CN115824932B - Method and kit for detecting concentration of sulfate reducing bacteria - Google Patents
Method and kit for detecting concentration of sulfate reducing bacteria Download PDFInfo
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Abstract
The application discloses a method and a kit for detecting concentration of sulfate reducing bacteria. The method for detecting the concentration of the sulfate reducing bacteria comprises the steps of applying voltage to a liquid to be detected, enabling particles in the liquid to be detected to pass through micropores, generating voltage or current pulses, obtaining the particle size and concentration of the particles in the liquid to be detected through pulse signals of the voltage or current, and selecting the concentration of the particles with the particle size range of 500-1300 nm as the concentration of the sulfate reducing bacteria. The method for detecting the concentration of the sulfate reducing bacteria utilizes the pulse signals generated by the sulfate reducing bacteria passing through micropores to detect the particle size and the concentration, and has the advantages of high accuracy, low detection limit and the like; the detection method is simple and easy to operate, can be used for detecting the concentration of the sulfate reducing bacteria of various samples, and is particularly suitable for detecting and monitoring the sulfate reducing bacteria in special environments such as aquaculture, oilfield pipelines and the like.
Description
Technical Field
The application relates to the technical field of sulfate reducing bacteria concentration detection, in particular to a method and a kit for detecting the concentration of sulfate reducing bacteria.
Background
Sulfate-reducing bacteria (SRB) are anaerobic microorganisms which have various forms and various nutrition types and can utilize Sulfate or other oxidized sulfides as electron acceptors to dissimilate organic substances, and the biological corrosion of the SRB brings great harm and loss.
Currently, there are several methods for concentration testing of SRB bacteria in a sample: (1) And (3) performing parallel sterilization dilution, namely performing parallel culture on different test tubes after performing multiple ratio dilution on the sample, observing sample sediment by adopting a microscope, and judging the concentration of SRB cells according to the sediment and the dilution multiple of the test tubes. But this method generally requires 14 days or more of incubation. (2) Immunological methods, an APS reductase is found in the SRB cell, which is a characteristic enzyme of SRB; APS reductase is capable of catalyzing the reduction of adenosine-5' -phosphate sulfate reductase and producing the specific product of interest; the bacterial concentration of the SRB can be judged by utilizing the intensity of the color reaction between the product and the color reagent. However, this method fails to test a relatively low concentration of bacterial sample. (3) Adenosine Triphosphate (ATP), a compound found in all living biological cells, is in proportion to the cell concentration. However, the method can only detect the total number of microorganisms, cannot specifically detect SRB, is easily interfered by other microorganisms, and is inaccurate in test. (4) The PCR method is to break bacteria in a sample, perform PCR amplification on bacterial DNA, and reversely deduce the concentration of bacteria in the sample through a PCR test Ct value. However, the complex composition of the sample of SRB bacteria generally affects the amplification effect of PCR and thus the test results.
Therefore, no better method for accurately testing the concentration of sulfate-reducing bacteria exists at present. How to rapidly and accurately detect the concentration of sulfate-reducing bacteria is still a problem to be solved in the field.
Disclosure of Invention
The application aims to provide a novel method and a kit for detecting the concentration of sulfate reducing bacteria.
The application adopts the following technical scheme:
One aspect of the application discloses a method for detecting concentration of sulfate-reducing bacteria, which comprises the steps of applying voltage to a liquid to be detected, enabling particles in the liquid to be detected to pass through micropores, generating voltage or current pulses, obtaining particle size and concentration of the particles in the liquid to be detected by measuring pulse signals of the voltage or the current, and selecting concentration of the particles with particle size range of 500 nm-1300 nm as concentration of sulfate-reducing bacteria.
The application creatively adopts an electrical impedance method, utilizes the coulter principle to detect the concentration of the liquid to be detected of the sulfate reducing bacteria, and takes the concentration of particles with the particle diameter ranging from 500nm to 1300nm in the liquid to be detected as the concentration of the sulfate reducing bacteria according to research and analysis. The detection method can rapidly and accurately detect the concentration of the sulfate reducing bacteria; in principle, as long as the liquid to be detected contains a sulfate reducing bacterium, a pulse signal is generated when the sulfate reducing bacterium passes through the micropores, and the sulfate reducing bacterium can be detected, and has high sensitivity and low detection limit.
It should be noted that, the test solution of the present application generally adopts a culture product of sulfate reducing bacteria or a supernatant obtained by centrifuging the culture product, and the test solution generally does not contain other particles except sulfate reducing bacteria; therefore, the particles detected in the test solution are generally sulfate-reducing bacteria. However, in order to exclude other possible interferences, the concentration of the particles with a specific particle size range is used as the concentration of sulfate reducing bacteria; the specific particle size range is a particle size range which can cover most of the size of sulfate reducing bacteria and is obtained through a great deal of research and practical analysis, namely 500 nm-1300 nm.
In one implementation mode of the application, the mode of applying voltage to the liquid to be detected comprises the steps of adopting two liquid tanks with electrodes, placing the liquid to be detected in one liquid tank, placing electrolyte in the other liquid tank, communicating the two liquid tanks through micropores, applying voltage to the electrodes of the two liquid tanks, and enabling particles in the liquid to be detected to enter the other liquid tank through the micropores, so that a pulse signal of voltage or current is generated on the electrodes.
In one implementation mode of the application, the method for detecting the concentration of the sulfate reducing bacteria further comprises the steps of carrying out acid solution titration on a sample to be detected until a clear solution is obtained, centrifuging the clear solution, and taking supernatant as the liquid to be detected.
It should be noted that, the sample to be tested in the application is generally a sulfate reducing bacteria culture product of the collected sample, some precipitation may be generated during the sulfate reducing bacteria culture process, and in order to avoid the influence of the precipitation on the detection result, in the improved scheme of the application, the sample to be tested is firstly titrated with an acidic solution, and after the titration is completed, the supernatant is centrifugally taken as the liquid to be tested. It is understood that during the cultivation of sulfate-reducing bacteria, the culture solution becomes turbid due to the generation of sediment, and the culture product becomes clear gradually upon titration with an acidic solution. Of course, since sulfate-reducing bacteria remain suspended in the culture broth and supernatant; thus, the acidic solution is not in a completely clear state after titration, but in a relatively clear state.
In one implementation of the present application, the acidic solution includes at least one of hydrochloric acid and nitric acid.
The acid solution titration of the application needs to be capable of dissolving and precipitating on one hand, and avoids adverse effects on sulfate reducing bacteria on the other hand as much as possible; therefore, hydrochloric acid or nitric acid is preferably used. It is understood that other acidic solutions of similar function may be employed within the inventive concepts of the present application, not limited to hydrochloric acid, nitric acid.
In one implementation of the application, the centrifugation conditions are centrifugation for 5-15 minutes at a rotational speed of not more than 3000 rpm.
It should be noted that the purpose of the present application is mainly to remove some possible large particle precipitation; however, in order to avoid that sulfate-reducing bacteria are also precipitated by centrifugation, the present application preferably performs centrifugation at a rotational speed of not more than 3000 rpm.
In one implementation of the application, the pore diameter of the micropores is 2000 nm-40000 nm.
The micropores are mainly used for enabling sulfate reducing bacteria to pass through so as to generate a pulse signal of voltage or current; thus, the pore size of the micropores depends on the size of the sulfate-reducing bacteria of the present application.
In one implementation mode of the application, the method for detecting the concentration of the sulfate reducing bacteria further comprises the steps of carrying out gradient dilution on a sample to be detected, and respectively carrying out constant-temperature culture on the samples subjected to gradient dilution; then, respectively detecting the concentration of sulfate reducing bacteria on the culture products; and according to the gradient dilution multiple, the corresponding sulfate reducing bacteria detection concentration and the culture time, calculating the original sulfate reducing bacteria concentration of the sample to be detected.
It should be noted that, the concentration of sulfate reducing bacteria is detected after the sample to be detected is subjected to gradient dilution and constant temperature culture, so as to ensure that the sample to be detected contains enough sulfate reducing bacteria. It is understood that the object of the application for constant temperature cultivation is sulfate reducing bacteria; therefore, the conventional sulfate-reducing bacteria culture solution can be used for the constant temperature culture, and is not particularly limited herein.
In one implementation of the application, the original sulfate-reducing bacteria concentration of the sample to be measured is calculated, which comprises determining the culture amplification factor of the sulfate-reducing bacteria corresponding to the culture time according to the growth curve of the sulfate-reducing bacteria, calculating the original sulfate-reducing bacteria concentration of the sample to be measured according to the following formula,
Original sulfate reducing bacteria concentration of sample to be tested = sulfate reducing bacteria detection concentration x dilution factor/(culture amplification factor).
In one implementation mode of the application, the original sulfate-reducing bacteria concentration of the sample to be measured is calculated, and the method further comprises the steps of respectively calculating the original sulfate-reducing bacteria concentration of the sample to be measured according to each gradient dilution sample, and taking the average value of calculation results of each gradient dilution sample as the final original sulfate-reducing bacteria concentration of the sample to be measured.
It should be noted that, in principle, the original sulfate-reducing bacteria concentration of the sample to be measured can be directly calculated according to any one of the gradient diluted samples; however, in order to ensure accuracy, the application preferably uses the average value of the calculation results of each gradient dilution sample as the original sulfate-reducing bacteria concentration of the final sample to be tested.
In one implementation mode of the application, the constant temperature culture condition is that the culture is carried out for 12-100 hours at 35 ℃.
In one implementation mode of the application, the conditions of constant temperature culture are controlled to ensure that the culture amplification factor of the culture product is more than 10 times.
The application draws a growth curve of 0 to 117 hours of culture of the sulfate reducing bacteria, and the result shows that the culture amplification factor increases well with the increase of the culture time within the period from the start of the culture to about 100 hours of culture; after 100 hours of incubation, the culture expansion factor increased, but was relatively slow. Therefore, the constant temperature culture time of the present application is preferably 12 to 100 hours. In principle, the culture amplification factor of the culture product is more than 10 times, so that a better detection result can be obtained; therefore, the culture can basically meet the requirement for 12 to 100 hours. As for the culture temperature, it is generally 35℃and, of course, the culture temperature may be adjusted within the allowable range of the test according to the need. In addition, the detection method provided by the application can meet the detection requirement by only needing a short culture time even if the sample to be detected is subjected to gradient dilution and constant-temperature culture, and has the advantage of short culture time.
In one implementation mode of the application, the culture solution for constant temperature culture is a culture solution without particulate matters, and the culture solution without particulate matters consists of NaCl、K2HPO4、MgSO4、Fe(NH4)2(SO4)2、FeSO4、NH4Cl、Na2SO4、CaCl2、70％ lactic acid, yeast extract and water.
In one implementation mode of the application, the concentration of each component in the culture solution without the particulate matters is NaCl4g/L、K2HPO4 0.5g/L、MgSO4 1.5g/L、Fe(NH4)2(SO4)2 1g/L、FeSO4 1g/L、NH4Cl1g/L、Na2SO4 0.5g/L、CaCl2 0.2g/L、70％ g/L of lactic acid and 1g/L of yeast extract.
In principle, the conventional sulfate-reducing bacteria culture solution can be applied to the present application; however, the concentration detection method of the present application requires that the culture product contains as little sediment as possible, and that the culture solution itself should be free of particulate matter; therefore, the application optimizes and improves the culture solution. The culture solution without the particulate matters, which is prepared by adopting the improved formula, can lead the sediment in the culture product to be as little as possible, thereby reducing the detection interference caused by the sediment and improving the detection accuracy. It will be appreciated that the specific concentrations of the above components are those specifically employed in one implementation of the present application, and that the concentrations of the components may be appropriately adjusted without affecting the performance and culture effect of the culture medium.
In one implementation mode of the application, the gradient dilution of the sample to be tested is carried out by adopting a culture solution cultured at constant temperature, namely the culture solution without the particulate matters is preferably adopted.
In one implementation of the present application, the multiples of the gradient dilution include in particular 10, 100, 1000, 10000 and 100000 times.
In one embodiment of the application, the electrolyte is likewise a culture medium for constant temperature culture, i.e. preferably a culture medium without particulate matter according to the application.
It should be noted that the electrolyte is used for providing a liquid environment for the electrical impedance method and is also used for detecting sulfate reducing bacteria; therefore, a culture broth cultivated at constant temperature, i.e., a culture broth free of particulate matter of the present application, is preferably used.
In one implementation mode of the application, the method for detecting the concentration of the sulfate reducing bacteria further comprises the step of judging and calculating the accuracy of the original sulfate reducing bacteria concentration of the sample to be detected according to the linear correlation of the sulfate reducing bacteria detection concentration and the dilution multiple of each gradient dilution sample, wherein the better the linear correlation is, the higher the accuracy is.
The original sulfate reducing bacteria concentration of the sample to be detected is calculated and obtained according to the sulfate reducing bacteria detection concentration, dilution factor and culture amplification factor; therefore, if the linear correlation between the sulfate-reducing bacteria detection concentration and the dilution factor is better, the original sulfate-reducing bacteria concentration of the sample to be detected, which is directly obtained through formula calculation, is proved to be more accurate.
In one implementation mode of the application, the method for detecting the concentration of the sulfate reducing bacteria further comprises the step of adopting a negative reference sample and/or a positive reference sample for a control test; wherein the negative reference sample is a solution which does not contain bacteria and has the same components as the liquid to be detected; the positive reference sample is bacterial suspension with known sulfate reducing bacteria concentration and the same components as the liquid to be tested.
In the implementation mode of the application, the negative reference sample and the positive reference sample are subjected to gradient dilution, constant temperature culture, acid solution titration and sulfate reducing bacteria concentration detection according to the same method as the sample to be detected; therefore, the detection results of the negative reference sample and the positive reference sample can be used as quality control and reference of the whole detection process. For example, in one implementation of the application, the background concentration is removed directly from the sulfate-reducing bacteria detection concentration of the negative reference sample. Or correcting the calculation result of the original sulfate reducing bacteria concentration of the sample to be detected according to the calculation result of the original sulfate reducing bacteria concentration of the positive reference sample.
The application further discloses a kit for detecting the concentration of sulfate-reducing bacteria, which comprises a culture solution without particulate matters, wherein the culture solution without particulate matters consists of NaCl、K2HPO4、MgSO4、Fe(NH4)2(SO4)2、FeSO4、NH4Cl、Na2SO4、CaCl2、70％ lactic acid, yeast extract and water.
In one implementation mode of the application, the concentration of each component in the culture solution without the particulate matters of the kit is NaCl 4g/L、K2HPO4 0.5g/L、MgSO4 1.5g/L、Fe(NH4)2(SO4)2 1g/L、FeSO41g/L、NH4Cl 1g/L、Na2SO4 0.5g/L、CaCl2 0.2g/L、70％ g/L of lactic acid and 1g/L of yeast extract.
When the sulfate reducing bacteria concentration of the sample to be detected is detected, the culture solution without the particulate matters in the kit is adopted to culture the sample to be detected at a constant temperature, so that not only can the sulfate reducing bacteria with enough quantity be obtained by culture, but also the sediment in the culture product is relatively less, the interference on the detection result can be reduced, and the detection accuracy is improved. The culture solution without the particulate matters can also be used as a dilution solution for gradient dilution and an electrolyte for an electrical impedance method, and has multiple functions and purposes, and is simple and convenient to use.
The application has the beneficial effects that:
The method for detecting the concentration of the sulfate reducing bacteria utilizes the pulse signals generated by the sulfate reducing bacteria passing through micropores to detect the particle size and the concentration, and has the advantages of high accuracy, low detection limit and the like; the detection method is simple and easy to operate, can be used for detecting the concentration of the sulfate reducing bacteria of various samples, and is particularly suitable for detecting and monitoring the sulfate reducing bacteria in special environments such as aquaculture, oilfield pipelines and the like.
Drawings
FIG. 1 is a schematic diagram of a detection assembly according to an embodiment of the present application;
FIG. 2 is a graph showing the particle size concentration distribution of SRB cultured for 12 hours with 10-fold dilution of the sample in the example of the present application;
FIG. 3 is a graph showing the particle size concentration distribution of SRB cultured for 12 hours with a 100-fold dilution of the sample according to the example of the present application;
FIG. 4 is a distribution chart of the particle size concentration of SRB cultured for 12 hours with a 1000-fold dilution of the sample in the example of the present application;
FIG. 5 is a distribution diagram of the particle size concentration of SRB cultured for 12 hours with 10000-fold dilution of the sample in the example of the present application;
FIG. 6 is a distribution diagram of the particle size concentration of SRB cultured for 12 hours from 100000-fold diluted sample in the example of the present application;
FIG. 7 is a distribution diagram of the particle size concentration of SRB cultured for 12 hours in the positive control group sample according to the embodiment of the present application;
FIG. 8 is a linear plot of the detected concentration of each gradient diluted sample incubated for 12 hours in an example of the present application;
FIG. 9 is a distribution chart of the particle size concentration of SRB cultured for 96 hours with 10-fold dilution of the sample in the example of the present application;
FIG. 10 is a distribution chart of the particle size concentration of SRB cultured for 96 hours with 100-fold dilution of the sample in the example of the present application;
FIG. 11 is a graph showing the particle size concentration distribution of SRB cultured for 96 hours with 1000-fold dilution of the sample in the example of the present application;
FIG. 12 is a distribution diagram of the particle size concentration of SRB cultured for 96 hours with 10000-fold dilution of the sample in the example of the present application;
FIG. 13 is a distribution chart of particle size concentration of SRB cultured for 96 hours with 100000-fold diluted sample in the example of the present application;
FIG. 14 is a linear plot of the detected concentration of each gradient diluted sample incubated for 96 hours in an example of the present application;
FIG. 15 is a graph showing growth of sulfate-reducing bacteria in examples of the present application.
Detailed Description
The existing sulfate reducing bacteria concentration detection method has the defects and defects of high detection cost, low sensitivity, low accuracy and the like.
Therefore, the application designs a brand new detection method based on single particle detection for the content of the sulfate reducing bacteria by adopting a micropore electrical impedance method, namely, applying voltage to the liquid to be detected, enabling particles in the liquid to be detected to pass through micropores to generate voltage or current pulses, obtaining the particle size and concentration of the particles in the liquid to be detected by measuring pulse signals of the voltage or the current, and selecting the concentration of the particles with the particle size range of 500 nm-1300 nm as the concentration of the sulfate reducing bacteria.
In one implementation mode of the application, the detection can be carried out by directly adopting the Coulter particle size analyzer and two liquid tanks with electrodes, and the application has the advantages of high accuracy, low detection limit and the like, and has low detection cost, simplicity and easy operation. Even if the constant temperature culture is needed, the detection method of the application only needs to culture for 12-100 hours, and the culture time is short. The sulfate reducing bacteria concentration detection method can be used for detecting the sulfate reducing bacteria concentration of various samples, and is particularly suitable for detecting the sulfate reducing bacteria in aquaculture, oilfield pipeline monitoring and the like.
The application is described in further detail below with reference to specific examples and figures. The following examples are merely illustrative of the present application and should not be construed as limiting the application.
Examples
1. Sulfate reducing bacteria sample culture
1.1 Preparation of culture solution
The special culture solution for the sulfate reducing bacteria is prepared in the example, the formula of the culture solution is shown in table 1, and after the components are dissolved in water according to the proportion, the culture solution is sterilized by an autoclave for standby.
TABLE 1 culture broth without particulate matter
| Composition of the components | Content (g/L) | Composition of the components | Content (g/L) |
| NaCl | 4 | NH4Cl | 1 |
| K2HPO4 | 0.5 | Na2SO4 | 0.5 |
| MgSO4·7H2O | 1.5 | CaCl2 | 0.2 |
| Fe(NH4)2(SO4)2·6H2O | 1 | 70% Lactic acid | 5 |
| FeSO4 | 1 | Yeast paste | 1 |
1.2 Gradient dilution
In the sample to be tested SRB, prepared culture solution is used for carrying out 10 times, 100 times, 1000 times, 10000 times and 100000 times of 5 gradient dilution respectively, and the final volume is controlled at 100mL.
1.3 Inoculation
The 5 concentration gradient diluted SRB bacterial solutions were inoculated into 5 100mL culture flasks, respectively. Another 100mL culture flask was taken, and 100mL of sterilized blank culture solution was added as a negative control group, namely a negative reference sample. 100mL of SRB bacterial liquid which is cultured for 10 days is taken into another 100mL culture flask to be used as a positive control group, namely a positive reference sample.
1.4 Cultivation
The 7 flasks were sealed, incubated in a 35℃incubator, and 1mL of the incubated product was taken out for 12 hours and 96 hours, respectively, for determination of SRB particle concentration.
2. Sulfate reducing bacteria sample processing
2.1 Sampling
In this example, after 12 hours and 96 hours of culture, 1mL of each of 5 bacterial liquid experiments, negative control groups and positive control groups were taken and placed in 7 1.5mL centrifuge tubes.
2.2 Titration
1Mol/L hydrochloric acid was used, slowly added dropwise to the sampled 7 centrifuge tubes, and the solution was allowed to clear.
2.3 Centrifugation
And (3) placing the sample titrated by the acid solution into a centrifugal machine, centrifuging for 10 minutes at a rotating speed of 3000 rpm, and taking the supernatant as a liquid to be tested.
3. Sulfate Reducing Bacteria (SRB) concentration determination
3.1 Test instrument
The method adopts a nano Coulter particle size analyzer developed and produced by Shenzhen technology Co., ltd, and adopts a micropore electrical impedance method for detection, and the detection principle is as follows: etching a micropore with the aperture of about 3000nm on a silicon-based chip, loading the chip into a detection assembly, adding electrolyte on two sides of the detection assembly, applying voltage on two sides of the detection assembly, generating a potential pulse when particles pass through the micropore, and collecting and analyzing a potential pulse signal by an instrument to obtain particle size and concentration data of the via hole particles. The detection assembly is shown in fig. 1, and consists of a sample tank 1, an electrolyte tank 2, a sample tank electrode 11, an electrolyte tank electrode 21 and a silicon-based chip 3, wherein the silicon-based chip 3 is provided with micropores 31.
3.2 Sample application
In this example, 200. Mu.L of electrolyte is added to one liquid tank of the detection assembly, and 200. Mu.L of liquid to be detected is added to the other liquid tank, namely the detection card sample tank.
3.2 Detection
And 200mv of voltage is input on the operation software, particles pass through micropores, and the software automatically measures the particle size distribution and concentration of SRB in a sample.
4. Data analysis
4.1 Concentration correction
And removing background concentration according to the concentration test data of the negative control group, and eliminating concentration errors caused by culture and titration processes.
4.2 Particle size correction
According to the theoretical particle size of the SRB, judging that the particles with the particle size of 500-1300nm are real SRB, removing the particles out of the range, and combining with 4.1 to obtain the real concentration of the SRB.
4.3 Test results for 12 hours of incubation
After 12 hours of incubation, the test results of the experimental group, the negative control group, and the positive control group are shown in table 2 and fig. 2 to 7, and the concentration linearity chart is shown in fig. 8.
TABLE 2 results of SRB particle size and concentration tests for 12 hours of incubation
| Sample name | SRB mean particle size (nm) | SRB concentration (particles/mL) |
| Dilution by 10 times | 788 | 1.26E+09 |
| 100-Fold dilution | 800 | 1.32E+08 |
| Dilution by 1000 times | 790 | 1.09E+07 |
| 10000 Times dilution | 768 | 1.24E+06 |
| 100000 Times dilution | 801 | 1.16E+05 |
| Negative control | / | 0 |
| Positive control | 797 | 3.32E+11 |
Fig. 2 is a SRB particle size concentration profile of a 10-fold diluted sample, fig. 3 is a SRB particle size concentration profile of a 100-fold diluted sample, fig. 4 is a SRB particle size concentration profile of a 1000-fold diluted sample, fig. 5 is a SRB particle size concentration profile of a 10000-fold diluted sample, fig. 6 is a SRB particle size concentration profile of a 100000-fold diluted sample, and fig. 7 is a SRB particle size concentration profile of a positive control group sample.
4.4 Test results for 96 hours of incubation
After 96 hours of incubation, the test results of the experimental group, the negative control group, and the positive control group are shown in table 3 and fig. 9 to 13, and the concentration linearity chart is shown in fig. 14.
TABLE 3 results of SRB particle size and concentration test for 60 hours of incubation
| Sample name | SRB mean particle size (nm) | SRB concentration (particles/mL) |
| Dilution by 10 times | 805 | 6.18E+09 |
| 100-Fold dilution | 799 | 5.94E+08 |
| Dilution by 1000 times | 786 | 5.67E+07 |
| 10000 Times dilution | 810 | 5.70E+06 |
| 100000 Times dilution | 786 | 6.10E+05 |
| Negative control | / | 0 |
| Positive control | 801 | 1.51E+12 |
Fig. 9 is a SRB particle size concentration profile of a 10-fold diluted sample, fig. 10 is a SRB particle size concentration profile of a 100-fold diluted sample, fig. 11 is a SRB particle size concentration profile of a 1000-fold diluted sample, fig. 12 is a SRB particle size concentration profile of a 10000-fold diluted sample, and fig. 13 is a SRB particle size concentration profile of a 100000-fold diluted sample.
5. Test determination
First, according to the growth condition of SRB bacteria, a growth curve of the amplification factor changing with the culture time is drawn. The growth of the samples was tested according to the growth of 10-fold dilution samples, and the growth curves were drawn according to the multiple of each time period relative to the culture for 0 hour, i.e., the culture amplification multiple. The results are shown in FIG. 15.
As can be seen from the growth curve, the culture amplification factor corresponding to 12 hours of culture was 11.41, and the culture amplification factor corresponding to 96 hours of culture was 52.03.
Calculating the original sulfate reducing bacteria concentration of the sample to be measured according to the formula,
Original sulfate reducing bacteria concentration of sample to be detected = sulfate reducing bacteria detection concentration x dilution multiple ≡culture amplification multiple
The calculation results of 5 gradient dilution samples of 12 hours, 10 times, 100 times, 1000 times, 10000 times and 100000 times are sequentially as follows: 1.10E+09, 1.16E+09, 9.55E+08, 1.09E+09, 1.02E+09, and the average value of 5 concentration gradient dilution calculation results was 1.06E+09.
The calculation results of 5 gradient dilution samples of 96 hours, 10 times, 100 times, 1000 times, 10000 times and 100000 times are sequentially as follows: 1.19E+09, 1.14E+09, 1.09E+09, 1.10E+09, 1.17E+09, the average value of 5 concentration gradient dilution calculation results was 1.14E+09.
The results show that the original sulfate reducing bacteria concentration data of the sample to be tested obtained through final calculation are equivalent after 12 hours and 96 hours of culture, and the accurate test result is shown. As can also be seen from fig. 8 and 14, the linear relationship between the measured concentration value and the dilution factor of each gradient diluted sample is good, and the linear correlation dilution of the measured concentration value and the dilution factor for 12 hours of culture R 2 =0.9999 and the linear correlation dilution of the measured concentration value and the dilution factor for 96 hours of culture R 2 = 0.9991 also indicate that the original sulfate-reducing bacteria concentration of the sample to be measured obtained by the calculation of the formula is high in accuracy.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.
Claims (17)
1. A method for detecting sulfate-reducing bacteria concentration, characterized by: the method comprises the steps of carrying out gradient dilution on a sample to be detected, and respectively carrying out constant-temperature culture on the samples subjected to gradient dilution; then, respectively detecting the concentration of sulfate reducing bacteria on the culture products; according to the gradient dilution multiple, the corresponding sulfate reducing bacteria detection concentration and the culture time, the original sulfate reducing bacteria concentration of the sample to be detected is calculated;
The sulfate reducing bacteria concentration detection is carried out on the culture product, which comprises the steps of carrying out acid solution titration on the culture product until clear solution is obtained, centrifuging the clear solution, and taking supernatant as a liquid to be detected; applying voltage to the liquid to be measured, enabling particles in the liquid to be measured to pass through micropores to generate voltage or current pulses, obtaining the particle size and concentration of the particles in the liquid to be measured through pulse signals of the voltage or current, and selecting the concentration of the particles with the particle size range of 500-1300 nm as the concentration of sulfate reducing bacteria;
The method for applying voltage to the liquid to be measured comprises the steps of adopting two liquid tanks with electrodes, placing the liquid to be measured in one liquid tank, placing electrolyte in the other liquid tank, communicating the two liquid tanks through micropores, applying voltage to the electrodes of the two liquid tanks, and enabling particles in the liquid to be measured to enter the other liquid tank through the micropores, so that a pulse signal of voltage or current is generated on the electrodes;
And the centrifugation condition is that the centrifugal force is applied for 5-15 minutes at the rotating speed of not more than 3000 rpm.
2. The method according to claim 1, characterized in that: the acidic solution comprises at least one of hydrochloric acid and nitric acid.
3. The method according to claim 1, characterized in that: the aperture of the micropore is 2000 nm-40000 nm.
4. The method according to claim 1, characterized in that: the calculation of the original sulfate reducing bacteria concentration of the sample to be measured comprises determining the culture amplification times of the sulfate reducing bacteria corresponding to the culture time according to the growth curve of the sulfate reducing bacteria, calculating the original sulfate reducing bacteria concentration of the sample to be measured according to the following formula,
Original sulfate reducing bacteria concentration of sample to be tested = sulfate reducing bacteria detection concentration x dilution factor/(culture amplification factor).
5. The method according to claim 4, wherein: the method comprises the steps of calculating the original sulfate reducing bacteria concentration of a sample to be measured, and further comprises the steps of calculating the original sulfate reducing bacteria concentration of the sample to be measured according to each gradient dilution sample, and taking the average value of calculation results of each gradient dilution sample as the final original sulfate reducing bacteria concentration of the sample to be measured.
6. The method according to claim 1, characterized in that: the constant temperature culture condition is that the culture is carried out for 12-100 hours at 35 ℃.
7. The method according to claim 1, characterized in that: and controlling the conditions of the constant temperature culture to ensure that the culture amplification factor of the culture product is more than 10 times.
8. The method according to claim 1, characterized in that: the culture solution for constant temperature culture is a culture solution without particulate matters, and the culture solution without particulate matters consists of NaCl、K2HPO4、MgSO4、Fe(NH4)2(SO4)2、FeSO4、NH4Cl、Na2SO4、CaCl2、70% lactic acid, yeast extract and water.
9. The method according to claim 8, wherein: the concentration of each component in the culture solution without the particulate matters is NaCl 4g/L、K2HPO4 0.5g/L、MgSO4 1.5g/L、Fe(NH4)2(SO4)2 1g/L、FeSO4 1g/L、NH4Cl 1g/L、Na2SO4 0.5g/L、CaCl2 0.2g/L、70% g/L of lactic acid and 1g/L of yeast extract.
10. The method according to claim 9, wherein: the gradient dilution adopts the culture solution without the particulate matters.
11. The method according to claim 10, wherein: the multiples of the gradient dilution include 10-fold, 100-fold, 1000-fold, 10000-fold and 100000-fold.
12. The method according to claim 9, wherein: the electrolyte adopts the culture solution without the particulate matters.
13. The method according to claim 1, characterized in that: and judging and calculating the accuracy of the original sulfate reducing bacteria concentration of the sample to be detected according to the linear correlation of the sulfate reducing bacteria detection concentration and the dilution multiple of each gradient dilution sample, wherein the better the linear correlation is, the higher the accuracy is.
14. The method according to any one of claims 1-13, wherein: the method also comprises the step of performing a control test by adopting a negative reference sample and/or a positive reference sample; the negative reference sample is a solution which does not contain bacteria and has the same components as the liquid to be detected; the positive reference sample is bacterial suspension with known sulfate reducing bacteria concentration and the same components as the liquid to be tested.
15. The method according to claim 14, wherein: and removing the background concentration according to the sulfate reducing bacteria detection concentration of the negative reference sample.
16. A kit for detecting the concentration of sulfate-reducing bacteria, characterized in that: comprises a culture solution without granular matters, wherein the culture solution without granular matters consists of NaCl、K2HPO4、MgSO4、Fe(NH4)2(SO4)2、FeSO4、NH4Cl、Na2SO4、CaCl2、70% lactic acid, yeast extract and water.
17. The kit of claim 16, wherein: the concentration of each component in the culture solution without the particulate matters is NaCl 4g/L、K2HPO4 0.5g/L、MgSO4 1.5g/L、Fe(NH4)2(SO4)2 1g/L、FeSO4 1g/L、NH4Cl 1g/L、Na2SO4 0.5g/L、CaCl2 0.2g/L、70% g/L of lactic acid and 1g/L of yeast extract.
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