Method for detecting and counting relative content of microorganism
11505817 · 2022-11-22
Assignee
Inventors
Cpc classification
C12Q1/64
CHEMISTRY; METALLURGY
International classification
Abstract
Provided is a method for detecting and counting the relative content of a microorganism, comprising: adding a redox indicator to a growth medium to produce an indicating growth medium; the range of color variation of the redox indicator comprising three or more colors that can be easily recognized by the naked eye; diluting a sample to be tested, configuring multiple degrees of dilution, configuring multiple parallels for each degree of dilution, and growing the diluted test sample using the indicating growing medium; reading the color or absorbance of the indicating growth medium while growing and/or when growing is completed; and producing the relative content of a microorganism in the test sample on the basis of the level of color variation or the value of absorbance variation of the indicating growth medium.
Claims
1. A method for determining the relative content of a microorganism, comprising the following steps: i) adding a redox indicator to a medium to obtain an indicator medium; the color change range of the redox indicator includes three or more colors; ii) diluting a test sample 3-15 times with 3-15 replicates of each dilution to produce diluted test samples, and culturing the diluted test samples using the indicator medium; iii) reading the color or absorbance of the indicator medium in each diluted test sample during culture and/or after the completion of culture; and iv) calculating the relative content of the microorganism in the test sample according to color change or absorbance change value of the indicator medium by: according to the correlation between the color of the indicator medium and the content of microorganism, reading the color of the indicator medium in each diluted test sample and giving different color a numerical value, and the numerical value of the reading is positively correlated with the content of microorganism; and calculating the relative content of the microorganism in the test sample by a formula of:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)×Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)×Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)×Y.sub.m, wherein, Z is the relative content of the microorganism in the test sample; m is the m.sup.th dilution, 3≤m≤15; n is the n.sup.th replicate sample, 3≤n≤15; X is the value of a color reading, X≥0; X.sub.m-n represents the value of the reading of the n.sup.th replicate sample of the m.sup.th dilution; Y represents a dilution coefficient, Y>0; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥Y.sub.2≥Y.sub.1; or, according to the absorbance change value of the indicator medium, calculating the relative content of the microorganism in the test sample by a formula of:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)×Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)×Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)×Y.sub.m, wherein, Z is the relative content of the microorganism in the test sample; m is the m.sup.th dilution, 3≤m≤15; n is the n.sup.th replicate sample, 3≤n≤15; X is the absorbance change value of the indicator medium; X.sub.m-n represents the absorbance change value of the n.sup.th replicate sample of the m.sup.th dilution; Y represents a dilution coefficient, Y>0; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥Y.sub.2≥Y.sub.1.
2. The method for determining the relative content of a microorganism according to claim 1, wherein the redox indicator is resazurin or methyl blue.
3. The method for determining the relative content of a microorganism according to claim 1, wherein reading is reading the color of the indicator medium in each diluted test sample during the culture and after the completion of the culture, and the reading is performed at least twice during the culture and after the completion of the culture; and calculating the relative content of the microorganism in the test sample according to the correlation between the color of the indicator medium and the content of microorganism, recording the color of the indicator medium as a number, and the number is positively correlated with the content of microorganism; calculating the final value of one reading by a formula of:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)×Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)×Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)×Y.sub.m, wherein, Z.sub.i is the final value of one reading; i is the times of color reading of the indicator medium during the culture and after the completion of the culture, i≥2; m is the m.sup.th dilution, 3≤m≤15; n is the n.sup.th replicate sample, 3≤n≤15; X is the value of a color reading, X≥0; X.sub.m-n represents the reading of the n.sup.th replicate sample of the m.sup.th dilution; Y represents a dilution coefficient, Y>0; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥Y.sub.2≥Y.sub.1; and calculating the relative content of the microorganism in the test sample by a formula of:
Z=Z.sub.1+ . . . +Z.sub.i, wherein, Z is the relative content of the microorganism in the test sample; Z.sub.i is the final value of one reading; i is the times of color reading of the indicator medium during the culture and after the completion of the culture, i≥2.
4. The method for determining the relative content of a microorganism according to claim 1, wherein reading is reading the absorbance of the indicator medium in each diluted test sample during the culture and after the completion of the culture, and the reading is performed at least twice during the culture and after the completion of the culture; and calculating the relative content of the microorganism in the test sample comprises, according to the absorbance change value of the indicator medium, calculating the final value of one reading of the test sample by a formula of:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)×Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)×Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)×Y.sub.m, wherein, Z.sub.i is the final value of one reading; i is the times of reading the absorbance change value of the indicator medium during the culture and after the completion of the culture, i≥2; m is the m.sup.th dilution, 3≤m≤15; n is the n.sup.th replicate sample, 3≤n≤15; X is the absorbance change value; X.sub.m-n represents the absorbance change value of the n.sup.th replicate sample of the m.sup.th dilution, Y represents a dilution coefficient, Y>0; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥Y.sub.2≥Y.sub.1; and calculating the relative content of the microorganism in the test sample by a formula of:
Z=Z.sub.1+ . . . +Z.sub.i, wherein, Z is the relative content of the microorganism in the test sample; Z.sub.i is the final value of one reading; i is the times of reading the absorbance change value of the indicator medium during the culture and after the completion of the culture, i≥2.
5. The method for determining the relative content of a microorganism according to claim 1, wherein the wavelength for detecting the absorbance is the maximum absorption wavelength of the redox indicator.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
(8) The present disclosure discloses a method for determining the relative content of a microorganism. A person having ordinary skill in the art can learn from the content of the present disclosure and appropriately improve the process parameters to achieve it. It is necessary to point out that all similar alternatives and modifications which are obvious to those having ordinary skill in the art are considered to be included in the present invention. The method and the application of the present disclosure have been described with reference to the preferred embodiments and it is obvious that the method and application described herein may be modified or appropriately changed and combined without departing from the content, spirit and scope of the present invention to implement and apply the techniques of the present invention.
(9) The present disclosure provides a method for determining the relative content of a microorganism, comprising the following steps:
(10) 1) selecting an indicator having a significant color change range for microorganism growth, and adding the indicator to a medium;
(11) 2) following the operation of the conventional MPN method, setting multiple dilutions as needed, setting multiple replicates for each dilution, and culturing;
(12) 3) performing reading after culturing for a period of time, recording the change degree of the indicator for microorganism growth when reading;
(13) 4) setting multiple reading time points according to the culture time of the microorganism, are performing multiple readings; and
(14) 5) calculating the possible content of the microorganism in the sample using the data of the change degree of the indicator obtained in each reading and the result of multiple readings.
(15) Wherein, Step 3) and Step 4) need not to be carried out at the same time, the effect of improving the accuracy of the MPN method can be achieved when Step 3) or Step 4) is carried out alone, but the result is better when they are combined.
(16) Preferably, the microorganism growth indicator having a significant color change range in Step 1) includes resazurin, etc.
(17) Preferably, the change degree of the microorganism growth indicator is read by visual observation or by absorbance measurement using a spectrophotometer.
(18) Preferably, according to the standard that the color difference can be easily recognized by the naked eyes, the color change range of the indicator is divided into three or more colors, the color of the indicator is recorded when reading, and a small to large number is used to represent the indicator color that varies from the color of the indicator itself to the complete color change. When no microorganism grows, a relatively small number is used for the color of the indicator itself, a relatively large number is used when a large number of microorganisms grow and the color of the indicator changes completely.
(19) Preferably, the maximum absorption wavelength of the indicator is used as the light wavelength for the measurement. The absorbance of the culture medium at the beginning of the culture is measured using a spectrophotometer. After culturing for a period of time, the absorbance of the culture medium is measured, and the absorbance change is recorded.
(20) Preferably, the reading time point in Step 4) is 2 or more.
(21) Preferably, the method of calculation in Step 5) is performed by setting coefficient for each dilution, and the coefficient set for the low dilution≤the coefficient set for the high dilution. For the naked eye observation method, the reading of each replicate sample of a certain dilution is multiplied by the coefficient corresponding to the dilution and added together to obtain the result of the dilution. The result of the reading point is obtained by adding the result of each dilution. The final result of the sample is obtained by adding the results of all reading points.
(22) Preferably, the method of calculation in Step 5) is performed by setting coefficient for each dilution, and the coefficient set for the low dilution≤the coefficient set for the high dilution. For the absorbance measurement method, the value of absorbance change obtained by measuring each replicate sample of a certain dilution is multiplied by the coefficient corresponding to the dilution and added together to obtain the result of the dilution. The result of the reading point is obtained by adding the result of each dilution. The final result of the sample is obtained by adding the results of all reading points.
(23) The reagents and instruments used in the method for determining the relative content of microorganism provided by the present disclosure are all commercially available.
(24) The present disclosure is further illustrated below with references to the examples.
Example 1
(25) The samples of this example were collected above an oil and gas trap of a sea area. The marine sediment was collected and the hydrocarbon-oxidizing bacteria in it were detected in order to study the developmental condition of the hydrocarbon-oxidizing bacteria above the oil and gas trap of the sea area.
(26) The specific implementation steps of the method for determining the relative content of microorganism of the present disclosure were as follows:
(27) 1. According to the trap structure discovered by geophysical methods, sampling points were arranged in a uniform grid, and 42 samples were collected. The distance between the sample collection sites was 500 m, and the collection depth was 20 cm. The samples were quickly frozen after collection and frozen, and then transported to a testing laboratory where it was stored in a frozen state.
(28) 2.5 g of sample was weighed and put into 20 ml of hydrocarbon-oxidizing bacteria culture medium and mixed to prepare a sample stock solution. The culture medium was referred to the examples in U.S. Pat. No. 5,093,236 (Victoria Gonzales-Prevatt, 1992). The microorganism growth indicator was resazurin.
(29) 3. 1 ml of the sample stock solution of Step 2 was pipetted and added to 9 ml of sterilized culture medium, and mixed to prepare a low dilution culture medium. Then, 1 ml of the low dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a medium dilution culture medium. Then, 1 ml of the medium dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a high dilution culture medium. Three replicate samples were set for each dilution to form a MTN detection system with three dilutions and three replicate samples for each dilution.
(30) 4. The MTN detection system was set up as described in steps 2 and 3 for each of the 42 samples. After the completion of the inoculation, the samples were cultured at 24° C. for 15 days.
(31) 5. A reading was performed every 3 days. The color of the culture medium was observed when reading. Blue was scored as 0, purple was scored as 1, magenta was scored as 2, red was scored as 3 and colorless was scored as 4.
(32) 6. Each reading result was calculated according to the following formula:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)×Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)×Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)×Y.sub.m,
(33) wherein, Z is the final value of the reading; m is the m.sup.th dilution; n is the n.sup.th replicate sample; X.sub.m-n represents the value of the reading of the n.sup.th replicate sample of the m.sup.th dilution; Y represents a dilution coefficient; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥ . . . Y.sub.2≥Y.sub.1.
(34) For example, for a certain reading, the reading scores of all replicate samples of the 1.sup.st dilution (the low dilution) were added together and multiplied by the coefficient 1 of the 1.sup.st dilution; the reading scores of all replicate samples of the 2.sup.nd dilution (the medium dilution) were added together and multiplied by the coefficient 2 of the 2.sup.nd dilution; the reading scores of all replicate samples of the 3.sup.rd dilution (the high dilution) were added together and multiplied by the coefficient 3 of the 3.sup.rd dilution. Then the result of the reading was obtained by adding together all the dilution results of multiplying the sum of the replicate samples by the coefficient of each dilution.
(35) The three replicate samples of the 1.sup.st dilution (the low dilution) were read as 3-3-2, the three replicate samples of the 2.sup.nd dilution (the medium dilution) were read as 2-2-1, and the three replicate samples of the 3.sup.rd dilution (the high dilution) were read as 1-1-0. Then the result of the reading was calculated as (3+3+2)×1+(2+2+1)×2+(1+1+0)×3=24.
(36) 7. The reading was performed every 3 days. Each of a total of five readings was processed according to step 6. Then the results of the five readings were added together to obtain the result of the relative content of microorganism of the sample.
(37) 8. Conventional MPN method was used as a control. Only the last reading, i.e. the result of the reading on the 15.sup.th day, was recorded; and only positive culture medium (purple, magenta, red or colorless) or negative culture medium (blue) was recorded. After recording, the MPN value was obtained by referring to the MPN table.
(38) 9. The data recorded and processed according to the above-mentioned steps 5 to 7 of the present disclosure were compared with the data recorded and processed according to the above-mentioned step 8. The results are shown in
(39) It can be seen from
Example 2
(40) The samples of this example were collected above an oil and gas reservoir confirmed by drilling of a sea area. The marine sediment was collected and the hydrocarbon-oxidizing bacteria in it were detected in order to study the developmental condition of the hydrocarbon-oxidizing bacteria above the oil and gas trap of the sea area.
(41) The specific implementation steps of the method for determining the relative content of microorganism of the present disclosure were as follows:
(42) 1. For an oil and gas reservoir confirmed by drilling of a sea area, sampling points were arranged by survey line, and 23 samples were collected. The distance between the sample collection sites was 500 m, and the collection depth was 20 cm. The samples were quickly frozen after collection and frozen, and then transported to a testing laboratory where it was stored in a frozen state.
(43) 2. 5 g of sample was weighed and put into 20 ml of hydrocarbon-oxidizing bacteria culture medium and mixed to prepare a sample stock solution. The culture medium was referred to the examples in U.S. Pat. No. 5,093,236 (Victoria Gonzales-Prevatt, 1992). The microorganism growth indicator was resazurin.
(44) 3. 1 ml of the sample stock solution of Step 2 was pipetted and added to 9 ml of sterilized culture medium, and mixed to prepare a low dilution culture medium. Then, 1 ml of the low dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a medium dilution culture medium. Then, 1 ml of the medium dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a high dilution culture medium. Three replicate samples were set for each dilution to form a MPN detection system with three dilutions and three replicate samples for each dilution.
(45) 4. The MPN detection system was set up as described in steps 2 and 3 for each of the 23 samples. After the completion of the inoculation, the samples were cultured at 24° C. for 15 days.
(46) 5. A reading was performed after the completion of culture on the 15.sup.th day. Blue was scored as 0, purple was scored as 1, magenta was scored as 2, red was scored as 3 and colorless was scored as 4.
(47) 6. The reading result on the 15.sup.th day was calculated according to the following formula.
(48) The reading scores of all replicate samples of the low dilution were added together and multiplied by the coefficient 1 of the low dilution; the reading scores of all replicate samples of the medium dilution were added together and multiplied by the coefficient 2 of the medium dilution; the reading scores of all replicate samples of the high dilution were added together and multiplied by the coefficient 3 of the high dilution. Then the result of the reading was obtained by adding together all the dilution results of multiplying the sum of the replicate samples by the coefficient of each dilution, that is, the relative content of microorganism in the sample.
(49) The three replicate samples of the 1.sup.st dilution (the low dilution) were read as 3-3-2, the three replicate samples of the 2.sup.nd dilution (the medium dilution) were read as 2-2-1, and the three replicate samples of the 3.sup.rd dilution (the high dilution) were read as 1-1-0. Then the result of the reading was calculated as (3+3+2)×1+(2+2+1)×2+(1+1+0)×3=24.
(50) 7. Conventional MPN method was used as a control. Only the last reading, i.e. the result of the reading on the 15.sup.th day, was recorded; and only positive culture medium (purple, magenta, red or colorless) or negative culture medium (blue) was recorded. After recording, the MPN value was obtained by referring to the MPN table.
(51) 8. The data recorded and processed according to the above-mentioned steps 5 to 6 of the present disclosure were compared with the data recorded and processed according to the above-mentioned step 7. The results are shown in
(52) The results show that, confirmed by drilling, the samples numbered 7 to 17 were collected from the oil and gas reservoir area. From the counting result of the method of the present disclosure and the counting result of the MPN method, the counting result of the method of the present disclosure can better reflect the oil and gas reservoir area located between the samples numbered 7 to 17. Samples numbered 7, 8, 10, 13, 14, and 15 gave relatively low values in MPN counting method, while the same samples gave relatively high values in the method of the present disclosure, which was consistent with the actual situation.
Example 3
(53) 1. Acinetobacter isolated from sea was activated. The activation steps were: the strain was first streaked in a solid culture medium for marine hydrocarbon-oxidizing bacteria, then a single colony was picked and inoculated into the marine hydrocarbon-oxidizing bacteria liquid medium for enrichment culture.
(54) 2. The Acinetobacter after the enrichment culture was diluted with sterile water to prepare a series of Acinetobacter dilutions with 15 different concentrations. The cell numbers in different concentrations were counted by a hemocytometer under a microscope, and the concentrations of Acinetobacter in this series of 15 different concentrations were obtained, expressed in unit of cell/ml.
(55) 3. 1 ml of the Acinetobacter solution of Step 2 was pipetted and added to 9 ml of sterilized culture medium, and mixed to prepare a low dilution culture medium. Then, 1 ml of the low dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a medium dilution culture medium. Then, 1 ml of the medium dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a high dilution culture medium. Three replicate samples were set for each dilution to form a MPN detection system with three dilutions and three replicate samples for each dilution. The culture medium was referred to the examples in U.S. Pat. No. 5,093,236 (Victoria Gonzales-Prevatt, 1992). The microorganism growth indicator was resazurin.
(56) 4. The MPN detection system was set up as described in step 3 for 15 Acinetobacter solutions with different concentrations. After the completion of the inoculation, the samples were cultured at 24° C. for 15 days.
(57) 5. A reading was performed every 3 days. The color of the culture medium was observed when reading. Blue was scored as 0, purple was scored as 1, magenta was scored as 2, red was scored as 3 and colorless was scored as 4.
(58) 6. Each reading result was calculated according to the following formula:
Z=(X.sub.1-1+X.sub.1-2+ . . . +X.sub.1-n)*Y.sub.1+(X.sub.2-1+X.sub.2-2+ . . . +X.sub.2-n)*Y.sub.2+ . . . +(X.sub.m-1+X.sub.m-2+ . . . +X.sub.m-n)*Y.sub.m,
(59) wherein, Z is the final value of the reading; m is the m.sup.th dilution; n is the n.sup.th replicate sample; X.sub.m-n represents the value of the reading of the n.sup.th replicate sample of the m.sup.th dilution; Y represents a dilution coefficient; Y.sub.m represents the dilution coefficient of the m.sup.th dilution, and Y.sub.m≥Y.sub.m-1≥Y.sub.m-2≥ . . . Y.sub.2≥Y.sub.1.
(60) For example, for a certain reading, the reading scores of all replicate samples of the 1.sup.st dilution (the low dilution) were added together and multiplied by the coefficient 1 of the 1.sup.st dilution; the reading scores of all replicate samples of the 2.sup.nd dilution (the medium dilution) were added together and multiplied by the coefficient 2 of the 2.sup.nd dilution; the reading scores of all replicate samples of the 3.sup.rd dilution (the high dilution) were added together and multiplied by the coefficient 3 of the 3.sup.rd dilution. Then the result of the reading was obtained by adding together all the dilution results of multiplying the sum of the replicate samples by the coefficient of each dilution.
(61) The three replicate samples of the 1.sup.st dilution (the low dilution) were read as 3-3-2, the three replicate samples of the 2.sup.nd dilution (the medium dilution) were read as 2-2-1, and the three replicate samples of the 3.sup.rd dilution (the high dilution) were read as 1-1-0. Then the result of the reading was calculated as (3+3+2)×1+(2+2+1)×2+(1+1+0)×3=24.
(62) 7. The reading was performed every 3 days. Each of a total of five readings was processed according to step 6. Then the results of the five readings were added together to obtain the result of the relative content of microorganism of the sample.
(63) 8. Conventional MPN method was used as a control. Only the last reading, i.e. the result of the reading on the 15.sup.th day, was recorded; and only positive culture medium (purple, magenta, red or colorless) or negative culture medium (blue) was recorded. After recording, the MPN value was obtained by referring to the MPN table.
(64) At the same time, the numbers of Acinetobacter in the above-mentioned samples were counted by microscopic counting.
(65) The comparison of the results of microscopic counting, method of the present disclosure and MPN method of the Acinetobacter solution with 15 different concentrations are shown in Table 1 and
(66) TABLE-US-00001 TABLE 1 Counting Results of Acinetobacter by each method Microscopic Counting The Present MPN Method No. (cells/ml) Disclosure (MPN Value/ml) 1 8080000 47 240 2 7272000 46 240 3 6464000 41 240 4 5656000 41 240 5 4848000 32 43 6 4040000 30 43 7 3232000 26 43 8 2424000 10 23 9 1616000 9 9.2 10 808000 7 3.6 11 404000 3 0 12 202000 2 0 13 101000 1 0 14 50500 0 0 15 25250 0 0
(67) It can be seen from the above-mentioned figure and table that the results obtained by the method of the present disclosure are significantly better than that of the MPN method, and are more consistent with the results of Acinetobacter cell numbers obtained by microscopic counting, the correlation coefficient between the two reached 0.9734, while the correlation coefficient between standard MPN method and microscopic counting was only 0.8218.
Example 4
(68) The samples of this example were collected above an oil and gas reservoir confirmed by drilling of a sea area. The marine sediment was collected and the hydrocarbon-oxidizing bacteria in it were detected in order to study the developmental condition of the hydrocarbon-oxidizing bacteria above the oil and gas trap of the sea area.
(69) The specific implementation steps of the method for determining the relative content of microorganism of the present disclosure were as follows:
(70) 1. For an oil and gas reservoir confirmed by drilling of a sea area, sampling points were arranged by survey line, and 23 samples were collected. The distance between the sample collection sites was 500 m, and the collection depth was 20 cm. The samples were quickly frozen after collection and frozen, and then transported to a testing laboratory where it was stored in a frozen state.
(71) 2. 5 g of sample was weighed and put into 20 ml of hydrocarbon-oxidizing bacteria culture medium and mixed to prepare a sample stock solution. The culture medium was referred to the examples in U.S. Pat. No. 5,093,236 (Victoria Gonzales-Prevatt, 1992). The microorganism growth indicator was resazurin. The amount of resazurin was adjusted so that the absorbance of the culture medium at 600 nm wavelength was 1.000.
(72) 3. 1 ml of the sample stock solution of Step 2 was pipetted and added to 9 ml of sterilized culture medium, and mixed to prepare a low dilution culture medium. Then, 1 ml of the low dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a medium dilution culture medium. Then, 1 ml of the medium dilution culture medium was taken and added to another 9 ml of the sterilized culture medium and mixed to prepare a high dilution culture medium. Three replicate samples were set for each dilution to form a MPN detection system with three dilutions and three replicate samples for each dilution.
(73) 4. The MPN detection system was set up as described in steps 2 and 3 for each of the 23 samples. After the completion of the inoculation, the samples were cultured at 24° C. for 15 days.
(74) 5. A reading was performed after the completion of culture on the 15.sup.th day. The absorbance of the culture medium at 600 nm was recorded at the time of reading, and the absorbance value measured after the completion of culture was subtracted from the initial absorbance of 1.000 to obtain an absorbance change value.
(75) 6. The reading result on the 15.sup.th day was calculated according to the following formula.
(76) The absorbance change values of all replicate samples of the low dilution were added together and multiplied by the coefficient 10 of the low dilution; the absorbance change values of all replicate samples of the medium dilution were added together and multiplied by the coefficient 20 of the medium dilution; the absorbance change values of all replicate samples of the high dilution were added together and multiplied by the coefficient 30 of the high dilution. Then the result of the reading was obtained by adding together all the dilution results of multiplying the sum of the replicate samples by the coefficient of each dilution, that is, the relative content of microorganism in the sample.
(77) The absorbance change values of three replicate samples of the 1.sup.st dilution (the low dilution) were 0.752-0.733-0.509; the absorbance change values of three replicate samples of the 2.sup.nd dilution (the medium dilution) were 0.560-0.480-0.376; absorbance change values of three replicate samples of the 3.sup.rd dilution (the high dilution) were 0.224-0.269-0.109. Then the result of the reading was calculated as (0.752+0.733+0.509)×10+(0.560+0.480+0.376)×20+(0.224+0.269+0.109)×30=66.32.
(78) 7. Conventional MPN method was used as a control. Only the last reading, i.e. the result of the reading on the 15.sup.th day, was recorded; and only positive culture medium (purple, magenta, red or colorless) or negative culture medium (blue) was recorded. After recording, the MPN value was obtained by referring to the MPN table.
(79) 8. The data recorded and processed according to the above-mentioned steps 5 to 6 of the present disclosure were compared with the data recorded and processed according to the above-mentioned step 7. The results are shown in
(80) The results show that, confirmed by drilling, the samples numbered 7 to 17 were collected from the oil and gas reservoir area. From the counting result of the method of the present disclosure and the counting result of the MPN method, the counting result of the method of the present disclosure can better reflect the oil and gas reservoir area located between the samples numbered 7 to 17. Samples numbered 7, 8, 10, 13, 14, and 15 gave relatively low values in MPN counting method, while the same samples gave relatively high values in the method of the present disclosure, which was consistent with the actual situation.
(81) The above descriptions are only preferred embodiments of the present disclosure. It should be pointed out that a number of modifications and refinements may be made by those having ordinary skill in the art without departing from the principles of the present invention, and such modifications and refinements are also considered to be within the protection scope of the present invention.