CN105907636B - Automatic instrument for microorganism high-flux culture and large concentration range real-time detection - Google Patents

Automatic instrument for microorganism high-flux culture and large concentration range real-time detection Download PDF

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CN105907636B
CN105907636B CN201610341529.9A CN201610341529A CN105907636B CN 105907636 B CN105907636 B CN 105907636B CN 201610341529 A CN201610341529 A CN 201610341529A CN 105907636 B CN105907636 B CN 105907636B
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sample bottle
sample
detection
concentration
probe
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CN105907636A (en
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万俊
蒋佩琪
齐伯
齐一伯
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Jie Ling instrument manufacturing (Tianjin) Co., Ltd.
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Jie Ling Instrument Manufacturing (tianjin) Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Abstract

The invention provides an automatic instrument for microorganism high-flux culture and large concentration range real-time detection. According to the automatic instrument, a transmission light path and a back scattering light path are combined together and share one light source (a light emitting diodes) a light signal receiver (a photodiode) of the transmission light path is arranged in a direction directly facing the light source; a light signal receiver (another photodiode) of the back scattering light path is arranged on a side same as the light source. When in detection, light is emitted from the light source, the two signal receivers receive light intensity signals (transmission light and back scattering light) simultaneously, the relationship between the light signals of the two signal receivers is compared, an appropriate detection light path is selected to measure the concentration of bacterium suspension, and thus the growth situation of microorganism culture can be accurately obtained. Due to adoption of the transmission light path and the back scattering light path of the concentration detection device, a double-detector composite light path can be constructed, defects of a single detection method is avoided, and the automatic instrument is particularly applicable to concentration detection on a test tube container in high-flux culture. Due to adoption of high-flux liquid stirring and a sample bottle tray transmission device in the automatic instrument, and the synergism of a brushless motor and a stepper motor, stirring and concentration detection on a liquid culture medium are organically combined, full-automatic operation is achieved, and multiple defects of a conventional method are avoided.

Description

A kind of automatic for microorganism high-pass culture and its big concentration range real-time detection Change instrument
Technical field
This technology belongs to Microbiological Lab's research field, is that one kind is capable of microorganism high-pass culture and big concentration The self-reacting device of scope real-time detection.
Background technology
Microorganism, as a big class biotic population on the earth, has been directed to the every aspect of human lives, is widely used in The fields such as food, medicine, industrial chemicals, biological product, feedstuff, pesticide, environmental conservation, are also used for weaving, process hides, stone now Oil exploitation and fermentation, the field such as metallurgy, macromolecular compound.
The growth and breeding of microorganism is synthesis under inside and outside various environmental factorss interact for the metabolism of its own Reflection, therefore, the data of relevant growth and breeding can as the important indicator of the various physiology of research, biochemistry and genetic problem, and then Understand the environmental factorss of microbial growth rule and its growth of impact.In the nutrition of microorganism, breathing, substance metabolism, life In the research such as relation between length, breeding, heredity and variation, evolution and different microorganisms, due to microorganism individuality very little, grind The individual growth studying carefully them is highly difficult, so in most cases, studies its population growth by amplification culture.Will be a certain amount of micro- Bacterization a closing, in container filling certain volume fluid medium, keep certain temperature, pH and dissolved oxygen Amount, microorganism growth and breeding wherein, the quantity that microorganism in result becomes many by few, again by tailing off after peaking, very more To dead Changing Pattern.With the logarithm of micro organism quantity or growth rate as vertical coordinate, with growth time as abscissa, draw The curve becoming is referred to as reproduction curve or the growth curve of microorganism.The growth rate of various microorganisms is different, each microorganism There is each distinctive growth curve.In microbial cultivation process, understand cultivating microorganism growth curve variation tendency in real time, can Determine the growth rhythm of various microorganisms, research and production tool is of great significance.
Measure bacteria suspension in micro organism quantity one side can according to microbial cell amount, microbial body volume or quality Lai Direct measurement is it is also possible to indirectly be measured with the content or certain metabolic activity intensity of certain cellular material.Traditional direct survey Amount method has：(spectrophotometry de termination or turbidity are surveyed for stain smear counting method, blood counting chamber counting method, photodensitometry Mensuration), plate total plate count algoscopy etc..Traditional indirect measurement method has：ATP biloluminescence method, impedance bioelectrical measurement method, connect Catalase measurement method, DNA quantitative method etc..
Wherein, for the measurement of the bacteria suspension bacterium number of unicellular microorganism, photodensitometry is relative most convenient, quick And a kind of method being widely used, its principle is：After muddy bacteria suspension, the intensity of light is subtracted the light of a certain wavelength Weak, incident illumination is related with thickness to the intensity ratio of transmitted light and the concentration of bacteria suspension, if bacteria suspension thickness is certain, light is close Degree (OD) is related to bacteria suspension concentration.Because thalline is light tight, within the specific limits, suspension concentration and transmittance are inversely proportional to, with Optical density (OD) is directly proportional, and cell number is more, and optical density (OD) is bigger, therefore, if with traditional spectrophotometric determination bacterium The optical density (OD) of suspension, you can record the concentration of this bacteria suspension.Bacterium by the bacteria suspension of unknown cell number and known cell number Suspension optical density compares, and can obtain the cell number contained by unknown bacteria suspension, i.e. spectrophotometry de termination.Wherein, spectrophotometric What the conventional spectrophotometers used by measurement method, microplate reader used is all transmission light measurements method, i.e. light emitting diode and photoelectricity two The angle of pole pipe is 180 degree, and the wavelength of light emitting diode is typically in 600nm.The scope of this method measurement is less, typically All in 1.0OD600Within, if bacteria suspension concentration surpasses gone beyond the scope, need to carry out gradient dilution to sample.
Additionally, the concentration measuring bacteria suspension using transmissometer is also a kind of conventional photodensitometry, its principle is： A branch of directional light is propagated in transparency liquid, if no any particle exists in liquid, then light beam is in straightline propagation Direction will not be changed；If there being particle, whether not transparent tube particle is, and light beam will change direction when running into granule, and this is just Form scattered light, granule is more, and turbidity is higher, and scattering of light is stronger.Scopometer emits beam, and light is through granule in sample After material scattering, detector on some scattered light entrance and incident illumination direction in 90 degree, the intensity of detectors measure scattered light, The concentration of particulate matter in the intensity counter sample of scattered light.Wherein, the light path that this transmissometer is usually used is one and lights Diode and a photodiode, typically between 800-900nm, the diameter of measurement bottle is generally the wavelength of light emitting diode 25mm, and light emitting diode and photodiode around sample cell and are in same level, and both are generally 90 by angle Degree, the turbidimetry scope of this angle is also limited, so when being applied to the measurement of bacteria suspension, there is also measurement range limited Shortcoming.
In practice, photodensitometry needs timing sampling from culture vessel, then using spectrophotometer specific Detect the optical density (OD) of solution it is impossible to accomplish real-time detection under wavelength；Using transmissometer measurement be also required to timing by test tube from Take out in incubator and measure again, nor accomplish that real-time online detects.And because spectrophotometer or transmissometer are using single Light transmission or an angle of 90 degrees scattering method detected, useful range is limited, typically in 0.1-2.0 OD600Between, and most absolutely Number microorganism culturing has often been above the effective dose of spectrophotometer or transmissometer to middle and late stage optical densities (OD) or turbidity Journey (for example grow in test tube and obtain ratio antibacterial faster, final OD600Value can exceed that 10), need to carry out gradient to culture fluid dilute Release and detect again, testing result is multiplied by optical density (OD) or the turbidity that extension rate obtains reality, finally draws out growth of microorganism Time corresponding optical density (OD) or the growth curve of turbidity.
Under the trend that current cultivating system milligram ammonia, high pass quantify, this offline inspection side of traditional photodensitometry There are a lot of drawbacks in method, such as：Need frequently to sample during measurement bacteria suspension concentration, destroy cultivating system and training to a certain extent The stability of foster environment；Frequently sampling increases the probability of pollution；During big flux culture, operability is not strong；Cannot be in culture body System be in shaking state or have visible ray environment in detected；Obtained testing result all has temporal delayed Property it is impossible to reflect in real time the state of cultivating system；Measurement result is easily subject to interference of coloring matter, impurity etc. etc..
Additionally, when carrying out microorganism culturing, because most of microbe needs to consume oxygen in metabolic process, discharging The gaseous products such as carbon dioxide, ceaselessly shake the gas exchange that culture fluid can be accelerated in culture fluid, so that microorganism is obtained more Many oxygen, make nutrient substance be uniformly distributed, and microorganism also can be made to be in suspended state always, thus allowing microorganism to be in than conveniently Growing environment in.Traditional method is that the culture vessels such as test tube, triangular flask are placed in shaking incubator, but because culture is held Device is constantly in shaking state, and culture vessel is not easy to be popped one's head in Concentration Testing and integrates.
At present, some microbe researches person use full-automatic microplate reader or similar products be used for high-flux microorganism culture and Microorganism concn detection in real time, the Cleaning Principle of this kind of product is similar to spectrophotometer, microwell plate is used as microorganism culturing Container, instrument can provide isoperibol for microwell plate, and in incubation, instrument persistently can be shaken to microwell plate, and fixed When detect the concentration of bacteria suspension in each hole.But because the similar products such as microplate reader can not reach preferable shaking effect, and And transparent sealing lid can only be used above microwell plate, it is difficult to breathe freely, easily cause periphery hole and centre bore oxygen exchange efficiency Difference, the microorganism in micropore cannot be in preferably in growing environment.And when detecting, use transmission light measurements method, survey That measures is limited in scope.So this kind of product is not appropriate for high flux culture and the real-time concentration detection of microorganism.
Researcher is had to measure the concentration of bacteria suspension in microwell plate, example using the method that forward scattering and back scattering combine As, Chinese patent application 201080009279, denomination of invention " equipment of analysis biological sample " disclose a kind of using front to Scattering Measurement measure microwell plate in bacteria suspension concentration technology, this invention be applied to microwell plate bacteria suspension concentration qualitative Measurement, thus realizing whether there is antibacterial quickly and in high throughput testing sample, and completes typing or antibacterial inspection.Due to this There is air, the height between lid for micro plate and bacteria suspension liquid level can affect measurement knot between bright lid for micro plate and bacteria suspension liquid level Really, the method there is also the shortcoming similar with microplate reader simultaneously, and therefore this invention is only used for the qualitative detection of culture, that is, uses In medical diagnosis purposes, the skill of the high-flux microorganism culture and real-time microorganism concn detection needing at present still cannot be solved Art needs.
Researcher is had to develop non-intrusion type Biomass on-line measuring device for shake-flask culture, this device uses turbidity The method of measurement, realizes the on-line checking of shake-flask culture bacterial concentration using back scattering optical measurement techniques, can not interrupt training In the case of foster equipment, the concentration of bacteria suspension in shaking flask is detected.For example, Chinese patent application 201510551900, send out A kind of bright title " non-intrusion type Biomass on-line measuring device for shake-flask culture ", however this device use single backward Scattering photo measure, when bacteria suspension concentration is less, is vulnerable to the interference of chamber wall reflected light, is only suitable for the larger shaking flask of volume, Be not suitable for the conventional test tube of high flux culture, and each container needs to install a detection means additional, this is for high pass For amount, the culture of miniaturization, that is, increased equipment cost, increase systematic error, also have great difficulty on the implementation.
Chinese utility model patent 201420223505, denomination of invention " detection means of culture fluid " disclose a kind of being suitable for The detection means of bacteria suspension culture in real time in triangular flask, this device is sent out by arranging light in culture bottle overthe openings to be checked simultaneously Emitter and optical receiver, and light-reflecting portion is set in culture fluid, thus being formed through culture fluid to be checked and without culture bottle wall Light path, it is possible to achieve the examinations to culture fluid, avoid simultaneously because the uneven thickness of culture bottle or nutrient solution volume solid Determine and cause the inaccurate problem of result.But although this invention achieves the effect of real-time detection microorganism concn, but this Bright use to measure bacteria suspension concentration similar to spectrophotometric transmitted light principle, and need for single culture bottle It is respectively provided with detection means and reflex reflector, therefore do not enable the effect of high flux high concentration real-time detection.
" one kind being capable of detection container internal specimen for disclosed Chinese patent application 201480033621, denomination of invention recently It is strong that the detection means of middle microorganism presence, system and method " disclose microorganism concn and fluorescence in a kind of real-time detection container The device of degree, this device detects microorganism concn using single rear orientation light, thus realizing the effect of real-time detection.So And, this device utilizes single rear orientation light measurement method, and the part reflected light still not solving inboard wall of test tube also can enter light Electric diode, causes to detect in low concentration inaccurate, a device must adhere to single container simultaneously, does not therefore enable One device detects multiple testing containers simultaneously, thus not enabling the effect of high flux high concentration real-time detection.
Zhao Mingfu etc. report a kind of using light biomass concentration detecting line sensor (《Optical precision engineering》, The 4th phase of volume 15 in 2007), the light source of this sensor has selected 760nm near infrared light.Using an incident optical (light source) and One accepts optical fiber (detector), in conjunction with two reflectings surface, reaches the purpose of measurement light absorbs and light scattering.However, this sensing Device uses single light source and single detector, transmitted light and rear orientation light simultaneously by same detectors measure it is impossible to distinguish Transmitted light and scattered light signal, and due to the increase with bacteria suspension concentration, optical signal transmissive is gradually weakening, and dissipates backward Penetrate optical signal gradually strengthening, the maximum OD value of its detection is just above 0.4, and far smaller than conventional microbiological culture can reach Concentration, therefore can not meet the needs of the examinations of high flux high concentration bacterium solution.
As immediate prior art, Chinese patent application 201010148891, a kind of denomination of invention " microbial cell The online observation system and method for suspension culture " disclose the dress using the concentration of suspension cell in infrared light real-time detection container Put, wherein infrared light supply is set in culture vessel side-wall outer side, turbidity transducer is set in bottom, the outfan of detection data leads to Cross data wire to be connected with computer, thus realizing the on-line checking automation equipment of microbial cell suspension culture.This invention Final principle is similar with common transmissometer principle, and that is, between light source and detector, angle is 90 degree, detects that it measures incident illumination 90 Scattered light intensity on degree direction.This is visible, and this inventive nature still falls within single light path turbidimetry, measurement of concetration model It is with limit, high flux culture will be realized, need to integrate other shaking equipment, and be not carried out single detection probe Multi-example Aulomatizeted Detect.
Additionally, the Chinese invention patent application 201310246480, " shaking table of contactless multichannel on-line checking fermentation liquid Device and method of testing " all discloses the device realizing that using reflected light multichannel real-time detection cultivates bacterium solution, this device profit With laser transmitted light and computer processing technology, enable the growth of the multiple cultivating microorganism of high flux real-time detection.However, should Method still falls within traditional transmission optical detection technique, simultaneously need to arranging generating laser and reception in each test bottle side Device, while realizing high throughput testing, also results in testing equipment technological deficiency excessive, with high costs.
Additionally, when high flux is cultivated, the sample size once cultivated is all tens, so being essentially all using examination Cultivating, if using single transmission light measurements method, detectable concentration is limited in scope pipe；If using single rear orientation light Measurement method, due to test tube diameter less, in the bacteria suspension of low concentration, except scattered light can enter photodiode, in test tube The part reflected light of wall also can enter photodiode, causes to detect in low concentration inaccurate；If using 90 degree of angles and The method that back scattering measurement method combines, can increase the complexity of detection probe, if detection full-automation, machinery will be realized Part also can become more sophisticated.
To sum up, either using spectrophotometer or turbidimetric conventional offline measuring method, or existing exist in real time Line detecting method, all cannot meet and carry out real-time online measuring high-concentration bacterial suspension when cultivating using test tube class container high flux. Culture for high flux, miniaturization detects it should make every effort to a Concentration Testing probe to multiple samples, reduces dense as far as possible Degree detection probe quantity, and realize the automatization of whole detection process.If a equipment can carry out high-flux microorganism training Support, real-time microorganism concn detection can be carried out moreover it is possible to make microorganism grow under ideal conditions again, microorganism will be greatly facilitated Research work.
Therefore, for problem above, need one kind can carry out high-flux microorganism culture and micro- life in real time at present simultaneously The product of thing Concentration Testing or method, with realize microorganism concn detection full-automation, high flux, high accuracy.
Content of the invention
Problem to be solved by this invention is：When carrying out microorganism culturing, because most of microbe is in metabolism Need in journey to consume oxygen, discharge the gaseous products such as carbon dioxide, ceaselessly shaking culture fluid can accelerate the gas in culture fluid Body exchanges, and makes microorganism obtain more oxygen, so that nutrient substance is uniformly distributed, and microorganism also can be made to be in suspended state always, from And allow microorganism to be in than in growing environment conveniently.Traditional method is that the culture vessels such as test tube, triangular flask are placed on shaking In incubator, but because culture vessel is constantly in shaking state, culture vessel is not easy to be popped one's head in Concentration Testing and integrates.Institute With for the detection product of high-flux microorganism culture and real-time microorganism concn it is therefore desirable to realize Concentration Testing probe right Multiple samples are detected, as far as possible minimizing Concentration Testing probe quantity, and realize automatization and the high flux of whole detection process, Thus avoiding the defect that prior art exists：Or with low cost accurate with result, but high throughput testing cannot be realized；Or it is real Existing high throughput testing, but cost is excessively high；Or achieve cost economy and the purpose of high flux real-time detection, but can not expire The needs of sufficient high-flux microorganism culture.
Therefore, the invention aims to overcoming the weak point of existing equipment, provide a kind of full-automatic, high accuracy, High flux, the microorganism high-pass culture of directly perceivedization and its self-reacting device of concentration real-time detection.
In order to realize above-mentioned technical purpose, the present invention is on the basis of traditional single transmitted light or single scattering light detection method On, transmitted light and back scattering light detection method are combined, using a light emitting diode and two photoelectricity two poles by creativeness The combination of pipe, the distance optimizing light source and photodiode in back scattering detection is it is achieved that transmission signal and back scattering The joint-detection of signal.
Therefore, one of principle of the invention is, the present invention passes through for transmitted light to detect light path and rear orientation light detection light Road group is combined in same detection probe, thus completing to be simultaneously achieved the joint-detection of transmission signal and backscatter signal.Tool For body, the principle of the invention is to be combined together transmitted light path and backward astigmatism road, and the two shares a light source (luminous two Pole pipe), the optical signal receiver (photodiode) of transmitted light path is arranged on light source just to direction, the light letter of back scattering light path Number receptor (photodiode) is arranged on light source homonymy.Light source luminescent when detection, two signal receivers accept simultaneously Light intensity signal (transmitted light, rear orientation light), and by comparing the relation between the optical signal of the two, select suitable detection light Road measuring the concentration of bacteria suspension, thus accurately obtaining the growing state of microorganism culturing.
The two of the principle of the invention are, in the technology of conventional magnetic stirring, have invented and have been applied to high flux liquid agitation With the technology of sample bottle pallet transmission it is achieved that high flux culture under homogeneous stirring condition for each sample is visited with single detection The automatic detection to multiple samples for the head, it is to avoid single incubator is required to arrange the defect of detection probe.
The three of the principle of the invention are, adopt in conjunction with the technology such as accurate temperature control and culture vessel gas exchange and data Collection, analysis, output software it is achieved that microorganism high-pass culture, concentration real-time detection data analysis full-automation.
Therefore, first purpose of the present invention is to provide a kind of detection probe 1 of big concentration range real-time detection, including： One infrarede emitting diode 104, the first photodiode 105, the second photodiode 108, U-bracket 102, windowpane 106, wherein,
Probe one side is respectively perpendicular and is provided with light emitting diode 104 and the first photodiode 105, light emitting diode 104 Corresponding side surface be provided with the second photodiode 108, the two is centrally located in same horizontal line, thus light emitting diode 104 He Second photodiode 108 defines transmitted light detection light path, and light emitting diode 104 and the first photodiode 105 are with regard to shape Become back scattering detection light path；
Probe upper end is provided with for fixing U-bracket 102, and this support includes the first probe arm 103 and the second probe arm 107, fix light emitting diode 104 and the first photodiode 105 respectively, and the second photodiode 108；
Transmitting or reception in described light emitting diode 104, the first photodiode 105 and the second photodiode 108 End is designed with windowpane 106, plays a protective role.
In any of the above-described embodiment, in the vertical direction of light emitting diode 104 and the first photodiode 105 Central point distance is 6-10mm.In another embodiment, described central point distance be 6,7,8,9mm, wherein preferably 6 Or 7mm.
In any of the above-described embodiment, three windowpanes 106 are located at infrarede emitting diode 104 and photodiode 105 (108) transmitting or receiving terminal, are mainly used for avoiding the impact infrarede emitting diode such as dust, cut and photoelectricity two pole The performance of pipe, with lens paper cleaning glazing surface, user only need to can ensure that detection light road is not subject to before each is used miscellaneous Matter affects.
In any of the above-described embodiment, described light emitting diode 104 is peak wavelength scope 800-1400nm's Infrarede emitting diode, shuttle is the cylindrical cuvette of diameter 25-35-50mm.In a specific embodiment, described Test tube bottle wall be 0.5-3mm, preferably 1,1.5,2,3mm.
In any of the above-described embodiment, this probe is also included for U-bracket 102 is fixed to Concentration Testing probe The fixed handle 101 of support 305.In a specific embodiment, described Concentration Testing probe bracket 305 can fix one Or multiple Concentration Testing probe 1.
In other any of the above-described embodiments, described probe is 0-0.8 using the OD value scope of transmitted light detection, makes OD scope with rear orientation light detection is 0.8-15.0,1.2-8.0,3.5-6.5,4.0-5.8,4.5-5.0.Concrete at one Embodiment in, described rear orientation light detection OD scope is preferably 0.8-15.0.
The present invention second purpose is to provide the automatization of a kind of microorganism high-pass culture and big concentration range real-time detection Device, including above-mentioned Concentration Testing probe 1, high flux liquid stirring device, sample bottle tray transmission, wherein,
(1) high flux liquid stirring device, including sample bottle pallet 203, magnetic stir bar 204, is used for carrying sample bottle Permanent magnet fixed column 205, sample bottle basetray 216, wherein on sample bottle basetray 216 arrange loop configuration sample Product bottle holder disk 203, this pallet outward flange arranges the sample bottle fixing hole 226 of annular array, and it is solid that tray center end is embedded in setting On fixed column 205, when being positioned over magnetic stir bar 204 in sample bottle, in the presence of permanent magnet fixed column 205, magnetic agitation Son 204 constant agitation in sample bottle 201, therefore realizes high flux in multiple sample bottles by multiple magnetic stir bars and stirs Mix.
In any of the above-described embodiment, described sample bottle pallet can arrange 1 layer or multilamellar, example according to actual needs 1-10 layer in this way, preferably 1,2,3,4,5 or more, thus forming the agitating device of multi-layer annular structure.In another embodiment party In case, every layer of sample bottle pallet, one or more sample bottle fixing holes can be arranged as required to, e.g. 2-48, preferably 4th, 8,16,24 or more.In a specific embodiment, the agitating device of described multi-layer annular structure, is can be free The modular device of assembly and disassembly, thus realize increasing or decreasing the multi-layer annular structure of agitating device.In another specific embodiment party In case, described agitating device also includes sample bottle tray support post 214, support column fixed plate 212, therefore both can be in every layer of ring The sample bottle tray bottom of shape is respectively provided with a basetray it is also possible to by sample bottle tray support post and support column fixed plate In the presence of, only one basetray is set in fixing column bottom.In other specific embodiments, by by permanent magnet 206 are arranged in fixed column 205, make fixed column become permanent magnet, and make its magnetic field N and S extreme direction be in horizontal distribution.Also at it In his specific embodiments, wherein sample bottle is the cylindrical cuvette of diameter 25-35-50mm.
(2) sample bottle tray transmission, including motor 211, motor fixing plate 220, wherein motor passes through motor shaft 207 It is connected with permanent magnet fixed column 205, and motor and permanent magnet fixed column 205, sample bottle basetray 216, sample bottle pallet 203rd, the center of circle setting of support column fixed plate 212 is located on same vertical line, thus Motor drive permanent magnet fixed column 205 is revolved Turn, the magnetic stir bar 204 of the rotating excitation field of generation and then drive sample bottle 201 bottom rotates, and finally realizes magnetic stir bar 204 agitated liquid culture medium.
In any of the above-described embodiment, motor fixing plate 220 also sets up motor 222, big bearing 221, Driving gear 218, driven gear 217, wherein, motor 222 is arranged on below motor fixing plate 220 vertically upward, and leads to Cross that step motor shaft 219 is vertical upwards with driving gear 218 to be connected, driving gear pass through the driven gear 217 of horizontal direction with Sample bottle basetray 216 connects.When motor 222 drives driving gear 218 to rotate, and order about driven gear 217 and rotate, And then drive sample bottle basetray 216 and sample bottle pallet 203 to rotate, finally realize sample bottle 201 and fix around permanent magnet Post 205 axis rotates at a certain angle, and this rotation angle is the angle between two neighboring samples fixing holes 227.This angle should When ensureing light path line transmission, suitable angle can be obtained according to the sample bottle quantitative proportion of 360 degree and every layer.
In any of the above-described specific embodiments, the big bearing fixed seat of annular is set above motor fixing plate 220 209, motor shaft 207 passes through from the center of circle of big bearing fixed seat 209, arranges big bearing 221, greatly on big bearing fixed seat 209 Bearing 221 inner ring is nested in the outward flange of big bearing fixed seat 209；Big bearing sleeve 208 is nested in the outer ring of big bearing 221； Driven gear 217 is arranged on above big bearing sleeve 208, and driving gear 218 and driven gear 217 are mutually twisted；Sample bottle holder Seat 216 of trying to get to the heart of a matter is arranged on above big bearing sleeve 208；Little bearing 213 inner ring is nested in permanent magnet fixed column top 229；Little axle Cartridge 215 is arranged on above sample bottle basetray 216, and top inner side is nested in the outer ring of little bearing 213；Sample bottle pallet Support column 214 is arranged between two sample bottle pallets 203；Support column fixed plate 212 is arranged on little bearing sleeve 215 and sample The top of bottle holder disk support column 214.In a more particular embodiment, described motor 211 is brushless electric machine.
In any of the above-described specific embodiments, permanent magnet fixed column 205 is fixed on the inner ring of little bearing 213, sample Product bottle holder disk 203 is fixed on little bearing by sample bottle basetray 216, little bearing sleeve 215, support column fixed plate 212 On 213 outer rings, the rotation of such permanent magnet fixed column 205 and sample bottle pallet 203 is independent mutually, is independent of each other.
(3) Concentration Testing probe 1 is fixed on rustless steel intracavity bottom and top from rustless steel inner chamber by probe bracket 305 Marginal position, and make the vertical central axis line right through sample bottle 201 for the transmitted light path of Concentration Testing probe 1, sample bottle pallet During 203 rotation, the sample bottle 201 on sample bottle pallet 203 can just pass through between probe left arm 103 and probe right arm 107.
In any of the above-described embodiment, in Concentration Testing probe 1, the described U-bracket 102 inner side lucky energy of spacing The sample bottle 201 holding certain diameter passes perpendicularly through from centre, and U-bracket 102 inner side spacing does not surpass more than sample bottle 201 external diameter Cross 1 millimeter.When described sample bottle 201 stops in U-bracket 102, infrarede emitting diode 104 and its just to photoelectricity two pole The axis line of pipe (right) 108 just overlaps with the diameter of sample bottle 201.
The present invention the 3rd purpose is to provide the automatization of a kind of microorganism high-pass culture and big concentration range real-time detection Attemperating unit, the temperature control of pop one's head in including above-mentioned Concentration Testing 1, high flux liquid stirring device, sample bottle tray transmission Device 3 processed, wherein,
Described temperature control equipment 3 is included in the rustless steel for cincture or sealing above-mentioned agitating device and actuating device etc. Chamber 302, electric boiling plate 301, copper pipe 303, heat-insulation layer 304, cooling water circulation machine 501.Wherein electric boiling plate 301 is attached to rustless steel Inside the upper and lower outer surface of inner chamber 302 and Qianmen 311；Copper pipe 303 wraps up the left and right and rear side appearance of rustless steel inner chamber 302 Face；Wrap up heat-insulation layer 304 in the outside of electric boiling plate 301 and copper pipe 303；Copper pipe water inlet 309 and outlet 310 lead to insulation Outside layer 304, and cooling water circulation machine 501 connects.
In any of the above-described embodiment, in temperature control equipment 3, take the photograph in room temperature+5-70 when needing temperature control When family name spends, heated using the upper and lower outer surface of rustless steel inner chamber 302 and the electric boiling plate 301 of Qianmen 311 inner side；When needing temperature Degree controls in+5 degrees Celsius of 10- room temperature, is passed through coolant using cooling water circulation machine 501 keeps stainless in copper pipe 303 Steel inner chamber 302 low temperature.
In any of the above-described embodiment, described automation equipment also includes gas exchange device 4 inside and outside sample bottle, bag Include sample bottle cap 403 with holes, hydrophobic permeable membrane 401, silicagel pad with holes 402.In sample bottle cap wherein with holes, 403 divide from the inside to surface Do not install diameter and sample bottle cap internal diameter identical circle hydrophobic permeable membrane 401 and silicagel pad with holes 402 additional.
In any of the above-described specific embodiments, in gas exchange device 4 inside and outside sample bottle, hydrophobic permeable membrane 401 Aperture is less than 0.45 micron, and the gas such as oxygen, carbon dioxide can freely pass through, and vapor and microorganism cannot easily penetrate. Silica gel pad 402 with holes can the gas transmission rate as needed size to choose aperture.
In any of the above-described embodiment, wherein Concentration Testing probe 1, high flux liquid agitation and the transmission of sample bottle pallet Device 2 and temperature control equipment 3 are controlled by software.On computer 506, computer 506 passes through RS232 connection to this software installation 505 are connected with instrument host 504.Following functions can be realized on software：
1) target temperature of incubator is set；
2) motor speed is set；
3) the setting Concentration Testing probe assay intervals time；
4) the independent curve showing each sample concentration corresponding time in real time, each sample has independent display window；
5) displays temperature corresponds to the change curve of time in real time；
6) curve of a plurality of concentration corresponding time is contrasted；
7) it is directed to variable concentrations unit, there is automatic calibration function；
8) each sample can be labeled；
9) target concentration value is set, after reaching target concentration value, has prompting；
10) it is directed to the sample size of each detection, corresponding curve display window can be selected by sample placement location；
11) testing result can be exported into Excel form by testing result output function, the numerical value of each testing result, The information such as unit, time all record in form.
In one embodiment, this software can also according to tested microorganism concentration range, select suitable transmission or Backward scattered detection pattern, and automatically calibrated.
The 4th purpose of the present invention is to provide a kind of microorganism high-pass that carries out using any of the above-described device to cultivate and big The method of concentration range real-time detection, step includes：
(1) temperature control：Temperature control equipment 3 makes the temperature of rustless steel inner chamber 302 maintain microorganism culturing in advance to be needed The level wanted, then testing sample, aseptic liquid nutrient medium and sterilized magnetic stirrer 204 is added in sample bottle 201, covers Gas exchange device 4 inside and outside sample bottle, are placed in sample bottle fixing hole 227；
(2) stir culture：Permanent magnet fixed column 205 is driven to rotate by motor 211, the rotating excitation field of generation drives sample Magnetic stir bar 204 in bottle 201 rotates, thus promoting growth and breeding in the stirring of microorganism liquid medium within；
(3) Concentration Testing：Motor 222 drives sample bottle pallet 203 to rotate to an angle, Concentration Testing probe 1 inspection Survey first group of sample concentration；After detection finishes, motor 222 drives sample bottle pallet 203 to rotate to an angle, second group of sample Product enter Concentration Testing probe 1 and start to detect；After detection finishes, motor 222 drives sample bottle pallet 203 to continue rotation Certain angle, the 3rd group of sample enters Concentration Testing probe 1 and starts to detect；So Concentration Testing probe 1 and motor 222 Alternation, to the last one group of sample detection finish, motor quits work.
(4) duplicate detection：With certain time for interval, continuous repeat step (3) successively, such motor 211, motor 222 and Concentration Testing pop one's head in 1 three's collaborative work, real-time detection concentration change in microbial cultivation process, until experiment knot Bundle.
In any of the above-described embodiment, described light emitting diode 104 is peak wavelength scope 800-1400nm's Infrarede emitting diode, shuttle is the cylindrical cuvette of diameter 25-35-50mm.In a specific embodiment, described Test tube bottle wall be 0.5-3mm, preferably 1,1.5,2,3mm.
In any of the above-described embodiment, in measuring samples bottle 201 during bacteria suspension concentration, light emitting diode 104 Light, light passes through windowpane 106 and the side wall of sample bottle 201 enters bacteria suspension 202, and partly light is by microorganism in bacteria suspension 202 Grain absorbs or scatters, and the light not absorbed or scattering enters the second photodiode 108, and some scattered light enters the first photoelectricity two Pole pipe 105, two photodiodes change into the faint signal of telecommunication by receiving faint optical signal, and the faint signal of telecommunication is through putting Big circuit is enlarged into the signal of telecommunication that can easily identify.Using signal magnitude according to detected by two kinds of diodes for the single-chip microcomputer, and Compare built-in calibration curve, draw bacteria suspension concentration value, automatically select effective concentration value further according to the concentration limit setting.? In one specific embodiments, suitable transmission can be selected or dissipates backward by software according to tested microorganism concentration range The detection pattern penetrated, and automatically calibrated.
In any of the above-described embodiment, in high flux liquid agitation and sample tray actuating device 2, permanent magnet The central spot of 206 central point and magnetic stir bar 204 in same level, the magnetic stir bar of sample bottle 201 bottom 204 length are less than the internal diameter of sample bottle 201, and difference is less than or equal to 2 millimeters, when permanent magnet 206 is in the center of circle, sample bottle 201 Rounded when being uniformly distributed, the magnetic force that the magnetic stir bar 204 of all samples bottle 201 bottom is subject to is identical, permanent magnet During 206 rotation, magnetic stir bar 204, can be in the effect of the rotating excitation field of permanent magnet 206 due to being constrained by sample bottle 201 Lower horizontal rotation, and then reach the purpose of agitated liquid culture medium 202.In certain range of speeds, permanent magnet 206 rotary speed Faster, magnetic stir bar 204 also rotates faster, and fluid medium 202 stirs more violent, gas exchange above liquid and liquid Speed is also faster, is more conducive to the growth of aerobic microbiological.Sample agitating function and detection function are incorporated into one by described device Rise, each sample bottle pallet 203, only need one group of Concentration Testing probe 1, you can realize several to tens sample bottles 201 Culture and Concentration Testing process are full-automatic, and only need to increase sample bottle pallet 203 and Concentration Testing probe 1 quantity, you can Culture flux is extended.
In any of the above-described embodiment, described attemperating unit 4 to be controlled by the way of three faces cool down and three faces are heated The temperature of rustless steel inner chamber 302 processed.Adopt electric boiling plate in the upper and lower of rustless steel inner chamber 302 and 311 3, Qianmen outer surface 301 controlling the target temperature of more than+5 degrees Celsius of room temperature, left and right and rear three bread wraps up in copper pipe in rustless steel inner chamber 302 303, copper pipe 303 connects cooling water circulation machine 501, and the coolant pump of uniform temperature is entered copper pipe 303 by cooling water circulation machine 501, Rustless steel inner chamber 302 temperature is enable to be less than+5 degrees Celsius of room temperature.
Also in any of the above-described embodiment, inside and outside sample bottle, gas exchange device 4 is to accelerate inside and outside sample bottle 201 The upper lid of gas exchange.Upper lid is from outside to inside by sample bottle cap 403 with holes, hydrophobic permeable membrane 401 and silica gel pad with holes 402 Composition.The aperture of hydrophobic permeable membrane 401 is less than 0.45 micron, and the gas such as oxygen, carbon dioxide can freely pass through, and vapor Cannot easily penetrate with microorganism.Silica gel pad 402 with holes has Packed effect, it is to avoid cross-contamination, and pore size can be determined Fixed ventilative speed.
In any of the above-described embodiment, the Detection wavelength of described transmitted light detection and back scattering detection method is 850nm, described probe uses the OD of transmitted light detection600Value scope is 0-0.8, using the OD of rear orientation light detection600Scope For 0.8-15.0,1.2-8.0,3.5-6.5,4.0-5.8,4.5-5.0.In a specific embodiment, described dissipate backward Penetrate light detection OD600Scope is preferably 0.8-15.0.In conjunction with transmission light measurements method (OD600Value scope 0-0.8), therefore, the present invention Can be in 0-15OD600In the range of obtain accurate measurement result.
Term and definition
Term " spectrophotography " refers to, by measuring the suction of measured matter light in certain wave strong point or a wavelength range Receipts degree, carries out qualitative and quantitative analysis to this material.Different types of spectrophotometric ultimate principle is similar, is all to utilize one The individual light source that can produce multiple wavelength, by serial light-dividing device, thus produce the light source of specific wavelength.Light source is through test Sample rear portion light splitting source absorbed, by the light absorption value of measuring samples, through calculating the concentration that can change into sample.Sample Light absorption value be directly proportional to the concentration of sample.
Term " spectrophotometer " refers to measure the instrument of sample using above-mentioned spectrophotography.Common spectrophotometer Difference according to wavelength and application can be divided into：
Visible spectrophotometer, total range of wavelength is the visible region of 400-760nm；
Ultraviolet spectrophotometer, total range of wavelength is the ultraviolet region of 200-400nm；
Infrared spectrophotometer, total range of wavelength is the infrared light district more than 760nm；
Spectrofluorophotometer, for scanning the fluorescence spectrum that liquid phase fluorescent marker is sent；
Atomic absorption spectrophotometer, light source sends tested characteristic spectrum radiation, by the sample after atomizer Element ground state atom to be measured in steam is absorbed, and obtains containing of tested element by measuring the absorbed size of characteristic radiation Amount.
Purposes in biological sample for the spectrophotometer is mainly：
The quantitation of nucleic acid：Wavelength using 260nm can be dissolved in the oligonucleotide of buffer, single-stranded, double-strand with quantitative measurement The concentration of DNA and RNA.
The quantitation of protein：This method is in 280nm wavelength direct test proteins concentration.
Bacterial cell density：During antibacterial culturing, not add the culture fluid of bacterium solution under 600nm or other appropriate wavelengths As blank solution, the bacteria suspension concentration after measurement culture.
Term " big concentration range "：Refer to detected bacteria suspension concentration range and include detecting low concentration bacteria suspension and highly concentrated Degree bacteria suspension (OD600Value>10).
Term " central point distance "：Refer to two on light emitting diode 104 and the vertical direction of the first photodiode 105 Air line distance between caliber central point.On the one hand because light path sends from light emitting diode 104, directly to being transmitted to the of opposite On the other hand, there is back scattering in the optical path in two photodiodes 108, and backward in oblique entrance diode 105.Therefore, Diode 105 is only located in suitable central point distance range, fully could receive optical signal with effective.As can be seen here, originally Although invention represents three relations between light source emitter and receptor with central point distance, any other statement is above-mentioned The identical or equivalent limiting mode of relation, for example, pass through to limit horizontal linear and the diode 105 and 108 of diode 104 and 108 Oblique line between angle limiting mode, or by limiting horizontal range and the testing sample bottle (example of diode 104 and 108 As volume, bottle wall, density), the limiting mode such as the power of light source emitter/receptor or sensitivity, but as long as described three light Between source emitter and receptor, position relationship is identical, then these other limiting modes all fall within the identical or equivalent of the present invention Protection domain.
Term " rotation angle "：Angle between referring to as two neighboring samples fixing holes 227, this angle is used for ensureing sample Bottle rotates every time fixed angle, and in detection, the light path of transmitted light passes through the vertical central axis of sample bottle after ensureing to rotate every time Line.This angle should be guaranteed that light path line transmission, can obtain suitable folder according to the sample bottle quantitative proportion of 360 degree and every layer Angle.Such as one layer has 12 sample bottles, and rotation angle can be 360/12=30 degree.In the present invention, when being designed as 16 samples Product bottle, rotation angle can be 360/16=22.5 degree.
Brief description：
Fig. 1 is Concentration Testing probe 1 side schematic view.
Fig. 2 Concentration Testing parallel plastic plate medial surface schematic diagram of probe.
Fig. 3 is that the detection light path of the present invention looks squarely structural representation.
Fig. 4 is the detection light path overlooking the structure diagram of the present invention.
Fig. 5 is high flux liquid agitation and sample bottle tray transmission 2 structural representation of the present invention.
Fig. 6 is sample bottle tray substrate structural representation.
Fig. 7 is sample bottle fixed plate structure schematic diagram.
Fig. 8 is permanent magnet fixed column structural representation.
Fig. 9 is attemperating unit 3 structural representation.
Figure 10 is copper tube design figure.
Figure 11 is copper pipe scheme of installation (casing cross section).
Figure 12 is copper pipe and electric boiling plate scheme of installation (casing longitudinal section).
Figure 13 is gas exchange device 4 structural representation inside and outside sample bottle.
Figure 14 is the position top view that probe bracket is relative to rustless steel inner chamber.
Figure 15 is instrument integrated connection mode.
Marginal data：
1：Concentration Testing is popped one's head in
101：Fixed handle；102U shape support；103：First probe arm；104：Light emitting diode；105：First photoelectricity two pole Pipe (left)；106：Windowpane；107：Second probe arm；108：Second photodiode；
2：High flux liquid agitation and sample bottle tray transmission
201：Sample bottle；202：Fluid medium；203：Sample bottle pallet；204：Magnetic stir bar；205：Permanent magnet is solid Fixed column；206：Permanent magnet；207：Motor shaft；208：Big bearing sleeve；209：Big bearing fixed seat；210：Support column；211：Electricity Machine；212：Support column fixed plate；213：Little bearing；214：Sample bottle tray support post；215：Little bearing sleeve；216：Sample bottle Basetray；217：Driven gear；218：Driving gear；219：Step motor shaft；220：Motor fixing plate；221：Big bearing； 222：Motor；223：Base plate；224：Sample bottle pallet connecting hole；225：Sample bottle tray substrate；226：Sample bottle pallet Fixing hole；227：Sample bottle fixing hole；228：Sample bottle fixed plate；229：Permanent magnet fixed column top；230：Permanent magnet is fixed Hole；231：Permanent magnet fixed column top；
3：Attemperating unit
301：Electric boiling plate；302：Rustless steel inner chamber；303：Copper pipe；304：Heat-insulation layer；305：Concentration Testing probe bracket； 306：Bearing plate；307：Support column；308：Surface-mounted integrated circuit；309：Copper pipe water inlet；310：Copper tube water outlet；311：Qianmen；
4：Gas exchange device inside and outside sample bottle
401：Hydrophobic permeable membrane；402：Silica gel pad with holes；403：Sample bottle cap with holes；
5：Instrument is connected mode
501：Cooling water circulation machine；502：Cooling water circulation machine outlet pipe；503：Cooling water circulation machine oral siphon；504：Instrument Device main frame；505：RS232 connection；506：Computer；507：RS485 connection
Technique effect：
(1) concentration detection apparatus of the present invention use transmission and back scattering light path, constitute the compound of a dual detector Light path, it is to avoid the deficiency of single detection method, is particularly suitable for carrying out Concentration Testing to test tube container during high flux culture.
(2) the high flux liquid agitation in instrument and sample bottle tray transmission pass through brushless electric machine and motor Collaborative work, the stirring of fluid medium and Concentration Testing are combined, and have accomplished full-automation, it is to avoid tradition The shortcomings of method.
(3) temperature control equipment in instrument installs electric boiling plate and copper pipe additional using rustless steel inner chamber outer surface and combines cold But the mode of water circulation machine carries out temperature control to rustless steel inner chamber.Because psychrophile culture only accounts for small part, if so entered Row Low- temperature culture, only needs the cooling water circulation machine that an external laboratory is commonly used, and need not add inside self-reacting device The refrigeration plants such as dress compressor, lowering apparatus cost.
(4) inside and outside the sample bottle in described self-reacting device, gas exchange device 4 uses hydrophobic permeable membrane and silica gel with holes Pad combination, can make the gas free exchange inside and outside sample bottle, provide oxygen for growth of microorganism, be also avoided that in sample bottle Liquid excessive vaporization, makes microorganism be in suitable growing environment.
(5) this self-reacting device passes through novel design, using composite light path detection probe, stirs in conjunction with high pass quantity of fluid Mix, sample transmission, the technology such as temperature control and sample bottle gas exchange and data acquisition, analysis, output software is it is achieved that microorganism High flux culture, the full-automation of concentration real-time detection data analysis.Solve high flux in current microbe research, in real time Concentration Testing, liquid agitation, temperature control can not be compatible on a large scale a difficult problem.Significantly reduce the working strength of operator, Research for microorganism provides the very efficient instrument of one kind.
(6) in Concentration Testing probe 1, by using transmission and back scattering light path, constitute answering of a dual detector Closing light road, simultaneously using Single-chip Controlling, according to the concentration limit (can be corresponding magnitude of voltage or other relative value) setting Automatically select applicable concentration detection method, improve power of test in larger concentration range for the detection probe.Described light path knot The work of structure is controlled by infrared light emission circuit and mid-infrared optical receiving circuit, and infrared light emission circuit adopts special constant current source core Piece controls, and both can guarantee that use safety and the service life of infrarede emitting diode, and also can guarantee that the infrared light work(of transmitting simultaneously Rate is stable.Infrared light emission circuit has light emitting diode defencive function, and power supply is reversed or when electric current is excessive, light emitting diode is all Can be effectively protected.Mid-infrared optical receiving circuit adopts high-end amplifier chip, builds high magnification numbe low noise across resistance amplifying circuit, can So that extremely faint optical signal to be converted to the signal of telecommunication.Use power supply decoupling, analog filtering and coaxial cable in circuit simultaneously The technology such as transmission signal are it is ensured that the signal to noise ratio of signal and circuit anti-interference ability.
Specific embodiment：
The present invention is further described with reference to the accompanying drawings and examples.But described below, is only the preferable reality of the present invention Apply example, not the present invention made with any pro forma restriction, thus every without departing from technical solution of the present invention content, according to this Any simple modification, equivalent variations and modification that the technical spirit of invention is made to above example, all still fall within skill of the present invention In the range of art scheme.
As Figure 1-4, the structure of Concentration Testing probe and light path schematic diagram.Wherein, light emitting diode 104 is peak value ripple , in the infrarede emitting diode of 800-1400nm, the first photodiode 105 and the second photodiode 108 are using high for long scope Sensitivity, high stability, the High Linear and wavelength of peak sensitivity photoelectricity two in the range of light emitting diode 104 peak wavelength Pole pipe, model is identical.At the line of centres of light emitting diode 104 and the second photodiode 108 in the same horizontal line, light Diode 104 and the first photodiode 105 are on same vertical line, and the distance of two diode center point is 6mm.So Light emitting diode 104 and the second photodiode 108 are the formation of transmitted light detection light path；Light emitting diode 104 and the first light Electric diode 105 is the formation of back scattering detection light path.
First probe arm 103 is wide, high consistent with the second probe arm 107, width be less than 15mm, highly at least above 90mm.The spacing of the first probe arm 103 and the second probe arm 107 is 36mm, is easy to the sample bottle 201 energy level that external diameter is 35mm By the gap between two flat boards.Above U-bracket, photoswitch is installed, in conjunction with the catch on sample tray, can control Turning of motor 222 processed stops.Concentration Testing probe 1 is fixed on Concentration Testing probe bracket 305 by fixed handle 101, dense Degree detection probe 1 is connected with surface-mounted integrated circuit 308 with probe bracket 305 through fixed handle 101 with the circuit of photoswitch.
As shown in Fig. 9 and 3-4, probe bracket 305 is fixed on rustless steel intracavity bottom and top near rustless steel inner chamber side Edge position, Concentration Testing probe 1 is fixed on probe bracket 305, make Concentration Testing pop one's head in 1 transmitted light path just and sample bottle The diameter of pallet 203 overlaps, and during sample bottle pallet 203 rotation, the sample bottle 201 on sample bottle pallet 203 can just be from first Pass through between probe arm 103 and the second probe arm 107.
Concentration Testing probe 1 in measuring samples bottle 201 during sample concentration, send out according to modulating frequency by light emitting diode 104 Light, the second photodiode 108 and the first photodiode 105 receive transmitted light and back scattering optical signal respectively, and pass through Pre-amplification circuit, filter circuit, demodulator circuit output direct current signal, input single-chip microcomputer calculates dense according to Concentration Testing principle Degree.If the measurement result of transmitted light is less than or equal to 0.8OD600, then the measurement result of system default transmission beam method is effective；As The measurement result of fruit scattered light is more than 0.8OD600, then the measurement result by system default scattering method is effective, the measurement of scattering method Maximum magnitude can exceed 15.0OD600.
As viewed in figures 5-8, apparatus subject partial internal structure is mainly by Concentration Testing probe, high flux liquid agitation and sample Product bottle tray transmission and control circuit composition, wherein,
Sample bottle tray substrate 225 is annular metal sheet, and there is the hole of diameter 80mm at center.In sample bottle fixed plate 228 it is in Circular distribution has the sample bottle fixing hole 226 of 16 diameter 35.5mm, and the angle between each hole is 22.5 degree.Sample bottle pallet Base plate 225 and sample bottle fixed plate 228 are fixed together by sample bottle pallet connecting hole 224 above, and make between the two Interval 20mm.What so sample bottle 201 can consolidate is placed on above, and that is, each layer can place 16 sample bottles 201, each The magnetic stir bar 204 that length is 30mm is placed in sample bottle 201.By increasing sample bottle pallet 203 and permanent magnet fixed column The quantity of the permanent magnet 206 on 205, can increase sample bottle 201 capacity of whole support.It is applied to three layers of sample bottle pallet 203 permanent magnet fixed column 205 structural representation is as shown in Figure 8.The distance between permanent magnet fixing hole 230 is 110mm.Brushless Motor 211 drives permanent magnet fixed column 205 to rotate, and permanent magnet 206 rotates the rotating excitation field producing and then drives magnetic stir bar 204 rotations, finally realize magnetic stir bar 204 agitated liquid culture medium.The rotating speed of controlled motor 211, can control magnetic force to stir Mix the mixing speed of son 204.Motor 222 drives driving gear 218 to rotate, and driving gear 218 drives driven gear 217 to revolve Turn, and then drive big bearing sleeve 208, sample bottle basetray 216, sample bottle pallet 203 to rotate, finally realize sample bottle 201 revolve 22.5 degree around permanent magnet fixed column 205 axis.Permanent magnet fixed column 205 is fixed on the inner ring of little bearing 213, sample Bottle holder disk 203 is fixed on little bearing 213 by sample bottle basetray 216, little bearing sleeve 215, support column fixed plate 212 On outer ring, the rotation of such permanent magnet fixed column 205 and sample bottle pallet 203 is independent mutually, is independent of each other.
As shown in figs9-12, in temperature control equipment, culture box cavity is stainless steel, and inside dimensions are (long × wide × high) it is 370mm × 370mm × 420mm, top, bottom and three, Qianmen outer surface install the heating that power is 200W respectively additional Resistance wire, left side, right side and rear side three bread wrap up in copper pipe, and make copper pipe water inlet and outlet be located at rear side.Water inlet and going out The mouth of a river is connected with water inlet with the outlet of cooling water circulation machine respectively.Take the photograph in room temperature+5-70 when microorganism culturing is temperature required During family name's degree scope, temperature control is carried out to casing by the resistive heater in rustless steel inner chamber three face, when temperature required less than room temperature+5 Degree Celsius when, using cooling water circulation machine for casing provide coolant control spin manifold temperature.
As shown in Figure 13 and 3, in sample bottle and its inside and outside gas exchange device, sample bottle 201 body is cylinder, outward Footpath 35mm, internal diameter 32mm, high 85mm, volume is about 50mL, carries screw socket, and material is quartz glass.Sample bottle 201 install additional resistance to Autoclaved plastic sample bottle cap 403, the circular hole of diameter 1cm is arranged at sample bottle cap 403 top, from upper inside sample bottle cap 403 To lower pad hydrophobic permeable membrane 401 and silica gel pad with holes 402 respectively.Hydrophobic permeable membrane 401 aperture is less than 0.45 micron, oxygen The gases such as gas, carbon dioxide can freely pass through, and vapor and microorganism cannot easily penetrate.Silica gel pad 402 with holes can basis The size to choose aperture for the gas transmission rate needing.
As shown in figure 14, probe bracket 305 is fixed on rustless steel intracavity bottom and top near rustless steel luminal border position Put, and make the vertical central axis line right through sample bottle 201 for the transmitted light path of Concentration Testing probe 1, sample bottle pallet 203 rotates When, the sample bottle 201 on sample bottle pallet 203 can just pass through between probe left arm 103 and probe right arm 107.
As shown in figure 15, in high-flux microorganism growth analyses system integrated connection mode, instrument host 504 is connected and passes through RS232 connection 505 is connected with computer 506.Instrument host 504 passes through water inlet pipe 57 and outlet pipe 58 and cooling water circulation machine 56 Connect, and both are connected by data wire 62, with RS485 communication protocol communication.
Embodiment one：The method carrying out microorganism high-pass culture and concentration real-time detection using any of the above-described device
Step includes：
(1) determine the temperature of incubator, i.e. the growth temperature of microorganism, so that it is determined that still making using cooling water circulation machine Use electric boiling plate temperature control.The temperature of setting incubator, sample message, incubation time, Concentration Testing on the software of computer in advance The parameters such as interval time, the rotating speed of brushless electric machine.After parameter setting is good, incubator begins to warm up or cools down, default to reach Temperature.
(2) fluid medium, sterilized magnetic stir bar and micro- life to be cultivated are added in sterilized sample bottle Thing bacterial strain, covers the sample lid equipped with ventilated membrane and silica gel pad, and each sample is numbered.
(3) after incubator temperature reaches preset temperature, sample bottle is put into successively in sample bottle fixing hole, in sample bottle Magnetic stir bar start rotate, drive liquid be in vortex flow.Motor drives sample tray automatically to rotate, and reaches starting point Position, sample bottle is located at Concentration Testing probe center.Then detection function is started on the software of computer.
(4) in whole incubation, brushless electric machine continuous firing, microorganism growth and breeding in sample bottle, work as culture After continuing an assay intervals time, the concentration of sample in Concentration Testing probe measurement sample bottle, measurement data is shown in real time In computer software.Measure a sample after one second, motor drives whole sample tray to rotate 22.5 degree of angles, next Sample bottle is popped one's head in just into Concentration Testing, and between two parties.After one second, in Concentration Testing probe measurement sample bottle, sample is dense Degree, measurement data is shown in computer software in real time.After having measured one second, motor works on, and drives whole sample Pallet rotates 22.5 degree of angles.After such 16 detections, motor and Concentration Testing probe quit work, until next detection Cycle starts.After such 16 detections, each sample creates a data, next detection cycle, and each sample produces again A raw data, constantly repeats like this, after incubation time cut-off, system stalls, and the data that each sample produces The growth curve of this sample corresponding time can be formed.These individual datas or whole curve can carry out post analysis.
Embodiment two：The impact to measurement range for the different vertical distance of light emitting diode and photodiode
According to the method for embodiment one, for the saccharomyces cerevisiae (Saccharomyces of culture 2-96 hour Cerevisiae), when light emitting diode is different below the parallel installation of same vertical, difference real-time testing with photodiode The impact to measurement range for the central point distance, the measured value under all 850nm in wavelength leads to what the spectrophotography of comparison recorded OD600Method is corrected, and is accordingly converted into OD600Value scope.
As can be seen here, when central point distance is for 6mm, larger model can be reached using back scattering photo measure bacteria suspension concentration Enclose, in conjunction with transmission light measurements method (OD600Value scope 0-0.8), can be in 0-15OD600In the range of obtain accurate measurement result. Increase with distance, the range of linearity of measurement can drastically reduce, and is not suitable for the Concentration Testing of interior bacteria suspension on a large scale.

Claims (19)

1. a kind of detection probe (1) for microorganism big concentration range real-time detection, including：Infrarede emitting diode (104), First photodiode (105), the second photodiode (108), U-bracket (102), wherein,
Probe one side is respectively perpendicular and is provided with light emitting diode (104) and the first photodiode (105), light emitting diode (104) corresponding side surface is provided with the second photodiode (108), and the two is centrally located in same horizontal line, thus light-emitting diodes Pipe (104) and the second photodiode (108) define transmitted light detection light path, and light emitting diode (104) and the first photoelectricity Diode (105) is the formation of back scattering detection light path；
Probe upper end is provided with for fixing U-bracket (102), and this support includes the first probe arm (103) and the second probe arm (107), light emitting diode (104) and the first photodiode (105) are fixed respectively, and the second photodiode (108)；
Vertically downward, opening inner side spacing can hold the sample bottle of certain diameter to the opening of described U-bracket (102) just (201) pass perpendicularly through from centre, and make described sample bottle (201) stop at U-bracket (102) interior when, infrarede emitting diode (104) and its just to photodiode (right)（ 108）Axis line just overlap with the diameter of sample bottle (201)；
Described light emitting diode (104) is infrarede emitting diode, and peak wavelength scope is in 800-1400nm.
2. the detection probe described in claim 1, wherein light emitting diode (104) and the first photodiode (105) vertical Central point distance on direction is 6-10mm, and described peak wavelength scope is 800-1000nm.
3. the detection probe described in claim 1 or 2, wherein light emitting diode (104) and the first photodiode (105) hang down Nogata central point distance upwards is 6,7,8 or 9mm, described peak wavelength scope is 850-950nm, and probe also includes using In the fixed handle (101) that U-bracket (102) is fixed to Concentration Testing probe bracket (305), and in described light emitting diode (104), the transmitting of the first photodiode (105) and the second photodiode (108) or receiving terminal are designed with windowpane (106).
4. the detection probe described in claim 3, wherein said Concentration Testing probe bracket (305) can fix one or many Individual Concentration Testing is popped one's head in (1), and described central point distance is 6 or 7mm.
5. the automation equipment of a kind of microorganism high-pass culture and big concentration range real-time detection, including：
(1) high flux liquid stirring device, including sample bottle pallet (203), magnetic stir bar (204), is used for carrying sample bottle Permanent magnet fixed column (205), sample bottle basetray (216), wherein ring junction is set sample bottle basetray (216) is upper The sample bottle pallet (203) of structure, this pallet outward flange arranges the sample bottle fixing hole (226) of annular array, and tray center end covers It is embedded in setting fixed column (205), when being positioned over magnetic stir bar (204) in sample bottle, in permanent magnet fixed column (205) Under effect, magnetic stir bar (204) constant agitation in sample bottle (201), therefore realized many by multiple magnetic stir bars High flux stirring in individual sample bottle；
(2) sample bottle tray transmission, including motor (211), motor fixing plate (220), wherein motor passes through motor shaft (207) be connected with permanent magnet fixed column (205), and motor and permanent magnet fixed column (205), sample bottle basetray (216), Sample bottle pallet (203), the center of circle setting of support column fixed plate (212) are located on same vertical line, thus Motor drive permanent magnetism Body fixed column (205) rotates, magnetic stir bar (204) rotation of the rotating excitation field of generation and then drive sample bottle (201) bottom, Finally realize magnetic stir bar (204) agitated liquid culture medium；
(3) probe bracket is passed through in the detection probe described in any one of Claims 1-4 (1), wherein Concentration Testing probe (1) (305) it is fixed on rustless steel intracavity bottom and top from rustless steel luminal border position, and make the transmission of Concentration Testing probe (1) Light path right through the vertical central axis line of sample bottle (201), during sample bottle pallet (203) rotation, on sample bottle pallet (203) Sample bottle (201) can just pass through between probe left arm (103) and probe right arm (107).
6. the automation equipment described in claim 5, wherein said sample bottle pallet may be configured as 1-10 layer freely as needed The agitating device of assembly and disassembly modular loop configuration, and every layer of sample bottle pallet, can be arranged as required to 1 or 2-48 Individual sample bottle fixing hole.
7. the automation equipment described in claim 5 or 6, wherein said agitating device also includes sample bottle tray support post (214), support column fixed plate (212), can be respectively provided with a basetray every layer of annular sample bottle tray bottom, or borrow In the presence of helping sample bottle tray support post and support column fixed plate, only one basetray is set in fixing column bottom.
8. the automation equipment described in claim 7, wherein passes through permanent magnet (206) is arranged in fixed column (205), makes solid Fixed column becomes permanent magnet, and makes its magnetic field N and S extreme direction be in horizontal distribution.
9. the automation equipment described in claim 8, wherein also sets up motor (222), big on motor fixing plate (220) Bearing (221), driving gear (218), driven gear (217), motor (222) is arranged on motor fixing plate vertically upward (220) below, and it is connected by step motor shaft (219) is vertical upwards with driving gear (218)；Driving gear passes through level side To driven gear (217) be connected with sample bottle basetray (216).
10. the automation equipment described in claim 9, wherein when motor (222) drives driving gear (218) rotation, and Order about driven gear (217) rotation, and then drive sample bottle basetray (216) and sample bottle pallet (203) rotation, finally Realize sample bottle (201) to rotate at a certain angle around permanent magnet fixed column (205) axis, this rotation angle is two neighboring samples Angle between fixing hole (227).
Automation equipment described in 11. claim 10, arranges the big bearing of annular wherein above motor fixing plate (220) and fixes Seat (209), motor shaft (207) passes through from the center of circle of big bearing fixed seat (209), big in the upper setting of big bearing fixed seat (209) Bearing (221), big bearing (221) inner ring is nested in the outward flange of big bearing fixed seat (209)；Big bearing sleeve (208) is nested Outer ring in big bearing (221)；Driven gear (217) is arranged on above big bearing sleeve (208), driving gear (218) and from Moving gear (217) is mutually twisted；Sample bottle basetray 216) it is arranged on above big bearing sleeve (208)；In little bearing (213) Circle is nested in permanent magnet fixed column top (229)；Little bearing sleeve (215) is arranged on above sample bottle basetray (216), top It is nested in the outer ring of little bearing (213) inside end；Sample bottle tray support post (214) is arranged on two sample bottle pallets (203) Between；Support column fixed plate (212) is arranged on little bearing sleeve (215) and the top of sample bottle tray support post (214).
Automation equipment described in 12. claim 11, wherein permanent magnet fixed column (205) are fixed on the interior of little bearing (213) On circle, sample bottle pallet (203) passes through sample bottle basetray (216), little bearing sleeve (215), support column fixed plate (212) It is fixed on little bearing (213) outer ring, the rotation of such permanent magnet fixed column (205) and sample bottle pallet (203) is only mutually Vertical, it is independent of each other.
Automation equipment described in 13. claim 11 or 12, wherein also includes gas exchange device inside and outside sample bottle (4), bag Include sample bottle cap (403) with holes, hydrophobic permeable membrane (401), silicagel pad with holes (402), in this sample bottle cap with holes, (403) are from inner Install diameter and sample bottle cap internal diameter identical circle hydrophobic permeable membrane to outer respectively additional（401) and silicagel pad with holes (402).
Automation equipment described in 14. claim 13, in gas exchange device (4) wherein inside and outside sample bottle, hydrophobic, air-permeability Film (401) aperture is less than 0.45 micron, and the gas such as oxygen, carbon dioxide can freely pass through, and vapor and microorganism are not allowed Easily pass through, and silica gel pad (402) with holes can the gas transmission rate as needed size to choose aperture.
A kind of 15. microorganism high-pass cultures and the automatic temperature-controlled device of big concentration range real-time detection, including：
(1) automation equipment described in any one of claim 5-14；
(2) temperature control equipment (3), wherein,
Described temperature control equipment (3) includes the rustless steel inner chamber for cincture or sealing above-mentioned agitating device and actuating device etc. (302), electric boiling plate (301), copper pipe (303), heat-insulation layer (304), cooling water circulation machine (501)；Wherein electric boiling plate (301) It is attached to inside upper and lower outer surface and Qianmen (311) of rustless steel inner chamber (302)；Copper pipe (303) parcel rustless steel inner chamber (302) Left and right and rear side outer surface；In the outside of electric boiling plate (301) and copper pipe (303) parcel heat-insulation layer (304)；Copper pipe enters water Mouth (309) and outlet (310) lead to outside heat-insulation layer (304), and cooling water circulation machine (501) connects；
When needing temperature control in+5-70 degree Celsius of room temperature, using upper and lower outer surface and the Qianmen of rustless steel inner chamber (302) (311) electric boiling plate (301) heating inside；When needing temperature control in+5 degrees Celsius of 10- room temperature, using cooling water circulation Machine (501) is passed through coolant to keep rustless steel inner chamber (302) low temperature in copper pipe (303).
Automatic temperature-controlled device described in 16. claim 15, wherein Concentration Testing probe (1), high flux liquid agitation and sample Product bottle tray transmission (2) and temperature control equipment (3) are controlled by automated software, to determine that cultivation temperature, motor turn Speed, probe assay intervals time, displays temperature correspond to the change curve of time, mark sample, reduced concentration and time graph, set Put and point out sample concentration value, select detection pattern and automatic calibration data, and output data testing result.
Automation equipment described in a kind of 17. utilization any one of 5-14, or the automatic temperature-controlled described in claim 15 or 16 The method to carry out microorganism high-pass culture and big concentration range real-time detection for the device, step includes：
(1) temperature control：Temperature control equipment (3) makes the temperature of rustless steel inner chamber (302) maintain microorganism culturing in advance to be needed The level wanted, then testing sample, aseptic liquid nutrient medium and sterilized magnetic stirrer (204) is added in sample bottle (201), Cover gas exchange device inside and outside sample bottle (4), be placed in sample bottle fixing hole (227)；
(2) stir culture：Permanent magnet fixed column (205) rotation is driven by motor (211), the rotating excitation field of generation drives sample Magnetic stir bar (204) rotation in bottle (201), thus promote growth and breeding in the stirring of microorganism liquid medium within；
(3) Concentration Testing：Motor (222) drives sample bottle pallet (203) to rotate to an angle, and Concentration Testing pops one's head in (1) Detect first group of sample concentration；After detection finishes, motor (222) drives sample bottle pallet (203) to rotate to an angle, the Two groups of samples enter Concentration Testing probe (1) and start to detect；After detection finishes, motor (222) drives sample bottle pallet (203) continue to rotate to an angle, the 3rd group of sample enters Concentration Testing probe (1) and start to detect；So Concentration Testing is visited Head (1) and motor (222) alternation, to the last one group of sample detection finish, motor quits work；
(4) duplicate detection：With certain time for interval, continuous repeat step (3) successively, such motor (211), motor (222) and Concentration Testing probe (1) three's collaborative work, real-time detection concentration change in microbial cultivation process, until real Test end.
The method of 18. claim 17, wherein said attemperating unit (4) to control by the way of three faces cool down and three faces are heated The temperature of rustless steel inner chamber (302)；At (311) three, the upper and lower and Qianmen of rustless steel inner chamber (302), outer surface adopts electrical heating Plate (301) is controlling the target temperature of more than+5 degrees Celsius of room temperature, left and right and rear three bread is wrapped up in rustless steel inner chamber (302) Copper pipe (303), copper pipe (303) connects cooling water circulation machine (501), and cooling water circulation machine (501) is by the coolant of uniform temperature Pump enters copper pipe (303) so that rustless steel inner chamber (302) temperature can be less than+5 degrees Celsius of room temperature.
The method of 19. claim 17 or 18, wherein the detectable concentration scope of testing sample cultivating microorganism are 0-15OD600, and And shuttle is the cylindrical cuvette of diameter 25-35-50mm, described test tube bottle wall is 0.5,1,1.5,2 or 3mm.
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Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106290162B (en) * 2016-10-10 2019-09-06 承启医学（深圳）科技有限公司 A kind of device and method of continuous detection bacterium growth curve
CN106872538A (en) * 2017-03-28 2017-06-20 海南大学 A kind of high-flux electric chemical analysis system based on smart mobile phone
US10435662B1 (en) * 2018-03-29 2019-10-08 Inscripta, Inc. Automated control of cell growth rates for induction and transformation
US20210322912A1 (en) * 2018-08-29 2021-10-21 Rapid Micro Biosystems, Inc. Use of clean and dry gas for particle removal and assembly therefor
CN109609376B (en) * 2018-12-11 2022-03-01 新绛县威顿建材外加剂有限公司 Intelligent integrated magnetic stirring device
CN109554292A (en) * 2019-01-28 2019-04-02 长沙爱扬医药科技有限公司 Manifold Light Way device and microorganism high-pass culture and detection device
CN112697654B (en) * 2020-12-11 2023-08-25 南京工业大学 OD detector sensor
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CN112725237A (en) * 2021-01-27 2021-04-30 川北医学院附属医院 Intestinal bacteria culture observation method
CN113186082A (en) * 2021-04-01 2021-07-30 周文龙 Microorganism detection system

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102519898A (en) * 2011-12-29 2012-06-27 上海智城分析仪器制造有限公司 Device utilizing single light source to detect fermentation liquid of shake table
CN103234921A (en) * 2013-03-27 2013-08-07 中国科学院安徽光学精密机械研究所 Rapid online detection apparatus and detection method for water body bacterial microorganisms
CN203838050U (en) * 2014-05-04 2014-09-17 天津市基理科技有限公司 Culture solution detection device
CN104877900A (en) * 2015-05-26 2015-09-02 大连理工大学 High-flux quick-detection microfluidic chip directed towards pathogenic microorganism and preparation method for microfluidic chip
CN105044038A (en) * 2015-08-31 2015-11-11 广州中国科学院先进技术研究所 Non-invasive biomass online detection device used for shake-flask culture

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102519898A (en) * 2011-12-29 2012-06-27 上海智城分析仪器制造有限公司 Device utilizing single light source to detect fermentation liquid of shake table
CN103234921A (en) * 2013-03-27 2013-08-07 中国科学院安徽光学精密机械研究所 Rapid online detection apparatus and detection method for water body bacterial microorganisms
CN203838050U (en) * 2014-05-04 2014-09-17 天津市基理科技有限公司 Culture solution detection device
CN104877900A (en) * 2015-05-26 2015-09-02 大连理工大学 High-flux quick-detection microfluidic chip directed towards pathogenic microorganism and preparation method for microfluidic chip
CN105044038A (en) * 2015-08-31 2015-11-11 广州中国科学院先进技术研究所 Non-invasive biomass online detection device used for shake-flask culture

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