CN113897411A - Method for quickly, simply and conveniently evaluating microbial safety of source water and drinking water - Google Patents
Method for quickly, simply and conveniently evaluating microbial safety of source water and drinking water Download PDFInfo
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- CN113897411A CN113897411A CN202010637957.2A CN202010637957A CN113897411A CN 113897411 A CN113897411 A CN 113897411A CN 202010637957 A CN202010637957 A CN 202010637957A CN 113897411 A CN113897411 A CN 113897411A
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 29
- 235000020188 drinking water Nutrition 0.000 title claims abstract description 26
- 239000003651 drinking water Substances 0.000 title claims abstract description 26
- 230000000813 microbial effect Effects 0.000 title claims abstract description 24
- 230000012010 growth Effects 0.000 claims abstract description 20
- 230000001580 bacterial effect Effects 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 244000005700 microbiome Species 0.000 claims abstract description 15
- 238000011156 evaluation Methods 0.000 claims abstract description 7
- 238000011081 inoculation Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 7
- 238000010586 diagram Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000003763 carbonization Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000012512 characterization method Methods 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000007405 data analysis Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 238000007619 statistical method Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 2
- 239000000460 chlorine Substances 0.000 claims 2
- 229910052801 chlorine Inorganic materials 0.000 claims 2
- 238000006386 neutralization reaction Methods 0.000 claims 2
- 238000002474 experimental method Methods 0.000 claims 1
- 239000005416 organic matter Substances 0.000 claims 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims 1
- 235000019345 sodium thiosulphate Nutrition 0.000 claims 1
- 239000008399 tap water Substances 0.000 abstract description 4
- 235000020679 tap water Nutrition 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000003894 drinking water pollution Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000034655 secondary growth Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Abstract
The invention relates to a method for quickly, simply and conveniently evaluating the microbial safety of source water and drinking water. According to the invention, the microbial safety evaluation of source water and drinking water is realized by detecting the Bacterial Growth Potential (BGP) of a water sample by utilizing the relationship between the growth rate of microorganisms in water and biodegradable organic matters. The method takes the native flora of the water source water which is simple and easy to obtain as the test microorganism, inoculates the native microorganism to the sample to be tested, measures the total number of culturable bacteria after cultivation, measures the concentration of biodegradable organic matters in the sample to be tested, and compares and verifies the two to realize the evaluation of the microbial safety of the sample. Compared with the traditional determination method, the method has the advantages of simple operation, short time consumption and high accuracy. The method can be used for measuring water samples at important positions of drinking water supply systems such as water intakes, pipe network nodes, household tap water and the like, realizes quick and simple microbial safety evaluation, and practically ensures the biological safety of the drinking water of residents.
Description
Technical Field
The invention belongs to a drinking water microbial pollution monitoring method, particularly relates to a method for quickly, simply and conveniently evaluating microbial safety of source water and drinking water, and belongs to the technical field of drinking water quality monitoring.
Background
Clean drinking water is the main reason for greatly improving the life of human beings, and the biological safety of the drinking water is the basic requirement for water quality. In recent years, water pollution accidents and normal pollution of source water frequently occur, and the safety of drinking water in China faces a serious challenge. 1996 + 2015, the public health incident of the sudden drinking water pollution in China is up to 219, wherein the exceeding standard factors of microorganisms account for 26 percent.
Drinking water sources are generally polluted to a certain degree, and the polluted water bodies have higher content of organic matters, nitrogen and phosphorus, so that nutrient substances are provided for the growth of microorganisms. After the source water is treated by a water plant, the water still contains a certain level of biodegradable organic matters. The organic matters and inorganic nutrient elements such as nitrogen and phosphorus support heterotrophic and autotrophic microorganisms in the pipe network, including the growth of some pathogenic microorganisms, form a pipe network biomembrane and suspended secondary growth microorganisms, and induce pipe wall corrosion and scaling. The aging and falling of the biological membrane can cause the water quality deterioration of users, the increase of chromaticity and turbidity, and the threat to the human health and the water delivery safety. Therefore, it is necessary to evaluate the microbial safety of source water and drinking water.
The existing evaluation method has high requirements on experimental conditions and personnel operation, consumes long time and cannot quickly warn the risk of microorganisms. Therefore, a method for evaluating the microbial safety of source water and drinking water, which is simple in operation, short in time consumption and high in accuracy, needs to be developed, and can provide technical support for solving the early warning of sudden drinking water pollution events and guaranteeing the microbial safety of the drinking water.
Disclosure of Invention
The invention aims to provide a method for quickly, simply and conveniently evaluating the microbial safety of source water and drinking water. According to the invention, the relationship between the growth rate of microorganisms in water and biodegradable organic matters is utilized, and the microbial safety evaluation of source water and drinking water is realized by rapidly and efficiently detecting the growth potential of bacteria, so that the timely monitoring and early warning are realized, and the water safety is ensured.
The method comprises the following steps:
(1) preparing inoculation liquid:
500 mL of source water containing a large amount of indigenous bacteria was filtered through a 2 μm filter and cultured at 20 ℃ for 5 days to consume the organic carbon therein. After the bacterial number of the inoculation liquid reaches a platform level, the inoculation liquid can be stored for one month in a refrigerator at the temperature of 4 ℃ for standby;
(2) pretreatment of a water sample to be detected:
and filtering the water sample to be detected by using a 0.22 mu m filter membrane. The filtering method comprises the following steps: the solution is first purified by about 500 mL and discarded. Then filtering the water sample, discarding 200 mL of filtrate before 150-;
(3) inoculation:
adding 2 mL of inoculation liquid into 200 mL of water sample to be detected (volume ratio: 1: 100), covering the cover, shaking uniformly, and adjusting the inoculation amount and the inoculation volume ratio according to the condition to make the initial concentration of bacteria in the system about 101-102 CFU/mL；
(4) And (3) culture counting:
and (4) putting the inoculated water sample into a constant-temperature incubator, and culturing for 5d at 20 ℃. Diluting water sample with 0.9% sterile physiological saline, and coating 100 μ L of the diluted solution on R2And counting colonies on the culture medium A, wherein the effective counting interval is 30-300, and three parallel plates are arranged. The bacterial concentration (CFU/mL) is the bacterial growth potential value (BGP);
(5) and (3) comparison and verification:
after the water sample to be tested is cultured for 3 days, a 50 ml injector is used for sampling, and after the water sample is filtered by a 0.45 mu m filter membrane, the DOC and BDOC water samples before and after the culture are measured3=DOC0 – DOC3；
(6) And (3) data analysis:
drawing the bacterial content changes of the water sample to be detected and the ultrapure water on the same curve graph, using origin statistical analysis to draw a graph, taking the culture days as an abscissa and the bacterial content as an ordinate, drawing a curve, and drawing the platform-stage bacterial content and the pair BDOC3And comparing, drawing a scatter diagram, and obtaining the correlation verification of the scatter diagram and the scatter diagram. The water temperature is 20 ℃, and BDOC is less than 0.15 mg/L; the water temperature is 15 ℃, BDOC is less than 0.2 mg/L, the biological stability of the water quality is better, and the BGP threshold value can be calculated.
Remarks explanation:
all glassware was treated as carbonless. No carbonization: firstly, carrying out non-carbonization treatment in a muffle furnace at 550 ℃ for 2 h; soaking the potassium dichromate washing solution for 4 hours, washing the solution with tap water, washing the solution with distilled water for 3 times, and washing the solution with ultrapure water for 1 time;
soaking non-glassware (such as pipette tip) in dilute acid, washing with tap water, distilled water and ultrapure water for 3 times, sterilizing under high pressure, and oven drying at 60 deg.C.
The invention has the advantages that:
(1) the time consumption is short: the traditional method needs more than 14 days for AOC and more than 28 days for BDOC; the inoculation culture method adopted by the invention can obtain results only in 7 days as shortest as possible, thereby greatly shortening the detection time;
(2) the accuracy is high: the traditional method such as AOC only uses two strains of pseudomonas fluorescens P17 and spirillum NOX, and the number and types of available carbon sources are limited. According to the invention, indigenous mixed bacteria are used as inoculated bacteria, so that the nutrient medium in a water sample is utilized more fully, the determination result is more accurate, and the potential of the water body for supporting the growth of bacteria can be reflected more sensitively;
(3) the cost is low: there is no need for large instruments such as a TOC meter, nor for expensive strains such as P17 and NOx. The method can be operated in a common laboratory, and has low requirements on experimental conditions and personnel.
The basic principle of the invention is as follows:
organic matters and other nutrients in the water sample provide conditions for the growth of microorganisms. The higher the organic content, the greater the potential to support microbial growth. The growth potential of bacteria is an important characterization index for measuring organic matters and biological stability of the water body. The method takes indigenous microorganisms in a water sample as inoculation strains, counts the water sample after proper culture, and expresses the potential of organic matters in the water sample for supporting the bacterial regrowth under different inorganic limiting factor conditions by the bacterial concentration (CFU/mL). BGP is high, which indicates that the potential of the water body for supporting the growth of microorganisms is high, and the risk of sudden microbial pollution is increased. Therefore, the method can be used for rapidly evaluating the microbial safety of the water body by measuring the BGP in the source water and the drinking water and is simple to operate.
Drawings
FIG. 1 is a schematic diagram of a method for detecting the growth potential of bacteria.
Fig. 2 is a graph of BGP over time.
Fig. 3 is a graph of a correlation fit of BDOC to BGP.
The specific implementation mode is as follows:
the technical solution of the present invention is further described in detail with reference to specific examples below:
500 mL of source water is taken as an inoculation liquid source, filtered by 2 mu m and cultured for 5 days at 20 ℃ to obtain the inoculation liquid. The numbers of the water samples to be tested are 0 (ultrapure water), 1 (example one), 2 (example two), 3 (example three), 4 (example four) and 5 (example five). Adding 2 mL of the inoculated liquid into 200 mL of water sample to be detected, culturing the inoculated water sample for 5d at 20 ℃, and performing R2And A, culture counting of the culture medium. BDOC was detected according to a conventional standing suspension culture assay. Data were recorded by daily sampling. The bacterial content of the ultrapure water and 5 sample water samples was plotted against time in the same graph using origin software, see table 1 and figure 1. And drawing a scatter diagram by using BDOC as an abscissa and BGP on the 5 th day as an ordinate, and fitting a corresponding equation by using origin software, wherein the equation is shown in figure 2.
TABLE 1 bacterial content as a function of time (CFU/mL)
To further verify the feasibility of the test method of the present invention, the water samples to be tested were also assayed using the conventional BDOC assay, and the results are shown in FIG. 2. The measurement result of the invention is similar to the result of the traditional method, and compared with the traditional method, the time consumption is short, and the measurement result is more accurate and sensitive. The water temperature is 20 ℃, and the BGP is less than 6.1 multiplied by 105CFU/mL; the water temperature is 15 ℃, and the BGP is less than 3.2 multiplied by 106CFU/mL, the biological safety of water quality is guaranteed. The invention can realize rapid and high-sensitivity microbial pollution risk evaluation on water samples of water sources such as water intake, important pipe network nodes, household tap water and the like and water samples of important positions of a drinking water system, and practically ensures the biological safety of drinking water of urban and rural residents.
TABLE 2 measurement results of conventional BDOC method and corresponding BGP
Claims (3)
1. A method for quickly, simply and conveniently evaluating the microbial safety of source water and drinking water is characterized in that the microbial safety evaluation of the source water and the drinking water is realized by detecting the growth potential of bacteria by utilizing the relationship between the growth rate of microbes in water and biodegradable organic matters.
2. The method for rapidly and easily evaluating the microbial safety of source water and drinking water according to claim 1, wherein the method comprises the following steps:
(1) preparing inoculation liquid:
collecting 500 mL of water containing large amount of indigenous bacteria, filtering with 2 μm filter membrane, culturing at 20 deg.C for 5 days to consume organic carbon therein,
after the bacterial number of the inoculation liquid reaches a platform level, the inoculation liquid can be stored for one month in a refrigerator at the temperature of 4 ℃ for standby;
(2) pretreatment of a water sample to be detected:
filtering a water sample to be detected by using a 0.22 mu m filter membrane, wherein the filtering method comprises the following steps: firstly, pure water is used for about 500 mL, the pure water is discarded, then a water sample is filtered, firstly, 150 mL of filtrate is discarded, 200 mL of filtrate is not used, and 200 mL of filtrate is filled into a 500 mL triangular flask, if residual chlorine exists in the water sample, a proper amount of sodium thiosulfate is added for neutralization before filtering, wherein the neutralization is generally 1.2 times of the equivalent of the residual chlorine;
(3) inoculation:
adding 2 mL of inoculation liquid into 200 mL of water sample to be detected (volume ratio: 1: 100), covering the cover, shaking uniformly, and adjusting the inoculation amount and the inoculation volume ratio according to the condition to make the initial concentration of bacteria in the system about 101-102 CFU/mL；
(4) And (3) culture counting:
placing the inoculated water sample into a constant temperature incubator, culturing at 20 deg.C for 5d, diluting the water sample with 0.9% sterile physiological saline in gradient, and coating 100 μ L of the water sample on R2Counting bacterial colonies on the culture medium A, wherein the effective counting interval is 30-300, three plates are arranged in parallel, and the bacterial concentration (CFU/mL) is the bacterial growth potentialForce value (BGP);
(5) and (3) comparison and verification:
after the water sample to be tested is cultured for 3 days, a 50 ml injector is used for sampling, and after the water sample is filtered by a 0.45 mu m filter membrane, the DOC and BDOC water samples before and after the culture are measured3=DOC0 – DOC3；
(6) And (3) data analysis:
drawing the bacterial content changes of the water sample to be detected and the ultrapure water on the same curve graph, using origin statistical analysis to draw a graph, taking the culture days as an abscissa and the bacterial content as an ordinate, drawing a curve, and drawing the platform-stage bacterial content and the pair BDOC3Comparing, drawing a scatter diagram, and obtaining correlation verification of the two, wherein the water temperature is 20 ℃, and the BDOC is less than 0.15 mg/L; the water temperature is 15 ℃, BDOC is less than 0.2 mg/L, the biological stability of the water quality is good, and the BGP threshold value can be calculated;
in the above operation steps, all the utensils are sterilized without carbonization, so that the experiment is not interfered by exogenous organic matters or microbes.
3. The method for rapidly and simply evaluating the microbial safety of source water and drinking water according to claim 1, which is based on the following principle: organic matters and other nutrient substances in the water sample provide conditions for the growth of microorganisms; the higher the organic content, the greater the potential to support microbial growth; the growth potential of bacteria is an important characterization index for measuring the organic matter and biological stability of the water body; the method takes indigenous microorganisms in a water sample as inoculation strains, counts the water sample after proper culture, and expresses the potential of organic matters in the water sample for supporting the regrowth of bacteria under different inorganic limiting factor conditions by the concentration of bacteria (CFU/mL); BGP height indicates that the potential of the water body for supporting the growth of microorganisms is high, and the risk of sudden microbial pollution is increased; therefore, the method can be used for rapidly evaluating the microbial safety of the water body by measuring the BGP in the source water and the drinking water and is simple to operate.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110272937A (en) * | 2019-06-28 | 2019-09-24 | 华润怡宝饮料（中国）有限公司 | The sensitive detection method of microorganism in a kind of packaging drinking water |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110272937A (en) * | 2019-06-28 | 2019-09-24 | 华润怡宝饮料（中国）有限公司 | The sensitive detection method of microorganism in a kind of packaging drinking water |
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Application publication date: 20220107 |