CN109655447A - Detection system and method for microorganism count - Google Patents
Detection system and method for microorganism count Download PDFInfo
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- CN109655447A CN109655447A CN201910080620.3A CN201910080620A CN109655447A CN 109655447 A CN109655447 A CN 109655447A CN 201910080620 A CN201910080620 A CN 201910080620A CN 109655447 A CN109655447 A CN 109655447A
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- 238000001237 Raman spectrum Methods 0.000 claims description 39
- 238000001069 Raman spectroscopy Methods 0.000 claims description 37
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- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 3
- 239000003651 drinking water Substances 0.000 claims description 3
- 235000020188 drinking water Nutrition 0.000 claims description 3
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 3
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Classifications
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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
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention provides a kind of detection systems for microorganism count, it includes: fluid pump, fluid control device and spectral detection system with fluid inlet and fluid outlet, wherein, the fluid control device includes mixed sample pool, the fluid circuit with first end and the second end and dilution liquid pool；Wherein, the mixed sample pool is in fluid communication with the fluid outlet, first end and dilution liquid pool respectively, and the fluid circuit includes inverted V type pipe unit, and the inverted V type pipe unit includes rising part and sloping portion, also, detection zone is equipped with close to the position of the second end；The spectral detection system includes light source and detection device, wherein the light source is arranged to generate the exciting light passed through from the detection zone；The detection device is arranged to receive and detect the optical signal generated from the detection zone, to generate spectrum.The present invention also provides a kind of methods for microorganism count comprising uses detection system of the invention.
Description
Technical field
The present invention relates to microorganism fields, more particularly to a kind of detection system and method for microorganism count.
Background technique
Food is all people's daily life necessity all the time, still because of its abundant, changeable mouthfeel and nutriment
Nutriment abundant also provides good condition for microorganism growth.Content of microorganisms also becomes one of assessment food safety
Important indicator.Currently, internationally recognized sanitary standard is using total number of bacteria, total number of fungi as index of security assessment.Various countries are
It ensures food safety, all formulates the Numeration of standardized testing process and total plate count accordingly.Standardized detection stream
Journey not only contributes to the safety of assessment food, and provides reliable scientific basis for foods supervision department.Standardization detection
Process also provides foundation for the food enterprises strict control product quality, can find substandard product in time, avoids not conforming to
Product comes into the market to people's lives and life zone and threatens.Currently, surveying the method master of inspection microorganism in chinese national standard
To use culture of microorganism: by using different culture mediums with orient or non-directional training method detection microorganism；It can
It intuitively observes and surveys inspection microbial count, the commonly required time is 24-72h, and part microorganism even more can just be examined long
It surveys as a result, required incubation time is long, operates step gathers cumbersome, heavy workload.Even some microorganisms escape from inspection because that can not cultivate
It surveys.The development that the introducing of new technology and method surveys inspection technology for microorganism is most important, and fluorescent labelling techniques, cell membrane electricity are raw
Reason technology and high resolution microscope make it possible our it is movable to microbial life it is direct explore, but various antibody,
Staining technique is not only expensive but also different degrees of influence or damage can be caused to the microbial cell under physiological status.It removes
Other than this, most of technology is there is also test operation complexity, many drawbacks such as time-consuming.
Micro Raman spectra technology is using low power laser, the holographic technique and confocal technology of high conversion efficiency, tool
Have detection sensitivity is high, the time is short, required sample size is small, sample prepare etc. without the means of other reagent or complexity it is excellent
Point, the above significant advantage are more and more widely used it in microorganism survey inspection analysis field.However, applying Raman light at present
Compose needs will test sample first when progress microbe colony sum detects is made small sample piece, the thickness of sample strip or suitable
Sample size is more difficult to control, if control is improper easily to generate direct influence to test result, then manufactured sample strip passes through Raman
When spectrum carries out microscope detection, need artificially to find microbiological specimens, it could be into determining microorganism within the vision
Row measurement, therefore limit the ability that Raman spectroscopy detects microorganism in the sample.
Therefore, this field still needs improved detection method and system, to realize accurate, quickly and conveniently microorganism
Detection counts.
Summary of the invention
The purpose of the present invention is to provide a kind of detections based on microbial count in light stream body technique detection stream body sample
System and method, by microorganism is carried out quickly and orderly arrangement and average rate by Raman spectrum, and to testing result
Statistical analysis is carried out, to realize the measurement of microbial count in fluid samples.
Therefore, in one aspect, the present invention provides a kind of for detecting or the system of enumeration of micro organisms, it includes:
Fluid pump 1 with fluid inlet and fluid outlet；
Fluid control device 2 comprising fluid circuit 21, mixed sample pool 22 with first end and the second end and dilute
Release liquid pool 23；Wherein, the mixed sample pool 22 is in fluid communication with the fluid outlet, first end and dilution liquid pool 23 respectively,
The fluid circuit 21 includes inverted V type pipe unit 211, and the inverted V type pipe unit 211 includes rising part 211a and sloping portion
211b, also, detection zone is equipped with close to the position of the second end；
Spectral detection system 3 comprising light source 31 and detection device 32, wherein the light source 31 be arranged to generate from
The exciting light that the detection zone passes through；The detection device 32 is arranged to receive and detect the light letter generated from the detection zone
Number, to generate spectrum.
In certain embodiments, the fluid circuit 21 includes the inverted V type pipe unit 211 of multiple fluid communication.At certain
In a little embodiments, the fluid circuit 21 includes the inverted V type pipe unit 211 of 3,4 or 5 fluid communication.
In certain embodiments, the angle that the rising part 211a and sloping portion 211b is formed is acute angle.It is preferred that
Ground, the range of the acute angle is between 45 to 75 degree.
In certain embodiments, the rising part 211a and horizontal angle are obtuse angle.Preferably, the obtuse angle
Range 100 to 135 degree between.
In certain embodiments, the rising part 211a and sloping portion 211b are isometric.
In certain embodiments, the internal diameter of the fluid circuit is 20 μm -50 μm, such as 20 μm -30 μm.
In certain embodiments, the fluid circuit 21 is made of the transparent material of exciting light.
In certain embodiments, the transparent material of the exciting light is selected from polymetylmethacrylate or poly- two
Methylsiloxane PDMS.
In certain embodiments, the first end of the fluid circuit 21 and its neighbouring part are horizontally extending.
In certain embodiments, the system also includes: for receiving the fluid source 4 of fluid to be measured sample, with institute
The fluid inlet for stating fluid pump 1 is in fluid communication.In certain embodiments, the fluid to be measured sample is liquid food, such as
Drink, liquid flavoring or drinking water.
In certain embodiments, the system also includes waste liquid pools 5, the second end with the fluid circuit 21
It is in fluid communication.
In certain embodiments, the system also includes valve 6, be used to control fluid from the mixed sample pool 22 to
The flowing of the fluid circuit 21.
In certain embodiments, the system includes at least two fluid control device 2, at least two fluid control
Mixed sample pool 22 included in device 2 is in fluid communication with each other.In certain embodiments, the system includes at least three fluid
Control device 2, mixed sample pool 22 included at least three fluid control device 2 are in fluid communication with each other.
In certain embodiments, the spectral detection system 3 further includes control device, with the light source 31 and
Detection device 32 carries out data communication.
In certain embodiments, the detection device 32 includes photoelectric converter.
In certain embodiments, the spectral detection system 3 is selected from Raman spectrum detection system, infrared spectroscopy detection system
System and ultraviolet spectra detection system.
In certain embodiments, the spectral detection system 3 is Raman spectrum detection system, and the detection device 32 is wrapped
Include CCD (charge coupled device).In certain embodiments, the detection device 32 further includes photomultiplier tube.In certain realities
It applies in scheme, the detection device 32 further comprises light splitting part, and the light splitting part is for dissipating Raman according to wavelength
Light is penetrated to be separated.For example, light splitting part can be diffraction grating.
On the other hand, the present invention provides the methods for the microorganism in detection or count samples comprising with
Lower step:
(i) make the fluid pump 1 of system and in fluid circuit 21 through the invention of the fluid to be measured sample containing microorganism
It is by first end to the second end direction and mobile with the flowing velocity of 5-1200 μ L/min, so that the fluid to be measured sample
In microorganism pass through the detection zone of the fluid circuit 21 one by one；
(ii) detection system 3 through the invention carries out spectroscopic methodology inspection to by the fluid to be measured sample of the detection zone
It surveys, to obtain the spectrum generated from the detection zone, the spectrum includes the thallus being attributed in the fluid to be measured sample
Map；
(iii) spectrum generated to the detection zone is analyzed, to obtain the map for being attributed to microorganism；
(iv) for statistical analysis to the map for being attributed to microorganism, to obtain the quantity of the microorganism.
In certain embodiments, in step (i), the fluid to be measured sample is applied to the fluid of present system
Source 4.
In certain embodiments, in step (ii), by Raman spectrum detection system to by the detection zone
Fluid to be measured sample carries out Raman spectroscopy detection, to obtain the Raman spectrum generated from the detection zone；Also, in step
(iii) in, the Raman spectrum generated to the detection zone is analyzed, to obtain the quantity for being attributed to microorganism.
In certain embodiments, the Raman spectroscopy is selected from confocal Raman spectra, Surface enhanced Raman spectroscopy, is concerned with
Anti stokes raman spectrum and laser tweezers Raman spectrum.
In certain embodiments, the Raman spectroscopy detection is carried out under the conditions of following one or more:
(i) acquisition time is 8ms-15s；
(ii) wavelength of exciting light is ultraviolet near infrared wavelength region；
(iii) laser power is 2-20mW；
In certain embodiments, the Raman spectroscopy detection is carried out under conditions of selected from following:
(a) acquisition time is 8ms, excitation light wave a length of 475nm, laser power 2mW；
(b) acquisition time is 15s, excitation light wave a length of 785nm, laser power 20mW；
(c) acquisition time is 2s, excitation light wave a length of 600nm, laser power 10mW；
(d) acquisition time is 100ms, excitation light wave a length of 500nm, laser power 2mW.
In certain embodiments, before step (i), the method also includes following steps: to described to flow measurement
Body sample is pre-processed.In certain embodiments, the pretreatment is selected from filtering, dilution, or any combination thereof.Certain
In embodiment, before step (i), the fluid to be measured sample is diluted using sterile water.
In certain embodiments, the fluid to be measured sample is liquid food, such as drink, liquid flavoring or is drunk
Water.In certain embodiments, the fluid to be measured sample is selected from soy sauce, milk, fruit juice and mineral water.
Advantageous effect of the invention
The present invention provides the detection system for microorganism count for the first time by fluid technique in conjunction with Raman spectroscopy
And method.Microorganism in fluid sample is carried out quickly and orderly arrangement by micro-pipe device by detection system/method of the invention,
So that microorganism passes through spectrometer one by one, the spectral information on micro-scale is obtained, and then analyze liquid in a short time
The biomolecular information of a large amount of microorganisms in sample, and statistical analysis is carried out to testing result, it realizes to bacterium in fluid sample
The detection of sum is fallen, to realize the purpose for detecting micro organism quantity in real time, dynamically, on a large scale.Detection system of the invention/
Method without film-making, select specified microorganisms bacterium colony and etc. can fast implement the detection of microbe colony in fluid sample,
The accuracy of testing result is improved, sample treatment and test process are simple, substantially increase detection speed.
Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but those skilled in the art
Member it will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.With reference to the accompanying drawings
With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art
It will be apparent.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the structural schematic diagram of the exemplary implementation scheme of detection system of the present invention.
Fig. 2 is the structural schematic diagram of the fluid circuit of detection system shown in FIG. 1.
Fig. 3 a-3b is that 1 Raman spectrum of experimental example and CCD count schematic diagram.
Fig. 4 a-4b is that 2 Raman spectrum of experimental example and CCD count schematic diagram.
Fig. 5 a-5b is that 3 Raman spectrum of experimental example and CCD count schematic diagram.
Fig. 6 a-6b is that 4 Raman spectrum of experimental example and CCD count schematic diagram.
Fig. 7 a-7b is that 1 Raman spectrum of comparative example and CCD count schematic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Below
Description only actually at least one exemplary embodiment be it is illustrative, never as to the present invention and its application or make
Any restrictions.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless specifically stated otherwise, positioned opposite, the digital table of the component and step that otherwise illustrate in these embodiments
It is not limited the scope of the invention up to formula and numerical value.Simultaneously, it should be appreciated that for ease of description, each portion shown in attached drawing
The size divided not is to draw according to actual proportionate relationship.For technology, side known to person of ordinary skill in the relevant
Method and equipment may be not discussed in detail, but in the appropriate case, and the technology, method and apparatus should be considered as authorizing explanation
A part of book.In shown here and discussion all examples, any occurrence should be construed as merely illustratively, and
Not by way of limitation.Therefore, the other examples of exemplary embodiment can have different values.It should also be noted that similar label
Similar terms are indicated in following attached drawing with letter, therefore, once it is defined in a certain Xiang Yi attached drawing, then subsequent attached
It does not need that it is further discussed in figure.
Embodiment 1. is used to detect or the detection system of enumeration of micro organisms
Fig. 1-Fig. 2 is respectively illustrated to be wrapped in the structural schematic diagram and the detection system of exemplary detection system of the present invention
The structural schematic diagram of the fluid circuit contained.
As shown in Figure 1, exemplary detection system of the invention include the fluid pump 1 with fluid inlet and fluid outlet,
Fluid control device 2, spectral detection system 3, the fluid source 4 for receiving fluid to be measured sample, waste liquid pool 5, valve 6.It is described
Exemplary detection system include 3 fluid control devices 2, respectively upper flow control device, central fluid control device and
Fluid beneath control device.
Fluid control device 2 mainly include the fluid circuit 21 with first end and the second end, mixed sample pool 22 and
Dilute liquid pool 23.The mixed sample pool 22 passes through pipeline fluid with the fluid outlet, first end and dilution liquid pool 23 respectively
Connection, for mixing fluid sample with the dilution diluted in liquid pool 23.The first end 21a of the fluid circuit 21 and its
Neighbouring part is horizontally extending, and valve 6 is partially equipped near first end 21a, for controlling fluid from described
Mixed sample pool 22 arrives the flowing of the fluid circuit 21.The second end 21b and waste liquid pool 5 of the fluid circuit 21 are in fluid communication,
Also, detection zone 21c is equipped in the position close to the second end 21b.
Fluid pump 1 makes fluid to be measured sample, and direction D shown in arrow is moved along Fig. 1 in fluid circuit 21.
As shown in Fig. 2, the fluid circuit 21 includes 4 inverted V type pipe units 211, the inverted V type pipe unit 211 includes
The angle that rising part 211a and sloping portion 211b, the rising part 211a and sloping portion 211b are formed is 60 degree, institute
Stating rising part 211a and horizontal angle is 120 degree, and the rising part 211a and sloping portion 211b are isometric.It is described
The internal diameter of fluid circuit 21 is 20 μm -30 μm, and the fluid circuit 21 is by polymetylmethacrylate or poly dimethyl silicon
Oxygen alkane PDMS is made.
Spectral detection system 3 comprising light source 31 and detection device 32, wherein the spectral detection system 3 is Raman light
Detection system is composed, therefore, the light source 31 is arranged to generate the raman excitation light passed through from the detection zone 21c, the inspection
It surveys device 32 to be arranged to receive and detect the optical signal generated from the detection zone 21c, to generate Raman spectrum.The light source
31 raman excitation lights generated form Raman optical path 33.
The course of work and working principle of above-mentioned example detection system are as follows:
The valve 6 being set in central fluid control device and fluid beneath control device is closed, at this point, fluid pump 1 will flow
Fluid to be measured sample in body source 4 is pumped into upper flow control device by pipeline.Wherein, fluid to be measured sample and diluting tank
Dilution in 23 is mixed in mixed sample pool 22, subsequently into fluid circuit 21.When just entering fluid circuit 21, in fluid to be measured sample
Microorganism at disordered state；When reaching at dog-ear, the motion path of fluid to be measured sample changes, at this point, being located at pipe
Also the variation of motion path can occur at the dog-ear for the most marginal microorganism in road, and reach next link of pipeline.It is rolling over
Motion path variation at angle can change the distance between microorganism flow direction and microorganism in fluid to be measured sample, through excessive
Motion path variation at a dog-ear, microorganism in fluid circuit 21 by ordered arrangement, and one by one by being generated by light source 31
Raman excitation light be formed by Raman optical path 33, due to raman excitation light have good penetrability, pass through transparent fluid
Pipeline 21 can be detected with the microorganism in direct irradiation pipeline, generated Raman diffused light or transmitted light by detection device 32,
Last fluid sample flows into waste liquid pool 5.
When micro organism quantity is very big in sample, can close set on upper flow control device and fluid beneath control dress
Valve 6 in setting, at this point, the fluid to be measured sample in fluid source 4 is pumped into central fluid control device by pipeline by fluid pump 1
In.Wherein, fluid to be measured sample is mixed in mixed sample pool 22 with the dilution in the diluting tank 23 in upper flow control device first
It closes, mixes subsequently into the mixed sample pool 22 in central fluid control device and with the dilution in diluting tank 23, hence into
Between fluid circuit 21 in fluid control device.The mistake of fluid circuit 21 of the fluid sample to be tested in central fluid control device
Journey is identical as the process of fluid circuit 21 in above-mentioned fluid control device above.
When raw quantity micro- in sample is still excessively high, can close set on upper flow control device and central fluid control
Valve 6 in device, at this point, the fluid to be measured sample in fluid source 4 is pumped into fluid beneath control dress by pipeline by fluid pump 1
In setting.Wherein, fluid to be measured sample is first with the dilution in the diluting tank 23 in upper flow control device in mixed sample pool 22
Mixing, mixes subsequently into the mixed sample pool 22 in central fluid control device and with the dilution in diluting tank 23, eventually enters into
Fluid circuit 21 in fluid beneath control device.Fluid circuit 21 of the fluid sample to be tested in fluid beneath control device
Process is identical as the process of fluid circuit 21 in above-mentioned fluid control device above.
The basic principle of detection system and method for the present invention has been shown and described above.The technical staff of the industry should
Solution, the present invention is not limited to the above embodiments, and the above embodiments and description only illustrate the principle of the present invention,
Without departing from the spirit and scope of the present invention, the present invention also has each changes and improvements, these changes and improvements are all fallen
In the range of entering claimed invention.
The measurement of film-forming yeast total plate count in 1. soy sauce of experimental example
Experimental group:
(1) preparation of standard bacterium solution
It takes film-forming yeast bacterium to cross on suitable solid medium respectively to cultivate, 30 DEG C of cultivation temperature, incubation time
48h is placed in 5mL sterile water, and vibrate 20s on the oscillator, is then existed with disposable transfer needle picking single colonie
10000rmp/min is centrifuged 1min, removes supernatant, and 5mL aseptic soy is added, vibrates 20s on the oscillator, and standard is made and produces film ferment
Female bacterium solution, thalline diameter are 30 μm, and set three in parallel.
(2) total plate count is detected using the detection system of embodiment 1
By above-mentioned film-forming yeast bacteria liquid sample be applied to embodiment 1 detection system (Raman spectrometer model is LS785,
Built-in CCD device model is PIXIS100BR) in, wherein microtubule diameter is 50 μm, and fluid flow rate is 5 μ L/min, Raman light
The determination condition of spectrum is set as: laser power range: 2mW, acquisition time: 8ms, surveys a length of 475nm of detection.
(3) data processing
(a) extract soy sauce background signal (see Fig. 3 a gray line)
(b) optical signal that extraction standard film-forming yeast bacterium solution is generated by Raman spectrum (see Fig. 3 a black line part)
(c) soy sauce background signal is deducted
(d) the black test point on Raman light is a bacterium, passes through the charge coupled device (CCD) of Raman spectrum
It collects computer and collects electric signal (black test point).It is for statistical analysis to the quantity of black test point, thus examination criteria
Total plate count in film-forming yeast bacterium solution (see Fig. 3 b).
The measurement of culture medium cultivation:
(1) the standard film-forming yeast bacterium solution of step (1) is used into the sum of culture medium cultivation measurement film-forming yeast, setting
Three parallel.The detailed step of culture medium cultivation measurement refers to national standard GB4789.2-2016.
(2) difference analysis: film-forming yeast sum, the medium therapy that light fluid detects are examined using SPSS (SPSS Inc.)
The film-forming yeast sum of survey is analyzed.
The result shows that: light fluid detection, by the analysis with soy sauce control group, by soy sauce background deduction in Fig. 3 a.And it will
Testing result splicing, black test point CCD device in Raman light with black stripe indicate thalline quantity, CCD dress in Raman light
It sets and saccharomycete sum (such as Fig. 3 b) in sample is counted by record black stripe number.Therefore the film-forming yeast sum of light fluid detection
It is 2.3 ± 0.2 × 103A, the film-forming yeast sum of medium therapy detection is 2.2 ± 0.1 × 103It is a.It is analyzed by SPSS, table
Mingguang City's stream detection and medium therapy detection film-forming yeast have no otherness.Culture medium detection method is to measure other sides as National Standard Method
Method standard, therefore light fluid detection method have very good accuracy in the detection method of film-forming yeast.
The measurement of bacillus sample total plate count in 2. milk of experimental example
Experimental group:
(1) prepared by standard bacterium solution
It takes bacillus to cross in suitable solid culture respectively to cultivate, 30 DEG C of cultivation temperature, incubation time 72h,
With disposable transfer needle picking single colonie, it is placed in 10mL sterile water, and vibrate 10s on the oscillator, then in 10000rmp/
Min is centrifuged 1min, removes supernatant, and 10mL germ-free milk is added and water vibrates 10s on the oscillator, standard bacillus bacterium is made
Liquid, thallus size are 10 μm, and set three in parallel.
(2) total plate count is detected using the detection system of embodiment 1
Above-mentioned bacillus liquid sample is applied in the detection system of embodiment 1, wherein the internal diameter of micro-pipe is 25 μm, stream
Body flow velocity is 1000 μ L/min, and the determination condition of Raman spectrum is set as: laser power range 20mW, acquisition time: 15s surveys inspection
Wavelength is 785nm.
(3) data processing
(a) extract milk background signal (see Fig. 4 a gray line)
(b) extraction standard bacillus generates optical signal by Raman spectrum (see Fig. 4 a black line)
(c) milk background signal is deducted
(d) the black test point on Raman light is a bacterium, passes through the charge coupled device (CCD) of Raman spectrum
It collects computer and collects electric signal (i.e. black test point).For statistical analysis to the quantity of black test point, thus detection is marked
Quasi- Bacillus colonies are total (see Fig. 4 b).
The measurement of culture medium cultivation:
(1) by the standard bacillus of step (1), the sum for determining bacillus is measured by culture medium cultivation, if
Fixed three parallel.With reference to such as experimental example 1.
(2) difference analysis: identical as experimental example 1.
The result shows that: light fluid detection, by the analysis with milk control group, by milk background deduction (method and experiment
Example 1 is identical).And will test result splicing, black test point CCD device in Raman light with black stripe indicates thalline quantity,
CCD device counts bacillus sum in sample by record black stripe number in Raman light (see Fig. 4 b).Therefore light stream physical examination
The bacillus sum of survey is 1.8 ± 0.1 × 103A, the bacillus sum of medium therapy detection is 1.7 ± 0.1 × 103It is a.
It is analyzed by SPSS, shows that light stream detection and medium therapy detection bacillus have no otherness.Culture medium detection method is as state
Mark method is to measure other method standards, therefore light fluid detection method has standard very well in the detection method of bacillus sum
True property.
The measurement of E. coli SampLes total plate count in 3. mineral water of experimental example
Experimental group:
(1) prepared by standard bacterium solution
It takes Escherichia coli to cross in suitable solid culture respectively to cultivate, 37 DEG C of cultivation temperature, incubation time 30h,
With disposable transfer needle picking single colonie, it is placed in 7mL sterile water, and vibrate 15s on the oscillator, then in 10000rmp/
Min is centrifuged 1min, removes supernatant, the sterile mineral water of 7mL is added, and vibrate 15s on the oscillator, standard E. coli sample is made
Product, thallus size are 15 μm, and set three in parallel.
(2) total plate count is detected using the detection system of embodiment 1
Above-mentioned E. coli SampLes are applied in the detection system of embodiment 1, wherein 20 μm of micro-pipe diameter, fluid stream
Speed: 800 μ L/min, the determination condition of Raman spectrum are set as: laser power range: 10mW, acquisition time: 2s, and it is a length of to survey detection
600nm。
(3) data processing
(a) extract mineral water background signal (see Fig. 5 a gray line)
(b) extraction standard Escherichia coli generate optical signal by Raman spectrum (see Fig. 5 a black line)
(c) mineral water background signal is deducted
(d) the black test point on Raman light is a bacterium, passes through the charge coupled device (CCD) of Raman spectrum
It collects computer and collects electric signal (i.e. black test point).It is for statistical analysis to the quantity of stain, thus examination criteria large intestine
Bacillus total plate count (see Fig. 5 b)
The measurement of culture medium cultivation:
(1) by the standard E. coli of step (1), culture medium cultivation measures the sum for determining Escherichia coli, setting three
It is a parallel.With reference to such as experimental example 1.
(2) difference analysis: the Escherichia coli sum and medium therapy that light fluid is detected using SPSS (SPSS Inc.)
The bacillus sum of detection is analyzed.
The result shows that: light fluid detection, by the analysis with mineral water control group, by mineral water background deduction (method with
Experimental example 1 is identical).And will test result splicing, black test point CCD device in Raman light with black stripe indicates thallus number
It measures, CCD device counts Escherichia coli sum in sample by record black stripe number in Raman light (see Fig. 5 b).Therefore light fluid
The Escherichia coli sum of detection is 2.5 ± 0.2 × 103A, the Escherichia coli sum of medium therapy detection is 2.4 ± 0.2 × 103
It is a.It is analyzed by SPSS, shows that light stream detection and medium therapy detection bacillus have no otherness.Culture medium detection method conduct
National Standard Method is to measure other method standards, therefore light fluid detection method has very well in the detection method of bacillus sum
Accuracy.
The measurement of total plate count in 4. samples of juice of experimental example
(1) prepared by hybrid standard bacterial strain
Aseptic juice fluid sample is accessed into saccharomycete, bacillus, Escherichia coli, takes 1ml after being mixed, be added 10mL without
In bacterium water, and 10s is vibrated on the oscillator, sample to be tested is made, and set three in parallel.
(2) total plate count is detected using the detection system of embodiment 1
Above-mentioned samples of juice is applied in the detection system of embodiment 1, wherein micro-pipe diameter is 30 μm, fluid flow rate:
500 μ L/min, the determination condition of Raman spectrum are set as: laser power range: 2mW, acquisition time: 100ms, and it is a length of to survey detection
500nm。
(3) data processing
(a) extract fruit juice background signal (see Fig. 6 a gray line)
(b) extraction standard Mixed Microbes generate optical signal by Raman spectrum (see Fig. 6 a black line)
(c) fruit juice background signal is deducted
(d) the black test point on Raman light is a bacterium, passes through the charge coupled device (CCD) of Raman spectrum
It collects computer and collects electric signal (i.e. stain).It is for statistical analysis to the quantity of stain, thus examination criteria Mixed Microbes bacterium colony
Always (see Fig. 6 b)
(4) culture medium cultivation measures: by the sample to be tested of step (1), culture medium cultivation measures micro- in sample to be tested
Biology total sets three in parallel.Method reference experiment example 1.
(5) difference analysis: Escherichia coli sum, the medium therapy that light fluid detects are examined using SPSS (SPSS Inc.)
The bacillus sum of survey is analyzed.
The result shows that: light fluid detection, by the analysis with fruit juice control group, by fruit juice background deduction (method and experiment
Example 1 is identical).And will test result splicing, black test point CCD device in Raman light with black stripe indicates thalline quantity,
CCD device is counted in sample by record black stripe number and mixes bacterium total plate count in Raman light (method is identical as experimental example 1).Cause
The Mixed Microbes sum of this light fluid detection is 400 ± 12, and the Mixed Microbes sum of medium therapy detection is 390 ± 10.Pass through
SPSS analysis shows that light stream detection and medium therapy detection bacillus have no otherness.Culture medium detection method is as National Standard Method
It is to measure other method standards, therefore light fluid detection method, with very good accuracy in the detection method of bacillus sum.
Comparative example 1.
(1) sample preparation
It takes film-forming yeast bacterium to cross on suitable solid medium respectively to cultivate, 30 DEG C of cultivation temperature, incubation time
48h is placed in 5mL sterile water, and vibrate 20s on the oscillator, is then existed with disposable transfer needle picking single colonie
10000rmp/min is centrifuged 1min, removes supernatant, and 5mL aseptic soy is added, vibrates 20s on the oscillator, and standard is made and produces film ferment
Female bacterium solution, thalline diameter are 30 μm, and set three in parallel.
(2) total plate count is detected using the detection system of embodiment 1
Above-mentioned film-forming yeast bacteria liquid sample is applied in the detection system of embodiment 1, but by micro-pipe (fluid hose therein
Road 21) straight line pipeline is changed into, 50 μm of internal diameter, fluid flow rate is 5 μ L/min, and the determination condition of Raman spectrum is set as: laser function
Rate range: 2mW, acquisition time: 8ms surveys a length of 475nm of detection.
(3) data processing
(a) extract soy sauce background signal (see Fig. 7 a gray line)
(b) optical signal that extraction standard film-forming yeast bacterium solution is generated by Raman spectrum (see Fig. 7 a black line)
(c) soy sauce background signal is deducted
(d) black test point is a bacterium on Raman light, is received by the charge coupled device (CCD) of Raman spectrum
Collect computer and collects electric signal (i.e. black test point).Black test point CCD device in Raman light with black stripe indicates bacterium
Body quantity, CCD device counts total plate count statistical analysis in sample by record black stripe number in Raman light, thus detection mark
Quasi- film-forming yeast sample total plate count (see Fig. 7 b)
The result shows that: when micro-pipe is rectilinear tubes, the film-forming yeast sum of light fluid detection is 1.1 ± 0.3 × 103It is a,
In contrast, medium therapy (National Standard Method is the standard for measuring other methods) detection film-forming yeast sum be 3.3 ± 0.2 ×
103It is a, it is analyzed by SPSS, shows that light stream detection and medium therapy detection film-forming yeast have significant difference.It can be seen that working as
Using light fluid detection method but when micro-pipe changes into straight line pipeline, do not have accuracy in the detection of film-forming yeast.It causes
The possible cause for stating phenomenon is that straight line pipeline causes film-forming yeast bacterium dispersibility that can reduce, and one or more bacterium are led to simultaneously at cenobium
Raman spectrum is crossed, is recorded into a bacterium to will cause Raman spectrum, causes to count inaccuracy, brings maximum error.
Although a specific embodiment of the invention has obtained detailed description, those skilled in the art will appreciate that root
According to all introductions announced, details can be carry out various modifications and be changed, and these change in guarantor of the invention
Within the scope of shield.Whole of the invention, which is divided into, to be given by the appended claims and any equivalents thereof.
Claims (12)
1. for detecting or the system of enumeration of micro organisms, it includes:
Fluid pump (1) with fluid inlet and fluid outlet；
Fluid control device (2) comprising fluid circuit (21), mixed sample pool (22) with first end and the second end and
It dilutes liquid pool (23)；Wherein, the mixed sample pool (22) is flowed with the fluid outlet, first end and dilution liquid pool (23) respectively
Body connection, the fluid circuit (21) include inverted V type pipe unit (211), and the inverted V type pipe unit (211) includes rising part
(211a) and sloping portion (211b), also, detection zone is equipped with close to the position of the second end；
Spectral detection system (3) comprising light source (31) and detection device (32), wherein the light source (31) is arranged to produce
The raw exciting light passed through from the detection zone；The detection device (32) is arranged to receive and detect to generate from the detection zone
Optical signal, to generate spectrum.
2. system described in claim 1, wherein the fluid circuit (21) includes the inverted V type pipe unit of multiple fluid communication
(211)；
Preferably, the fluid circuit (21) includes the inverted V type pipe unit (211) of 3,4 or 5 fluid communication.
3. system of any of claims 1 or 2, wherein the fluid circuit (21) has one or more in following characteristics:
(i) angle that the rising part (211a) and sloping portion (211b) are formed is acute angle；Preferably, the model of the acute angle
It is trapped among between 45 to 75 degree；
(ii) rising part (211a) and horizontal angle are obtuse angle；Preferably, the range at the obtuse angle is arrived 100
Between 135 degree；
(iii) rising part (211a) and sloping portion (211b) are isometric；
(iv) internal diameter of the fluid circuit is 20 μm -50 μm, such as 20 μm -30 μm；
(v) fluid circuit is made of the transparent material of exciting light；Preferably, the transparent material of the exciting light is selected from
PMMA or PDMS；
(vi) first end of the fluid circuit (21) and its neighbouring part are horizontally extending.
4. the described in any item systems of claim 1-3, wherein the system also includes selected from following one or more:
(1) for receiving the fluid source (4) of fluid to be measured sample, it is in fluid communication with the fluid inlet of the fluid pump (1)；It is excellent
Selection of land, the fluid to be measured sample are liquid food, such as drink, liquid flavoring or drinking water；
(2) waste liquid pool (5) are in fluid communication with the second end of the fluid circuit (21)；
(3) valve (6) are used to control flowing of the fluid from the mixed sample pool (22) to the fluid circuit (21).
5. the described in any item systems of claim 1-4, wherein the system includes at least two fluid control device (2), institute
Mixed sample pool (22) included at least two fluid control device (2) is stated to be in fluid communication with each other；
Preferably, the system includes at least three fluid control device (2), institute at least three fluid control device (2)
The mixed sample pool (22) for including is in fluid communication with each other.
6. the described in any item systems of claim 1-5, wherein the spectral detection system (3) has one in following characteristics
Item is multinomial:
(i) spectral detection system (3) further includes control device, with the light source (31) and detection device (32) into
Row data communication；
(ii) detection device (32) includes photoelectric converter；
(iii) spectral detection system (3) is selected from Raman spectrum detection system, infrared spectroscopy detection system and ultraviolet spectra inspection
Examining system.
7. system described in any one of claims 1-6, wherein the spectral detection system (3) is Raman spectrum detection system,
The detection device (32) includes CCD.
8. for detecting or the method for the microorganism in count samples comprising following steps:
(i) make the fluid to be measured sample containing microorganism in claim 1 in the fluid circuit by first end to the
Two end directions are simultaneously mobile with the flowing velocity of 5-1200 μ L/min, so that the microorganism in the fluid to be measured sample is one by one
Pass through the detection zone of the fluid circuit；
(ii) it is carried out by the described in any item detection systems of claim 1-7 to by the fluid to be measured sample of the detection zone
Spectroscopic methodology detection, to obtain the spectrum generated from the detection zone, the spectrum includes to be attributed in the fluid to be measured sample
Thallus map；
(iii) spectrum generated to the detection zone is analyzed, to obtain the map for being attributed to microorganism；
(iv) for statistical analysis to the map for being attributed to microorganism, to obtain the quantity of the microorganism,
Preferably, in step (i), the fluid to be measured sample is applied to fluid source (4) described in claim 4.
9. method according to any one of claims 8, in step (ii), by the detection system described in claim 6 to passing through
The fluid to be measured sample for stating detection zone carries out Raman spectroscopy detection, to obtain the Raman spectrum generated from the detection zone；And
And in step (iii), the Raman spectrum generated to the detection zone is analyzed, to obtain the feature for being attributed to microorganism
Peak；
Preferably, the Raman spectroscopy is selected from confocal Raman spectra, Surface enhanced Raman spectroscopy, coherent anti-Stokes Raman
Spectrum and laser tweezers Raman spectrum.
10. method as claimed in claim 9, wherein carry out the Raman spectroscopy detection under the conditions of following one or more:
(i) acquisition time is 8ms-15s；
(ii) wavelength of exciting light is ultraviolet near infrared wavelength region；
(iii) laser power is 2-20mW.
11. the described in any item methods of claim 8-10, before step (i), the method also includes to described to flow measurement
Body sample is pre-processed；
Preferably, the pretreatment is selected from filtering, dilution, or any combination thereof；
Preferably, before step (i), the fluid to be measured sample is diluted using sterile water.
12. the described in any item methods of claim 8-11, wherein the fluid to be measured sample is liquid food, such as drink,
Liquid flavoring or drinking water；
Preferably, the fluid to be measured sample is selected from soy sauce, milk, fruit juice and mineral water.
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