Method for rapidly detecting total number of microbial colonies
By combining microfluidic technology and fluorescent dye labeling with optical detection, the problems of long detection time and large error in total microbial colony count have been solved, achieving rapid and accurate detection of total colony count.
Patent Information
- Application Number
- CN202511408582.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-09-15
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-26
AI Technical Summary
Existing technologies for detecting total microbial colonies are time-consuming and have large errors, especially due to inaccuracies caused by colony overlap and manual counting methods.
Microfluidic technology is used to generate water-in-oil droplets. Combined with fluorescent dyes and optical detection, the concentration of viable bacteria is calculated using the Poisson distribution formula, enabling rapid and independent culture and fluorescent labeling of microorganisms. Fluorescence signals are then acquired using a fluorescence scanner for counting.
It significantly shortens the detection time, avoids colony overlap, improves the accuracy and sensitivity of detection, and enables rapid and accurate detection of total microbial colonies.
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Figure CN121207948A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of microorganism detection, and particularly relates to a method for rapidly detecting total number of microbial colonies. BACKGROUND
[0002] The total number of colonies refers to the total number of microbial colonies grown per gram (or milliliter) of sample under certain conditions (such as aerobic conditions, nutritional conditions, pH, culture temperature and time, etc.). By determining the total number of colonies, the degree of bacterial contamination of the sample can be determined.
[0003] At present, the total number of colonies is usually detected by using the national standard GB 4789.2-2022, and the flow chart is as shown in Figure 1 However, the following disadvantages exist:
[0004] 1) long culture time: 48h-72h for plate culture;
[0005] 2) pouring method causes errors in results: the standard specifies that 1mL of sample solution is taken and uniformly mixed in a sterile culture dish, then 15mL-20mL of liquid agar medium cooled to 46℃-50℃ is poured into the culture dish, the medium is mixed with the sample, and after solidification at room temperature, the culture is cultured, and the colonies growing on the surface are counted. This method can cause multiple bacteria to aggregate, resulting in overlapping of the cultured colonies, and multiple bacteria are counted as one colony; this method can also cause some microorganisms to grow in the agar medium, which cannot be found by the naked eye, resulting in errors in the number of microorganisms in the actual sample;
[0006] 3) errors in colony counting method: the national standard method specifies that "the naked eye observation method is used, sometimes a magnifying glass or a colony counter is used, and the dilution multiple and the corresponding number of colonies are recorded", and the errors caused by this manual counting method can also lead to inaccurate experimental results. SUMMARY
[0007] In view of the deficiencies of the prior art, the present application provides a method for rapidly detecting the total number of microbial colonies, comprising the following steps:
[0008] (1) mixing the sample to be tested with a diluent to obtain a sample solution;
[0009] (2) centrifuging the sample solution to remove the supernatant, and preparing a suspension with the precipitate using a liquid medium, adding a fluorescent dye and mixing uniformly to obtain a mixed solution;
[0010] (3) generating water-in-oil droplets by microfluidic droplet technology, and the particle size of the droplets is 25-100μm;
[0011] (4) placing the droplet in an incubator for a period of time to allow the microorganism in the droplet to proliferate rapidly, and the dye contained in the droplet to penetrate the cell membrane and stain the microorganism;
[0012] (5) scanning the incubated droplet with excitation light having a maximum excitation wavelength, and detecting the total number of droplets and the fluorescence intensity of each droplet at a maximum emission wavelength, and then calculating the viable bacterial concentration (i.e. the total number of colonies) by a Poisson distribution formula; the maximum excitation wavelength and the maximum emission wavelength are determined according to the characteristics of the fluorescent dye used, and the maximum excitation wavelength is consistent with the peak absorption wavelength of the selected fluorescent dye.
[0013] The present application mixes a sample liquid containing a sample to be tested with a culture solution and a fluorescent dye to obtain a culture solution system containing microorganisms and a fluorescent dye, and then divides the culture solution system into microdroplets by microfluidic technology, so that each droplet contains a very low concentration or a single microorganism. By short-time incubation of the droplets, the microorganisms in the droplets proliferate rapidly, the fluorescent dye contained therein can stain the viable microorganisms, and a fluorescent signal is generated. The fluorescence value is detected by specific excitation and emission spectrum, and the average fluorescence intensity + 10 times the standard deviation is used as the fluorescence threshold. The droplets with a fluorescence intensity higher than the threshold are positive droplets, and the droplets with a fluorescence intensity lower than the threshold are negative droplets. According to the proportion of positive droplets in the total number of droplets (λ = -ln(1-p), λ is the average number of bacteria in each droplet, and p is the proportion of positive droplets), the number of bacteria in the original droplet is calculated by using the Poisson distribution formula.
[0014] Preferably, in step (1), the diluent is sterile phosphate buffer or sterile normal saline;
[0015] When the sample to be tested is a liquid sample, the volume ratio of the sample to be tested to the diluent is 1:3-10;
[0016] When the sample to be tested is a solid sample, the mass-volume ratio of the sample to be tested to the diluent is 1g:3-10mL.
[0017] Preferably, in step (2), the centrifugation speed is 5000-10000xg, and the time is 5-10min.
[0018] Preferably, in step (2), the volume of the sample liquid is 1mL, the amount of the liquid culture medium is 75μL-225μL, and the amount of the fluorescent dye is 0.2μL-0.8μL. Research has found that the amounts of the liquid culture medium and the fluorescent dye are very critical. If the amount of the liquid culture medium is higher than the above range, there is a disadvantage of reducing the detection sensitivity, and if the amount is lower than the above range, there is a disadvantage of false negatives. If the amount of the fluorescent dye is higher than the above range, there are disadvantages of high background fluorescence, high false negatives, and high false positives, and if the amount is lower than the above range, there are disadvantages of weak fluorescence of positive droplets and high false negatives.
[0019] As preferred, in step (3), the specific process of the microfluidic droplet technology is as follows: taking the droplet generation oil as the oil phase and the mixed solution as the water phase, the water phase and the oil phase are injected into channel 1 and channel 2 of the microfluidic droplet generator respectively, and the water-in-oil droplet is obtained by controlling the pressure in channel 1 and channel 2.
[0020] As preferred, in step (4), the culture time is 3-5h.
[0021] As preferred, the fluorescent dye is SYTO9 dye;
[0022] The maximum excitation wavelength is 488nm, and the maximum emission wavelength is 500-530nm. SYTO 9 can penetrate the cell membrane, enter the inside of the cell, bind to the phosphate group in the nucleic acid molecule, and produce fluorescence, and the maximum excitation wavelength is 488nm and the emission wavelength is 500nm to 530nm.
[0023] The present application has the beneficial effects of:
[0024] 1) The method for rapidly detecting the total number of microbial colonies provided by the present application has the advantages that the single microorganism in the droplet is self-contained and is not disturbed by other bacteria, the nutrient medium in the droplet is transmitted at a high speed, the microorganism proliferates at a high speed, the microorganism individual can be recovered and proliferated through a short-time culture, the fluorescent dye can be used to mark the live microbial colonies, and the culture time is greatly shortened.
[0025] 2) The method for rapidly detecting the total number of microbial colonies provided by the present application has the advantages that the microbial colonies are independently distributed in single droplets, so that each microorganism is self-contained, the microorganism individual in the sample is more dispersed, the phenomenon of overlapping of the microbial colonies is avoided, and the counting is accurate.
[0026] 3) The method for rapidly detecting the total number of microbial colonies provided by the present application has the advantages that the accuracy and the sensitivity of the detection result are improved by optimizing the amount of the liquid medium and the fluorescent dye.
[0027] 4) The method for rapidly detecting the total number of microbial colonies provided by the present application has the advantages that the fluorescent signal is collected by selecting appropriate optical parameters to excite the fluorescence by using the fluorescence scanning counter, the counting and detection time is greatly shortened, the number of live microorganisms is absolutely quantified by using the Poisson distribution method, and the total number of microbial colonies is rapidly and accurately detected. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 The colony count test procedure is stipulated in GB 4789.2.
[0029] Figure 2A flow chart for generating water-in-oil droplets, staining, and scanning counting in Example 1.
[0030] Figure 3 A droplet fluorescence intensity graph for Example 1.
[0031] Figure 4 A droplet total number peak width peak height graph for Example 1.
[0032] Figure 5 A droplet peak area peak height graph for Example 1. DETAILED DESCRIPTION
[0033] The following examples are provided to illustrate the present application, but not to limit the scope of the present application. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.
[0034] In the following examples, the formula of the phosphate buffer used is as follows: sodium chloride (NaCl) 8.0 g, potassium chloride (KCl) 0.20 g, potassium dihydrogen phosphate (KH2PO4) 0.24 g, disodium hydrogen phosphate (Na2HPO4) 1.44 g, 1000 mL distilled water.
[0035] In the following examples, the microfluidic droplet generator used is derived from the microfluidic droplet preparation instrument of Zhejiang Dapu Biological Technology Co., Ltd., model DC400.
[0036] In the following examples, the scanning detector used is derived from the high-throughput droplet sorter of Zhejiang Dapu Biological Technology Co., Ltd., model DS1200.
[0037] Example 1
[0038] A method for rapidly detecting the total number of microbial colonies, the steps are as follows:
[0039] 1. Preparation of the sample to be tested: inoculate E. coli (CMCC 44102) in soy sauce (sterile after detection), the concentration is about 10 4 CFU / mL, to obtain the soy sauce sample to be tested.
[0040] 2. Sample detection: weigh 25 mL of the soy sauce sample to be tested, and place it in a sterile homogenization cup containing 225 mL of sterile phosphate buffer, mix well to prepare the sample liquid.
[0041] 3. Bacterial encapsulated droplet generation: take 1 mL of the sample liquid, centrifuge at 10000 x g for 7 min, remove the supernatant, and prepare the precipitate into a suspension with 75 μL of TSB liquid medium, mix well after adding 0.3 μL of SYTO9 dye.
[0042] The mixed solution was prepared into water-in-oil droplets with a particle size of 25–100 μm using a microfluidic droplet generator. (See [reference needed]). Figure 2 Specifically: using Dap Bio's universal droplet generating oil as the oil phase and the mixed solution as the water phase, the water phase and the oil phase are injected into channels 1 and 2 of the microfluidic droplet generator, respectively. The pressure in channels 1 and 2 is controlled by the microfluidic droplet generation to obtain water-in-oil droplets.
[0043] 4. Bacterial culture and staining: The prepared droplets are placed in an incubator and incubated at 36℃±1℃ for 3 hours; the microorganisms in the droplets proliferate rapidly and are stained by the SYTO9 dye contained in the droplets, which penetrates the cell membrane.
[0044] 5. Scanning Counting: After cultivation, the microdroplets were fed into the instrument. Excitation / emission wavelengths of 488nm / 530nm were selected, and fluorescence values were detected using a scanning detector to determine the total number of droplets and fluorescence intensity. Results are as follows: Figures 3-5 As shown, Figure 3 This is a droplet fluorescence intensity diagram from Example 1. Figure 3 Each fluorescence peak represents a droplet, through Figure 3 The average fluorescence intensity of the droplets was calculated to be 641, with a standard deviation of 27. The fluorescence threshold, 911, was set as the average fluorescence intensity plus 10 times the standard deviation. Peaks with fluorescence intensities above the threshold were considered positive droplets, while peaks below the threshold were considered negative droplets. Figure 4 This is a peak height (maximum fluorescence intensity) - peak width (duration) graph. Figure 5 This is a peak area (total fluorescence intensity) - slit width plot. Figure 4 , Figure 5 In the image, grayscale represents the number of droplets. (The text abruptly ends here.) Figure 5 The total number of droplets is 363,831, and the number of positive droplets is 134.
[0045] First, the average number of bacteria λ per droplet is calculated based on the proportion p of positive droplets in the total number of droplets. The result is 0.000368 (λ = -ln(1-p), where λ is the average number of bacteria per droplet and p is the proportion of positive droplets). Then, the concentration of viable bacteria in the sample is calculated using the Poisson distribution formula.
[0046] viable bacteria concentration = λ × n / V3 × (V1 + V2) / V1
[0047] Where: n is the total number of droplets;
[0048] V1 is the volume of the soy sauce sample to be tested (25 mL);
[0049] V2 is the volume of the diluent (225 mL);
[0050] V3 is the volume of the centrifuged sample liquid (1 mL);
[0051] 6. Results: The detection was completed within 6 hours from sample preparation to detection, and the viable bacterial concentration was 6x10 3 CFU / mL, and the total number of sample colonies verified by plate culture according to GB 4789.2-2022 was 8x10 3 CFU / mL, which was consistent with the above detection value in the order of magnitude. The detection limit was 10 CFU / mL.
[0052] Comparative Example 1
[0053] The difference compared with Example 1 is only that the amount of liquid medium TSB is 50 μL. As a result, the culture time of the droplet in the incubator is at least 10 hours to detect the fluorescence signal.
[0054] Comparative Example 2
[0055] The difference compared with Example 1 is only that the mixed solution is prepared into water-in-oil small droplets with a particle size of 20 μm by a microfluidic droplet generator. As a result, the scanning time of the scanning detector is 15-16 hours.
[0056] Comparative Example 3
[0057] The difference compared with Example 1 is only that the mixed solution is prepared into water-in-oil small droplets with a particle size of 250 μm by a microfluidic droplet generator. As a result, the detection limit of the method is increased to 200 cfu / mL.
[0058] The above examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by ordinary engineering technicians in the art shall fall within the protection scope determined by the claims of the present application.
Claims
1. A method for rapid detection of total microbial colony count, characterized in that, Includes the following steps: (1) Mix the sample to be tested with the diluent to obtain the sample solution; (2) Centrifuge the sample solution to remove the supernatant, prepare a suspension of the precipitate with liquid culture medium, add fluorescent dye and mix well to obtain a mixed solution; (3) The mixed solution is used to generate water-in-oil droplets using microfluidic droplet technology, and the droplet size is 25-100 μm; (4) Place the droplet in an incubator and incubate for a period of time; (5) Scan the cultured droplets with excitation light having the maximum excitation wavelength, and detect the total number of droplets and the fluorescence intensity of each droplet at the maximum emission wavelength. Then calculate the viable bacterial concentration using the Poisson distribution formula. The maximum excitation wavelength and the maximum emission wavelength are determined according to the characteristics of the fluorescent dye used.
2. The method for rapid detection of total microbial colony count according to claim 1, characterized in that, In step (1), the diluent is sterile phosphate buffer or sterile physiological saline; When the sample to be tested is a liquid sample, the volume ratio of the sample to the diluent is 1:3 to 10; When the test sample is a solid sample, the mass-to-volume ratio of the test sample to the diluent is 1g:3-10mL.
3. A method for rapid detection of total microbial colony count according to claim 1 or 2, characterized in that, In step (2), the centrifugation speed is 5000-10000×g and the time is 5-10min.
4. A method for rapid detection of total microbial colony count according to claim 1 or 2, characterized in that, In step (2), with the volume of the sample solution being 1 mL, the amount of liquid culture medium used is 75 μL to 225 μL, and the amount of fluorescent dye used is 0.2 μL to 0.8 μL.
5. A method for rapid detection of total microbial colony count according to claim 1 or 2, characterized in that, In step (3), the specific process of the microfluidic droplet technology is as follows: using the droplet-generating oil as the oil phase and the mixed solution as the water phase, the water phase and the oil phase are injected into the channels 1 and 2 of the microfluidic droplet generator respectively, and the pressure in the channels 1 and 2 is controlled by the microfluidic droplet generation to obtain water-in-oil droplets.
6. A method for rapid detection of total microbial colony count according to claim 1 or 2, characterized in that, In step (4), the culture time is 3 to 5 hours.
7. A method for rapid detection of total microbial colony count according to claim 1 or 2, characterized in that, The fluorescent dye is SYTO9 dye; The maximum excitation wavelength is 488 nm, and the maximum emission wavelength is 500–530 nm.