HJ 347.2-2018 PDF in English
HJ 347.2-2018 (HJ347.2-2018) PDF English
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Water quality - Determination of fecal coliform - Manifold zymotechnics
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Standards related to (historical): HJ 347.2-2018
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HJ 347.2-2018: PDF in English HJ 347.2-2018
ENVIRONMENTAL PROTECTION STANDARD
OF THE POPLE’S REPUBLIC OF CHINA
Partially replacing HJ/T 347-2007
Water quality - Determination of fecal coliform - Manifold
zymotechnics
ISSUED ON: DECEMBER 26, 2018
IMPLEMENTED ON: JUNE 01, 2019
Issued by: Ministry of Ecological Environment
Table of Contents
Foreword ... 4
1 Scope ... 6
2 Normative references ... 6
3 Terms and definitions... 6
4 Principles of the method ... 7
5 Interference and elimination ... 7
6 Reagents and materials ... 7
7 Instruments and equipment ... 9
8 Samples ... 9
9 Analytical procedures ... 10
10 Result calculation and presentation ... 12
11 Precision and accuracy ... 13
12 Quality assurance and quality control ... 14
13 Waste disposal ... 15
Appendix A (Informative) Most probable number (MPN) table ... 16
Appendix B (Informative) Recommended format for inspection records and reports of
fecal coliform ... 20
Water quality - Determination of fecal coliform - Manifold
zymotechnics
1 Scope
This standard specifies the manifold zymotechnics, for the determination of fecal
coliforms in water.
This standard applies to the determination of fecal coliforms, in surface water,
groundwater, domestic sewage, industrial wastewater.
The detection limit of this method is 3 MPN/L for the 12-pipe method; 20 MPN/L for
the 15-pipe method.
2 Normative references
This standard refers to the following documents or clauses thereof. For undated
references, the valid edition applies to this standard.
GB/T 14581 Water quality - Guidance on sampling techniques from lakes, natural
and man-made
HJ 494 Water quality - Guidance on sampling techniques
HJ/T 91 Technical specifications requirements for monitoring of surface water and
waste water
3 Terms and definitions
The following terms and definitions apply to this standard.
3.1
Fecal coliforms
Also known as thermotolerant coliforms. The aerobic and facultative anaerobic
Gram-negative A. budding bacteria, that can ferment lactose to produce acid and gas,
after being cultivated at 44.5 °C for 24 h.
3.2
Most probable number (MPN)
Also known as dilution culture counting, which is an indirect counting method,
based on Poisson distribution. Using statistical principles, according to the positive
number of target microorganisms, which are produced by a certain volume of
samples, at different dilutions after culturing, look up the Table to estimate the
number of target microorganisms, in a certain volume of samples (the most probable
number of target microorganisms per unit volume).
4 Principles of the method
The samples are added to the test tube, which contains lactose peptone medium. The
initial fermentation and enrichment culture is carried out at 37 °C. The coliform bacteria
grow and reproduces in the medium; decomposes lactose to produce acid and gas. The
generated acid changes the bromocresol purple indicator from purple to yellow; the gas
produced goes into the reverse tube, indicating gas production. After re-fermentation at
44.5 °C, the bile salt No.3 in the medium can inhibit the growth of Gram-positive
bacteria; the bacteria, that produced gas at the end, are determined to be fecal coliforms.
By checking the MPN Table, the fecal coliform concentration value is obtained.
5 Interference and elimination
5.1 Active chlorine has oxidative properties, which can destroy the enzyme activity in
microbial cells AND cause cell death. Sodium thiosulfate solution (6.7) can be added,
during sample collection (8.1), to eliminate the interference.
5.2The metal ions have cytotoxicity, which can destroy the enzymatic activity in the
microbial cells AND cause cell death. The disodium EDTA solution (6.8) can be added,
during sample collection (8.1), to eliminate the interference.
6 Reagents and materials
Unless otherwise stated, analytical reagents or biological reagents, that meet national
standards, are used in the analysis. The experimental water is distilled or deionized
water.
6.1 Lactose peptone medium.
Peptone: 10 g
Beef extract: 3 g
Lactose: 5 g
Weigh 15.7 g of sodium thiosulfate (6.5). Dissolve it in an appropriate amount of water.
Make its volume reach to 100 ml. Prepare it before use.
6.8 Disodium EDTA solution: ρ (C10H14N2O8Na2·2H2O) = 0.15 g/ml
Weigh 15 g of disodium EDTA (6.6). Dissolve it in an appropriate amount of water.
Make its volume reach to 100 ml. This solution can be stored for 30 days.
7 Instruments and equipment
7.1 Sampling bottle: 500 ml wide-mouth glass bottle with screw cap or ground stopper.
7.2 Autoclave: 115 °C, 121 °C adjustable.
7.3 Constant temperature incubator or water bath: The allowable temperature deviation
is 37 °C ± 0.5 °C, 44 °C ± 0.5 °C.
7.4 pH meter: Accurate to 0.1 pH unit.
7.5 Inoculation loop: 3 mm in diameter.
7.6 Test tubes: 300 ml, 50 ml, 20 ml.
7.7 Common laboratory instruments and equipment.
Note: Glassware and sampling equipment shall be wrapped, according to the requirements of
aseptic operation before the test; sterilized by high pressure steam at 121 °C for 20 minutes, for
later use.
8 Samples
8.1 Sample collection
The point layout and sampling frequency shall be implemented, in accordance with the
relevant provisions of GB/T 14581, HJ/T 494, HJ/T 91.
When collecting microbial samples, the sampling bottle (7.1) shall not be washed by
the sample. The sample shall be collected in a sterilized sampling bottle. The sampling
volume of clean water bodies is not less than 400 ml. The sampling volume of other
water bodies is not less than 100 ml.
When collecting surface water samples, such as rivers, lakes, reservoirs, it may hold the
bottom of the bottle, to directly insert the stoppered sampling bottle into the water, at
about 10 ~ 15 cm away from the water surface, with the bottle mouth facing the
direction of water flow. Pull out the stopper, to let the sample flow into the bottle. Then
cap the bottle. Remove the sampling bottle from the water. If there is no water flow, it
may hold the bottle, to push it horizontally forward. The sampling volume is generally
about 80% of the capacity of the sampling bottle. After the samples are collected, they
are quickly wrapped with sterile wrapping paper.
When collecting samples from the faucet device, do not select a leaking faucet. Before
collecting water, open the faucet to the maximum flow, to let the water run for 3 ~ 5
minutes. Then close the faucet. Use flame to burn it, for about 3 minutes, for
sterilization; OR use 70% ~ 75% alcohol to sterilize the faucet. Turn on the faucet to
the maximum, to let the water run for 1 min, to fully remove the retained impurities in
the water pipe. When sampling, control the water flow rate to carefully let it flow into
the bottle.
When collecting surface water, wastewater samples, samples at a certain depth,
sterilized special sampling devices can also be used for sampling.
When stratified sampling is carried out, at the same sampling point, it shall be carried
out from top to bottom, to avoid disturbance of different levels.
If a sample containing active chlorine is collected, add sodium thiosulfate solution (6.7),
before sterilizing the sampling bottle, to remove the inhibitory effect of active chlorine
on bacteria (add 0.1 ml of sodium thiosulfate solution per 125 ml volume). If the sample
with high heavy metal ion content is collected, add disodium EDTA solution (6.8),
before sterilizing the sampling bottle, to eliminate interference (add 0.3 ml of disodium
EDTA solution per 125 ml volume).
Note: 15.7 mg of sodium thiosulfate (6.5) can remove 1.5 mg of active chlorine in the sample;
the consumption of sodium thiosulfate can be adjusted, according to the actual active chlorine
content of the sample.
8.2 Sample preservation
After sampling, it shall be tested within 2 hours. Otherwise, it shall be refrigerated
below 10 °C but not more than 6 hours. After the laboratory receives the sample, if the
test cannot be carried out immediately, the sample shall be refrigerated below 4 °C and
tested within 2 hours.
9 Analytical procedures
9.1 Sample dilution and inoculation
9.1.1 15-pipe method
After thoroughly mixing the samples, add 10 ml of samples, to each of 5 test tubes (with
an inverted tube), which contain 5 ml of sterilized triple lactose peptone medium (6.2),
according to the requirements of aseptic operation. Add 1 ml of the sample, to each of
5 test tubes (with an inverted tube), which contains 10 ml of sterilized haploid peptone
9.2 Initial fermentation test
Incubate the test tube after inoculation (9.1) at 37 °C ± 0.5 °C, for 24 h ± 2 h.
When the color of the fermentation test tube turns yellow, it is acid production; when
there are air bubbles in the small inverted glass tube, it is gas production. Test tubes,
which produce acid and gas, indicate a positive test. If the gas production in the inverted
tube is not obvious, it may gently tap the test tube. If there are small bubbles rising, it
is positive.
9.3 Re-fermentation test
Gently shake the test tubes, that show positive or suspected positive (produces acid only,
without gas production), in the initial fermentation test (9.2). Use an inoculation loop
(7.5), which has been flame-burn sterilized and cooled, to respectively transfer the
cultures into the test tube, which contains the EC medium (6.3). Incubate it at 44.5 °C
± 0.5 °C, for 24 h ± 2 h. All test tubes must be placed in a constant temperature incubator
or water bath (7.3), within 30 min after transfer. Immediately observe it after the
incubation. If it produces gas in the inverted tube, it is confirmed to be positive for fecal
coliforms.
9.4 Control tests
9.4.1 Blank control
Perform a laboratory blank determination in accordance with steps 9.1 ~ 9.3, using
sterile water (6.4), for each test.
9.4.2 Positive and negative controls
Prepare the positive strains of fecal coliform (such as Escherichia coli) and negative
strains (such as Enterobacter aerogenes), into bacterial suspensions, which have a
concentration of 300 ~ 3000 MPN/L. Respectively take the corresponding volumes of
bacterial suspension, to inoculate it into the test tube, according to the requirements of
inoculation (9.1). Then culture it, according to the requirements of the initial
fermentation test (9.2) and the re-fermentation test (9.3). The positive strain shall show
a positive reaction; the negative strain shall show a negative reaction. Otherwise, the
measurement result of the sample is invalid. It shall find out the reason; make
determination again.
10 Result calculation and presentation
10.1 Result calculation
When inoculating 12 samples, check Table A.1 in Appendix A, to get the MPN value
of fecal coliforms per liter.
When inoculating 15 samples, check Table A.2 in Appendix A, to get the MPN value;
then convert the number of fecal coliform bacteria (MPN/L) in the sample, according
to formula (1):
10.2 Result representation
The determination result is reserved to the integer place, with two significant figures at
most. When the determination result is ≥ 100 MPN/L, it is expressed in scientific
notation. When the determination result is lower than the detection limit, it is expressed
as "undetected" or "< 3 MPN/L", for the 12-pipe method; it is expressed as "undetected"
or "< 20 MPN/L", for the 15-pipe method. See Appendix B, for the recommended
format of fecal coliform inspection records and reports.
11 Precision and accuracy
11.1 Precision
6 laboratories carry out 6 repeated determinations, for the actual samples and certified
standard samples of the fecal coliforms (concentration of 3670 MPN/L, acceptable
range of 330 ~ 7710 MPN/L), which have 3 different concentrations: low concentration
(groundwater, average concentration of 54 MPN/L), medium concentration (surface
water, average concentration of 2.7 x 104 MPN/L), high concentration (domestic
sewage, average concentration of 2.8 x 107 MPN/L): the relative standard deviation in
the laboratory ranges from 2.3% to 3.8%, 2.0% to 11%, 1.1% to 5.4%, 5.1% to 17%,
respectively; the inter-laboratory relative standard deviations are 3.4%, 11%, 1.6%,
5.4%, respectively; the inter-laboratory 95% confidence intervals are as shown in Table
2 .
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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