 in selective medium
( MUG) Enterobacteriaceae bacteria that release the fluorescent substance (4-methylumbelliferone).
3.4
The largest possible number of most probable number (MPN)
Also known as dilution culture count, it is an indirect counting method based on Poisson distribution. Using statistical principles, according to certain bodies
The number of target microorganisms produced by culturing samples with different dilutions, and the table to estimate the target microorganisms in a certain volume of samples
The quantity present (the maximum possible number of target microorganisms per unit volume).
4 Principle of the method
When cultured at a specific temperature for a specific period of time, total coliform, fecal coliform, and Escherichia coli can produce β-galactose
Glycosidase, which decomposes the colorless substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) in a selective medium into yellow
O-nitrophenol (ONP); Escherichia coli can also produce β-glucuronidase, 4- in selective medium
Methylumbelliferone-β-D-glucuronide (MUG) is decomposed into 4-methylumbelliferone, which fluoresces under ultraviolet light.
Count the number of positive reactions, check the MPN table, and calculate the concentration of total coliform, fecal coliform, and Escherichia coli in the sample.
5 interference and elimination
5.1 Active chlorine is oxidizing and can destroy the enzyme activity in microbial cells, leading to cell death.
The sodium thiosulfate solution ( 6.4) can be added during sample collection ( 8.1) to eliminate interference.
5.2 Heavy metal ions are cytotoxic and can destroy the enzyme activity in microbial cells, leading to cell death.
Add (2.8) to the disodium edetate solution ( 6.5) to eliminate interference.
6 reagents and materials
Unless otherwise stated, analytically pure reagents that meet national standards are used for analysis, and laboratory water should meet the requirements for tertiary water in GB/T 6682.
6.1 Medium.
This standard uses Minimal Medium ONPG-MUG medium. Use medium powder per 100 ml sample
2.7 g ± 0.5 g, the basic ingredients are as follows.
6.2 Sodium thiosulfate (Na2S2O3·5H2O).
6.3 Disodium edetate (C10H14N2O8Na2·2H2O).
6.4 Sodium thiosulfate solution. ρ(Na2S2O3) = 0.10 g/ml.
Weigh 15.7 g of sodium thiosulfate (6.2), dissolve in an appropriate amount of water, and dilute to 100 ml.
6.5 Ethylenediaminetetraacetic acid disodium solution. ρ(C10H14N2O8Na2·2H2O) = 0.15 g/ml.
Weigh 15 g of disodium edetate (6.3), dissolve in an appropriate amount of water, and dilute to 100 ml. The shelf life of this solution is 30 d.
6.6 97-well tray. 49 large holes and 48 small holes. Among them, each hole can hold 0.186 ml sample, large hole
The 48 large holes each hold 1.86 ml of sample and one top large hole accommodates 11 ml of sample. Commercially available finished products sterilized by ethylene oxide can also be used.
6.7 Standard positive colorimetric plate.
6.8 Sterile water. Take appropriate amount of experimental water and sterilize it by autoclaving at 121 °C for 20 min.
7 Instruments and equipment
7.1 Sampling bottle. 100 ml, 250 ml, 500 ml wide-mouth glass bottle with screw cap or grinder.
Note. Commercially available sterile sampling bottles or sterile sampling bags can be used when collecting samples that do not exist or do not consider residual oxygen or metal ion interference.
7.2 High pressure steam sterilizer. adjustable at 121 °C.
7.3 Constant temperature incubator. The temperature deviation is allowed to be 37 °C ± 1 °C, 44.5 °C ± 0.5 °C.
7.4 Programmable Quantitative Sealer. For the sealing of the 97-well dosing plate (6.6).
7.5 UV lamp. 365 ~ 366 nm.
7.6 Pipette. 1 ml ± 0.01 ml, 10 ml ± 0.1 ml. A metered adjustable pipette can also be used.
7.7 Triangle bottle. 100 ml.
7.8 Measuring cylinder. 100 ml ± 1 ml.
7.9 Common instruments and equipment used in general laboratories.
Note. Glassware and sampling instruments such as triangular bottles, pipettes, sampling bottles, etc. should be wrapped according to aseptic operation requirements before testing, autoclaved at 121 °C for 20 min, dried, and set aside.
8 samples
8.1 Sample Collection
The point layout and sampling frequency are carried out in accordance with the relevant provisions of GB/T 14581, HJ/T 494 and HJ/T 91.
When collecting microbial samples, the sampling bottle (7.1) should not be washed with the sample, and the sample should be collected in a sterile sampling bottle. 4
When collecting surface water samples such as rivers and lake banks, you can directly insert the bottle with the plug into the water by holding the lower part of the bottle, about water
10 to 15 cm, the bottle mouth is in the direction of water flow, pull the stopper, pour the sample into the bottle and close the stopper, and take the sample bottle from the water.
Take out. If there is no water flow, hold the bottle horizontally and push forward. The sampling amount is generally about 80% of the sample bottle capacity.
After the sample is collected, quickly insert the aseptic packaging paper.
When collecting samples from the faucet device, do not use the leaking faucet. Open the faucet to the maximum before collecting water, and drain the water for 3-5 minutes.
Then turn off the faucet, sterilize with a flame for about 3 minutes, or disinfect the faucet with 70% to 75% alcohol.
The water was again drained for 1 min to completely remove the retained impurities in the water pipe. Control the water flow rate during sampling and carefully connect to the bottle.
When collecting surface water, wastewater samples, and samples of a certain depth, it can also be sampled using a sterilized dedicated sampling device.
When stratified sampling is performed at the same sampling point, it should be performed from top to bottom to avoid different levels of disturbance.
If a sample containing active chlorine is collected, add sodium thiosulfate solution (6.4) before sterilization of the sample bottle to remove
Inhibition of bacteria by dechlorination of chlorine (0.1 ml of sodium thiosulfate solution per 125 ml volume);
For samples with high levels of heavy metal ions, add disodium edetate solution (6.5) before sterilization of the sample bottle to eliminate
Interference (0.3 ml of disodium edetate solution per 125 ml volume).
Note. 15.7 mg sodium thiosulfate (6.2) removes 1.5 mg of active chlorine from the sample. The amount of sodium thiosulfate can be adjusted according to the actual amount of active chlorine in the sample.
8.2 Sample storage
It should be tested within 2 h after sampling. Otherwise, it should be refrigerated below 10 °C but not more than 6 h. After the laboratory is sampled, it cannot
For immediate testing, samples should be refrigerated below 4 °C and tested within 2 h.
9 Analysis steps
9.1 Sample dilution
Determine the inoculum based on the degree of contamination of the sample (see Table 1), and avoid the 97-well plate (6.6) after inoculation of the sample.
All are positive or negative. When the inoculation amount is less than 100 ml, the sample should be diluted and inoculated. When the inoculation amount is 10 ml,
Add 10 ml of the sample to a triangular flask (7.7) containing 90 ml of sterile water (6.8) and mix to make a 1.10 diluted sample.
Dilution samples of other inoculum sizes are analogized. For unknown samples, multiple inoculum sizes can be used for testing.
Table 1 Sample inoculation amount reference table
9.2 Vaccination
Measure 100 ml of the sample or dilute the sample in a sterilized flask and add 2.7 g ± 0.5 g of medium (6.1) powder.
Mix thoroughly, completely dissolve, and pour all into the 97-well plate (6.6), and smooth the back of the 97-well plate with your hand.
Remove the bubbles from the holes and seal with a programmable quantitative sealer (7.4). Observe the color of the 97-well plate, if it appears similar or deep
In the color of the standard positive colorimetric plate (6.7), a series of factors such as sample, medium, and sterile water are required to terminate the test or re-operate.
Note. 9.1, 9.2 steps should avoid obvious local pollution sources when operating in the field. It is recommended to use disposable gloves, masks, alcohol lamps, etc.
9.3 Cultivation
For the determination of total coliforms and Escherichia coli, the 97-well plate after sealing was placed in a constant temperature incubator (7.3) for 24 h at 37 °C ± 1 °C.
When the fecal coliform group was measured, the sealed 97-well quantitative tray was placed in a constant temperature incubator at 44.5 ° C ± 0.5 ° C for 24 h.
9.4 Control test
9.4.1 Blank control
For each test, use laboratory water (6.8) to perform laboratory blank determination according to steps 9.1 to 9.3. 97 holes after cultivation
The quant disc must not have any color reaction. Otherwise, the sample measurement result is invalid, and the cause should be determined and then re-measured.
9.4.2 Negative and positive controls
Refer to Table 2 for negative, positive strains of total coliform, Escherichia coli, and fecal coliforms.
Table 2 Negative, positive strain reference table
10 Calculation and representation of results
10.1 Calculation of results
Total coliforms, fecal coliforms per 100 ml of sample were obtained from the 97-well quantitative disk method MPN table (see Appendix B)
The number of groups or the MPN value of Escherichia coli (see Appendix C for the confidence interval), and then according to the different dilutions of the sample,
Calculate the total coliform, fecal coliform or Escherichia coli concentration (MPN/L) in the sample according to formula (1).
10.2 Results are expressed
The measurement result retains two significant figures. When the measurement result is ≥100 MPN/L, it is expressed by scientific notation;
The wells are negative and can be reported as total coliform, fecal coliform or Escherichia coli not detected or < 10 MPN/L.
11 Precision and accuracy
11.1 Precision
6 laboratories for low concentration (groundwater, concentration mean 6.0×102 MPN/L), medium concentration (surface water,
The average concentration is 4.0×104 MPN/L) and the high concentration (domestic sewage, the average concentration is 9.0×107 MPN/L).
Samples with the total concentration of bacteria and certified standard samples (concentration.2000 MPN/L) were subjected to 6 replicates. Laboratory
The internal relative standard deviation ranges from 0.26% to 1.3%, 0.65% to 2.0%, 1.3% to 3.7%, and 0.68% to 2.8%, respectively.
The relative standard deviations between the chambers were 9.9%, 1.3%, 4.2% and 1.3%, respectively; the repeatability limits were 0.17, 0.18, 0.21 and 0.18;
Reproducibility limits are 1.8, 0.23, 0.38, and 0.20; interlaboratory confidence intervals are shown in Table 3. 7
Table 3 95% confidence interval table between the total coliforms laboratory
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