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HJ 1000-2018: Water quality - Determination of total bacteria - Plate count method
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Water quality - Determination of total bacteria - Plate count method
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HJ 1000-2018
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Basic data | Standard ID | HJ 1000-2018 (HJ1000-2018) | | Description (Translated English) | Water quality - Determination of total bacteria - Plate count method | | Sector / Industry | Environmental Protection Industry Standard | | Classification of Chinese Standard | Z16 | | Word Count Estimation | 12,180 | | 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 1000-2018: Water quality - Determination of total bacteria - Plate count method---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Water quality-Determination of total bacteria-Plate count method
National Environmental Protection Standard of the People's Republic
Determination of the total number of bacteria in water
Published on.2018-12-26
2019-06-01 Implementation
Ministry of Ecology and Environment
Content
Foreword...ii
1 Scope...1
2 Normative references...1
3 Terms and Definitions...1
4 Principles of the method...1
5 Interference and elimination...1
6 Reagents and materials...2
7 Instruments and Equipment...2
8 samples...2
9 Analysis steps...3
10 Results calculation and representation...4
11 Precision and Accuracy...5
12 Quality Assurance and Quality Control...5
13 Waste treatment...6
Appendix A (informative appendix) Total bacterial test record and report recommended format...7
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
The environment, the human health, the method for determining the total number of bacteria in the water, and the development of this standard.
This standard specifies the plate counting method for determining the total number of bacteria in surface water, groundwater, domestic sewage and industrial wastewater.
Appendix A of this standard is an informative annex.
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. Liaoning Provincial Environmental Monitoring Experimental Center.
This standard is verified by. Dalian Environmental Monitoring Center, Dandong Environmental Monitoring Center Station, Jinzhou Environmental Monitoring Center
Station, Liaoyang Environmental Monitoring Station, Shenyang Municipal Center for Disease Control and Prevention and Liaoning North Environmental Testing Technology Co., Ltd.
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.
Determination of the total number of bacteria in water
1 Scope of application
This standard specifies the plate counting method for determining the total number of bacteria in water.
This standard applies to the determination of the total number of bacteria in surface water, groundwater, domestic sewage and industrial wastewater.
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 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 bacteria total bacteria
The total number of aerobic and facultative anaerobic colonies grown on nutrient agar was 48 h at 36 °C.
3.2
Colony forming unit colony-forming units (CFU)
The total number of bacterial communities per unit volume of sample.
4 Principle of the method
The sample was inoculated on a nutrient agar medium and cultured under specific physical conditions (cultured at 36 ° C for 48 h).
The total number of aerobic and facultative anaerobic bacteria is the total number of bacterial colonies 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, which can be collected in samples (8.1)
Add sodium thiosulfate solution (6.5) to eliminate interference.
5.2 Heavy metal ions are cytotoxic and can destroy the enzyme activity in microbial cells, leading to cell death.
Add (VIII) Ethylenediaminetetraacetic acid disodium solution (6.6) to eliminate interference. 6 2
Reagents and materials
Analytically pure reagents or biological reagents that meet national standards are used for analysis, unless otherwise stated.
Water or deionized water.
6.1 Nutritional agar medium.
Dissolve the above ingredients or a commercially available product containing the above ingredients in 1000 ml of water, adjust the pH to 7.4 to 7.6, and dispense in
In a glass container, autoclaved at 121 ° C for 20 min, stored in a cool dark place. Protect from light, dry, if necessary
Store in a refrigerator at 5 ° C ± 3 ° C for no more than 1 month. The prepared nutrient agar medium cannot be subjected to multiple melting operations.
It is advisable to use a small amount of work. It should be discarded when the color of the medium changes or the dehydration is obvious.
6.2 Sterile water. Take appropriate amount of experimental water and sterilize it by autoclaving at 121 °C for 20 min.
6.3 Sodium thiosulfate (Na2S2O3·5H2O).
6.4 Disodium edetate (C10H14N2O8Na2·2H2O).
6.5 Sodium thiosulfate solution. (Na2S2O3) =0.10 g/ml
Weigh 15.7 g of sodium thiosulfate (6.3), dissolve in an appropriate amount of water, and dilute to 100 ml.
6.6 Ethylenediaminetetraacetic acid disodium solution. (C10H14N2O8Na2·2H2O) =0.15 g/ml
Weigh 15 g of disodium edetate (6.4), dissolve in an appropriate amount of water, and dilute to 100 ml. This solution can be stored for 30 days.
6.7 Glass beads. 3 to 8 mm in diameter.
7 Instruments and equipment
7.1 Sampling bottle. 250 ml wide-mouth glass bottle with screw cap or grinder.
7.2 High pressure steam sterilizer. 115 ° C, 121 ° C adjustable.
7.3 Constant temperature incubator. Allowable temperature deviation of 36 °C ± 1 °C.
7.4 Constant temperature water bath. 47 °C adjustable.
7.5 pH meter. Accurate to 0.1 pH unit.
7.6 Magnifier or colony counter.
7.7 Common instruments and equipment used in general laboratories.
Note. Glassware and sampling equipment should be wrapped according to aseptic operation before testing, and autoclaved at 121 °C for 20 min.
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. 3
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.
Open the faucet and let the water drain for 1 min to fully 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 (6.5) before sterilization of the sample bottle to remove
Inhibition of bacteria by deactivated chlorine (0.1 ml of sodium thiosulfate solution per 125 ml volume); if heavy is collected
For samples with higher metal ion content, add disodium edetate solution (6.6) before sterilization of the sample bottle.
To eliminate interference (add 0.3 ml of disodium edetate solution per 125 ml volume).
Note. 15.7 mg sodium thiosulfate (6.3) 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
The sample was shaken vigorously 20 to 25 times to disperse the possible bacterial coagulation. Determine dilution based on sample contamination
multiple. Pipette 10 ml of the thoroughly mixed sample aseptically and inject a triangular burn containing 90 ml of sterile water (6.2)
In the bottle (with appropriate amount of glass beads), mix to a 1.10 dilution sample. Draw a 1.10 dilution of the sample 10 ml into the well
Mix 90 ml of sterile water into a conical flask and mix to a 1.100 dilution sample. Dilute to 1.1000, 1.10000 in the same way
Release the sample. At least 3 suitable concentrations should be diluted for each sample.
Note. When pipetting different concentrations of diluent, the pipette must be replaced each time.
9.2 Vaccination
Pipette a well-mixed sample or dilute 1 ml of the sample in a sterile operation with a 1 ml sterile pipette.
In a dish, pour 15 to 20 ml of nutrient agar medium (6.1) cooled to 44 to 47 ° C, and immediately shake the plate to make a sample.
Mix the product or diluted sample with the medium. Pour 2 dishes for each sample or diluted sample.
9.3 Cultivation
After the nutrient agar medium in the plate is cooled and solidified, the plate is turned over to make the bottom surface upward (to avoid the uniform growth of bacteria due to surface moisture condensation).
The results were observed in a constant temperature incubator (7.3) for 48 h ± 2 h at 36 °C ± 1 °C.
9.4 Blank test
The laboratory blank is determined by using sterile water. There shall be no colony growth on the plate after cultivation. Otherwise, the sample is determined.
Invalid, should be re-measured after the cause is identified.
10 Calculation and representation of results
10.1 Interpretation of results
When the plate has larger flaky colonies and exceeds half of the plate, the plate does not participate in counting.
When the flaky colonies are less than half of the plate, and the remaining half of the colonies are evenly distributed, the colonies with uniform distribution are counted.
Multiply by 2 to represent the total number of colonies.
Appearance (morphology or color) similar, colonies that are similar but not touching, as long as the distance between them is not less than the minimum
The diameter of the colony is counted. Colonies that are in close contact and have different appearances are counted.
10.2 Calculation of results
Multiply the dilution number by the total number or average of each plate colony (the average number of two replicate plates of the same dilution)
To calculate the total number of bacteria in a 1 ml sample. The calculation of various situations is as follows.
Preference is given to counting the average number of colonies between 30 and 300, when there is only one dilution factor of the average colony
When the number satisfies this range, the average number of colonies multiplied by the dilution factor is the measured value of the total number of bacteria (see Table 1 Example 1).
If there are two dilution factors, the average number of colonies is between 30 and 300, calculate the ratio of the two (multiplely multiplied by their dilution)
After a multiple, the ratio of the larger value to the smaller value). If the ratio is less than 2, the average of the two is the measured value of the total number of bacteria;
When the ratio is greater than or equal to 2, the total number of colonies with a small dilution factor is the total number of bacteria (see Table 1, Example 2, Example 3, and Example 4).
If the average number of colonies of all dilution factors is greater than 300, multiply the dilution number by the average number of colonies with the largest dilution factor.
The number is the total number of bacteria measured (see Table 5, Example 5).
If the average number of colonies in all dilutions is less than 30, multiply the dilution number by the average number of colonies with the smallest dilution factor.
The number is the total number of bacteria measured (see Table 1 Example 6).
If the average number of colonies in all dilutions is not between 30 and 300, the average colony is the closest to 300 or 30.
The number is multiplied by the dilution factor to determine the total number of bacteria (see Table 1 Example 7).
Table 1 Dilution multiple selection and total number of colonies
10.3 Results Representation
The measurement result is retained to the whole number, and at most two significant digits are retained. When the measurement result is ≥100 CFU/ml,
Refer to the method of counting; if the undiluted stock solution is aseptically grown on the plate, use the "not detected" or "< 1 CFU/ml" table.
Show. See Appendix A for the recommended format of the total bacterial test record and report.
11 Precision and accuracy
11.1 Precision
6 laboratories for low concentration (groundwater, average concentration of 39 CFU/ml), medium concentration (surface water, concentration, respectively)
The value is 2.5×103 CFU/ml) and the high concentration (domestic sewage, the average concentration is 1.3×105 CFU/ml)
Samples of total bacteria and certified standard samples (concentration 95 MPN/ml, acceptable range 22 to 168 MPN/ml)
Six repeated measurements were performed. the relative standard deviations in the laboratory ranged from 2.4% to 6.2%, 0.6% to 1.8%, and 0.3%, respectively.
1.3% and 1.7% to 5.6%; the relative standard deviations between laboratories were 18%, 4.7%, 2.4%, and 3.7%, respectively;
The 95% confidence interval is shown in Table 2.
Table 2 95% confidence interval between laboratories
12 Quality Assurance and Quality Control
12.1 Culture test
Positive strain testing should be performed when changing different batches of media to ensure they meet the requirements. Commonly used positive standard strains are 6
Escherichia coli, Staphylococcus aureus, Bacillus subtilis
Bacillus subtilis, Enterococcus faecalis, and the like. The above standard strain is formulated to a concentration of 30~
300 CFU/ml of bacterial suspension, mix well and take 1ml of bacterial suspension according to inoculation (9.2) and culture (9.3), flat
30 to 300 colonies were uniformly produced in the dish, indicating that the batch medium was qualified.
12.2 Blank test
Laboratory blanks (9.4) are performed for each test to check the sterility of dilution water, glassware and other utensils.
13 Waste treatment
The used waste and utensils must be autoclaved at 121 ° C for 30 min or using a liquid disinfectant (home made or commercially available)
Sterilize. After sterilization, the utensils can be cleaned and the waste disposed as general waste. 7
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