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HJ 755-2015: Water quality. Determination of total coliforms and fecal coliforms. Paper strip method
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Water quality. Determination of total coliforms and fecal coliforms. Paper strip method
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HJ 755-2015
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Standard similar to HJ 755-2015 HJ 511 HJ 945.3 HJ 943 Basic data | Standard ID | HJ 755-2015 (HJ755-2015) | | Description (Translated English) | Water quality. Determination of total coliforms and fecal coliforms. Paper strip method | | Sector / Industry | Environmental Protection Industry Standard | | Word Count Estimation | 15,150 | | Date of Issue | 2015-10-22 | | Date of Implementation | 2015-12-01 | | Regulation (derived from) | Ministry of Environment Announcement 2015 No.62 | | Issuing agency(ies) | Ministry of Ecology and Environment | | Summary | This standard specifies the determination of total coliforms and fecal coliforms in water of paper rapid method. This standard applies to surface water, Rapid Determination of total coliforms and fecal coliforms. The detection limit of this standard 20MPN/L. | HJ 755-2015: Water quality. Determination of total coliforms and fecal coliforms. Paper strip method
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Water quality.Determination of total coliforms and fecal coliforms.Paper strip method
National Environmental Protection Standard of the People's Republic
Determination of total coliform and fecal coliforms in water quality
Paper fast method
Water quality Determination of total coliforms and fecal coliforms
Paper strip method
Published on.2015-10-22
2015-12-01 Implementation
Release Ministry
Content
Foreword.ii
1 Scope.1
2 Terms and Definitions 1
3 principle of the method.1
4 reagents and materials 1
5 Instruments and equipment 2
6 sample 2
7 Analysis steps.3
8 Calculation and representation of results. 5
9 precision and accuracy. 5
10 Quality Assurance and Quality Control..6
11 Waste treatment.6
Appendix A (informative appendix) results decision reference picture.7
Appendix B (informative) Maximum Possible Number (MPN) Table.8
Foreword
To implement the Environmental Protection Law of the People's Republic of China and the Law of the People's Republic of China on Water Pollution Prevention and Control, to protect the environment,
This standard is formulated to ensure human health and to regulate the determination of total coliforms and fecal coliforms in water.
This standard specifies the rapid method for the determination of total coliforms and fecal coliforms in surface water and wastewater.
This standard is the first release.
Appendix A and Appendix B of this standard are informative annexes.
This standard was formulated by the Science and Technology Standards Department of the Ministry of Environmental Protection.
This standard is mainly drafted by. Changzhou Environmental Monitoring Center and Environmental Standards Research Institute of the Ministry of Environmental Protection.
The standard verification unit. Shanghai Environmental Monitoring Center, Jiangsu Environmental Monitoring Center, Zhejiang Environmental Monitoring Center,
Suzhou Environmental Monitoring Center Station, Nanjing Environmental Monitoring Center Station and Taizhou Environmental Monitoring Center Station.
This standard was approved by the Ministry of Environmental Protection on October 22,.2015.
This standard has been implemented since December 1,.2015.
This standard is explained by the Ministry of Environmental Protection.
1 Determination of total coliforms and fecal coliforms in water quality
1 Scope of application
This standard specifies a rapid method for the determination of total coliforms and fecal coliforms in water.
This standard applies to the rapid determination of total coliforms and fecal coliforms in surface water and wastewater.
The detection limit of this method is 20 MPN/L.
2 Terms and definitions
The following terms and definitions apply to this standard.
2.1 total coliforms total coliforms
Cultured at 37 ° C, can ferment lactose to produce acid-producing aerobic and facultative anaerobic Gram-negative Bacillus sp.
2.2 fecal coliforms fecal coliforms
Cultured at 44.5 ° C, can ferment lactose to produce acid-producing aerobic and facultative anaerobic Gram-negative Bacillus sp.
2.3 The maximum possible number of most probable number, MPN
Microbial test The commonly used fermentation method, also known as the dilution method, is a method for quantitatively detecting the concentration of microorganisms using statistical principles. It is based on not
The frequency of the presence or absence of the tested microorganisms in a certain volume of the same dilution, the table looks for the concentration of microorganisms in the sample, and directly reports the number of colonies.
The plate count method is different, it finally reports the target microorganism concentration most likely to exist in the sample, which is the maximum possible concentration,
It is called the most probable number (abbreviated as MPN).
3 Principle of the method
According to the MPN method, a certain amount of water sample is inoculated by aseptic operation to adsorb the appropriate amount of indicator (bromocresol purple and 2,3,5-chlorinated three
Phenyltetrazolium (TTC) and lactose and other nutrients on sterile filter paper, cultured at a specific temperature (37 ° C or 44.5 ° C) for 24 h, when fine
When the bacteria grow and multiply, acid production lowers the pH value, and the bromocresol purple indicator changes from purple to yellow. At the same time, the corresponding dehydrogenase in the gas production process is suitable.
In the pH range, the catalytic substrate is dehydrogenated to reduce TTC to form red insoluble tribendamidine (TTF), which can be displayed under the yellow background after acid production.
Red spots (or blush). Through the color change of the above indicator, it is possible to judge whether or not the acid is produced, thereby determining whether there is a total
Intestinal flora or fecal coliforms exist, and the concentration of the corresponding total coliform or fecal coliforms can be obtained by examining the MPN table.
4 reagents and materials
Analytical purification reagents that meet national standards were used for analysis unless otherwise stated.
4.1 Commercial water quality total coliform and fecal coliform test paper. 10 ml water sample paper, 1 ml water sample paper
The quality is identified as follows, and can be used after meeting the requirements.
(1) The outer aluminum foil packaging bag should be sealed and the inner packaging polypropylene plastic film bag is not damaged.
(2) The appearance of the paper sheet should be neat and burr-free, without damage, and it will be evenly yellowish green. It will be purple after adding deionized water or distilled water, no matter adding water and
2 No, there should be no mottling spots, no obvious deformation, and the surface is flat.
(3) The paper is added to the corresponding water sample. After fully infiltrating and absorbing, the inner packaging polypropylene plastic film bag is inverted, and the mouth of the bag should be suspended without water drops.
(4) After the paper sheet is sufficiently wetted with deionized water or distilled water, the pH should be in the range of 7.0 to 7.4.
(5) Paper and inner packaging polypropylene plastic film bags should be sterile, add the appropriate amount of sterile water, culture at 37 ° C ± 1 ° C for 24 h, the paper should be no
Microbial growth, its purple color remains unchanged, and no erythema appears.
(6) According to the method in 7.4.2, the total coliform and fecal coliforms are negative and positive standard strains are tested, and their characteristics should meet the requirements.
4.2 Sterile water
Prepare with freshly prepared deionized water or distilled water, autoclave at 121 °C for 20 min, and set aside.
4.3 Sodium thiosulfate solution. ρ(Na2S2O3)=0.10 g/ml
Weigh 10 g of sodium thiosulfate, dissolve in an appropriate amount of distilled water (or deionized water), dilute to 100 ml, and mix.
4.4 Ethylenediaminetetraacetic acid disodium (EDTA-Na2) solution. ρ(C10H14N2O8Na2·2H2O)=0.15 g/ml
Weigh 15 g of EDTA-Na2, dissolve it in an appropriate amount of distilled water (or deionized water), dilute to 100 ml, and the shelf life of this solution is 30 d.
5 Instruments and equipment
5.1 Constant temperature incubator. 37 °C ± 1 °C.
5.2 Constant temperature incubator. 44.5 ° C ± 0.5 ° C.
5.3 High pressure steam sterilizer. 121 ° C, 101.3 kpa.
5.4 Refrigerator. 0 ~ 4 °C.
5.5 Pipette. 1 ± 0.01 ml, 10 ± 0.1 ml.
5.6 Tube. ø15 mm × 150 mm.
5.7 Sampling bottle. 250 ml.
Note 1. Glassware and sampling instruments such as pipettes, test tubes, and sampling bottles should be wrapped according to aseptic operation before testing, and sterilized by autoclaving at 121 °C for 20 min, and dried.
spare.
6 samples
6.1 Sample Collection
When sampling with other items, first collect the microbial samples separately. The sampling bottles should not be washed with water samples, and the water should be collected according to the requirements of aseptic operation.
Approximately.200 ml is sampled in a sterile sample bottle.
Paper without water and water
3 When collecting surface water samples from rivers, rivers, lakes, reservoirs, etc., you can directly insert the bottle with the plug into the water, 10-15 cm from the water surface.
At the same time, the bottle mouth is in the direction of water flow, the glass stopper is pulled out, the water sample is poured into the bottle and the bottle stopper is covered, and the sampling bottle is taken out from the water. If there is no water flow,
Hold the bottle horizontally and push forward. After taking the water sample, quickly put on 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, drain the water for 3 to 5 minutes; then the dragon
The head is closed, sterilized by flame burning for about 3 minutes, the faucet is opened, and water is discharged for 1 min to completely remove the retained impurities in the water pipe. Sampling control
Water flow rate, carefully connected to the bottle.
When collecting surface water, wastewater samples, and water samples of a certain depth, they can 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.
6.2 Sample storage
Test within 2 hours after sampling, otherwise, it should be refrigerated below 10 °C and should not exceed 6 h. After the laboratory is sampled, it cannot be tested immediately.
The sample was placed in a refrigerator at 0 to 4 ° C and measured within 2 h.
6.3 Interference and elimination
If the sample contains residual chlorine or chlorinated water, add sodium thiosulfate solution (4.3) 0.2 ml before sterilization of the sample bottle;
If a water sample with a high content of heavy metal ions is collected, add EDTA-Na2 solution (4.4) before sterilization of the sample bottle.
0.6 ml to eliminate interference. The sampling bottle filled with the interference eliminator is autoclaved at 121 ° C for 20 min, and the outer wall of the sampling bottle and the wrapping paper are dried.
For sample collection. For acidic samples, the pH of the sample should be adjusted to 7.0-8.0 as required by aseptic processing prior to analysis.
Note 2. 10 mg sodium thiosulfate can ensure the removal of 1.5 mg of residual chlorine in the water sample. The amount of sodium thiosulfate can be adjusted according to the actual residual chlorine content of the water sample.
7 Analysis steps
The following steps were carried out under aseptic conditions.
7.1 Preparation for inoculation of water samples
When the amount of water inoculated per sheet is 10 ml or 1 ml, mix the water sample thoroughly.
When the amount of water inoculated per sheet is less than 1 ml, the water sample should be used as a diluted sample. When the inoculation amount is 0.1 ml and 0.01 ml, respectively
A 1.10 diluted sample was made and a 1.100 diluted sample was prepared. Dilution samples of other inoculum sizes are analogized.
The 1.10 diluted sample was prepared by taking 1 ml of water and injecting it into a test tube containing 9 ml of sterile water and mixing it to make a 1.10 dilution.
sample. Dilution samples of other dilutions were made in the same manner.
7.2 Water sample inoculation
7.2.1 Inoculum size
Each sample was inoculated with three different inoculation doses of 10 times, and each inoculum was inoculated with 5 sheets of paper, and a total of 15 sheets were inoculated.
According to the degree of pollution of the water sample, the inoculation amount should be determined as much as possible. The five papers with the largest inoculation amount should be positive, and the five papers with the smallest inoculation amount should be
Negative, avoiding all three different inoculations. A total of 15 sheets were all positive or all negative.
The reference inoculum for clean water samples is 10 ml, 1 ml, 0.1 ml, respectively. The reference inoculum of contaminated water samples can be inoculated with 1 ml according to the degree of contamination.
0.1 ml, 0.01 ml or 0.1 ml, 0.01 ml, 0.001 ml, etc., see Table 1 below.
7.2.2 Vaccination
Clean the water sample, the total amount of water sample is 55.5 ml, 5 sheets of 10 ml water sample, 10 ml of water sample per infusion, 1 ml water sample paper 10
One of the five samples was inoculated with 1 ml of water, and the other 5 were inoculated with 1 ml of diluted water sample of 1.10. Contaminated water samples, inoculated with 3 different dilutions
5 ml of 1 ml dilution water sample.
4 Inoculation water sample should be evenly added to the paper, the paper is fully infiltrated, absorb the water sample, and gently smooth the outside of the polypropylene plastic film bag by hand.
mark.
Note 3. After the paper is added to the water sample, it will turn yellow or fade in a short time, indicating that the water sample has acid or oxidant interference, which needs to be removed according to “6.3 Interference and Elimination”.
Table 1 Water sample inoculation amount reference table
Water sample type
Inoculum size (ml)
10 1 0.1 10-2 10-3 10-4 10-5 10-6
Lake water, water source ▲ ▲ ▲
River water ▲ ▲ ▲
Domestic sewage ▲ ▲ ▲
Medical institutions discharge sewage (after treatment) ▲ ▲ ▲
Waste water discharged from livestock and poultry farming ▲ ▲ ▲
7.3 Cultivation
When the total coliform group was detected, the results were observed after incubation at 37±1 °C for 18-24 hours; when detecting fecal coliforms, at 44.5±0.5 °C
The results were observed after 18 to 24 hours of culture.
Note 4. When detecting fecal coliforms, the paper should be placed in a constant temperature incubator at 44.5±0.5°C immediately after inoculation. If it is left at room temperature for a long time, it will affect the detection knot.
The accuracy of the fruit.
7.4 Controlled trial
7.4.1 Blank control
The whole process blank is determined by using sterile water, and there is no color reaction on the paper after the cultivation. Otherwise, the measurement result of the sample is invalid.
The cause should be identified and re-measured.
7.4.2 Positive and negative controls
The positive strain determined by total coliform is Escherichia coli, and the negative strain is Staphylococcus aureus.
(Staphylococcus aureus); the positive strain determined by fecal coliform is Escherichia coli, and the negative strain is produced.
Enterobacter aerogenes.
The above standard strains are all prepared into a suspension of bacteria at a concentration of 300-3000 cells/ml, and the bacterial suspensions of the corresponding amounts of water are respectively inoculated into the paper sheets, positively and
5 negative strains, according to the "7.3 culture" requirements, Escherichia coli should be positive; Staphylococcus aureus, Enterobacter aerogenes
A negative reaction is present. Otherwise, the measurement result of this sample is invalid, and the cause should be determined and then re-measured.
Note 5. A higher concentration of bacterial suspension can be prepared first, and the concentration of the bacterial suspension can be determined by a blood cell counter under a microscope, and then diluted with sterile water according to the actual situation.
Up to 300 to 3000/ml.
7.5 Interpretation of results
(1) An erythema or blush appears on the paper and the surroundings turn yellow, which is positive.
(2) The whole piece of paper turns yellow, without erythema or redness, and is positive.
(3) The part of the paper turned yellow, and there was no erythema or redness, which was negative.
(4) An erythema or blush appears on the purple background of the paper, and the surroundings are not yellow and negative.
(5) The paper has no change and is negative.
The results of the reference picture are shown in Appendix A.
58 Calculation and representation of results
8.1 Calculation of results
According to the number of positive papers with different inoculation amount, check the MPN table (Appendix B) to get the MPN value (MPN/100 ml) according to formula (1).
Convert and report the total coliform or fecal coliform population in 1 L water sample.
M100C (1)
In the formula.
C--Water total coliform or fecal coliform concentration (MPN/L)
M--Check MPN value obtained by MPN table (MPN/ 100 ml)
Q--Maximum inoculum of actual water sample (ml)
100-- is 10×10 ml, wherein 10 converts the MPN value of the unit MPN/100 ml into MPN/L, and 10 ml is in the MPN table.
Maximum inoculum
8.2 Results representation
The measurement result retains two significant figures. When it is greater than or equal to 100, it is expressed by scientific notation. The unit of the result is MPN/L. Average value
What is the average calculation.
9 Precision and accuracy
The microbiological test data is skewed. According to its statistical analysis requirements, the test results are all converted by logarithm (base 10).
Under analysis.
9.1 precision
6 laboratories have certified standard samples (15600 MPN/L), low concentrations (4.0×102 MPN/L), medium concentrations (1.0×104 MPN/L),
The total coliforms of the high concentration (8.0×104 MPN/L) actual samples were determined, and the relative standard deviations in the laboratory were. 4.5%~
7.5%, 3.5% to 12.4%, 4.5% to 6.9%, 2.8% to 5.8%; the relative standard deviations between laboratories were 3.3%, 12.6%, 4.6%, respectively.
1.6%; repeatability limits are 0.61, 0.67, 0.67, 0.64; reproducibility limits are 1.09, 0.81, 0.66, 0.69.
6 laboratories have certified standard samples (12100 MPN/L), low concentrations (1.0×102 MPN/L), medium concentrations (4.0×103 MPN/L),
The high-concentration (5.0×104 MPN/L) actual samples of fecal coliforms were measured, and the relative standard deviations in the laboratory were. 4.5%~
11.3%, 11.4% to 31.3%, 2.8% to 21.5%, 3.9% to 13.2%; the relative standard deviations between laboratories were 5.2%, 14.8%, 11.9%, respectively.
8.2%. The repeatability limits were 0.93, 0.83, 0.85, and 0.77; the reproducibility limits were 1.28, 1.44, 1.35, and 0.89.
9.2 Accuracy
Six laboratories tested the standard sample of total coliform bacteria (15600 MPN/L), and the relative error range in the laboratory was -5.5%~
-12.8%, the final value of the relative error is -8.8% ± 6.0%.
Six laboratories tested the standard sample of fecal coliforms (12100 MPN/L), and the relative error range in the laboratory was -3.8%~
-16.0%, the final value of the relative error is -10.3% ± 9.2%.
610 Quality Assurance and Quality Control
10.1 A quality-qualified piece of paper must be used.
10.2 Each batch of samples was subjected to a full procedure blank assay according to the “7.4 Control Test” and a positive and negative control trial was performed using a certified standard strain.
11 Waste treatment
After use, the utensils and wastes must be sterilized by autoclaving at 121 °C for 20 min. The vessels can be cleaned and the wastes disposed as general waste.
7 Appendix A
(informative appendix)
Result determination reference picture
Positive
(Red spots or blush on the paper and yellowing around)
Positive negative
(The whole piece of paper turns yellow, no erythema or blush) (no change in the paper)
negative
(The part of the paper turns yellow without erythema or blush)
negative
(The erythema or blush appears on the purple background of the paper, and the surroundings are not yellow)
8 Appendix B
(informative appendix)
Maximum possible number (MPN) table
(The water sample inoculation amount is 5 parts 10 ml, 5 parts 1 ml, 5 parts 0.1 ml)
Positive number of each inoculum
MPN/100ml
95% confidence limit, number of positive inoculations
MPN/100ml
95% confidence limit
10ml 1ml 0.1ml lower limit upper limit 10ml 1ml 0.1ml lower limit
0 0 0 < 2 3 0 0 8 1 19
0 0 1 2 < 0.5 7 3 0 1 11 2 25
0 0 2 4 < 0.5 7 3 0 2 13 3 31
0 1 0 2 < 0.5 7 3 1 0 11 2 25
0 1 1 4 < 0.5 11 3 1 1 14 4 34
0 1 2 6 < 0.5 15 3 1 2 17 5 46
0 1 3 7 3 1 3 20 6 60
0 2 0 4 < 0.5 11 3 2 0 14 4 34
0 2 1 6 < 0.5 15 3 2 1 17 5 46
0 2 2 7 3 2 2 20 6 60
0 3 0 6 < 0.5 15 3 3 0 17 5 46
0 3 1 7 3 3 1 21 7 63
0 4 0 8 3 4 0 21 7 63
0 4 1 9 3 4 1 24 8 72
9 number of positive inoculations
MPN/100ml
95% confidence limit, number of positive inoculations
MPN/100ml
95% confidence limit
10ml 1ml 0.1ml lower limit upper limit 10ml 1ml 0.1ml lower limit
0 5 0 9 3 5 0 25 8 75
1 0 0 2 < 0.5 7 4 0 0 13 3 31
1 0 1 4 < 0.5 11 4 0 1 17 5 46
1 0 2 6 < 0.5 15 4 0 2 21 7 63
1 0 3 8 1 19 4 0 3 25 8 75
1 1 0 4 < 0.5 11 4 1 0 17 5 46
1 1 1 6 < 0.5 15 4 1 1 21 7 63
1 1 2 8 1 19 4 1 2 26 9 78
1 2 0 6 < 0.5 15 4 2 0 22 7 67
1 2 1 8 1 19 4 2 1 26 9 78
1 2 2 10 2 23 4 2 2 32 11 91
1 3 0 8 1 19 4 3 0 27 9 80
1 3 1 10 2 23 4 3 1 33 11 93
1 3 2 12 4 3 2 39 13 110
Positive number of each inoculum
MPN/100ml
95% confidence limit, number of positive inoculations
MPN/100ml
95% confidence limit
10ml 1ml 0.1ml lower limit upper limit 10ml 1ml 0.1ml lower limit
1 4 0 11 2 25 4 4 0 34 12 93
1 4 1 13 4 4 1 40 14 110
1 5 0 13 4 5 0 41 16 120
2 0 0 5 < 0.5 13 5 0 0 23 7 70
2 0 1 7 1 17 5 0 1 31 11 89
2 0 2 9 2 21 5 0 2 43 15 110
2 0 3 12 3 28 5 0 3 58 19 140
2 0 4 14 5 0 4 76 24 180
2 1 0 7 1 17 5 1 0 33 11 93
2 1 1 9 2 21 5 1 1 46 16 120
2 1 2 12 3 28 5 1 2 63 21 150
2 1 3 14 5 1 3 84 26.200
2 1 4 17 5 1 4 110
2 1 5 19 5 1 5 130
2 2 0 9 2 21 5 2 0 49 17 130
2 2 1 12 3 28 5 2 1 70 23 170
2 2 2 14 4 34 5 2 2 94 28 220
2 2 3 17 5 2 3 120 33 280
2 2 4 19 5 2 4 150 38 370
2 2 5 22 5 2 5 180 44 520
2 3 0 12 3 28 5 3 0 79 25 190
2 3 1 14 4 34 5 3 1 110 31 250
Positive number of each inoculum
MPN/100ml
95% confidence limit, number of positive inoculations
MPN/100ml
95% confidence limit
10ml 1ml 0.1ml lower limit upper limit 10ml 1ml 0.1ml lower limit
2 3 2 17 5 3 2 140 37 340
2 3 3 20 5 3 3 180 44 500
2 3 4 22 5 3 4 210 53 670
2 3 5 25 5 3 5 250 77 790
2 4 0 15 4 37 5 4 0 130 35 300
2 4 1 17 5 4 1 170 43 490
2 4 2 20 5 4 2 220 57 700
2 4 3 23 5 4 3 280 90 850
2 4 4 25 5 4 4 350 120 1000
2 4 5 28 5 4 5 430 150 1200
2 5 0 17 5 5 0 240 68 750
2 5 1 20 5 5 1 350 120 1000
2 5 2 23 5 5 2 540 180 1400
2 5 3 26 5 5 3 920 300 3200
2 5 4 29 5 5 4 1600 640 5800
2 5 5 32 5 5 5 ≥2400 800
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