A method for rapidly quantifying the concentration of a bacterial solution
Patent Information
- Application Number
- CN202510445385.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2026-10-20
AI Technical Summary
[0003]目前，菌液浓度常用的测定方法有平板计数法、比浊法，其中平板计数法虽然可准确计得活菌数，但一般需要进行系列稀释，选取3～4个适宜的稀释度（细菌菌落最佳计数范围30～300个）进行倾注法或涂布法测定，所需操作复杂，操作时间合并培养时间，周期长，通量小
[0021]本发明采用酶标仪直接测定待测菌液在625nm处的吸光度，根据吸光度和菌液浓度之间的关系曲线对待测菌液进行快速定量。与浊度法相比，不需要标准比浊管且操作简单、通量高。与平板计数方法相比，本发明建立的快速定量菌液浓度的方法通量高，采用96孔平底板可以同时测定多个待测菌液，且操作简便，无需多次稀释接种及培养，快速高效。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a method for rapid quantitative determination of bacterial concentration. Background Technology
[0002] In microbiological experiments on pharmaceuticals and medical devices, commonly used test bacteria include Staphylococcus aureus, Escherichia coli, Clostridium sporogenes, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans, Bacillus subtilis, Burkholderia cepacia, Burkholderia neocaridia, and Burkholderia cepacia mysteryensis. The preparation of multiple standard bacterial solutions is crucial in these experiments, forming the foundation for all procedures. Therefore, rapid quantitative methods for various bacterial solutions are particularly important.
[0003] Currently, the commonly used methods for determining bacterial concentration include plate counting and turbidimetry. While plate counting can accurately count viable bacteria, it generally requires serial dilutions, selecting 3-4 suitable dilutions (the optimal bacterial colony counting range is 30-300) for pour or spread assays. This method is complex, time-consuming, and has a long cycle due to the combined operation and incubation time, resulting in low throughput. Turbidimetry requires specific equipment such as a turbidimeter and standard turbidimetric tubes, has low throughput, and is not widely used in routine laboratories, presenting numerous inconveniences.
[0004] This patent provides a method for rapid quantification of bacterial concentration, which is simple to measure and can be used for rapid quantification of large batches of microbial samples. Summary of the Invention
[0005] The present invention aims to provide a simple, stable and efficient method for rapid quantification of bacterial culture using an enzyme-linked immunosorbent assay (ELISA) reader.
[0006] To achieve the requirements of the above invention, the present invention provides the following technical solution:
[0007] This invention provides a method for rapidly quantifying bacterial suspension concentration. The bacterial suspension is diluted a certain factor, and the absorbance of the test bacterial suspension at a specific wavelength is measured using a multifunctional microplate reader. A curve relating the absorbance to the bacterial suspension concentration is established, and the concentration of the test bacterial suspension is calculated.
[0008] (1) Preparation of bacterial culture
[0009] The bacterial strain is activated and inoculated into a solid or liquid culture medium suitable for its growth, and then cultured for a specific time under specified conditions.
[0010] (2) Establish the relationship between bacterial absorbance and concentration.
[0011] A series of bacterial suspensions of different concentrations were prepared by diluting with a diluent. The absorbance (OD) values of the suspensions were measured at a specific wavelength using an ELISA reader. At the same time, the concentrations of the series of bacterial suspensions were counted using the plate count method. A relationship curve between the absorbance value and the concentration of the bacterial suspensions was established with the bacterial suspension concentration as the x-axis and the bacterial suspension absorbance value as the y-axis, and the corresponding regression equation for the relationship curve was obtained.
[0012] (3) Calculate the concentration of the bacterial solution.
[0013] Dilute the bacterial solution to be tested so that the concentration of the diluted solution is within the range of the relationship curve. Measure the absorbance (OD) value of the diluted bacterial solution at a specific wavelength using an ELISA reader. Calculate the concentration of the bacterial solution to be tested based on the corresponding relationship curve and regression equation.
[0014] In step (1), the solid and liquid culture media suitable for the growth of the strain are tryptic soy agar medium and tryptic soy liquid medium, which are prepared and sterilized according to the instructions.
[0015] In step (1), the strain is activated by inoculating the strain into sterilized tryptic soy peptone liquid culture medium at an inoculation rate of 1% to 2%, and then incubating it in a biochemical incubator at 30 to 35°C until the logarithmic growth phase of the strain, which takes about 16 to 24 hours.
[0016] In step (1), after activation, 0.1-0.2 ml of the strain is streaked onto tryptic soy agar medium and incubated in a biochemical incubator at 30-35°C for 16-24 hours. The culture is then eluted with 2-5 ml of 0.9% sterile sodium chloride solution and collected to obtain the bacterial suspension.
[0017] In step (2), take 0.5-1 ml of bacterial suspension and dilute it with 0.9% sterile sodium chloride solution at a ratio of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to prepare a series of bacterial suspension concentrations. Use 0.9% sterile sodium chloride solution as a blank control.
[0018] In step (2), the absorbance (OD) values of a series of diluted bacterial solutions at 625 nm were measured using an ELISA reader, and the concentration of each series of diluted bacterial solutions was measured using the pouring method. A curve showing the relationship between bacterial solution absorbance and bacterial solution concentration was established with bacterial solution concentration as the abscissa and bacterial solution absorbance as the ordinate, and the corresponding regression equation for the relationship curve was obtained.
[0019] Based on the measured OD value and
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] This invention uses an ELISA reader to directly measure the absorbance of the bacterial suspension at 625 nm, and rapidly quantifies the bacterial suspension based on the relationship curve between absorbance and bacterial concentration. Compared with the turbidimetric method, it does not require standard turbidity tubes and is simple to operate with high throughput. Compared with the plate counting method, the rapid quantitative method for bacterial concentration established in this invention has high throughput, can simultaneously measure multiple bacterial suspensions using a 96-well plate, and is simple to operate, requiring no multiple dilutions, inoculations, or incubations, making it rapid and efficient. Attached Figure Description Figure 1 This is the curve showing the relationship between the concentration of Staphylococcus aureus and absorbance in Example 1. Figure 2 This is the curve showing the relationship between Escherichia coli concentration and absorbance in Example 2. Figure 3 This is the curve showing the relationship between Pseudomonas aeruginosa concentration and absorbance in Example 3. Detailed Implementation
[0022] The present invention will be described in detail below with reference to the embodiments, but the present invention is not limited to the following embodiments. The implementation conditions in the embodiments can be further adjusted according to different bacterial strains.
[0023] Example 1
[0024] This embodiment provides a method for rapid quantification of Staphylococcus aureus, the specific steps of which are as follows:
[0025] (1) Preparation of Staphylococcus aureus bacterial suspension. The bacterial strain was inoculated into sterile tryptic soy agar medium at a concentration of 1%–2%, and incubated in a biochemical incubator at 30–35℃ until the logarithmic growth phase, approximately 16–24 hours. After activation, 0.1–0.2 ml of the strain was streaked onto tryptic soy agar medium and incubated in a biochemical incubator at 30–35℃ for 16–24 hours. The suspension was then eluted with 2–5 ml of 0.9% sterile sodium chloride solution and collected to obtain the bacterial suspension. 0.5–1 ml of the bacterial suspension was diluted with 0.9% sterile sodium chloride solution at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to prepare a series of bacterial suspension concentrations. The 0.9% sterile sodium chloride solution served as a blank control.
[0026] (2) Absorbance determination. A series of diluted bacterial culture samples and 0.9% sterile sodium chloride solution were added to 96-well plates, 0.1 ml / well, with 3 replicates for each sample. The absorbance value at 626 nm was measured by microplate reader, and the average absorbance OD value was taken.
[0027] (3) Plate count. Dilute the bacterial suspension samples at a series of dilutions, and select 3 to 4 dilutions for pour plate counting. Select the dilution level with an average colony count of less than 300 CFU as the basis for reporting the bacterial count. If the average colony count of two plates at the same dilution level is not less than 15, the colony counts of the two plates should not differ by more than 100%. Report according to the lowest dilution level that complies with the bacterial count reporting rules.
[0028] (4) Establishing the relationship between bacterial absorbance and concentration. Based on the obtained average absorbance value and plate count results, the relationship curve between the two is obtained, as shown in the figure. Figure 1 As shown, the linear regression equation is y = 0.0167x + 0.0847, r 2 =0.9998.
[0029] (5) Take the Staphylococcus aureus bacterial suspension to be tested, and perform a 10-fold serial dilution with 0.9% sterile sodium chloride solution for plate counting. Based on the counting results, dilute the Staphylococcus aureus bacterial suspension to be tested accordingly, and measure its absorbance value at 625 nm using an enzyme-linked immunosorbent assay (ELISA) reader to obtain the concentration of the Staphylococcus aureus sample to be tested calculated by absorbance method. See Table 1.
[0030] Table 1 sample Plate count concentration (cfu / ml) Dilution factor <![CDATA[Diluted OD 625nm > Calculation of bacterial concentration (cfu / ml) using absorbance method Bacterial solution 1 <![CDATA[1.73×10 9 ]]> 10 0.111 <![CDATA[1.99×10 9 ]]> Bacterial solution 2 <![CDATA[2.62×10 9 ]]> 16 0.111 <![CDATA[2.66×10 9 ]]> Bacterial solution 3 <![CDATA[1.66×10 8 ]]> Undiluted 0.113 <![CDATA[1.78×10 8 ]]>
[0032] The commonly used concentration of bacterial suspensions in pharmacopoeias is generally required to be 10. 8 Based on the results in Table 1, the absorbance of Staphylococcus aureus at 625 nm is approximately 0.108–0.134, therefore, an ELISA reader can be used to measure the OD. 625nm The method is to count quickly.
[0033] Example 2
[0034] This embodiment provides a method for rapid quantification of Escherichia coli, the specific steps of which are as follows:
[0035] (1) Preparation of Escherichia coli bacterial suspension. The bacterial strain was inoculated into sterilized tryptic soy agar medium at a concentration of 1%–2%, and incubated in a biochemical incubator at 30–35°C until the logarithmic growth phase, approximately 16–24 hours. After activation, 0.1–0.2 ml of the bacterial strain was streaked onto tryptic soy agar medium and incubated in a biochemical incubator at 30–35°C for 16–24 hours. The culture was then eluted with 2–5 ml of 0.9% sterile sodium chloride solution and collected to obtain the bacterial suspension. 0.5–1 ml of the bacterial suspension was diluted with 0.9% sterile sodium chloride solution at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to prepare a series of bacterial suspension concentrations. The 0.9% sterile sodium chloride solution served as a blank control.
[0036] (2) Absorbance determination. A series of diluted bacterial culture samples and 0.9% sterile sodium chloride solution were added to 96-well plates, 0.1 ml / well, with 3 replicates for each sample. The absorbance value at 626 nm was measured by microplate reader, and the average absorbance OD value was taken.
[0037] (3) Plate count. Dilute the bacterial suspension samples at a series of dilutions, and select 3 to 4 dilutions for pour plate counting. Select the dilution level with an average colony count of less than 300 CFU as the basis for reporting the bacterial count. If the average colony count of two plates at the same dilution level is not less than 15, the colony counts of the two plates should not differ by more than 100%. Report according to the lowest dilution level that complies with the bacterial count reporting rules.
[0038] (4) Establishing the relationship between bacterial absorbance and concentration. Based on the obtained average absorbance value and plate count results, the relationship curve between the two is obtained, as shown in the figure. Figure 2 As shown, the linear regression equation is y = 0.0168x + 0.1064, r 2 =0.9969.
[0039] (5) Take the Escherichia coli bacterial solution to be tested and perform a 10-fold serial dilution with 0.9% sterile sodium chloride solution for plate counting. Based on the counting results, dilute the Escherichia coli bacterial solution to be tested and measure its absorbance value at 625 nm using an enzyme-linked immunosorbent assay (ELISA) reader to obtain the concentration of the Escherichia coli sample to be tested calculated by absorbance method. See Table 2.
[0040] Table 2 sample Plate count concentration (cfu / ml) Dilution factor <![CDATA[OD after dilution 625nm > Calculation of bacterial concentration (cfu / ml) using absorbance method Bacterial solution 1 <![CDATA[3.26×10 9 ]]> 20 0.135 <![CDATA[3.40×10 9 ]]> Bacterial solution 2 <![CDATA[2.22×10 9 ]]> 20 0.128 <![CDATA[2.57×10 9 ]]> Bacterial solution 3 <![CDATA[4.60×10 8 ]]> 2 0.155 <![CDATA[5.79×10 8 ]]>
[0042] The commonly used concentration of bacterial suspensions in pharmacopoeias is generally required to be 10. 8 Based on the results in Table 2, the absorbance of Escherichia coli at 625 nm is approximately 0.128–0.155, therefore, an ELISA reader can be used to determine the OD value. 625nm The method is to count quickly.
[0043] Example 3
[0044] This embodiment provides a method for rapid quantification of Pseudomonas aeruginosa, the specific steps of which are as follows:
[0045] (1) Preparation of Pseudomonas aeruginosa bacterial suspension. The bacterial strain was inoculated into sterile tryptic soy agar medium at a concentration of 1%–2%, and incubated at 30–35°C in a biochemical incubator until the logarithmic growth phase, approximately 16–24 hours. After activation, 0.1–0.2 ml of the strain was streaked onto tryptic soy agar medium and incubated at 30–35°C for 16–24 hours. The culture was then eluted with 2–5 ml of 0.9% sterile sodium chloride solution, and the eluted culture was collected to obtain the bacterial suspension. 0.5–1 ml of the bacterial suspension was diluted with 0.9% sterile sodium chloride solution at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to prepare a series of bacterial suspension concentrations. The 0.9% sterile sodium chloride solution served as a blank control.
[0046] (2) Absorbance determination. A series of diluted bacterial culture samples and 0.9% sterile sodium chloride solution were added to 96-well plates, 0.1 ml / well, with 3 replicates for each sample. The absorbance value at 626 nm was measured by microplate reader, and the average absorbance OD value was taken.
[0047] (3) Plate count. Dilute the bacterial suspension samples at a series of dilutions, and select 3 to 4 dilutions for pour plate counting. Select the dilution level with an average colony count of less than 300 CFU as the basis for reporting the bacterial count. If the average colony count of two plates at the same dilution level is not less than 15, the colony counts of the two plates should not differ by more than 100%. Report according to the lowest dilution level that complies with the bacterial count reporting rules.
[0048] (4) Establishing the relationship between bacterial absorbance and concentration. Based on the obtained average absorbance value and plate count results, the relationship curve between the two is obtained, as shown in the figure. Figure 3 As shown, the linear regression equation is y = 0.0192x + 0.0986, r 2 =0.9992.
[0049] (5) Take the Pseudomonas aeruginosa bacterial suspension to be tested, and perform a 10-fold serial dilution with 0.9% sterile sodium chloride solution for plate counting. Based on the counting results, dilute the Pseudomonas aeruginosa bacterial suspension to be tested accordingly, and measure its absorbance value at 625 nm using an ELISA reader to obtain the concentration of the Pseudomonas aeruginosa sample calculated by absorbance method. See Table 3.
[0050] Table 3 sample Plate count concentration (cfu / ml) Dilution factor <![CDATA[Diluted OD 625nm > Calculation of bacterial concentration (cfu / ml) using absorbance method Bacterial solution 1 <![CDATA[4.12×10 9 ]]> 64 0.113 <![CDATA[4.80×10 9 ]]> Bacterial solution 2 <![CDATA[1.07×10 9 ]]> 10 0.127 <![CDATA[1.59×10 9 ]]> Bacterial solution 3 <![CDATA[1.45×10 8 ]]> 4 0.107 <![CDATA[1.71×10 8 ]]>
[0052] The commonly used concentration of bacterial suspensions in pharmacopoeias is generally required to be 10. 8 Based on the results in Table 3, the absorbance of *Pseudomonas aeruginosa* at 625 nm is approximately 0.120–0.130. Therefore, an ELISA reader can be used to measure the OD value. 625nmThe method is to count quickly.
Claims
1. A method for rapidly quantifying bacterial concentration, characterized in that... The rapid quantitative method for bacterial culture includes the following steps: (1) Preparation of bacterial culture. (2) Establish the relationship between the absorbance and concentration of the bacterial solution. (3) Calculate the bacterial concentration.
2. The bacterial culture preparation according to claim 1, characterized in that: In step (1), the strain is activated and inoculated into a solid or liquid culture medium suitable for the growth of the strain, and cultured for a specific time under specified conditions.
3. The bacterial culture preparation according to claim 1, characterized in that: In step (1), the bacterial strain is activated by inoculating the strain into sterilized tryptic soy peptone liquid culture medium at an inoculation rate of 1% to 2%, and then culturing it in a biochemical incubator at 30 to 35°C until the logarithmic growth phase of the bacterial culture, which takes about 16 to 24 hours.
4. The bacterial culture preparation according to claim 1, characterized in that: In step (1), the activated bacterial strain is streaked 0.1 to 0.2 ml onto tryptic soy agar medium and incubated in a biochemical incubator at 30 to 35°C for 16 to 24 hours. The culture is then eluted with 2 to 5 ml of 0.9% sterile sodium chloride solution and collected to obtain the bacterial suspension.
5. The method for establishing the relationship between bacterial absorbance and concentration according to claim 1, characterized in that: In step (2), the absorbance (OD) values of a series of diluted bacterial solutions at 625 nm are measured using an ELISA reader, and the concentration of each series of diluted bacterial solutions is measured using the pouring method. A relationship curve between the absorbance value and the concentration of the bacterial solution is established with the bacterial solution concentration as the abscissa and the bacterial solution absorbance value as the ordinate, and the corresponding regression equation for the relationship curve is obtained.
6. The method for establishing the relationship between bacterial absorbance and concentration according to claim 1, characterized in that: In step (2), the series of diluted bacterial solutions are prepared by taking 0.5-1 ml of bacterial suspension and diluting it with 0.9% sterile sodium chloride solution at a ratio of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to prepare a series of bacterial solution concentrations, with 0.9% sterile sodium chloride solution as a blank control.
7. The method for calculating bacterial concentration according to claim 1, characterized in that: In step (3), the operation is to dilute the bacterial solution to be tested so that the concentration of the diluted bacterial solution is within the range of the relationship curve, and to measure the absorbance OD value of the diluted bacterial solution at a specific wavelength using an enzyme-linked immunosorbent assay (ELISA) reader. The concentration of the bacterial solution to be tested is calculated based on the corresponding relationship curve and regression equation.
8. The method for rapid quantitative determination of bacterial concentration according to claim 1, characterized in that... This paper presents a rapid bacterial quantification method that is simple to operate, has high throughput, is highly practical, stable, and effective, and can be used for the rapid quantification of large batches of microbial samples. The commonly used bacterial suspension concentration in pharmacopoeias is 10... 8 CFU corresponds to the OD values of various strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 625nm Approximately 0.108–0.134, 0.128–0.155, and 0.120–0.130.