CN117384995A - Method for rapidly detecting viable bacteria in water - Google Patents
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- CN117384995A CN117384995A CN202311462632.5A CN202311462632A CN117384995A CN 117384995 A CN117384995 A CN 117384995A CN 202311462632 A CN202311462632 A CN 202311462632A CN 117384995 A CN117384995 A CN 117384995A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 103
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 52
- 239000012528 membrane Substances 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 238000005070 sampling Methods 0.000 claims abstract description 21
- 239000008223 sterile water Substances 0.000 claims abstract description 7
- 238000000967 suction filtration Methods 0.000 claims abstract description 7
- 238000002386 leaching Methods 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 13
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 239000011550 stock solution Substances 0.000 claims description 8
- 108060001084 Luciferase Proteins 0.000 claims description 6
- 239000005089 Luciferase Substances 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 238000005415 bioluminescence Methods 0.000 claims description 6
- 230000029918 bioluminescence Effects 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 238000000540 analysis of variance Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 2
- 238000002795 fluorescence method Methods 0.000 abstract description 7
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 50
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 50
- 244000005700 microbiome Species 0.000 description 7
- 231100000582 ATP assay Toxicity 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 4
- 238000010219 correlation analysis Methods 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 235000015073 liquid stocks Nutrition 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical class S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000126 substance Substances 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
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Abstract
The invention is applicable to the technical field of water detection, and provides a method for rapidly detecting living bacteria in water, which comprises the following steps: collecting a water sample to be detected, taking 3 groups of samples from a water intake, and sampling 250ml by a sampling bottle; after each group of water samples in the ultra-clean bench are subjected to suction filtration by adopting a 0.45-micrometer sterile filter membrane, washing a sampling bottle by using 100ml sterile water, and then leaching and extracting living bacteria on the side wall of the filter bottle and the filter membrane for three times, so that the living bacteria are all trapped on the filter membrane and the extracellular ATP interference is completely eliminated; after the live bacteria of the wiping filter membrane covered by the wiper are wiped, the wiping wiper is quickly put into an ATP fluorometer, RLU1 is detected within a set time, the wiping is performed again, RLU2 is read, and the RLU is the sum of the RLU1 and the RLU 2; taking the average value of 3 RLUs of 3 groups of samples, looking up a standard curve, and finding out the corresponding number of viable bacteria as the number of the viable bacteria. Therefore, the invention accelerates the detection speed of the ATP fluorescence method, eliminates ATP outside the living bacteria, and has more accurate result.
Description
Technical Field
The invention relates to the technical field of water detection, in particular to a method for rapidly detecting living bacteria in water.
Background
Currently, the detection of living bacteria in water is specified in standard GB5750 by a plate counting method (Heterotrophic Plate Count, HPC) and an enzyme substrate method, and because of the long sampling test time, a plurality of living bacteria exist in a living but non-culturable state, so that the plate counting method can seriously underestimate the quantity of dormant living bacteria, sometimes even in an order of magnitude, when the underestimated bacteria meet a proper environment, explosive growth is caused, the miscontrol of production during the bacterial test is caused, a plurality of defective yields are seriously caused, and great loss is caused for enterprises. An obvious example is that the bacteria count of mineral water source water from underground cannot be detected by using a GB flat plate method, and the underground mineral water is mistakenly regarded as sterile water, and the mineral water is actually rapidly increased after 7 days. The market demand is pressing for a rapid and accurate bacterial assay. Among the current methods for rapid detection of microorganisms in water, ATP detection has the most potential, and the basic principle is based on the firefly luminescence principle, and Adenosine Triphosphate (ATP) is rapidly detected by using a "luciferase-luciferin system". Since all living cells contain a constant amount of ATP, the ATP content clearly indicates the presence of microorganisms in the water.
Even though the ATP fluorescence method is slightly applied at present, the main problems in the detection of bacteria in water are that the water sampling amount is too small, and meanwhile, the problem of interference of ATP outside the fine living bacteria in water caused by using disinfectants such as chlorine and ozone is ignored, so that the test result is increased virtually, and the distortion is caused.
In summary, the prior art has obvious inconvenience and defects in practical use, so that improvement is needed.
Disclosure of Invention
In view of the above-mentioned drawbacks, the present invention aims to provide a method for rapidly detecting living bacteria in water, which accelerates the detection speed of the ATP fluorescence method, and eliminates ATP outside the living bacteria, resulting in more accurate results.
In order to achieve the above object, the present invention provides a method for rapidly detecting living bacteria in water, the method comprising the steps of:
1) Treatment of water to be detected
Collecting a water sample to be detected, taking 3 groups of samples from a water intake, and sampling 250ml of samples from each sampling bottle;
and after each group of water samples are subjected to suction filtration by adopting a 0.45-micrometer sterile filter membrane on an ultra-clean bench, washing the side wall of the suction filter bottle and living bacteria on the filter membrane by using 100ml of sterile water, and leaching and extracting for three times to ensure that the living bacteria are all trapped on the filter membrane and completely eliminate the interference of ATP outside bacteria.
2) Extraction of viable bacteria and RLU numerical detection
And after wiping living bacteria of the filter membrane with the complete coverage by using an ATP fluorometer, rapidly placing the wiping swab into the ATP fluorometer, detecting RLU1 in a specified time, wiping again, and reading RLU2, wherein the RLU is the sum of the RLU1 and the RLU 2.
3) Analysis of living bacteria in water to be detected
Taking the average value of 3 RLUs of the 3 groups of samples, looking up a standard curve, and finding out the corresponding number of viable bacteria as the number of the viable bacteria.
According to the method for rapidly detecting the living bacteria in the water, the water sample is cultured in the constant temperature incubator at 37 ℃ for 24 hours to increase the bacteria, and the water sample after the bacteria increase is a culture stock solution.
According to the method for rapidly detecting the living bacteria in the water, the stock solution of the culture is diluted by 10 times of physiological saline.
According to the method for rapidly detecting living bacteria in water, the detection time of the step (2) is 30 minutes.
According to the method for rapidly detecting viable bacteria in water, the wiping is performed by transversely and repeatedly wiping for 10 times and longitudinally and repeatedly wiping for 10 times for sampling.
According to the method for rapidly detecting the living bacteria in the water, the wiped wiper is inserted into a biological fluorescence test tube, rapidly squeezed into lysate and luciferase, and fully mixed and oscillated.
According to the method for rapid detection of viable bacteria in water of the present invention, the 3 sets of samples determine 3 dilution gradients for plate count, at least one of which has a viable bacteria count in the range of 30 to 300 aspirate 1ml of the set of samples.
According to the method for rapidly detecting the living bacteria in the water, the 3 samples are respectively subjected to three times of wiping detection in parallel.
According to the method for rapidly detecting the living bacteria in the water, the analysis of variance and linear correlation of the factors of the RLU numerical value detection in the steps adopt an SPSS11.5 software package.
The invention provides a method for rapidly detecting living bacteria in water, which comprises the steps of filtering and cleaning a water sample, wiping the living bacteria on a filter membrane, rapidly detecting the living bacteria in the water by an ATP fluorescent method after wiping the living bacteria, taking 3 groups of samples from a water intake, sampling 250ml of samples from each sampling bottle, carrying out suction filtration on each group of the water samples by adopting a 0.45-micrometer sterile filter membrane on an ultra-clean bench, cleaning the water sample, washing the sampling bottle by 100ml of sterile water, and then leaching and extracting the living bacteria on the side wall of the filter bottle and the filter membrane in three times; after the live bacteria of the filter membrane are wiped twice by using an ATP fluorometer, respectively and rapidly placing the wiped swabs of the two times into the ATP fluorometer, detecting and reading the wiped values of the two times in a specified time, wherein RLU is the sum of the wiped values of the two times; the number of viable bacteria corresponding to the average of 3 RLUs for the 3 groups of samples was found on the standard curve. Therefore, the method for rapidly detecting the living bacteria in the water accelerates the detection speed of the ATP fluorescence method, eliminates ATP outside the living bacteria, and has more accurate results.
Drawings
FIG. 1 is a graph showing the correspondence between plate counts and light values in a method for rapidly detecting viable bacteria in water according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a method for rapidly detecting living bacteria in water, which is used for preparing standard curves of the number of the living bacteria or the bacterial colony and RLU according to a dilution method and a flow cytometry of the living bacteria or the bacterial colony with a certain number range and the following methods. The method comprises the following steps:
1) Treatment of water to be detected
Collecting a water sample to be detected, taking 3 groups of samples from a water intake, and sampling 250ml of samples from each sampling bottle.
And after each group of water samples are subjected to suction filtration by adopting a 0.45-micrometer sterile filter membrane on an ultra-clean bench, washing the side wall of the suction filter bottle and living bacteria on the filter membrane by using 100ml of sterile water, and leaching and extracting for three times to ensure that the living bacteria are all trapped on the filter membrane and completely eliminate the interference of ATP outside bacteria.
2) Extraction of viable bacteria and RLU numerical detection
And after the live bacteria of the filter membrane are wiped by using the wiper matched with the ATP fluorometer, the wiper is quickly placed into the ATP fluorometer, RLU1 is detected in a specified time, the wiper is wiped again, RLU2 is read, and the RLU is the sum of the RLU1 and the RLU 2. The number of living bacteria measured by flow cytometry has a good linear relation with the ATP of the living bacteria, so that the number of the living bacteria can be measured only by measuring the ATP of the living bacteria. The average ATP concentration per viable cell is 1.39 x 10-11NM according to the global experimental results. The membrane suction filtration method is adopted, the water intake is large, the situation of living bacteria or bacterial colonies in water is truly reflected, and the data are more accurate.
3) Analysis of living bacteria in water to be detected
Taking the average value of 3 RLUs of the 3 groups of samples, looking up a standard curve, and finding out the corresponding number of viable bacteria as the number of the viable bacteria.
ATP is an energy unit of living cells such as bacteria, saccharomycetes and mould cells, and all living microorganisms are rich in ATP, so that the ATP can be detected to reflect the quantity of the microorganisms, and the ATP of the microorganisms in a water sample emits light under the action of luciferase and luciferin after being extracted; the amount of light is proportional to the ATP and can be detected by a fluorometer, the more microorganisms are viable, the more ATP is produced and the greater the amount of light is produced, thereby detecting the microbial condition in the sample. Since all living cells contain a constant amount of ATP, the ATP content can clearly indicate the amount of microorganisms in the water sample for use in determining sanitary conditions.
The ATP bioluminescence method principle is that the ATP bioluminescence method is characterized in that luciferase is utilized to catalyze oxydecarboxylation of luciferin under the participation of magnesium ions, ATP and oxygen to generate activated oxyluciferin, photons are emitted, and 560nm fluorescence is generated: under the action of a lysate, the ATP released after bacterial lysis participates in an enzymatic reaction, and a fluorescence detector is used for quantitatively measuring a luminescence value, so that the content of ATP is obtained, and the bacterial content is reflected; the ATP fluorometer displays the results as RLU (relative light units) values. Specifically, light generated by the ATP reaction occurring in the ATP sampling rod diverges in the form of photons. Photons are tiny energy beams and are basic units of light, the photons are directly displayed in the form of RLU values after being detected by an ATP detector, the larger the number of photons detected by the ATP detector is, the larger the number of the RLU is, and the linear proportion enables a user to easily judge the number of living bacteria in water.
The experimental steps of the invention are as follows:
and (3) preparing the water sample before the test, culturing the water sample in a constant temperature incubator at 37 ℃ for 24 hours for enrichment, taking the water sample after enrichment as a stock solution of a culture, slightly placing the water sample in a slant culture medium, and culturing the water sample in the incubator at 37 ℃ for 24 hours for enrichment.
The culture stock solution was diluted 10-fold by 10-fold with physiological saline, and after 1 day before counting, the bacterial slant culture was scraped with an inoculating loop and inoculated into a large test tube containing 10ml of nutrient broth, and cultured overnight at 37℃with shaking, which was the culture stock solution, and the culture stock solution was diluted 10-fold by physiological saline.
Preferably, the time of the detection of step (2) is 30 minutes.
Preferably, the wiping is performed by applying 10 times transversely to and from the applicator, and 10 times longitudinally to and from the applicator.
Preferably, the wiped wiper is inserted into a bioluminescence test tube, rapidly squeezed into a lysate and luciferase, and thoroughly mixed and shaken.
Preferably, 3 dilution gradients for plate count are determined for the 3 groups of samples, 1ml of the groups of samples are sucked in the range of 30-300 by at least one of the viable bacteria counts of the gradients, namely plate count, 3 dilution gradients for plate count are determined, 1ml of viable bacteria count of at least one gradient is sucked in the range of 30-300, the dilution gradients are added into a glass sterile empty culture dish, three plates are inverted in parallel for each gradient, blank control is simultaneously performed, the culture is performed for 48 hours at 37 ℃, and the dilution with viable bacteria count evenly dispersed in the range of 30-300 is selected as the plate count and averaged.
Preferably, the 3 samples are respectively and parallelly subjected to three times of wiping detection, relative light unit values (Relative Light Unit, RLU) are measured, living bacteria liquid with different dilutions are respectively injected into 100ml of distilled water, and after hundred-fold concentration is carried out through a filter membrane, a sys-tem II type ATP fluorometer is started, the instrument starts to carry out 60 seconds of self calibration, after the calibration is successfully completed, an ultra snap biological fluorescence test tube is opened, a special cotton swab is taken out to smear a 0.45um filter membrane after a concentrated water sample, the same tester is smeared for 10 times in a transverse round-trip manner, the longitudinal round-trip smearing is carried out for 10 times for sampling, the sampled cotton swab is inserted into the biological fluorescence test tube, the lysis liquid and the luciferase are rapidly extruded, and the operation time of all dilutions of the sample is consistent as much as possible. The cotton swab was placed in a sys-tem SURE II type ATP fluorometer, the lid was closed, the body was tested vertically, the results were displayed after 15 seconds, the readings were recorded, and the same sample was run three times in parallel. The same ATP biological fluorescence method is used for measuring the RLU of the bacterial liquid stock solution, and the RLU is continuously carried out for 3 times to serve as a positive control; RLU values of clean water were determined using ATP bioluminescence as a negative control.
Preferably, the factor variance analysis and linear correlation analysis of RLU numerical value detection in the step uses SPSS11.5 software package, and the test uses SPSS11.5 software package to perform single factor variance analysis and linear correlation analysis on each batch of data of ATP assay and plate count method, to verify whether the data in tables 1 to 4 below are from the same population and to perform correlation analysis on Lg light value and Lg bacteria total number value.
TABLE 1 ATP assay and plate count method lot 1 data
TABLE 2 ATP assay and plate count method lot 2 data
TABLE 3 ATP assay and plate count method lot 3 data
TABLE 4 ATP assay and plate count method lot 4 data
According to the data in tables 1 to 4, a linear correlation curve was plotted by x.y scatter plot with the logarithmic value of the average number of light values as the Y axis and the average value of plate count as the X axis, and r= 0.98525, indicating that the correlation between the total number of Lg bacteria and the Lg light value was high. Sd=0.3308, p <0.0001, the difference being statistically significant. As can be seen in FIG. 1, the plate count was positively correlated with the bioluminescence value over the range of 10-1/2cfu/ml total bacteria.
The results were analyzed by one-way variance using the SPSS11.5 statistical software package and the results are shown in Table 5 below. The Lg light value and the Lg total bacteria number are analyzed by a single factor variance analysis, and P is larger than 0.05, which indicates that four groups of result data come from the same normal overall, and linear correlation analysis can be performed.
TABLE 5 analysis of variance results
As the total bacterial count is not the total bacterial count, the bacterial types are different at different places and different times, the curve can not be used as a whole area, the all-weather linear relation, namely the linear slope at all times is changed, and the application range of the method is the test range at the same place, and the method is used for sampling and detecting rapidly. The linear relationship is obvious at this time, and the test time is fast.
Under the condition that the method is applicable, the total number of the viable bacteria is larger than the total number of the bacterial colonies, and the difference is that some bacteria in the total number of the bacterial colonies cannot be cultivated according to the GB test method of the total number of the bacterial colonies, and the ratio relationship also changes at any time and any place. The total number of colonies is tracked, and the comparison before and after the process is performed in a short time is effective. It is effective to keep track of the total number of viable bacteria for a long time.
In summary, the method for rapidly detecting the viable bacteria in the water is provided, filtering and cleaning are carried out on a water sample, and wiping of the viable bacteria on a filter membrane is carried out, the viable bacteria in the water is rapidly detected through an ATP fluorescence method after wiping, the water sample filtering process comprises the steps of taking 3 groups of samples from a water intake, sampling 250ml of samples from each sampling bottle, carrying out suction filtration on each group of water samples by adopting a 0.45-micrometer sterile filter membrane on an ultra-clean bench, cleaning the water sample, washing the sampling bottle by 100ml of sterile water, and then leaching and extracting the viable bacteria on the side wall of the filter bottle and the filter membrane in three times; after the live bacteria of the filter membrane are wiped twice by using an ATP fluorometer, respectively and rapidly placing the wiped swabs of the two times into the ATP fluorometer, detecting and reading the wiped values of the two times in a specified time, wherein RLU is the sum of the wiped values of the two times; the number of viable bacteria corresponding to the average of 3 RLUs for the 3 groups of samples was found on the standard curve. Therefore, the method for rapidly detecting the living bacteria in the water accelerates the detection speed of the ATP fluorescence method, eliminates ATP outside the living bacteria, and has more accurate results.
Of course, various other embodiments of the invention are possible, such as by using Flow Cytometry (FCM) FCM-RLU standard curve formulation, as well as a more accurate and rapid method of determining living bacteria. Various modifications and variations may be made in the present invention by those skilled in the art without departing from the spirit and substance of the invention, and such modifications and variations are intended to be within the scope of the appended claims.
Claims (9)
1. A method for rapidly detecting viable bacteria in water, which is characterized by comprising the following steps:
1) Treatment of water to be detected
Collecting a water sample to be detected, taking 3 groups of samples from a water intake, and sampling 250ml of samples from each sampling bottle;
and after each group of water samples are subjected to suction filtration by adopting a 0.45-micrometer sterile filter membrane on an ultra-clean bench, washing the side wall of the suction filter bottle and living bacteria on the filter membrane by using 100ml of sterile water, and leaching and extracting for three times to ensure that the living bacteria are all trapped on the filter membrane and completely eliminate the interference of ATP outside bacteria.
2) Extraction of viable bacteria and RLU numerical detection
And after wiping living bacteria of the filter membrane with the complete coverage by using an ATP fluorometer, rapidly placing the wiping swab into the ATP fluorometer, detecting RLU1 in a specified time, wiping again, and reading RLU2, wherein the RLU is the sum of the RLU1 and the RLU 2.
3) Analysis of living bacteria in water to be detected
Taking the average value of 3 RLUs of the 3 groups of samples, looking up a standard curve, and finding out the corresponding number of viable bacteria as the number of the viable bacteria.
2. The method for rapidly detecting living bacteria in water according to claim 1, wherein the water sample is cultured in a constant temperature incubator at 37 ℃ for 24 hours for bacterial enrichment, and the water sample after bacterial enrichment is a culture stock solution.
3. The method for rapid detection of viable bacteria in water according to claim 2, wherein the stock culture solution is diluted 10-fold by physiological saline.
4. The method for rapid detection of viable bacteria in water according to claim 1, wherein the time of the detection of step (2) is 30 minutes.
5. The method for rapid detection of viable bacteria in water according to claim 1, wherein the wiping is performed by applying 10 times transversely and 10 times longitudinally.
6. The method according to claim 5, wherein the wiped wiper is inserted into a bioluminescence tube, squeezed into a lysate and luciferase rapidly, and mixed thoroughly.
7. The method for rapid detection of viable bacteria in water of claim 1, wherein said 3 sets of samples define 3 dilution gradients for plate count, viable bacteria count of at least one of said gradients ranging from 30 to 300 aspirate 1ml of said sets of samples.
8. The method for rapidly detecting viable bacteria in water according to claim 1, wherein the 3 samples are subjected to three wiper tests in parallel.
9. The method for rapid detection of viable bacteria in water according to any of claims 1 to 8, wherein the analysis of variance and linear correlation of the RLU values detected in step (a) employs the SPSS11.5 software package.
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