CN101302557A - Quantitative determination method of attached type sulfate reducing bacteria and used biological film sampling apparatus - Google Patents
Quantitative determination method of attached type sulfate reducing bacteria and used biological film sampling apparatus Download PDFInfo
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- CN101302557A CN101302557A CNA2008100649026A CN200810064902A CN101302557A CN 101302557 A CN101302557 A CN 101302557A CN A2008100649026 A CNA2008100649026 A CN A2008100649026A CN 200810064902 A CN200810064902 A CN 200810064902A CN 101302557 A CN101302557 A CN 101302557A
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Abstract
The invention discloses a quantificational detection method for adhesive sulfate reducing bacteria and a biomembrane sampling device thereof, which relate to the quantificational detection method for sulfate reducing bacteria and the special biomembrane sampling device in the method. The invention solves the problem that the prior detection method fails to realize quick and more accurate quantificational detection of the adhesive sulfate reducing bacteria. The method has the main steps of: biomembrane removal by using the biomembrane sampling device, bacteria preparation, multiple proportion dilution, PCR operation, augmentation, electrophoretic detection, and meter reading and number recording. A probe (1) orderly passes through a probe compression assembly (5) and a body (4), and is fixed on the upper end surface of the body (4) through a fixing part (2), a shoulder (1-1) at the upper end of the probe (1) is lapped on the upper end surface of the probe compression assembly (5), and a control valve (3) is arranged on the side wall of the lower end of the body (4). The method has more accurate number recording and shortens counting time; the application of the device can effectively obtain adhesive biomembranes of tank walls and pipe walls.
Description
Technical field
The present invention relates to biological film sampling apparatus special-purpose in a kind of sulphate reducing bacteria quantitative detecting method and this method.
Background technology
At present, the detection method of sulphate reducing bacteria (SRB) mainly concentrates on three aspects: the one, and traditional detection method, comprise culture method and microscope direct-counting method, the former passes through selective medium, under the suitable culture condition, the bacterium in the sample is cultivated the counting of the bacterium that lives, mainly comprise the method for plate culture count, most probable rate counting process (most probable number (MPN) also is called coubling dilution) etc.; And the microscope direct-counting method is to measure bacteria total amount in good time, quickly and accurately, mainly comprises technology such as opticmicroscope, fluorescent microscope, turbidity (than turbid) counting process, electrical impedance method and cells were tested by flow cytometry method etc.; The 2nd, the gene aspect, based on the qualitative detection of the 16S rDNA sequence of SRB kind, as gene chip and FISH technology to SRB quantitatively and qualitative detection, APS reductase gene and alienation type sulfite reductase gene carry out qualitative detection; The 3rd, the albumen aspect mainly is that the principle by immunosorption is achieved, as the quick detection kit based on the SRB of APS reductase enzyme.Above-mentioned conventional detection is unsuitable for the detection by quantitative of attached type sulfate reducing bacteria.
In the oil field system counting of sulphate reducing bacteria mainly be at the sulphate reducing bacteria-non-attached type sulfate reducing bacteria of drainage flow in mutually.At present mainly carry out the China National Petroleum industry standard: " oilfield injection water bacteria analyzing method---disappearance dilution method " (ministerial standard), SY-T0532-93 for the counting of sulphate reducing bacteria (SRB) in the oil field system of non-attached type.The problem that aforesaid method exists is: detect the time that needs fortnight, because sense cycle is longer, can not effectively, immediately instruct production practice, some strict anaerobism SRB bacterium can't survive in actual mechanical process, cause the numeration result of SRB bacterium inaccurate, can not reflect the production practical situation really, testing cost is higher simultaneously.
Granted publication number is the quantitative detecting method that the patent of invention on November 4th, 2007 has proposed a kind of water quality in oil field through sulfate reducting bacteria for CN 100348731C, Granted publication day; Granted publication number is that the patent of invention on March 5th, 2008 has proposed the direct doubling dilution PCR of a kind of sulphate reducing bacteria fast quantitative measurement method for detecting for CN100372943C, Granted publication day; All do not mention the quantitative detecting method of attached type sulfate reducing bacteria in the above-mentioned prior art.Because of there is the long detection by quantitative that causes problems such as the numeration result is inaccurate can't be suitable for attached type sulfate reducing bacteria of sense cycle in the quantitative detecting method of sulphate reducing bacteria (SRB) in the oil field system of non-attached type, do not have the special quantitative detecting method that proposes attached type sulfate reducing bacteria in the prior art again, therefore also do not carry out at the detection of the sulphate reducing bacteria-attached type sulfate reducing bacteria on tank skin and the tube wall.Yet the sulphate reducing bacteria of attached type is the major reason that causes oil field corrosion and sulfide to produce in the production of reality.Therefore, the detection to attached type sulfate reducing bacteria has great importance more.
Summary of the invention
The present invention for solve existing sulphate reducing bacteria detection method can't realize attached type sulfate reducing bacteria fast, detection by quantitative, and existing sulphate reducing bacteria detection method expense problem of higher more accurately; And a kind of quantitative determination method of attached type sulfate reducing bacteria and used biological film sampling apparatus are provided.
The technical scheme that technical solution problem of the present invention is adopted is: quantitative determination method of attached type sulfate reducing bacteria is to realize according to following steps: step 1, take off the microbial film that contains sulphate reducing bacteria that adheres on tank body or the inboard wall of tube body with biological film sampling apparatus: on the sidewall of tank body or body, get through the hole earlier, again biological film sampling apparatus is installed in described through hole, and make the probe on the biological film sampling apparatus concordant with the inner side-wall of tank body or body, and keep online installation 10-20 days, treat that sulphate reducing bacteria fully can take off microbial film after the film forming on the described probe; The thalline of step 2, sulphate reducing bacteria prepares: the microbial film on the described probe is peeled off to the solution of preparation made thalline; Step 3, the thalline for preparing in the step 2 is carried out doubling dilution; Step 4, the PCR that carries out on ice operate; Step 5, the amplification of PCR on the amplification instrument; Step 6, amplified production are 1.0% agarose electrophoresis detection in mass concentration; Step 7, result observe the numeration of tabling look-up.
The realization of method noted earlier must could realize by biological film sampling apparatus, this sampling unit comprises main body, probe compresses assembly, mounting block and control valve, described sampling unit also comprises probe, the upper end of described probe is provided with shoulder, compress the upper end that assembly is positioned at main body, probe passes probe successively and compresses assembly, main body, probe is packed on the upper surface of main body by mounting block, the shoulder of probe upper end is overlapped on probe and compresses on the upper surface of assembly, probe compresses assembly and is installed in main body and is provided with on the locating shoulder, and control valve is installed on the lower end sidewall of main body.
The present invention has following beneficial effect: the present invention realized attached type sulfate reducing bacteria fast, detection by quantitative more accurately.Method numeration of the present invention is more accurate, can be as accurate as a thalline, shortened gate time effectively, whole invention only needs four hours from making sample to going out the result, be far smaller than the numeration time of fortnight, real illness that has not attacked the vital organs of the human body the quantity of SRB bacterium of reality of water sample, reacted production practical situation at that time accurately, can directly effectively instruct production, also reduced testing cost simultaneously.Utilization apparatus of the present invention can effectively obtain the attached biological film of tank skin and tube wall, and apparatus of the present invention also have advantage of simple structure.The fast quantification that the present invention is suitable for attached type sulfate reducing bacterias (SRB) such as tube wall, tank skin in the water treatment procedure of conventional sewage, polymer-bearing waste-water, surface technology in the oil-field water system detects, and also can be used for various microbial films, the medium detection that contains the environmental samples of attached type sulfate reducing bacteria of works.
Description of drawings
Fig. 1 is the structural representation of biological film sampling apparatus of the present invention, Fig. 2 is the structural representation after the probe of biological film sampling apparatus of the present invention takes out from pipeline, Fig. 3 is the structural representation of probe, and Fig. 4 is the on-the-spot process flow diagram of specific embodiments of the invention.
Embodiment
Embodiment one: the described quantitative determination method of attached type sulfate reducing bacteria of present embodiment is realized according to following steps: step 1, take off the microbial film that contains sulphate reducing bacteria that adheres on tank body or the inboard wall of tube body with biological film sampling apparatus: on the sidewall of tank body or body, get through the hole earlier, again biological film sampling apparatus is installed in described through hole, and make the probe on the biological film sampling apparatus concordant with the inner side-wall of tank body or body, and keep online installation 10-20 days, treat that sulphate reducing bacteria fully can take off microbial film after the film forming on the described probe; The thalline of step 2, sulphate reducing bacteria prepares: the microbial film on the described probe is peeled off to the solution of preparation made thalline; Step 3, the thalline for preparing in the step 2 is carried out doubling dilution; Step 4, the PCR that carries out on ice operate; Step 5, the amplification of PCR on the amplification instrument; Step 6, amplified production are 1.0% agarose electrophoresis detection in mass concentration; Step 7, result observe the numeration of tabling look-up.The described step 2 of present embodiment to step 7 is a prior art, Granted publication number is that the patent of invention on March 5th, 2008 has proposed a kind of " the direct doubling dilution PCR of sulphate reducing bacteria fast quantitative measurement method for detecting " for CN100372943C, Granted publication day, in this patent step 2 to the content of step 7 is all had disclosure.
Embodiment two: the described quantitative determination method of attached type sulfate reducing bacteria of present embodiment is realized according to following steps:
The thalline of step 2, sulphate reducing bacteria prepares: the microbial film on the described probe is peeled off to the solution of preparation made thalline; Specifically may further comprise the steps: a, the microbial film that takes off put into the 1mL deionized water is housed and through the centrifuge tube of 1.5mL of sterilization; B, will be on the centrifuge tube earthquake device abundant concussion 5min, the centrifugal 1min of 13000r/min, from pipe gently abandon supernatant liquor 0.9mL; C, in remaining 0.1mL, add " thalline pretreatment buffer liquid " (DNA pretreatment buffer liquid) 0.9mL, abundant concussion 5min on the earthquake device; The centrifugal 2min of d, 13000r/min abandons supernatant liquor 0.98mL (remaining 20 μ l), fully shakes 2min, preparation finish in the short period of time (0～1 day) under 4 ℃ of conditions, preserves and gets final product, (greater than 1 day) not be used under-20 ℃ of conditions and preserves for a long time; The mother liquor composition of described " thalline pretreatment buffer liquid " is: 70g NaCi, 2g Kci, 12g Na
2HPO
4, 2g KH
2PO
4, 1 ‰ SDS; The mother liquor dilution is become the thalline pretreatment buffer liquid for 10 times; The pH of control working fluid is 7.5, gets final product behind the 15～30min that sterilizes under 121 ℃ of conditions.
Embodiment three: present embodiment keeps online last 15 day of sidewall that is installed in tank body or body at biological film sampling apparatus described in the step 1.Other step is identical with embodiment one or two.
Embodiment four: present embodiment adopts three pipes or five pipe parallel methods, 3 or 5 samples of each gradient in the PCR operating process of carrying out on ice.Method test result of the present invention shows, the result of three pipe parallel methods records no matter under the situation of water sample lower concentration or high density, on the order of magnitude with five pipe parallel method indifferences.Importantly be that the result of five pipe parallel methods records is more accurate, degree of confidence is greater than 99%.The result of five pipe parallel method SRB-MPN-PCR methods numerations with compare with 14 days numeration result of sulphate reducing bacteria Bacteria Detection reagent bottle (chemical reagent factory of Beijing Huaxing) on the order of magnitude on average big by 10
2, more can react in the water sample quantity of actual SRB bacterium.Other step is identical with embodiment one, two or three.Granted publication number is that the patent of invention on November 4th, 2007 has proposed a kind of " quantitative detecting method of water quality in oil field through sulfate reducting bacteria " for CN100348731C, Granted publication day; Granted publication number is that the patent of invention on March 5th, 2008 has proposed a kind of " the direct doubling dilution PCR of sulphate reducing bacteria fast quantitative measurement method for detecting " for CN 100372943C, Granted publication day; Above-mentioned two patents have all disclosed the technical scheme of three pipes or five pipe parallel methods.
Embodiment five: shown in Fig. 1～3, the biological film sampling apparatus of present embodiment comprises main body 4, probe compresses assembly 5, mounting block 2 and control valve 3 (sluice of band turnbuckle), described sampling unit also comprises probe 1, the upper end of described probe 1 is provided with shoulder 1-1, compress the upper end that assembly 5 is positioned at main body 4, probe 1 passes probe successively and compresses assembly 5, main body 4, probe 1 is packed on the upper surface of main body 4 by mounting block 2, the shoulder 1-1 of 1 upper end of popping one's head in is overlapped on probe and compresses on the upper surface of assembly 5, probe compresses assembly 5 and is installed in main body 4 and is provided with on the locating shoulder 4-1, and control valve 3 is installed on the lower end sidewall of main body 4.The effect that probe compresses assembly 5 is to prevent to pop one's head in 1 will be popped one's head in by the pressure of the liquid in tank body or the body 6 and 1 eject.In use, earlier on the sidewall of tank body or body 6, get through the hole, biological film sampling apparatus being installed in described through hole makes the probe on the biological film sampling apparatus concordant with the inner side-wall of tank body or body again, and keep online installation 10-20 days, treat that sulphate reducing bacteria fully can take off microbial film after the film forming on the described probe.Mycoderm on the probe is peeled off to the solution of preparation, and preserve the concussion back.Biological film sampling apparatus is that the detection of attached type sulfate reducing bacteria fast quantification is achieved key point, and it has solved the difficult problem that attached type sulfate reducing bacteria can't be taken a sample, and is the precondition of subsequent detection.
Specific embodiment:
Test is a test point with three factories north three or the three dehydrating station Ground Processing Systems that recover the oil, and has carried out the research of tube wall and tank skin attached type SRB assay.On-the-spot test suite (comprising biological film sampling apparatus) is installed in respectively to settle out mouthful in north three or three dehydrating station slurry tanks outlet and inlet, north 313 and inlet, north 31 outer water delivery headers outlets and enter the mouth on, on-the-spot technical process is referring to Fig. 4, and test result sees Table 1.Adopt the SRB substratum after improveing to count.
According to the SRB content in the growth result calculating mycoderm, on-the-spot attached type SRB assay result, by table 1 as seen, a large amount of SRB bacterium apposition growths is arranged on the tube wall, show that to a certain extent the quantity of tube wall SRB bacterium is relatively greater than the quantity in the sewage, by being installed in the proofing unit and the coupling of disappearance dilution method (MPN) on the tube wall in the same pipeline, realized the detection by quantitative of the attached type SRB bacterium on tube wall and the tank skin, this technology is applied aborning.The colleague also is the attached type SRB that only monitors in the present bacterium numeration in the sewage, in the moving phase, do not monitor attached type SRB, the scene is adhered to SRB assay result and is shown, a large amount of SRB apposition growths is arranged on the tube wall of system, this result of study has also confirmed the conclusion of indoor SRB flora kind and growth characteristics research, promptly has the SRB of apposition growth and the SRB of non-apposition growth in the ground system of oil field.System control method for specifying sulphate reducing bacteria has very important significance, and has changed the problem that " sterilization scheme " in the past cured the symptoms, not the disease.
The table 1 oil recovery three attached type SRB of factory content measuring results
Claims (3)
1, a kind of quantitative determination method of attached type sulfate reducing bacteria, it is characterized in that it realizes according to following steps: step 1, take off the microbial film that contains sulphate reducing bacteria that adheres on tank body or the inboard wall of tube body with biological film sampling apparatus: on the sidewall of tank body or body, get through the hole earlier, again biological film sampling apparatus is installed in described through hole, and make the probe on the biological film sampling apparatus concordant with the inner side-wall of tank body or body, and keep online installation 10-20 days, treat that sulphate reducing bacteria fully can take off microbial film after the film forming on the described probe; The thalline of step 2, sulphate reducing bacteria prepares: the microbial film on the described probe is peeled off to the solution of preparation made thalline; Step 3, the thalline for preparing in the step 2 is carried out doubling dilution; Step 4, the PCR that carries out on ice operate; Step 5, the amplification of PCR on the amplification instrument; Step 6, amplified production are 1.0% agarose electrophoresis detection in mass concentration; Step 7, result observe the numeration of tabling look-up.
2, quantitative determination method of attached type sulfate reducing bacteria according to claim 1 is characterized in that keeping online last 15 day of sidewall that is installed in tank body or body at biological film sampling apparatus described in the step 1.
3, realize the biological film sampling apparatus in the described method of claim 1, it is characterized in that described sampling unit comprises main body (4), probe compresses assembly (5), mounting block (2) and control valve (3), it is characterized in that described sampling unit also comprises probe (1), the upper end of described probe (1) is provided with shoulder (1-1), compress the upper end that assembly (5) is positioned at main body (4), probe (1) passes probe successively and compresses assembly (5), main body (4), probe (1) is packed on the upper surface of main body (4) by mounting block (2), the shoulder (1-1) of probe (1) upper end is overlapped on probe and compresses on the upper surface of assembly (5), probe compresses assembly (5) and is installed in main body (4) and is provided with on the locating shoulder (4-1), and control valve (3) is installed on the lower end sidewall of main body (4).
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| CNA2008100649026A CN101302557A (en) | 2008-07-11 | 2008-07-11 | Quantitative determination method of attached type sulfate reducing bacteria and used biological film sampling apparatus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706688A (en) * | 2012-07-10 | 2012-10-03 | 哈尔滨工业大学 | Intelligent ball pipeline growth ring sampler without stopping water |
| CN102925348A (en) * | 2012-11-11 | 2013-02-13 | 中国船舶重工集团公司第七二五研究所 | Device for detecting concentration of sulfate-reducing bacteria (SRB) |
| CN103983636A (en) * | 2014-05-20 | 2014-08-13 | 中国石油化工股份有限公司 | Rapid detecting method of sulphate reducing bacteria and kit thereof |
| CN105567552A (en) * | 2016-03-15 | 2016-05-11 | 吉林建筑大学 | Biomembrane outdoor culture and collection device |
| CN107400699A (en) * | 2017-07-13 | 2017-11-28 | 上海市水利工程设计研究院有限公司 | A kind of pipeline biomembrane sampling system and the method for sampling based on driven by power |
-
2008
- 2008-07-11 CN CNA2008100649026A patent/CN101302557A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706688A (en) * | 2012-07-10 | 2012-10-03 | 哈尔滨工业大学 | Intelligent ball pipeline growth ring sampler without stopping water |
| CN102706688B (en) * | 2012-07-10 | 2014-06-11 | 哈尔滨工业大学 | Intelligent ball pipeline growth ring sampler without stopping water |
| CN102925348A (en) * | 2012-11-11 | 2013-02-13 | 中国船舶重工集团公司第七二五研究所 | Device for detecting concentration of sulfate-reducing bacteria (SRB) |
| CN102925348B (en) * | 2012-11-11 | 2013-10-09 | 中国船舶重工集团公司第七二五研究所 | Device for detecting concentration of sulfate-reducing bacteria (SRB) |
| CN103983636A (en) * | 2014-05-20 | 2014-08-13 | 中国石油化工股份有限公司 | Rapid detecting method of sulphate reducing bacteria and kit thereof |
| CN103983636B (en) * | 2014-05-20 | 2016-06-22 | 中国石油化工股份有限公司 | A kind of sulfate reducting bacteria method for quick and test kit thereof |
| CN105567552A (en) * | 2016-03-15 | 2016-05-11 | 吉林建筑大学 | Biomembrane outdoor culture and collection device |
| CN105567552B (en) * | 2016-03-15 | 2017-11-03 | 吉林建筑大学 | A kind of biomembrane field culture harvester |
| CN107400699A (en) * | 2017-07-13 | 2017-11-28 | 上海市水利工程设计研究院有限公司 | A kind of pipeline biomembrane sampling system and the method for sampling based on driven by power |
| CN107400699B (en) * | 2017-07-13 | 2024-03-15 | 上海市水利工程设计研究院有限公司 | Pipeline biological film sampling system and sampling method based on electric drive |
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Open date: 20081112 |