CN109655447B - Detection system and method for microbial enumeration - Google Patents

Detection system and method for microbial enumeration Download PDF

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Publication number
CN109655447B
CN109655447B CN201910080620.3A CN201910080620A CN109655447B CN 109655447 B CN109655447 B CN 109655447B CN 201910080620 A CN201910080620 A CN 201910080620A CN 109655447 B CN109655447 B CN 109655447B
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fluid
detection
sample
raman
tested
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CN109655447A (en
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刘辉
童星
周斌
孙爱华
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Foshan Haitian Flavoring and Food Co Ltd
Foshan Haitian Gaoming Flavoring and Food Co Ltd
Guangdong Haitian Innovation Technology Co Ltd
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Foshan Haitian Flavoring and Food Co Ltd
Foshan Haitian Gaoming Flavoring and Food Co Ltd
Guangdong Haitian Innovation Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block

Abstract

The present invention provides a detection system for microbial enumeration, comprising: the system comprises a fluid pump with a fluid inlet and a fluid outlet, a fluid control device and a spectrum detection system, wherein the fluid control device comprises a sample mixing pool, a fluid pipeline with a first end and a second end and a diluent pool; the sample mixing pool is respectively in fluid communication with the fluid outlet, the first end and the diluent pool, the fluid pipeline comprises an inverted V-shaped pipe unit, the inverted V-shaped pipe unit comprises an ascending part and a descending part, and a detection area is arranged at a position close to the second end; the spectral detection system comprises a light source and a detection device, wherein the light source is arranged to generate excitation light that passes through the detection zone; the detection device is configured to receive and detect the optical signal generated from the detection zone to generate a spectrum. The invention also provides a method for microbial enumeration comprising use of the detection system of the invention.

Description

Detection system and method for microbial enumeration
Technical Field
The present invention relates to the field of microorganisms, in particular to a detection system and method for microbial enumeration.
Background
The food is a necessity of daily life of people because of rich and variable taste and nutrient substances, but the rich nutrient substances also provide good conditions for the growth of microorganisms. The microbial content also becomes an important index for evaluating food safety. At present, the internationally accepted hygienic standard takes the total number of bacteria and the total number of fungi as safety evaluation indexes. In order to guarantee food safety, various countries correspondingly establish standardized detection procedures and counting methods of total bacterial colonies. The standardized detection process is not only beneficial to evaluating the safety of food, but also provides reliable scientific basis for food supervision departments. The standardized detection process also provides a basis for food production enterprises to strictly control the product quality, unqualified products can be found in time, and the situation that the unqualified products flow into the market to threaten the life and lives of people is avoided. At present, the method for detecting microorganisms in the national standard of China mainly adopts a microorganism culture method: detecting the microorganisms by using different culture media in a directional or non-directional culture manner; the total number of detected microorganisms can be visually observed, the required time is generally 24-72h, detection results can be obtained by partial microorganisms even longer, the required culture time is long, the operation steps are complicated, and the workload is large. Even some microorganisms cannot be cultured and thus escape detection. The introduction of new technologies and methods is crucial to the development of microbial detection technologies, and fluorescent labeling technologies, cell membrane electrophysiological technologies and high-resolution microscopes make it possible to directly explore the life activities of microorganisms, but various antibodies and staining technologies are expensive and can affect or damage microbial cells in physiological states to different degrees. Besides, most of the techniques have the disadvantages of complicated test operation, long time consumption and the like.
The micro-Raman spectrum technology adopts a low-power laser, a holographic technology with high conversion efficiency and a confocal technology, has the advantages of high detection sensitivity, short time, small required sample amount, no need of additional reagents or complex means for preparing samples and the like, and has the remarkable advantages of more and more extensive application in the field of microorganism detection and analysis. However, when the raman spectroscopy is used to detect the total number of microbial colonies at present, a detection sample needs to be made into a small sample plate, the thickness or the appropriate sample amount of the sample plate is difficult to control, and if the control is not proper, the test result is easily directly affected, and then when the manufactured sample plate is subjected to the microscope detection through the raman spectroscopy, a microbial sample needs to be manually searched, and the microbes in a determined visual field range can be detected, so that the capability of detecting the microbes in the sample by the raman spectroscopy is limited.
Accordingly, there remains a need in the art for improved detection methods and systems to achieve accurate, rapid, and convenient detection or enumeration of microorganisms.
Disclosure of Invention
The invention aims to provide a detection system and a detection method for detecting the total number of microorganisms in a fluid sample based on a photo-fluidic technology.
Accordingly, in one aspect, the present invention provides a system for detecting or enumerating microorganisms, comprising:
a fluid pump 1 having a fluid inlet and a fluid outlet;
a fluid control device 2 including a fluid line 21 having a first end and a second end, a sample mixing well 22, and a diluent well 23; the sample mixing pool 22 is respectively in fluid communication with the fluid outlet, the first end and the diluent pool 23, the fluid pipeline 21 comprises an inverted V-shaped pipe unit 211, the inverted V-shaped pipe unit 211 comprises an ascending part 211a and a descending part 211b, and a detection area is arranged at a position close to the second end;
a spectral detection system 3 comprising a light source 31 and a detection device 32, wherein the light source 31 is arranged to generate excitation light passing through the detection zone; the detection device 32 is arranged to receive and detect the light signal generated from the detection zone to generate a spectrum.
In certain embodiments, the fluid line 21 comprises a plurality of inverted V-tube units 211 in fluid communication. In certain embodiments, the fluid line 21 comprises 3, 4, or 5 inverted V-tube units 211 in fluid communication.
In certain embodiments, the angle formed by the rising portion 211a and the falling portion 211b is acute. Preferably, the acute angle ranges between 45 and 75 degrees.
In some embodiments, the rising portion 211a is at an obtuse angle to the horizontal. Preferably, the obtuse angle ranges between 100 and 135 degrees.
In certain embodiments, the rising portion 211a is of equal length as the falling portion 211 b.
In certain embodiments, the fluid line has an inner diameter of 20 μm to 50 μm, such as 20 μm to 30 μm.
In certain embodiments, the fluid line 21 is made of a material that is transparent to excitation light.
In certain embodiments, the excitation light permeable material is selected from polymethylmethacrylate PMMA or polydimethylsiloxane PDMS.
In certain embodiments, the first end of the fluid line 21 and its vicinity extend in a horizontal direction.
In certain embodiments, the system further comprises: a fluid source 4 for receiving a fluid sample to be tested, in fluid communication with the fluid inlet of the fluid pump 1. In certain embodiments, the fluid sample to be tested is a liquid food product, such as a drink, liquid condiment, or drinking water.
In certain embodiments, the system further comprises: a waste reservoir 5 in fluid communication with a second end of the fluid line 21.
In certain embodiments, the system further comprises: a valve 6 for controlling the flow of fluid from the sample mixing basin 22 to the fluid line 21.
In certain embodiments, the system comprises at least 2 fluid control devices 2, the mixing wells 22 comprised in the at least 2 fluid control devices 2 being in fluid communication with each other. In certain embodiments, the system comprises at least 3 fluid control devices 2, the mixing wells 22 comprised in the at least 3 fluid control devices 2 being in fluid communication with each other.
In certain embodiments, the spectral detection system 3 further comprises a control device in data communication with the light source 31 and detection device 32.
In certain embodiments, the detection device 32 comprises a photoelectric converter.
In certain embodiments, the spectral detection system 3 is selected from the group consisting of a raman spectral detection system, an infrared spectral detection system, and an ultraviolet spectral detection system.
In certain embodiments, the spectroscopic detection system 3 is a raman spectroscopic detection system and the detection device 32 comprises a CCD (charge coupled device). In certain embodiments, the detection device 32 further comprises a photomultiplier tube. In certain embodiments, the detection device 32 further comprises a light splitting section for splitting the raman scattered light according to wavelength. For example, the spectroscopic portion may be a diffraction grating.
In another aspect, the present invention provides a method for detecting or enumerating microorganisms in a sample, comprising the steps of:
(i) passing a fluid sample to be tested containing microorganisms through the fluid pump 1 of the system of the present invention and moving in the fluid pipeline 21 in the direction from the first end to the second end and at a flow rate of 5-1200 μ L/min, so that the microorganisms in the fluid sample to be tested pass through the detection area of the fluid pipeline 21 one by one;
(ii) performing spectroscopic detection of a fluid sample to be detected passing through the detection zone by means of the detection system 3 of the present invention to obtain a spectrum generated from the detection zone, the spectrum comprising a spectrum attributed to bacteria in the fluid sample to be detected;
(iii) analyzing the spectrum produced by the detection zone to obtain a profile attributed to the microorganisms;
(iv) performing a statistical analysis on the profile attributed to the microorganism to obtain the number of the microorganism.
In certain embodiments, in step (i), the fluid sample to be tested is applied to the fluid source 4 of the system of the present invention.
In certain embodiments, in step (ii), raman spectroscopy detection is performed on the fluid sample to be tested passing through the detection zone by a raman spectroscopy detection system to obtain a raman spectrum generated from the detection zone; and, in step (iii), the raman spectrum generated at the detection zone is analyzed to obtain the amount attributed to the microorganism.
In certain embodiments, the raman spectroscopy is selected from the group consisting of confocal raman spectroscopy, surface enhanced raman spectroscopy, coherent anti-stokes raman spectroscopy, and laser tweezer raman spectroscopy.
In certain embodiments, the raman spectroscopy detection is performed under one or more of the following conditions:
(i) the acquisition time is 8ms-15 s;
(ii) the wavelength of the exciting light is in the wavelength range from ultraviolet to near infrared;
(iii) the laser power is 2-20 mW;
in certain embodiments, the raman spectroscopy detection is performed under conditions selected from the group consisting of:
(a) the acquisition time is 8ms, the wavelength of the excitation light is 475nm, and the laser power is 2 mW;
(b) the acquisition time is 15s, the wavelength of the excitation light is 785nm, and the laser power is 20 mW;
(c) the acquisition time is 2s, the wavelength of the excitation light is 600nm, and the laser power is10 mW;
(d) the collection time is100 ms, the wavelength of the excitation light is 500nm, and the laser power is 2 mW.
In certain embodiments, prior to step (i), the method further comprises the steps of: and preprocessing the fluid sample to be detected. In certain embodiments, the pretreatment is selected from filtration, dilution, or any combination thereof. In certain embodiments, prior to step (i), the fluid sample to be tested is diluted with sterile water.
In certain embodiments, the fluid sample to be tested is a liquid food product, such as a drink, liquid condiment, or drinking water. In certain embodiments, the fluid sample to be tested is selected from soy sauce, milk, juice, and mineral water.
Advantageous effects of the invention
The invention combines the fluid technology with the Raman spectrum technology for the first time, and provides a detection system and a detection method for counting microorganisms. The detection system/method of the invention arranges the microorganisms in the liquid sample rapidly and orderly through the microtube device, thereby leading the microorganisms to pass through the spectrometer one by one to obtain the spectral information on the micro scale, further analyzing the biomolecule information of a large number of microorganisms in the liquid sample in a short time, and carrying out statistical analysis on the detection result to realize the detection of the total number of bacterial colonies in the liquid sample, thereby realizing the purpose of detecting the number of the microorganisms in real time, dynamically and in a large scale. The detection system/method can quickly realize the detection of the microbial colonies in the liquid sample without the steps of flaking, selecting specific microbial colonies and the like, improves the accuracy of the detection result, has simple sample treatment and test processes, and greatly improves the detection speed.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a schematic structural view of an exemplary embodiment of a detection system of the present invention.
Fig. 2 is a schematic structural view of a fluid pipeline of the detection system shown in fig. 1.
FIGS. 3a-3b are schematic diagrams of Raman spectra and CCD statistics of Experimental example 1.
FIGS. 4a-4b are schematic diagrams of Raman spectra and CCD statistics of Experimental example 2.
FIGS. 5a-5b are schematic diagrams of Raman spectra and CCD statistics of Experimental example 3.
FIGS. 6a-6b are schematic diagrams of Raman spectra and CCD statistics of Experimental example 4.
FIGS. 7a-7b are schematic diagrams of Raman spectra and CCD statistics of comparative example 1.
Detailed description of the preferred embodiments
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, further discussion thereof is not required in subsequent figures.
Example 1 detection System for detecting or enumerating microorganisms
Fig. 1-2 show a schematic construction of an exemplary detection system of the present invention and a schematic construction of a fluid circuit included in the detection system, respectively.
As shown in fig. 1, the exemplary detection system of the present invention includes a fluid pump 1 having a fluid inlet and a fluid outlet, a fluid control device 2, a spectroscopic detection system 3, a fluid source 4 for receiving a fluid sample to be detected, a waste reservoir 5, and a valve 6. The exemplary detection system comprises 3 fluid control devices 2, an upper fluid control device, a middle fluid control device and a lower fluid control device.
The fluid control device 2 mainly comprises a fluid line 21 having a first end and a second end, a sample mixing basin 22 and a diluent basin 23. The sample mixing basin 22 is in fluid communication with the fluid outlet, the first end portion and the diluent basin 23 through pipelines, respectively, for mixing the fluid sample with the diluent in the diluent basin 23. The first end 21a of the fluid line 21 and its vicinity extend in the horizontal direction, and the vicinity of the first end 21a is provided with a valve 6 for controlling the flow of fluid from the mixing well 22 to the fluid line 21. The second end 21b of the fluid line 21 is in fluid communication with the waste reservoir 5, and a detection zone 21c is provided adjacent the second end 21 b.
The fluid pump 1 causes a fluid sample to be measured to move in the fluid line 21 in the direction D indicated by the arrow in fig. 1.
As shown in fig. 2, the fluid pipeline 21 includes 4 inverted V-shaped pipe units 211, the inverted V-shaped pipe units 211 include an ascending portion 211a and a descending portion 211b, an included angle formed by the ascending portion 211a and the descending portion 211b is 60 degrees, an included angle formed by the ascending portion 211a and a horizontal line is 120 degrees, and the ascending portion 211a and the descending portion 211b are equal in length. The inner diameter of the fluid pipeline 21 is 20-30 μm, and the fluid pipeline 21 is made of polymethyl methacrylate (PMMA) or Polydimethylsiloxane (PDMS).
A spectral detection system 3 comprising a light source 31 and a detection device 32, wherein the spectral detection system 3 is a raman spectral detection system, such that the light source 31 is arranged to generate raman excitation light passing through the detection zone 21c, and the detection device 32 is arranged to receive and detect an optical signal generated from the detection zone 21c to generate a raman spectrum. The raman excitation light generated by the light source 31 forms a raman optical path 33.
The working process and working principle of the above exemplary detection system are as follows:
the valves 6 provided in the intermediate fluid control device and the lower fluid control device are closed, and at this time, the fluid pump 1 pumps the fluid sample to be measured in the fluid source 4 into the upper fluid control device through the pipeline. Wherein, the fluid sample to be measured is mixed with the diluent in the dilution pool 23 in the sample mixing pool 22, and then enters the fluid pipeline 21. When the sample is initially put into the fluid pipeline 21, the microorganisms in the fluid sample to be detected are in a disordered state; when the fluid sample reaches the corner, the movement path of the fluid sample to be detected is changed, and at the moment, the microorganism positioned at the outermost edge of the pipeline can also change the movement path at the corner and reach the next link of the pipeline. The movement path change at the break can change the microorganism flow direction and the distance between the microorganisms in the fluid sample to be detected, the microorganisms are orderly arranged in the fluid pipeline 21 through the movement path change at a plurality of breaks, and pass through the Raman light path 33 formed by the Raman excitation light generated by the light source 31 one by one, because the Raman excitation light has good penetrability, the microorganisms in the pipeline can be directly irradiated through the transparent fluid pipeline 21, the generated Raman scattered light or transmitted light is detected by the detection device 32, and finally the fluid sample flows into the waste liquid pool 5.
When the number of microorganisms in the sample is large, the valves 6 disposed in the upper fluid control device and the lower fluid control device may be closed, and at this time, the fluid pump 1 pumps the fluid sample to be measured in the fluid source 4 into the intermediate fluid control device through the pipeline. The fluid sample to be measured is first mixed with the diluent in the dilution pool 23 of the upper fluid control device in the sample mixing pool 22, and then enters the sample mixing pool 22 of the middle fluid control device and is mixed with the diluent in the dilution pool 23, so as to enter the fluid pipeline 21 of the middle fluid control device. The flow of the fluid sample to be measured in the fluid line 21 of the intermediate flow control device is performed in the same manner as in the flow line 21 of the upper flow control device described above.
When the number of microorganisms in the sample is still too high, the valves 6 disposed in the upper fluid control device and the middle fluid control device may be closed, and at this time, the fluid pump 1 pumps the fluid sample to be measured in the fluid source 4 into the lower fluid control device through the pipeline. The fluid sample to be measured is first mixed with the diluent in the dilution pool 23 of the upper fluid control device in the sample mixing pool 22, then enters the sample mixing pool 22 of the middle fluid control device and is mixed with the diluent in the dilution pool 23, and finally enters the fluid pipeline 21 of the lower fluid control device. The flow of the fluid sample to be measured through the fluid line 21 in the lower fluid control device is the same as that through the fluid line 21 in the upper fluid control device described above.
The foregoing illustrates and describes the general principles of the detection system and method of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Experimental example 1 measurement of the Total number of Membrane-producing Yeast colonies in Soy sauce
Experimental groups:
(1) preparation of Standard bacterial liquid
Taking and streak-culturing membranous yeast bacteria on appropriate solid culture media respectively, culturing at the temperature of 30 ℃ for 48h, picking single bacterial colonies by using a disposable inoculating needle, placing the single bacterial colonies in 5mL of sterile water, oscillating the single bacterial colonies on an oscillator for 20s, then centrifuging the single bacterial colonies for 1min at 10000rmp/min, removing supernatant, adding 5mL of sterile soy sauce, oscillating the single bacterial colonies on the oscillator for 20s to prepare standard membranous yeast liquid, wherein the diameter of the bacterial colonies is 30 microns, and three parallel strains are set.
(2) Total colony count was detected using the detection system of example 1
The above-mentioned membrane-producing yeast liquid sample was applied to the detection system of example 1 (model LS785 for raman spectrometer, model PIXIS100BR for built-in CCD device), wherein the inner diameter of the microtube was 50 μm, the flow rate of the fluid was 5 μ L/min, and the measurement conditions for raman spectroscopy were: laser power range: 2mW, acquisition time: 8ms, the detection wavelength is 475 nm.
(3) Data processing
(a) Extracting soy sauce background signal (see fig. 3a gray line)
(b) Extracting light signal generated by Raman spectrum from standard membrane-producing yeast liquid (see black line of FIG. 3 a)
(c) Deducting soy sauce background signal
(d) The black detection points on the Raman light are all a bacterium, and an electric signal (black detection point) is collected by a Charge Coupled Device (CCD) collection computer of the Raman spectrum. The number of black spots was statistically analyzed to determine the total number of colonies in the standard membrane-producing yeast (see FIG. 3 b).
Determination by a culture medium method:
(1) and (3) determining the total number of the membrane-producing yeasts by using the standard membrane-producing yeast liquid obtained in the step (1) through a culture medium culture method, and setting three parallels. The detailed procedures of the culture medium culture method are referred to national standard GB 4789.2-2016.
(2) And (3) differential analysis: the total number of membrane-producing yeasts detected by the microfluidics and the total number of membrane-producing yeasts detected by the medium method were analyzed by SPSS (SPSS Co.).
The results show that: the background of soy sauce in figure 3a was subtracted by analysis with the soy sauce control group. And splicing the detection results, wherein the number of the thalli is represented by black bands at the black detection points by a CCD device in Raman light, and the total number of the yeasts in the sample is counted by the CCD device in Raman light by recording the number of the black bands (as shown in figure 3 b). Therefore, the total number of the membrane-producing yeasts detected by the optofluidic method is 2.3 +/-0.2 multiplied by 103The total number of the membrane-producing yeasts detected by a culture medium method is 2.2 +/-0.1 multiplied by 103And (4) respectively. SPSS analysis shows that there is no difference between the light stream detection and the culture medium method for detecting the membrane-producing yeast. Medium assay as a national standard is a measure of other assay criteria, so the optofluidic assay is very good in the detection of membrane-producing yeastAnd (4) accuracy.
Figure BDA0001960263330000101
EXAMPLE 2 determination of the Total number of colonies of a sample of Bacillus in milk
Experimental groups:
(1) preparation of standard bacterial liquid
Taking bacillus and respectively carrying out streak culture on appropriate solid culture at the culture temperature of 30 ℃ for 72h, picking a single colony by using a disposable inoculating needle, placing the single colony in 10mL of sterile water, oscillating the single colony on an oscillator for 10s, then centrifuging the single colony for 1min at 10000rmp/min, removing supernatant, adding 10mL of sterile milk, oscillating the single colony for 10s on the oscillator to prepare standard bacillus liquid, wherein the size of the bacillus is10 microns, and three parallel bacteria are set.
(2) Total colony count was detected using the detection system of example 1
The bacillus liquid sample was applied to the detection system of example 1, wherein the inner diameter of the microtube was 25 μm, the fluid flow rate was 1000 μ L/min, and the raman spectroscopy measurement conditions were as follows: laser power range 20mW, acquisition time: 15s, the detection wavelength is 785 nm.
(3) Data processing
(a) Extracting the background signal of milk (see fig. 4a gray line)
(b) Extraction of light signals from standard Bacillus species by Raman Spectroscopy (see FIG. 4a, black line)
(c) Deducting background signal of milk
(d) The black detection points on the Raman light are all a bacterium, and an electric signal (namely the black detection points) is collected by a Charge Coupled Device (CCD) collection computer of the Raman spectrum. The number of black spots was statistically analyzed, thereby detecting the total number of standard Bacillus colonies (see FIG. 4 b).
Determination by a culture medium method:
(1) the total number of the standard bacillus of the step (1) is determined by a culture medium culture method, and three parallels are set. See, e.g., experimental example 1.
(2) And (3) differential analysis: same as in experimental example 1.
The results show that: the light fluid detection, through the analysis with the milk control group, the milk background was subtracted (same method as experimental example 1). And splicing the detection results, wherein the number of the thalli is represented by black strips in a Raman light CCD device at black detection points, and the total number of the bacillus in the sample is counted by the Raman light CCD device by recording the number of the black strips (see figure 4 b). Therefore, the total number of bacilli detected by the optical fluid is 1.8 +/-0.1 multiplied by 103The total number of bacillus detected by a culture medium method is 1.7 +/-0.1 multiplied by 103And (4) respectively. SPSS analysis shows that the optical flow detection and the culture medium method for detecting the bacillus have no difference. The culture medium detection method is used as a national standard method for measuring other method standards, so that the optofluidic detection method has good accuracy in the detection method of the total number of bacillus.
Figure BDA0001960263330000121
EXAMPLE 3 determination of the Total number of colonies of E.coli samples in mineral Water
Experimental groups:
(1) preparation of standard bacterial liquid
Taking escherichia coli, respectively carrying out streak culture on appropriate solid culture at the culture temperature of 37 ℃ for 30h, picking single colony by using a disposable inoculating needle, placing the single colony in 7mL of sterile water, oscillating the single colony for 15s on an oscillator, then centrifuging the single colony for 1min at 10000rmp/min, removing supernatant, adding 7mL of sterile mineral water, oscillating the single colony for 15s on the oscillator, and preparing a standard escherichia coli sample, wherein the size of the thallus is 15 mu m, and three parallels are set.
(2) Total colony count was detected using the detection system of example 1
The above E.coli sample was applied to the detection system of example 1, in which the diameter of the microtube was 20 μm, the fluid flow rate: 800 μ L/min, and the Raman spectrum measurement conditions were: laser power range: 10mW, collecting time: 2s, the detection wavelength is 600 nm.
(3) Data processing
(a) Extracting mineral water background signal (see fig. 5a gray line)
(b) Extraction of Standard E.coli light signals by Raman Spectroscopy (see FIG. 5a black line)
(c) Deducting background signal of mineral water
(d) The black detection points on the Raman light are all a bacterium, and an electric signal (namely the black detection points) is collected by a Charge Coupled Device (CCD) collection computer of the Raman spectrum. The number of black spots was statistically analyzed, thereby detecting the total number of standard E.coli colonies (see FIG. 5b)
Determination by a culture medium method:
(1) and (3) determining the total number of the standard escherichia coli in the step (1) by a culture medium culture method, and setting three parallels. See, e.g., experimental example 1.
(2) And (3) differential analysis: the total number of Escherichia coli detected by the optical fluid and the total number of Bacillus detected by the medium method were analyzed by SPSS (SPSS Co.).
The results show that: optical fluid detection, mineral water background was subtracted by analysis with mineral water control (same method as in experimental example 1). And splicing the detection results, wherein the number of the thalli is represented by black strips at the black detection points by a CCD device in the Raman light, and the total number of the Escherichia coli in the sample is counted by the CCD device in the Raman light by recording the number of the black strips (see figure 5 b). Therefore, the total number of colibacillus detected by the optical fluid is 2.5 +/-0.2 multiplied by 103The total number of colibacillus detected by a culture medium method is 2.4 +/-0.2 multiplied by 103And (4) respectively. SPSS analysis shows that the optical flow detection and the culture medium method for detecting the bacillus have no difference. The culture medium detection method is used as a national standard method for measuring other method standards, so that the optofluidic detection method has good accuracy in the detection method of the total number of bacillus.
Figure BDA0001960263330000131
EXAMPLE 4 determination of the Total number of colonies in a fruit juice sample
(1) Mixed standard strain preparation
Inoculating the sterile fruit juice liquid sample into yeast, bacillus and escherichia coli, uniformly mixing, taking 1mL, adding 10mL of sterile water, oscillating for 10s on an oscillator to prepare a sample to be detected, and setting three parallels.
(2) Total colony count was detected using the detection system of example 1
The above juice sample was applied to the detection system of example 1, in which the diameter of the microtube was 30 μm, the fluid flow rate: 500. mu.L/min, and the Raman spectrum measurement conditions were as follows: laser power range: 2mW, acquisition time: 100ms, and the detection wavelength is 500 nm.
(3) Data processing
(a) Extracting juice background signal (see fig. 6a gray line)
(b) Extracting standard mixed bacteria and generating optical signal by Raman spectrum (see figure 6a black line)
(c) Deducting background signal of fruit juice
(d) The black detection points on the Raman light are all a bacterium, and an electric signal (namely a black point) is collected by a Charge Coupled Device (CCD) collection computer of the Raman spectrum. The number of black spots was statistically analyzed to thereby detect the number of standard mixed bacteria colonies (see FIG. 6b)
(4) Determination by a culture medium method: and (3) determining the total number of microorganisms in the sample to be detected in the step (1) by a culture medium culture method, and setting three parallels. The procedure is as in example 1.
(5) And (3) differential analysis: the total number of Escherichia coli and Bacillus bacteria detected by the microfluidics method and the medium method were analyzed by SPSS (SPSS Co.).
The results show that: the background of the juice was subtracted by analysis with a juice control (same procedure as in experimental example 1). And splicing the detection results, wherein the number of the thalli is represented by black strips at the black detection points by a CCD device in the Raman light, and the total number of the mixed bacterial colonies in the sample is counted by the CCD device in the Raman light by recording the number of the black strips (the method is the same as that of the experimental example 1). Therefore, the total number of the mixed bacteria detected by the optical fluid is 400 +/-12, and the total number of the mixed bacteria detected by the culture medium method is 390 +/-10. SPSS analysis shows that the optical flow detection and the culture medium method for detecting the bacillus have no difference. The culture medium detection method is used as a national standard method for measuring other method standards, so that the optofluidic detection method has good accuracy in the detection method of the total number of bacillus.
Figure BDA0001960263330000141
Comparative example 1.
(1) Sample preparation
Taking and streak-culturing membranous yeast bacteria on appropriate solid culture media respectively, culturing at the temperature of 30 ℃ for 48h, picking single bacterial colonies by using a disposable inoculating needle, placing the single bacterial colonies in 5mL of sterile water, oscillating the single bacterial colonies on an oscillator for 20s, then centrifuging the single bacterial colonies for 1min at 10000rmp/min, removing supernatant, adding 5mL of sterile soy sauce, oscillating the single bacterial colonies on the oscillator for 20s to prepare standard membranous yeast liquid, wherein the diameter of the bacterial colonies is 30 microns, and three parallel strains are set.
(2) Total colony count was detected using the detection system of example 1
The above-mentioned sample of the membrane-producing yeast liquid was applied to the detection system of example 1, but the microtube (fluid line 21) was changed to a straight line with an inner diameter of 50 μm and a fluid flow rate of 5 μ L/min, and the Raman spectrum measurement conditions were as follows: laser power range: 2mW, acquisition time: 8ms, the detection wavelength is 475 nm.
(3) Data processing
(a) Extracting soy sauce background signal (see fig. 7a gray line)
(b) Extracting the light signal generated by Raman spectrum from the standard membrane-producing yeast liquid (see the black line in FIG. 7 a)
(c) Deducting soy sauce background signal
(d) The black detection points on the Raman light are all a bacterium, and an electric signal (namely the black detection points) is collected by a Charge Coupled Device (CCD) collection computer of the Raman spectrum. Black detection point the number of cells was represented by black bands in a Raman light CCD device, and the total number of colonies in the standard membrane-producing yeast sample was determined by counting the number of black bands and counting the total number of colonies in the sample using a Raman light CCD device (see FIG. 7b)
The results show that: optical fluidic examination when the microtube is a straight tubeThe total number of the membrane-producing yeasts is 1.1 +/-0.3 multiplied by 103In contrast, the total number of the membrane-producing yeasts measured by the medium method (national standard method, which is a standard for measuring other methods) was 3.3. + -. 0.2X 103And SPSS analysis shows that the light stream detection and the culture medium method for detecting the membrane-producing yeast have significant difference. It can be seen that there is no accuracy in the detection of membrane-producing yeast when the optical fluid detection method is used but the microtubes are changed to the linear lines. The possible reasons for the above phenomena are that the dispersibility of the membrane-producing yeast is reduced due to the straight pipeline, and one or more bacteria form a bacteria mass and simultaneously pass through the Raman spectrum, so that the Raman spectrum is recorded into one bacteria, the counting is inaccurate, and great errors are brought.
Figure BDA0001960263330000151
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims (17)

1. A method for detecting or enumerating microorganisms in a sample, comprising the steps of:
(i) a system for detecting or enumerating microorganisms is provided, comprising:
a fluid pump (1) having a fluid inlet and a fluid outlet;
a fluid control device (2) comprising a fluid line (21) having a first end and a second end, a sample mixing basin (22), and a diluent basin (23); the sample mixing pool (22) is respectively in fluid communication with the fluid outlet, the first end and the diluent pool (23), the fluid pipeline (21) comprises an inverted V-shaped pipe unit (211), the inverted V-shaped pipe unit (211) comprises an ascending part (211a) and a descending part (211b), and a detection area is arranged at a position close to the second end;
wherein the fluid line (21) has the following features:
(a) the angle formed by the rising portion (211a) and the falling portion (211b) is an acute angle, and the acute angle ranges from 45 degrees to 75 degrees;
(b) the rising part (211a) forms an obtuse angle with the horizontal, and the obtuse angle ranges from 100 degrees to 135 degrees;
(c) the rising portion (211a) is equal in length to the falling portion (211 b);
(d) the inner diameter of the fluid pipeline is 20-50 μm;
(e) the fluid pipeline is made of a material which can be penetrated by exciting light;
(f) the first end of the fluid line (21) and its vicinity extend in a horizontal direction;
a raman spectroscopic detection system (3) comprising a light source (31) and a detection device (32), wherein the light source (31) is arranged to generate excitation light that passes through the detection zone; the detection means (32) being arranged to receive and detect light signals generated from the detection zone to generate a spectrum of light;
(ii) moving a fluid sample to be tested containing microorganisms in the fluid pipeline according to the direction from a first end to a second end and at the flow speed of 5-1200 mu L/min so as to enable the microorganisms in the fluid sample to be tested to pass through a detection area of the fluid pipeline one by one;
(iii) performing Raman spectroscopy detection on a fluid sample to be detected passing through the detection zone by the Raman spectroscopy detection system to obtain a Raman spectrum generated from the detection zone, the Raman spectrum including a spectrum attributed to bacteria in the fluid sample to be detected;
(iv) analyzing the raman spectrum generated at the detection zone to obtain a profile attributed to the microorganisms;
(v) performing a statistical analysis on the profile attributed to the microorganism to obtain the number of the microorganism.
2. The method of claim 1, wherein the system further comprises a fluid source (4) for receiving a fluid sample to be tested, which is in fluid communication with a fluid inlet of the fluid pump (1); in step (ii), the fluid sample to be tested is applied to the fluid source (4).
3. The method of claim 1, wherein the Raman spectroscopy is selected from confocal Raman spectroscopy, surface enhanced Raman spectroscopy, coherent anti-Stokes Raman spectroscopy, and laser tweezers Raman spectroscopy.
4. The method of claim 1, wherein the fluid line (21) comprises a plurality of inverted V-tube units (211) in fluid communication.
5. The method of claim 1, wherein the fluid line (21) comprises 3, 4 or 5 inverted V-tube units (211) in fluid communication.
6. The method of claim 1, wherein the excitation light permeable material is selected from PMMA or PDMS.
7. The method of claim 1, wherein the system further comprises:
(1) a waste reservoir (5) in fluid communication with a second end of the fluid line (21); and/or the presence of a gas in the gas,
(2) a valve (6) for controlling the flow of fluid from the sample mixing basin (22) to the fluid line (21).
8. The method according to claim 1, wherein the system comprises at least 2 fluid control devices (2), the mixing cells (22) comprised in the at least 2 fluid control devices (2) being in fluid communication with each other.
9. The method according to claim 1, wherein the system comprises at least 3 fluid control devices (2), the mixing cells (22) comprised in the at least 3 fluid control devices (2) being in fluid communication with each other.
10. The method of claim 1, wherein the raman spectroscopy detection system (3) is provided with one or more of the following features:
(i) the raman spectroscopy detection system (3) further comprises control means in data communication with the light source (31) and detection means (32);
(ii) the detection device (32) comprises a photoelectric converter;
(iii) the detection device (32) comprises a CCD.
11. The method of claim 1, wherein the Raman spectroscopy detection is performed under one or more of the following conditions:
(i) the acquisition time is 8ms-15 s;
(ii) the wavelength of the exciting light is in the wavelength range from ultraviolet to near infrared;
(iii) the laser power is 2-20 mW.
12. The method of claim 1, further comprising, prior to step (ii), pre-treating the fluid sample to be tested.
13. The method of claim 12, wherein the pre-treatment is selected from filtration, dilution, or any combination thereof.
14. The method of claim 1, wherein prior to step (ii), the fluid sample to be tested is diluted with sterile water.
15. The method of any one of claims 1-14, wherein the fluid sample to be tested is a liquid food.
16. The method of any one of claims 1-14, wherein the fluid sample to be tested is a drink, liquid flavoring, or drinking water.
17. The method of any of claims 1-14, wherein the fluid sample to be tested is selected from soy sauce, milk, fruit juice, and mineral water.
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