CN115824932A - 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 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 the concentration of the particles in the liquid to be detected through measuring voltage or current pulse signals, 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. According to the method for detecting the concentration of the sulfate reducing bacteria, the pulse signals generated by the sulfate reducing bacteria passing through the micropores are used for detecting the particle size and the concentration, and the method has the advantages of high accuracy, low detection limit and the like; in addition, 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, oil field 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 with various forms and nutrition types, and can utilize Sulfate or other oxidation state sulfides as electron acceptors to dissimilatory organic substances, and the biological corrosion of the SRB brings huge harm and loss.
Currently, for the concentration test of SRB bacteria in a sample, there are several methods: (1) And (3) a parallel sterilization dilution method, namely, after the sample is diluted in a multiple ratio, the sample is divided into different test tubes for parallel culture, then the sample precipitate is observed by adopting a microscope, and the concentration of the SRB cells is judged according to the precipitate and the dilution multiple of the test tubes. However, this method usually requires 14 days or more for the culture. (2) Immunological method, SRB intracellular has APS reductase, which is the characteristic enzyme of SRB; the APS reductase can catalyze adenosine-5' -phosphate sulfate reductase to perform reduction reaction and produce related specific products; the bacterial concentration of the SRB can be judged by utilizing the strength of the chromogenic reaction between the product and the color developing agent. However, this method cannot test a relatively low concentration of bacterial sample. (3) Adenosine Triphosphate (ATP), a compound present in all living biological cells, is present in an amount proportional to the cell concentration. However, the method can only detect the total number of microorganisms, cannot specifically detect the SRB, is easily interfered by other microorganisms, and is inaccurate in test. (4) And the PCR method is that bacteria in the sample are cracked, the bacteria DNA is subjected to PCR amplification, and the Ct value is tested by PCR to reversely deduce the concentration of the bacteria in the sample. However, the complexity of the components in the sample of SRB bacteria generally affects the amplification effect of PCR, and thus the test result.
Therefore, there is no good method for accurately testing the concentration of sulfate-reducing bacteria. How to rapidly and accurately detect the concentration of sulfate reducing bacteria still remains a problem to be solved in the field.
Disclosure of Invention
The purpose of the application is to provide a novel method and a kit for detecting the concentration of sulfate reducing bacteria.
The following technical scheme is adopted in the application:
one aspect of the application discloses a method for detecting concentration of sulfate reducing bacteria, which comprises the steps of applying voltage to 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 voltage or current pulse signals, and selecting the concentration of the particles with the particle size range of 500-1300nm as the concentration of the sulfate reducing bacteria.
It should be noted that, the present application creatively adopts an electrical impedance method, utilizes the coulter principle to detect the concentration of the solution to be detected of sulfate reducing bacteria, and uses the concentration of particles with the particle size range of 500nm to 1300nm in the solution to be detected as the concentration of sulfate reducing bacteria according to research and analysis. The detection method can quickly and accurately detect the concentration of the sulfate reducing bacteria; in principle, as long as the solution to be detected contains a sulfate reducing bacterium, a pulse signal is generated when the sulfate reducing bacterium passes through the micropores, so that the sulfate reducing bacterium can be detected, and the detection sensitivity is high and the detection limit is low.
It should be noted that the solution to be tested of the present application usually adopts a culture product of sulfate-reducing bacteria or a supernatant obtained by centrifuging the culture product, and the solution to be tested generally does not contain other particles except the sulfate-reducing bacteria; therefore, the particles detected in the solution to be detected are generally sulfate-reducing bacteria. However, to exclude other possible interferences, the present application refers to the concentration of particles in a particular size range as the concentration of sulfate-reducing bacteria; the specific particle size range is a particle size range which can cover the size of most sulfate reducing bacteria and is obtained through a great deal of research and practical analysis, namely 500-1300 nm.
In an implementation of this application, the mode of the applied voltage to the liquid that awaits measuring includes, adopts two cistern of taking the electrode, will wait to detect that the liquid is placed in one of them cistern, and electrolyte is placed to another cistern, and two cistocks are by the micropore intercommunication, and applied voltage on the electrode of two cistocks makes the granule in waiting to detect the liquid pass through the micropore and gets into in another cistern to produce the pulse signal of voltage or electric current on the electrode.
In an implementation manner of the present application, the method for detecting concentration of sulfate-reducing bacteria further includes titrating an acidic solution of a sample to be detected until a clear solution is obtained, centrifuging the clear solution, and taking a supernatant as a solution to be detected.
It should be noted that the sample to be tested is generally a sulfate reducing bacteria culture product of a collected sample, and some precipitates may be generated in the process of sulfate reducing bacteria culture, so as to avoid the influence of the precipitates on the detection result. It is understood that during the culture of sulfate-reducing bacteria, the culture solution is turbid due to the generation of precipitates, and the culture product is gradually cleared during the titration with an acidic solution. Of course, since sulfate-reducing bacteria are still suspended in the culture broth and the supernatant; thus, the acid 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.
It should be noted that, in the acid solution titration of the present application, on one hand, it is necessary to be able to dissolve the precipitate, and on the other hand, adverse effects on sulfate reducing bacteria should be avoided as much as possible; therefore, hydrochloric acid or nitric acid is preferably used. It is understood that other acidic solutions with similar functions, such as hydrochloric acid and nitric acid, can be used under the inventive concept of the present application.
In one implementation of the present application, the centrifugation conditions are centrifugation at a rotation speed of not more than 3000 rpm for 5 to 15 minutes.
It should be noted that the purpose of centrifugation of the supernatant in the present application is mainly to remove some possible large particle precipitates; however, to avoid sulfate-reducing bacteria also being precipitated by centrifugation, it is preferred in this application to carry out the centrifugation at a rotational speed not exceeding 3000 rpm.
In one implementation of the present application, the pore size of the micropores is 2000nm to 40000nm.
The micropores are mainly used for allowing 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 an implementation manner of the present application, the method for detecting concentration of sulfate-reducing bacteria further includes performing gradient dilution on a sample to be detected, and performing constant-temperature culture on the samples subjected to gradient dilution respectively; then, respectively carrying out sulfate reducing bacteria concentration detection on the culture products; and calculating the original sulfate reducing bacteria concentration of the sample to be detected according to the gradient dilution multiple, the corresponding sulfate reducing bacteria detection concentration and the culture time.
It should be noted that, the concentration of the sulfate-reducing bacteria is detected after the sample to be detected is subjected to gradient dilution and constant temperature culture, mainly to ensure that the sample to be detected contains a sufficient amount of sulfate-reducing bacteria. It can be understood that the object of the isothermal culture of the present application is the sulfate-reducing bacteria; therefore, the incubation can be carried out using a conventional sulfate-reducing bacteria culture solution, and is not particularly limited.
In one implementation of the present application, calculating the concentration of the original sulfate-reducing bacteria in the sample to be tested includes 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 concentration of the original sulfate-reducing bacteria in the sample to be tested according to the following formula,
original sulfate reducing bacteria concentration of a sample to be detected = sulfate reducing bacteria detection concentration x dilution times ÷ culture amplification times.
In one implementation manner of the application, the concentration of the original sulfate reducing bacteria of the sample to be detected is calculated according to each gradient dilution sample, and the average value of the calculation results of each gradient dilution sample is taken as the final concentration of the original sulfate reducing bacteria of the sample to be detected.
It should be noted that, in principle, the original concentration of the sulfate-reducing bacteria in the sample to be tested can be directly calculated by diluting the sample according to any gradient; however, in order to ensure the accuracy, the present application preferably uses the average value of the calculation results of each gradient dilution sample as the final original concentration of the sulfate-reducing bacteria in the sample to be tested.
In one implementation of the present application, the conditions of the constant temperature culture are 35 ℃ for 12 to 100 hours.
In one implementation of the present application, the conditions of the isothermal culture are controlled such that the culture amplification factor of the culture product is 10 times or more.
In the present application, a growth curve of sulfate-reducing bacteria was plotted for 0 to 117 hours of culture, and the results showed that the culture amplification factor increased relatively well as the culture time increased from the start of culture to about 100 hours of culture; after 100 hours of culture, the culture expansion rate is relatively slow although it is also increasing. Therefore, the preferred incubation time for this application is 12 to 100 hours. In principle, the culture amplification times of the culture products are more than 10 times, so that a better detection result can be obtained; therefore, the culture time of 12 to 100 hours can substantially satisfy the requirement. As for the culture temperature, it is generally 35 ℃ and, of course, it can be adjusted within the allowable range of the test as required. In addition, the detection method can meet the detection requirement only by 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 of the present application, the culture solution for constant temperature culture is a culture solution without particulate matter, and the culture solution without particulate matter is composed of NaCl and K 2 HPO 4 、MgSO 4 、Fe(NH 4 ) 2 (SO 4 ) 2 、FeSO 4 、NH 4 Cl、Na 2 SO 4 、CaCl 2 70% lactic acid, yeast extract and water.
In one implementation of the present application, the concentrations of the components in the culture medium without particulate matter are NaCl4g/L, K 2 HPO 4 0.5g/L、MgSO 4 1.5g/L、Fe(NH 4 ) 2 (SO 4 ) 2 1g/L、FeSO 4 1g/L、NH 4 Cl1g/L、Na 2 SO 4 0.5g/L、CaCl 2 0.2g/L, 5g/L of 70% lactic acid and 1g/L of yeast extract.
In principle, any conventional culture solution of sulfate-reducing bacteria can be applied to the present application; however, the concentration detection method of the present application requires that the culture product contains as little precipitate as possible, and the culture solution itself needs to be free of particulate matter; therefore, the culture solution is optimized and improved. By adopting the culture solution without the granular substances with the improved formula, the precipitation in the culture product can be reduced as much as possible, so that the detection interference caused by the precipitation is reduced, and the detection accuracy is improved. It is understood that the specific concentrations of the above components are those specifically adopted in one implementation manner of the present application, and the concentrations of the components can be appropriately adjusted without affecting the performance and culture effect of the culture solution.
In one implementation of the present application, the gradient dilution of the sample to be tested is performed using a constant temperature culture solution, i.e., preferably using the culture solution without particulate matter of the present application.
In one implementation of the present application, the fold-gradient dilution specifically includes 10 fold, 100 fold, 1000 fold, 10000 fold, and 100000 fold.
In one embodiment of the present application, the electrolyte is also a culture solution for isothermal cultivation, i.e. preferably a culture solution without particulate matter according to the present application.
The electrolyte has the functions of providing a liquid environment for an electrical impedance method and simultaneously providing a detection environment for sulfate reducing bacteria; therefore, it is preferable to use a culture solution for isothermal culture, i.e., a culture solution without particulate matter of the present application.
In an implementation manner of the present application, the method for detecting concentration of sulfate-reducing bacteria further includes determining accuracy of the original concentration of sulfate-reducing bacteria of the sample to be detected, which is obtained by calculation, according to linear correlation between the detected concentration of sulfate-reducing bacteria of each gradient dilution sample and the dilution multiple, where the linear correlation is better and the accuracy is higher.
The original sulfate reducing bacteria concentration of the sample to be detected is obtained by calculation according to the detection concentration, dilution multiple and culture amplification multiple of the sulfate reducing bacteria; therefore, if the linear correlation between the detected concentration of the sulfate reducing bacteria and the dilution multiple is better, the more accurate the original concentration of the sulfate reducing bacteria of the sample to be detected, which is directly obtained by calculation through a formula, is proved.
In one implementation manner of the present application, the method for detecting the concentration of sulfate-reducing bacteria further comprises performing a control test by using a negative reference sample and/or a positive reference sample; wherein, the negative reference sample is a solution which does not contain bacteria and has the same component as the liquid to be detected; the positive reference sample is a bacterial suspension with known concentration of sulfate reducing bacteria and the same component as the tested liquid.
It should be noted that, in an implementation manner of the present 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 present application, the background concentration is removed directly from the sulfate-reducing bacteria detection concentration of the negative reference sample. Alternatively, the calculation result of the original concentration of sulfate-reducing bacteria in the sample to be tested may be corrected based on the calculation result of the original concentration of sulfate-reducing bacteria in the positive reference sample.
Another aspect of the application discloses a kit for detecting the concentration of sulfate reducing bacteria, which comprises a culture solution without granular substances, wherein the culture solution without granular substances is composed of NaCl and K 2 HPO 4 、MgSO 4 、Fe(NH 4 ) 2 (SO 4 ) 2 、FeSO 4 、NH 4 Cl、Na 2 SO 4 、CaCl 2 70% lactic acid, yeast extract and water.
In one implementation of the present application, the concentrations of the components in the particulate matter-free culture solution of the kit of the present application are NaCl4g/L, K 2 HPO 4 0.5g/L、MgSO 4 1.5g/L、Fe(NH 4 ) 2 (SO 4 ) 2 1g/L、FeSO 4 1g/L、NH 4 Cl 1g/L、Na 2 SO 4 0.5g/L、CaCl 2 0.2g/L, 5g/L of 70% lactic acid and 1g/L of yeast extract.
It should be noted that, when the concentration of sulfate reducing bacteria is detected on a sample to be detected, the culture solution without particulate matter in the kit of the present application is used to perform constant temperature culture on the sample to be detected, so that not only can enough sulfate reducing bacteria be obtained by culture, but also the precipitate in the cultured product is relatively less, thereby reducing the interference on the detection result and improving the detection accuracy. In addition, the culture solution without the granular substances can also be used as a dilution solution for gradient dilution and an electrolyte for an electrical impedance method, has multiple functions and purposes, and is simple and convenient to use.
The beneficial effect of this application lies in:
according to the method for detecting the concentration of the sulfate reducing bacteria, the pulse signals generated by the sulfate reducing bacteria passing through the micropores are used for detecting the particle size and the concentration, and the method has the advantages of high accuracy, low detection limit and the like; in addition, 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, oil field pipelines and the like.
Drawings
FIG. 1 is a schematic structural diagram of a detection assembly according to an embodiment of the present application;
FIG. 2 is a graph showing the particle size distribution of SRB after culturing a 10-fold diluted sample for 12 hours in the examples of the present application;
FIG. 3 is a graph showing the particle size distribution of SRB after culturing a 100-fold diluted sample for 12 hours in the examples of the present application;
FIG. 4 is a graph showing the SRB particle size distribution of a 1000-fold diluted sample cultured for 12 hours in the examples of the present application;
FIG. 5 is a graph showing the distribution of the SRB particle size concentration of a 10000-fold diluted sample cultured for 12 hours in the examples of the present application;
FIG. 6 is a graph showing the distribution of the SRB particle size concentration of a 100000-fold diluted sample cultured for 12 hours in an example of the present application;
FIG. 7 is a graph showing the SRB particle size concentration distribution of a positive control sample cultured for 12 hours in the examples of the present application;
FIG. 8 is a linear plot of the detected concentration of each sample diluted in a gradient incubated for 12 hours in the examples of the present application;
FIG. 9 is a graph showing the particle size distribution of SRB after culturing a 10-fold diluted sample for 96 hours in the examples of the present application;
FIG. 10 is a graph showing the distribution of the SRB particle size concentration of 100-fold diluted samples cultured for 96 hours in the examples of the present application;
FIG. 11 is a graph showing the concentration of SRB particles at 96 hours after culturing a 1000-fold diluted sample in an example of the present application;
FIG. 12 is a graph showing the distribution of the SRB particle size concentration of a 10000-fold diluted sample cultured for 96 hours in the examples of the present application;
FIG. 13 is a graph showing the distribution of the SRB particle size concentration of a 100000-fold diluted sample cultured for 96 hours in the present example;
FIG. 14 is a line graph showing the detected concentration of each sample diluted in a gradient for 96 hours in the example of the present application;
FIG. 15 is a graph of the growth of sulfate-reducing bacteria in the examples of the present application.
Detailed Description
The existing sulfate reducing bacteria concentration detection method generally has the defects and shortcomings of high detection cost, low sensitivity, low accuracy and the like.
Therefore, the method adopts a micropore electrical impedance method, designs a brand-new method for detecting the content of the sulfate reducing bacteria based on single-particle detection, namely, applies voltage to the liquid to be detected, enables particles in the liquid to be detected to pass through micropores, generates voltage or current pulses, obtains the particle size and the concentration of the particles in the liquid to be detected by measuring voltage or current pulse signals, and selects the concentration of the particles with the particle size range of 500 nm-1300 nm as the concentration of the sulfate reducing bacteria.
In an implementation mode of the application, the coulter particle analyzer and two liquid tanks with electrodes can be directly adopted for detection, and the device not only has the advantages of high accuracy, low detection limit and the like, but also is low in detection cost, simple and easy to operate. Even if constant-temperature culture is required, the detection method only needs to be cultured for 12-100 hours, and the culture time is short. The method for detecting the concentration of the sulfate reducing bacteria can be used for detecting the concentration of the sulfate reducing bacteria of various samples, and is particularly suitable for detecting the sulfate reducing bacteria in aquaculture, oilfield pipeline monitoring and the like.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.
Examples
1. Sulfate reducing bacteria sample culture
1.1 preparation of culture solution
The present example prepares a culture solution for sulfate-reducing bacteria, the formula of which is shown in table 1, and the components are dissolved in water according to the mixture ratio and sterilized by an autoclave for standby.
TABLE 1 culture solution without particulate matter
| Composition (I) | Content (g/L) | Composition (I) | Content (g/L) |
| NaCl | 4 | NH 4 Cl | 1 |
| K 2 HPO 4 | 0.5 | Na 2 SO 4 | 0.5 |
| MgSO 4 ·7H 2 O | 1.5 | CaCl 2 | 0.2 |
| Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O | 1 | 70% lactic acid | 5 |
| FeSO 4 | 1 | Yeast cream | 1 |
1.2 gradient dilution
In this example, a sample SRB to be tested was taken and diluted with a prepared culture medium in 5 dilutions in 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold gradient volumes, respectively, to a final volume of 100mL.
1.3 inoculation
5 SRB bacterial liquids with concentration gradient dilution are respectively inoculated into 5 100mL culture flasks. Another 100mL culture flask was filled with 100mL of sterilized blank culture medium as a negative control, i.e., a negative reference sample. 100mL of the SRB bacterial liquid which has been cultured for 10 days is taken to be placed in another 100mL culture bottle to be used as a positive control group, namely a positive reference sample.
1.4 cultivation
The 7 culture flasks were sealed, placed in a 35 ℃ incubator, and cultured, and 1mL of the culture product was taken out for each of 12 hours and 96 hours of the incubation, and used for determination of the SRB particle concentration.
2. Sulfate reducing bacteria sample processing
2.1 sampling
In this example, after culturing for 12 hours and 96 hours, 1mL of each of 5 gradient dilution-cultured bacterial suspension experimental groups, negative control groups and positive control groups was taken and placed in 7 1.5mL centrifuge tubes.
2.2 titration
Slowly dropwise adding 1mol/L hydrochloric acid into 7 centrifuge tubes which have been sampled, and titrating until the solution is clear.
2.3 centrifugation
And (3) putting the titrated sample of the acid solution into a centrifuge, centrifuging for 10 minutes at the rotating speed of 3000 r/min, and taking the supernatant as the solution to be detected.
3. Sulfate Reducing Bacteria (SRB) concentration determination
3.1 test Instrument
The example adopts a nanometer Coulter particle analyzer developed and produced by Ruizhen Zhicheng (Shenzhen) science and technology Limited company, and uses a micropore electrical impedance method for detection, wherein the detection principle is as follows: a micropore with the aperture of about 3000nm is etched on a silicon-based chip, the chip is arranged in a detection assembly, two electrodes and two liquid tanks are arranged on two sides of the detection assembly, electrolyte is added into the two sides, after voltage is applied, a potential pulse is generated when particles pass through the micropore, and an instrument collects and analyzes the potential pulse signal to obtain the particle size and concentration data of the via hole particles. The detection assembly is shown in fig. 1, and comprises 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 Loading
In this embodiment, 200. Mu.L of the electrolyte solution is added to one of the liquid tanks of the detection assembly, and 200. Mu.L of the solution to be detected is added to the other liquid tank, i.e., the sample tank of the detection card.
3.2 detection
200mv voltage is input into operation software, particles pass through the micropores, and the software automatically measures the particle size distribution and concentration of the SRB in the sample.
4. Data analysis
4.1 concentration correction
According to the concentration test data of the negative control group, the background concentration is removed, and the concentration error caused by the culture and titration processes is eliminated.
4.2 particle size correction
And (3) according to the theoretical particle size of the SRB, judging that the particles of 500-1300nm are real SRB, removing the particles outside the range, and combining 4.1 to obtain the real concentration of the SRB.
4.3 incubation for 12 hours test results
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 linear graph of the concentration is shown in fig. 8.
TABLE 2 SRB particle size and concentration test results for 12 hours of culture
| Sample name | SRB average particle size (nm) | SRB concentration (particles/mL) |
| 10 times dilution | 788 | 1.26E+09 |
| 100 |
800 | 1.32E+08 |
| 1000 times dilution | 790 | 1.09E+07 |
| 10000 times dilution | 768 | 1.24E+06 |
| 100000 times diluted | 801 | 1.16E+05 |
| Negative control | / | 0 |
| Positive control | 797 | 3.32E+11 |
Fig. 2 is a SRB particle size concentration distribution diagram of a 10-fold diluted sample, fig. 3 is a SRB particle size concentration distribution diagram of a 100-fold diluted sample, fig. 4 is a SRB particle size concentration distribution diagram of a 1000-fold diluted sample, fig. 5 is a SRB particle size concentration distribution diagram of a 10000-fold diluted sample, fig. 6 is a SRB particle size concentration distribution diagram of a 100000-fold diluted sample, and fig. 7 is a SRB particle size concentration distribution diagram of a positive control sample.
4.4 incubation for 96 hours test results
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 linear graph of the concentration is shown in fig. 14.
TABLE 3 SRB particle size and concentration test results for 60 hours of incubation
| Sample name | SRB average particle size (nm) | SRB concentration (particles/mL) |
| 10 times dilution | 805 | 6.18E+09 |
| 100 times dilution | 799 | 5.94E+08 |
| 1000 times dilution | 786 | 5.67E+07 |
| 10000 times dilution | 810 | 5.70E+06 |
| 100000 times diluted | 786 | 6.10E+05 |
| Negative control | / | 0 |
| Positive control | 801 | 1.51E+12 |
Fig. 9 is a SRB particle size concentration profile for a 10-fold dilution sample, fig. 10 is a SRB particle size concentration profile for a 100-fold dilution sample, fig. 11 is a SRB particle size concentration profile for a 1000-fold dilution sample, fig. 12 is a SRB particle size concentration profile for a 10000-fold dilution sample, and fig. 13 is a SRB particle size concentration profile for a 100000-fold dilution sample.
5. Test determination
Firstly, according to the growth condition of SRB bacteria, a growth curve of the amplification factor along with the change of culture time is drawn. In this example, the growth of 10-fold diluted samples was measured for 0 to 117 hours of culture, and the growth curve was plotted based on the fold of each time period relative to 0 hour of culture, i.e., the culture amplification fold. The results are shown in FIG. 15.
From the growth curve, the amplification factor in culture was 11.41 for 12 hours and 52.03 for 96 hours.
Calculating the original sulfate reducing bacteria concentration of the sample to be detected according to a formula,
original sulfate reducing bacteria concentration of sample to be detected = sulfate reducing bacteria detection concentration x dilution times/culture amplification times
The calculation results of 5 gradient dilution samples cultured for 12 hours and 10 times, 100 times, 1000 times, 10000 times and 100000 times are as follows in sequence: the average value of the calculated results of 1.10E +09, 1.16E +09, 9.55E +08, 1.09E +09, 1.02E +09,5 concentration gradient dilutions is 1.06E +09.
The calculation results of 5 gradient dilution samples cultured for 96 hours and 10 times, 100 times, 1000 times, 10000 times and 100000 times are as follows in sequence: the average value of the results of 1.19E +09, 1.14E +09, 1.09E +09, 1.10E +09, 1.17E +09,5 concentration gradient dilutions is 1.14E +09.
The results show that the original sulfate-reducing bacteria concentration data of the sample to be tested obtained by final calculation are equivalent after the culture for 12 hours and the culture for 96 hours, which shows that the test result is accurate, and the concentration test and calculation method of the embodiment can accurately and stably measure the concentration and the particle size of the SRB in the sample to be tested. Furthermore, as can be seen from FIGS. 8 and 14, the linear relationship between the concentration values measured in the respective samples diluted in the respective gradients was good, and the dilution R was a linear correlation between the concentration values measured in the 12-hour incubation and the dilution factor 2 =0.9999, dilution R of the linear correlation between the concentration value measured at 96 hours of incubation and the dilution factor 2 And =0.9991, which also indicates that the concentration of the original sulfate-reducing bacteria of the sample to be tested obtained by calculation through the formula is high in accuracy.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A method for detecting the concentration of sulfate reducing bacteria is characterized by comprising the following steps: the method comprises the steps of applying voltage to 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 by measuring voltage or current pulse signals, and selecting the concentration of the particles with the particle size range of 500 nm-1300 nm as the concentration of sulfate reducing bacteria.
2. The method of claim 1, wherein: 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 of the liquid tanks, 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, thereby generating pulse signals of voltage or current on the electrodes.
3. The method of claim 2, wherein: carrying out acid solution titration on a sample to be detected until a clear solution is obtained, centrifuging the clear solution, and taking a supernatant as the liquid to be detected;
preferably, the acidic solution comprises at least one of hydrochloric acid and nitric acid;
preferably, the centrifugation condition is centrifugation for 5 to 15 minutes at a rotating speed of not more than 3000 revolutions per minute;
preferably, the pore diameter of the micropores is 2000nm to 40000nm.
4. The method of claim 3, wherein: the method also comprises the steps of carrying out gradient dilution on the sample to be detected, and respectively carrying out constant-temperature culture on the sample subjected to gradient dilution; then, respectively carrying out sulfate reducing bacteria concentration detection on the culture products; calculating the original sulfate reducing bacteria concentration of the sample to be detected according to the gradient dilution multiple, the corresponding sulfate reducing bacteria detection concentration and the culture time;
preferably, the calculating the concentration of the original sulfate-reducing bacteria of the sample to be detected comprises determining the culture amplification times of the sulfate-reducing bacteria corresponding to the culture time according to a growth curve of the sulfate-reducing bacteria, calculating the concentration of the original sulfate-reducing bacteria of the sample to be detected according to the following formula,
the original sulfate reducing bacteria concentration of a sample to be detected = sulfate reducing bacteria detection concentration x dilution times ÷ culture amplification times;
preferably, the calculating of the concentration of the original sulfate reducing bacteria of the sample to be detected further comprises calculating the concentration of the original sulfate reducing bacteria of the sample to be detected according to each gradient dilution sample, and taking the average value of the calculation results of each gradient dilution sample as the final concentration of the original sulfate reducing bacteria of the sample to be detected;
preferably, the constant temperature culture condition is 35 ℃ culture for 12-100 hours;
preferably, the conditions of the isothermal culture are controlled so that the culture amplification factor of the culture product is 10 times or more.
5. The method of claim 4, wherein: the culture solution cultured at constant temperature is a culture solution without granular substances, and the culture solution without granular substances is prepared from NaCl and K 2 HPO 4 、MgSO 4 、Fe(NH 4 ) 2 (SO 4 ) 2 、FeSO 4 、NH 4 Cl、Na 2 SO 4 、CaCl 2 70% lactic acid, yeast extract and water;
preferably, the concentration of each component in the culture solution without the particulate matter is NaCl4g/L and K 2 HPO 4 0.5g/L、MgSO 4 1.5g/L、Fe(NH 4 ) 2 (SO 4 ) 2 1g/L、FeSO 4 1g/L、NH 4 Cl 1g/L、Na 2 SO 4 0.5g/L、CaCl 2 0.2g/L, 5g/L of 70% lactic acid and 1g/L of yeast extract;
preferably, the gradient dilution is performed using the culture solution without the particulate matter;
preferably, the fold of gradient dilution includes 10 fold, 100 fold, 1000 fold, 10000 fold and 100000 fold;
preferably, the electrolyte adopts the culture solution without the particulate matter.
6. The method of claim 3, wherein: and judging and calculating the accuracy of the original sulfate reducing bacteria concentration of the sample to be detected according to the linear correlation between the sulfate reducing bacteria detection concentration of each gradient dilution sample and the dilution multiple, wherein the linear correlation is better and the accuracy is higher.
7. The method according to any one of claims 1-6, wherein: the method also comprises the step of carrying out 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 component as the liquid to be detected; the positive reference sample is a bacterial suspension with known concentration of sulfate reducing bacteria and the same component as the liquid to be detected.
8. The method of claim 7, wherein: and removing the background concentration according to the sulfate reducing bacteria detection concentration of the negative reference sample.
9. A kit for detecting the concentration of sulfate reducing bacteria is characterized in that: comprises a culture solution without granular substances, wherein the culture solution without the granular substances is prepared from NaCl and K 2 HPO 4 、MgSO 4 、Fe(NH 4 ) 2 (SO 4 ) 2 、FeSO 4 、NH 4 Cl、Na 2 SO 4 、CaCl 2 70% lactic acid, yeast extract and water.
10. The kit of claim 9, wherein: the concentration of each component in the culture solution without the granular substances is NaCl4g/L and K 2 HPO 4 0.5g/L、MgSO 4 1.5g/L、Fe(NH 4 ) 2 (SO 4 ) 2 1g/L、FeSO 4 1g/L、NH 4 Cl 1g/L、Na 2 SO 4 0.5g/L、CaCl 2 0.2g/L, 5g/L of 70% lactic acid and 1g/L of yeast extract.
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