Method for rapidly determining concentration of bacterial liquid based on pH value
By establishing a standard curve based on pH value, the bacterial concentration can be determined quickly and accurately, solving the problems of long detection cycle, complicated operation and high cost in traditional methods. This achieves efficient and low-cost bacterial concentration detection, and is suitable for rapid detection of a variety of bacteria.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHONGHANG INSPECTION TESTING & CERTIFICATION (ZHEJIANG) CO LTD
- Filing Date
- 2026-01-12
- Publication Date
- 2026-05-12
AI Technical Summary
Existing methods for detecting bacterial concentration suffer from problems such as excessively long detection cycles, cumbersome operations, high costs, and poor reproducibility of results. Furthermore, there is a lack of standardized rapid detection methods based on the negative correlation between bacterial pH and concentration.
By establishing a standard curve and using a precision pH meter to measure the pH value of the bacterial solution, combined with isothermal shaking culture and sample pretreatment, the bacterial solution concentration can be determined rapidly and accurately, including the gradient preparation, culture, pH measurement, and result correction of the standard bacterial solution.
It shortens the detection cycle to 2-4 hours, increases detection efficiency by 10 times, reduces costs and consumable consumption, and provides results with better accuracy and stability than traditional methods. It is suitable for the detection of antimicrobial materials, food hygiene, and environmental microorganisms.
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial detection technology, specifically a method for rapidly determining bacterial concentration based on pH value. Background Technology
[0002] In fields such as antimicrobial material performance testing, food hygiene and safety monitoring, and environmental microbial contamination control, accurate determination of bacterial concentration is a core technical aspect. For example, in the research and development of antimicrobial materials, the antimicrobial rate needs to be calculated by measuring the bacterial concentration before and after antimicrobial treatment to evaluate the antimicrobial performance of the material; in the food production process, the concentration of microorganisms in raw materials and finished products needs to be monitored to ensure that the products meet food safety standards.
[0003] Traditional methods for detecting bacterial concentration, represented by the plate count method, involve the following core steps: After the sample is in contact with the bacterial solution for a specified time, residual bacteria are eluted. The eluent is then serially diluted, inoculated onto solid culture plates, and incubated under suitable conditions for 24-48 hours. The bacterial concentration is then calculated by manually counting the number of colonies. While this method provides accurate results, it has several significant drawbacks: First, the detection cycle is too long, requiring 48-72 hours, which cannot meet the needs of rapid detection and emergency monitoring scenarios, such as real-time quality control on food production lines and rapid response to sudden microbial contamination incidents. Second, the operation is cumbersome, involving multiple steps including serial dilution, inoculation, incubation, and counting, requiring high levels of expertise from operators. Furthermore, manual counting is susceptible to subjective factors, leading to poor repeatability of the results. Third, the detection cost is high, consuming large amounts of consumables such as culture media and petri dishes, and occupying equipment resources such as incubators, making it unsuitable for large-scale batch testing of samples.
[0004] In existing technologies, it has been found that some bacteria produce acidic metabolites during their metabolism (such as lactic acid and acetic acid produced by Escherichia coli, and formic acid and acetic acid produced by Salmonella), causing the pH value of the bacterial solution to decrease systematically with the increase of bacterial count, i.e., there is a significant negative correlation between bacterial concentration and pH value. However, there is currently no standardized and scalable rapid detection method based on this characteristic: on the one hand, existing studies have not clearly defined unified culture conditions (such as temperature, rotation speed, and culture time), resulting in large differences in the correlation between pH value and bacterial concentration obtained by different experimenters; on the other hand, there is a lack of means to control interfering factors, such as external carbon dioxide and impurities in the bacterial solution, which can affect the accuracy of pH measurement and thus lead to concentration calculation errors; in addition, a supporting standardized detection procedure and dedicated equipment have not been developed, making it difficult to achieve large-scale application of the detection method. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for rapidly determining bacterial concentration based on pH value, thus solving the problems mentioned in the background section.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for rapidly determining bacterial concentration based on pH value, comprising the following steps: Establishment of the S1 standard curve: Select the target strain and prepare at least five standard bacterial solutions with varying concentrations, each ranging from 10 to 10. 3 ~10 9 CFU / mL, and the ratio of adjacent gradient concentrations is 10; Standard bacterial solutions of each gradient concentration were placed in sterile reaction containers and incubated for 2-4 hours under constant temperature shaking conditions of 35-37℃ and 180-220r / min. Using a precision pH meter with an accuracy ≤0.01pH, the pH value of standard bacterial solutions at each concentration gradient was measured after culturing. Each concentration group was measured in parallel 3 times, and the average value was taken as the pH value corresponding to that concentration. Plotting the logarithm of the standard bacterial concentration on the x-axis and the corresponding pH measurement value on the y-axis, a standard curve equation was established using linear regression analysis: y = ax + b, where y is the pH measurement value, x is the logarithm of the bacterial concentration, a is the regression coefficient, and b is the intercept. The coefficient of determination R0 of this standard curve is also calculated. 2 ≥0.98; S2 bacterial suspension concentration determination: The bacterial solution to be tested was aseptically pretreated to remove insoluble impurities and particulate matter that interfered with pH measurement. Following the culture conditions in step S1, the pretreated bacterial culture was subjected to constant temperature shaking culture. Using the same precision pH meter as in step S1, the pH value of the bacterial solution to be tested after cultivation was measured. The measurement was performed in parallel three times, and the average value was taken as the actual pH value of the bacterial solution to be tested. Substitute the measured pH value into the standard curve equation established in step S1 to calculate the logarithm of the concentration of the bacterial solution to be tested, and then convert it to obtain the actual concentration of the bacterial solution to be tested. S3 Result Verification and Correction: When the measured pH value of the bacterial solution to be tested exceeds the pH range of the standard curve, the bacterial solution to be tested is subjected to aseptic gradient dilution or concentration treatment so that the predicted pH value of the bacterial solution after treatment falls within the pH range of the standard curve, and then step S2 is repeated for measurement.
[0007] Furthermore, in step s1, the target strain is a bacterium that produces acidic substances during metabolism, including one or more of Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria.
[0008] Furthermore, in steps S1 and S2, a sealed reaction vessel is used during the cultivation process, and the ratio of headspace volume to bacterial culture volume in the reaction vessel is 1:3 to 1:5 to avoid interference from external carbon dioxide in pH measurement.
[0009] Furthermore, in step S2, the aseptic pretreatment includes centrifugation and filtration performed sequentially. The centrifugation conditions are 5000~8000 r / min for 5~10 minutes, and the filtration uses a 0.22~0.45 μm sterile filter membrane.
[0010] Furthermore, in steps S1 and S2, the pH meter needs to be calibrated at three points using standard buffer solutions with pH values of 4.00, 6.86, and 9.18 before measurement, with a calibration error ≤ ±0.02pH.
[0011] Furthermore, in step S1, the standard bacterial solution is prepared using the same culture medium as the test bacterial solution, and the concentration of the standard bacterial solution is calibrated by plate counting method to ensure the accuracy of the standard curve establishment.
[0012] Furthermore, in step S2, if the relative standard deviation of the parallel measured pH values is >3%, then resample and repeat the culture and pH measurement process of step S2 until the relative standard deviation is ≤3%.
[0013] This invention provides a method for rapidly determining bacterial concentration based on pH value, which has the following beneficial effects: This method for rapidly determining bacterial concentration based on pH value shortens the detection cycle of the traditional plate count method (48-72 hours) to 2-4 hours, increasing detection efficiency by more than 10 times. It can meet the needs of rapid screening of antimicrobial materials, real-time monitoring of food production lines, and emergency microbial testing, significantly shortening the detection cycle and improving work efficiency. It eliminates the need for cumbersome steps such as plate culture and colony counting; the entire detection process can be integrated using a dedicated device, requiring no specialized technicians. The method consumes few consumables, and the dedicated device is reusable, significantly reducing detection costs. A standardized detection process has been established by optimizing culture conditions (constant temperature oscillation, sealed environment) and controlling interference factors (sample pretreatment, pH meter calibration). The coefficient of determination R of the standard curve is [value missing]. 2 With a relative standard deviation (RSD) of ≤5% and an accuracy and stability that meets practical testing needs, the results show good consistency with the traditional plate count method. In addition to Escherichia coli, the standard curve can be adjusted to detect the concentration of other bacteria that produce acidic substances during metabolism (such as Salmonella and Staphylococcus aureus). Applicable scenarios include antimicrobial material testing, food hygiene monitoring, and environmental microbial testing. Detailed Implementation
[0014] The embodiments of the present invention will be described in further detail below with reference to examples. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.
[0015] Example 1: Detection of Escherichia coli concentration Establishment of S1 standard curve The target strain, *Escherichia coli* (ATCC25922, which produces acidic substances such as lactic acid and acetic acid during metabolism), was selected and cultured in the same LB liquid medium as the bacterial solutions to be tested until the logarithmic growth phase. The concentration was calibrated using the traditional plate count method, and graded standard bacterial solutions were prepared with concentrations of 10-1. 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, with an adjacent gradient concentration ratio of 10, meeting the requirement of "at least 5 gradients"; Take 10 mL of each gradient of standard bacterial solution and place them into 20 mL sealed sterile reaction containers. Place the reaction containers in a constant temperature shaking incubator and incubate for 3 hours under the specified conditions (37℃, 200 r / min). A precision pH meter (Mettler FE28) with an accuracy of 0.01 pH was selected. Before measurement, three-point calibration was performed using standard buffer solutions at pH values of 4.00, 6.86, and 9.18, with a calibration error ≤ ±0.02 pH. Subsequently, the pH values of the standard bacterial solutions after incubation at each concentration gradient were measured. Each concentration group was measured in triplicate, and the average value was taken as the corresponding pH value. The results are shown in Table 1 below. ; Table 1. Correspondence between the concentration of standard Escherichia coli bacterial solution and pH value. Plotting the logarithm of the standard bacterial concentration on the x-axis and the corresponding average pH value on the y-axis, a standard curve equation was established using linear regression analysis: y = -0.182x + 8.456. The coefficient of determination R of this standard curve is... 2 =0.992≥0.98.
[0016] S2 Test bacterial concentration determination Take the Escherichia coli bacterial suspension to be tested (source: eluent after antimicrobial material antimicrobial test) and perform aseptic pretreatment as required: first, centrifuge at 6000 r / min for 8 minutes to remove large particulate impurities; take the supernatant and filter it through a 0.22 μm sterile filter membrane to remove small particulate matter and impurities that interfere with pH measurement; Take 10 mL of the pretreated bacterial culture to be tested and place it in a 20 mL sealed sterile reaction container (headspace volume to bacterial culture volume ratio of 1:5). Incubate under constant temperature shaking for 3 hours according to the culture conditions of S1 (37℃, 200 r / min). Using the same precision pH meter as S1 (after three-point calibration), the pH value of the bacterial culture was measured after cultivation. The measurement was performed in parallel three times, and the values were 6.34, 6.36 and 6.35, respectively. The average value was 6.35. The relative standard deviation was calculated to be 0.16%≤3%, and there was no need to remeasure. This average value was taken as the actual pH value of the bacterial culture. Substituting the measured pH value of 6.35 into the standard curve equation established in S1: 6.35 = -0.182x + 8.456, we calculate x = (8.456 - 6.35) / 0.182 ≈ 11.57, and then convert it to obtain the actual concentration of the bacterial solution to be tested as 10. 11 . 57 ≈3.7×10 11 CFU / mL.
[0017] S3 Result Verification and Correction Based on the analysis, the measured pH value of 6.35 of the bacterial solution being tested corresponds to a concentration of 3.7 × 10⁻⁶. 11 The concentration (CFU / mL) exceeded the concentration range of the standard curve (10). 3 ~10 9 The test bacterial solution was aseptically serially diluted 100-fold (CFU / mL) to a concentration of approximately 3.7 × 10⁻⁶ CFU / mL. 9 (CFU / mL). The procedure for repeating S2 was as follows: the diluted bacterial culture was incubated at 37℃ and 200 r / min for 3 hours, and the average pH was measured to be 5.59. The relative standard deviation of parallel determinations was 0.21% ≤ 3%. Substituting the pH value of 5.59 into the standard curve equation, x = (8.456 - 5.59) / 0.182 ≈ 15.75. The corrected concentration of the diluted bacterial culture was approximately 3.9 × 10⁻¹⁰. 9 CFU / mL; multiplied by the dilution factor of 100, the final actual concentration of the test bacterial solution is 3.9 × 10⁻⁶. 11 CFU / mL.
[0018] Result Validation The concentration of the above-mentioned bacterial suspension was determined using the traditional plate count method, and the result was 3.7 × 10⁻⁶. 11 The CFU / mL measurement result obtained by the method of this invention has a relative error of 5.4% compared with the plate counting method, and the accuracy meets the actual detection requirements.
[0019] Example 2: Salmonella Concentration Detection Establishment of S1 standard curve The target strain, Salmonella (ATCC14028, which produces acidic substances such as formic acid and acetic acid through metabolism), was selected. It was cultured in the same nutrient broth medium as the test bacterial solution until the logarithmic growth phase. The concentration was calibrated using the plate count method to prepare a solution with a concentration of 10... 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 Standard bacterial suspension with CFU / mL; Take 8 mL of each gradient of standard bacterial solution and place it in a 24 mL sealed sterile reaction container. Incubate under constant temperature and shaking conditions (36℃, 190 r / min) for 3.5 hours. A precision pH meter with an accuracy of 0.01 pH (calibrated at three points with standard buffer solutions of pH=4.00, 6.86, and 9.18, with a calibration error ≤±0.02 pH) was used to measure the pH value of each standard bacterial solution. Each concentration was measured in parallel three times, and the average value was taken as the pH value. A standard curve equation was established with the logarithm of the standard bacterial concentration on the x-axis and the pH measurement value on the y-axis: y = -0.175x + 8.324, and the coefficient of determination R0. 2 =0.988≥0.98.
[0020] S2 Test bacterial concentration determination Take the Salmonella test solution from the food sample and perform aseptic pretreatment as required: centrifuge at 5000 r / min for 10 minutes, and filter the supernatant through a 0.45 μm sterile filter membrane; Take 8 mL of the pretreated bacterial culture to be tested and place it in a 24 mL sealed sterile reaction vessel (headspace volume to bacterial culture volume ratio of 1:3). Incubate under constant temperature and shaking conditions (36℃, 190 r / min) for 3.5 hours according to the culture conditions of S1. Using the same precision pH meter as S1, the pH value of the bacterial culture was measured after cultivation. The measurements were performed in three parallel runs, and the values were 6.40, 6.43, and 6.43, with an average value of 6.42 and a relative standard deviation of 0.24% ≤ 3%. These values were taken as the actual pH values. Substituting the measured pH value of 6.42 into the standard curve equation, we calculate x = (8.324 - 6.42) / 0.175 ≈ 10.88, which gives the actual concentration of the bacterial solution being tested as 10. 10 . 88 ≈7.6×10 10 CFU / mL.
[0021] S3 Result Verification and Correction The concentration of the bacterial suspension to be tested was 7.6 × 10⁻⁶. 10 CFU / mL exceeds the concentration range of the standard curve (10). 3 ~10 9 The concentration of the diluted bacterial culture (CFU / mL) was serially diluted 10-fold aseptically, and the S2 procedure was repeated. After culturing the diluted culture for 3.5 hours, the average pH was measured to be 6.59. Substituting these values into the standard curve equation, the calculated concentration after dilution was approximately 7.5 × 10⁻⁶. 9 CFU / mL, multiplied by a dilution factor of 10, yields a final actual concentration of 7.5 × 10⁻⁶ CFU / mL. 10 CFU / mL.
[0022] The concentration of the test bacterial suspension was determined to be 7.1 × 10⁻⁶ using the plate count method. 10 The CFU / mL measurement result obtained by the method of this invention has a relative error of 5.6% compared with the plate counting method, indicating good accuracy.
[0023] Example 3: Detection of Staphylococcus aureus concentration Establishment of S1 standard curve The target strain, Staphylococcus aureus (ATCC29213, which produces acidic metabolites), was selected. Using the same TSB liquid medium as the test culture, and calibrated by plate counting, 10... 3 ~10 9 .
[0024] CFU / mL gradient standard bacterial solutions (adjacent concentration ratio of 10); Take 10 mL of each gradient standard bacterial solution and place it in a 30 mL sealed sterile reaction container. Incubate under constant temperature shaking for 2.5 hours according to the conditions (37℃, 210 r / min). A precision pH meter calibrated at three points was used to measure the pH value of each standard bacterial solution. Each concentration was measured in triplicate, and the average value was taken. Establish the standard curve equation: y = -0.168x + 8.273, with a coefficient of determination R0. 2 =0.985≥0.98.
[0025] S2 Test bacterial concentration determination The Staphylococcus aureus test bacterial solution in the environmental monitoring sample was pretreated by centrifugation at 7000 r / min for 6 minutes and filtration through a 0.22 μm sterile filter membrane. Take 10 mL of the pretreated bacterial culture and incubate it at 37℃ and 210 r / min for 2.5 hours. The pH value was measured using the same precision pH meter. The values obtained from three parallel measurements were 6.61, 6.64, and 6.64, with an average value of 6.63 and a relative standard deviation of 0.23% ≤ 3%. These values were taken as the actual pH values. Substituting into the standard curve equation, we get x = (8.273 - 6.63) / 0.168 ≈ 9.78, which translates to an actual concentration of 10 in the test bacterial solution. 9 . 78 ≈6.0×10 9 The concentration of CFU / mL is within the range of the standard curve and requires no correction.
[0026] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.
Claims
1. A method for rapidly determining bacterial concentration based on pH value, characterized in that: Includes the following steps: Establishment of the S1 standard curve: Select the target strain and prepare at least five standard bacterial solutions with varying concentrations, each ranging from 10 to 10. 3 ~10 9 CFU / mL, and the ratio of adjacent gradient concentrations is 10; Standard bacterial solutions of each gradient concentration were placed in sterile reaction containers and incubated for 2-4 hours under constant temperature shaking conditions of 35-37℃ and 180-220r / min. Using a precision pH meter with an accuracy of ≤0.01pH, the pH value of standard bacterial solutions at each concentration gradient was measured after culturing. Each concentration group was measured in parallel three times, and the average value was taken as the pH value corresponding to that concentration. Plotting the logarithm of the standard bacterial concentration on the x-axis and the corresponding pH measurement value on the y-axis, a standard curve equation was established using linear regression analysis: y = ax + b, where y is the pH measurement value, x is the logarithm of the bacterial concentration, a is the regression coefficient, and b is the intercept. The coefficient of determination R0 of this standard curve is also calculated. 2 ≥0.98; S2 bacterial suspension concentration determination: The bacterial solution to be tested was aseptically pretreated to remove insoluble impurities and particulate matter that interfered with pH measurement. Following the culture conditions in step S1, the pretreated bacterial culture was subjected to constant temperature shaking culture. Using the same precision pH meter as in step S1, the pH value of the bacterial solution to be tested after cultivation was measured. The measurement was performed in parallel three times, and the average value was taken as the actual pH value of the bacterial solution to be tested. Substitute the measured pH value into the standard curve equation established in step S1 to calculate the logarithm of the concentration of the bacterial solution to be tested, and then convert it to obtain the actual concentration of the bacterial solution to be tested. S3 Result Verification and Correction: When the measured pH value of the bacterial solution to be tested exceeds the pH range of the standard curve, the bacterial solution to be tested is subjected to aseptic gradient dilution or concentration treatment so that the predicted pH value of the bacterial solution after treatment falls within the pH range of the standard curve, and then step S2 is repeated for measurement.
2. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that: In step s1, the target strain is a bacterium that produces acidic substances during metabolism, including one or more of Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria.
3. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that: In steps S1 and S2, a sealed reaction vessel is used during the cultivation process, and the ratio of headspace volume to bacterial culture volume in the reaction vessel is 1:3 to 1:5 to avoid interference from external carbon dioxide in pH measurement.
4. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that: In step S2, the aseptic pretreatment includes centrifugation and filtration performed sequentially. The centrifugation conditions are 5000~8000 r / min for 5~10 minutes, and the filtration uses a 0.22~0.45 μm sterile filter membrane.
5. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that: In steps S1 and S2, the pH meter needs to be calibrated at three points using standard buffer solutions with pH values of 4.00, 6.86, and 9.18 before measurement, with a calibration error ≤ ±0.02pH.
6. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that, In step S1, the standard bacterial solution is prepared using the same culture medium as the test bacterial solution, and the concentration of the standard bacterial solution is calibrated by plate counting method to ensure the accuracy of the standard curve establishment.
7. The method for rapidly determining bacterial concentration based on pH value according to claim 1, characterized in that: In step S2, if the relative standard deviation of the parallel pH measurement values is >3%, then resample and repeat the culture and pH measurement process of step S2 until the relative standard deviation is ≤3%.