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CN-122012667-A - Method for rapidly determining concentration of bacterial liquid based on pH value

CN122012667ACN 122012667 ACN122012667 ACN 122012667ACN-122012667-A

Abstract

The invention discloses a method for rapidly determining bacterial liquid concentration based on pH value, which relates to the technical field of microorganism detection and comprises the following steps of S1 standard curve establishment, wherein a target bacterial strain is selected, standard bacterial liquids with at least 5 gradient concentrations are prepared, the concentration range of each standard bacterial liquid with the gradient concentration is 10 3 ~10 9 CFU/mL, the ratio of adjacent gradient concentrations is 10, and the standard bacterial liquids with the gradient concentrations are respectively placed in a sterile reaction container and cultured for 2-4 hours under the constant-temperature oscillation condition of 35-37 ℃ and 180-220 r/min. According to the invention, the detection period of 48-72 hours of the traditional plate counting method is shortened to 2-4 hours, the detection efficiency is improved by more than 10 times, the requirements of scenes such as rapid screening of antibacterial materials, real-time monitoring of food production lines, emergency microorganism detection and the like can be met, the detection period is greatly shortened, and the working efficiency is improved.

Inventors

  • Qu Rongfang
  • XIE JIANFENG
  • LU CHENXI
  • Hu Rishang
  • WANG YIHAI
  • XU MIN
  • YE JIANBIN

Assignees

  • 中杭检验检测认证(浙江)有限公司

Dates

Publication Date
20260512
Application Date
20260112

Claims (7)

  1. 1. A method for rapidly determining the concentration of bacterial liquid based on pH value is characterized by comprising the following steps: S1, establishing a standard curve: Selecting a target strain, preparing standard bacterial solutions with at least 5 gradient concentrations, wherein the concentration range of the standard bacterial solution with each gradient concentration is 10 3 ~10 9 CFU/mL, and the ratio of adjacent gradient concentrations is 10; respectively placing standard bacterial solutions with gradient concentrations into a sterile reaction container, and culturing for 2-4 hours under the constant-temperature oscillation condition of 35-37 ℃ and 180-220 r/min; Respectively measuring the pH value of the standard bacterial liquid with gradient concentration after culture by adopting a precise pH meter with the precision less than or equal to 0.01, measuring each concentration group in parallel for 3 times, and taking the average value as a pH measurement value corresponding to the concentration; Taking the logarithm of the concentration of the standard bacterial liquid as an abscissa, the corresponding pH measured value as an ordinate, and adopting linear regression analysis to establish a standard curve equation, wherein y=ax+b, y is the pH measured value, x is the logarithm of the concentration of the bacterial liquid, a is a regression coefficient, b is an intercept, and the determination coefficient R 2 of the standard curve is more than or equal to 0.98; S2, measuring the concentration of the bacteria liquid to be measured: performing aseptic pretreatment on the bacterial liquid to be detected, and removing insoluble impurities and particulate matters interfering with pH measurement; carrying out constant-temperature shaking culture on the pretreated bacterial liquid to be tested according to the culture conditions in the step S1; Measuring the pH value of the bacteria liquid to be tested after culture by adopting a precise pH meter which is the same as that in the step S1, carrying out parallel measurement for 3 times, and taking the average value as the pH actual measurement value of the bacteria liquid to be tested; substituting the pH measured value into a standard curve equation established in the step S1, calculating to obtain the logarithm of the concentration of the bacteria liquid to be detected, and further converting to obtain the actual concentration of the bacteria liquid to be detected; And S3, verifying and correcting the result, namely when the pH measured value of the bacterial liquid to be detected exceeds the pH range of the standard curve, carrying out aseptic gradient dilution or concentration treatment on the bacterial liquid to be detected, enabling the pH predicted value of the bacterial liquid to be treated to fall within the pH range of the standard curve, and repeating the step S2 for measuring.
  2. 2. The method for rapidly determining a concentration of a bacterial liquid according to claim 1, wherein in the step s1, the target bacterial strain is a bacterium which generates acidic substances in a metabolic process, and the target bacterial strain comprises one or more of Escherichia coli, salmonella, staphylococcus aureus and Listeria.
  3. 3. The method for rapidly determining the concentration of the bacterial liquid based on the pH value, which is disclosed in claim 1, is characterized in that in the step S1 and the step S2, a sealed reaction container is adopted in the culture process, and the ratio of the headspace volume in the reaction container to the bacterial liquid volume is 1:3-1:5, so that the interference of external carbon dioxide on the pH determination is avoided.
  4. 4. The method for rapidly determining the concentration of the bacterial liquid based on the pH value according to claim 1, wherein in the step S2, the aseptic pretreatment comprises sequentially performing centrifugal treatment and filtration treatment, the centrifugal treatment condition is 5000-8000 r/min, 5-10 minutes, and the filtration treatment adopts an aseptic filter membrane of 0.22-0.45 μm.
  5. 5. The method for rapidly determining bacterial liquid concentration based on pH value according to claim 1, wherein in the step S1 and the step S2, the pH meter needs to adopt a standard buffer solution with pH=4.00, 6.86 and 9.18 for three-point calibration before measurement, and the calibration error is less than or equal to +/-0.02 pH.
  6. 6. The method for rapidly determining the concentration of the bacterial liquid based on the pH value according to claim 1, wherein in the step S1, the standard bacterial liquid is prepared by adopting a culture medium which is the same as the bacterial liquid to be detected, and the concentration of the standard bacterial liquid is calibrated by a plate counting method, so that the accuracy of establishing a standard curve is ensured.
  7. 7. The method for rapidly determining a bacterial liquid concentration according to claim 1, wherein in the step S2, if the measured pH value of the parallel measurement is greater than 3%, the culture and pH measurement procedures of the step S2 are resampled and repeated until the relative standard deviation is less than or equal to 3%.

Description

Method for rapidly determining concentration of bacterial liquid based on pH value Technical Field The invention relates to the technical field of microorganism detection, in particular to a method for rapidly determining the concentration of bacterial liquid based on a pH value. Background In the fields of antibacterial material performance detection, food sanitation and safety monitoring, environmental microorganism pollution prevention and control and the like, accurate determination of bacterial liquid concentration is a core technical link. For example, in the development process of antibacterial materials, the antibacterial rate is calculated by measuring the concentration of bacterial liquid before and after antibacterial treatment to evaluate the antibacterial performance of the materials, and in the production process of foods, the concentration of microorganisms in raw materials and finished products is monitored to ensure that the products meet the food safety standards. The traditional bacterial liquid concentration detection method is represented by a plate counting method, and the core process comprises the steps of fully contacting a sample to be detected with bacterial liquid for a specified time, eluting residual bacteria, carrying out gradient dilution on the eluent, then inoculating the eluent to a solid culture medium plate, culturing for 24-48 hours under a proper condition, and converting the bacterial liquid concentration by manually counting the number of bacterial colonies. The method has the defects that the detection result is accurate, the detection period is overlong, the whole process takes 48-72 hours, the requirements of scenes such as rapid detection, emergency monitoring and the like cannot be met, for example, real-time quality control on food production lines, rapid response of sudden microorganism pollution events and the like, the operation process is complicated, multiple steps such as gradient dilution, inoculation, culture and counting are involved, the professional skill requirement on operators is high, manual counting is easily influenced by subjective factors, the repeatability of the detection result is poor, the detection cost is high, a large amount of consumables such as culture media and culture dishes are consumed, and equipment resources such as an incubator are occupied, so that the method is not suitable for batch detection of large-scale samples. In the prior art, it has been found that some bacteria produce acidic metabolites (such as lactic acid and acetic acid produced by escherichia coli, formic acid and acetic acid produced by salmonella) during metabolism, which results in a regular decrease in pH of the bacterial liquid with increasing bacterial count, i.e., a significant negative correlation exists between the bacterial liquid concentration and pH. However, at present, a standardized and generalized rapid detection method based on the characteristics is not available, on one hand, the existing research does not clearly and uniformly determine culture conditions (such as temperature, rotating speed and culture time), so that the correlation difference between pH values obtained by different experimenters and the concentration of bacterial solutions is large, on the other hand, the control means aiming at interference factors such as external carbon dioxide, impurities in bacterial solutions and the like can influence the accuracy of pH measurement, further, concentration calculation errors are caused, and in addition, a matched standardized detection flow and a special device are not formed, so that the large-scale application of the detection method is difficult to realize. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a method for rapidly determining the concentration of bacterial liquid based on the pH value, which solves the problems in the prior art. The invention aims to realize the purpose by adopting the following technical scheme that the method for rapidly determining the concentration of the bacterial liquid based on the pH value comprises the following steps: S1, establishing a standard curve: Selecting a target strain, preparing standard bacterial solutions with at least 5 gradient concentrations, wherein the concentration range of the standard bacterial solution with each gradient concentration is 10 3~109 CFU/mL, and the ratio of adjacent gradient concentrations is 10; respectively placing standard bacterial solutions with gradient concentrations into a sterile reaction container, and culturing for 2-4 hours under the constant-temperature oscillation condition of 35-37 ℃ and 180-220 r/min; Respectively measuring the pH value of the standard bacterial liquid with gradient concentration after culture by adopting a precise pH meter with the precision less than or equal to 0.01, measuring each concentration group in parallel for 3 times, and taking the average value as a pH measurement value cor