CN101218507A - System for rapid analysis of microbiological materials in liquid samples - Google Patents
System for rapid analysis of microbiological materials in liquid samples Download PDFInfo
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- CN101218507A CN101218507A CNA2005800510056A CN200580051005A CN101218507A CN 101218507 A CN101218507 A CN 101218507A CN A2005800510056 A CNA2005800510056 A CN A2005800510056A CN 200580051005 A CN200580051005 A CN 200580051005A CN 101218507 A CN101218507 A CN 101218507A
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- 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
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
- G01N21/532—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke with measurement of scattering and transmission
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
A system for the rapid analysis of microbiological parameters includes a specimen container for containing a liquid sample, a housing having an enclosable chamber shaped for receiving the specimen container, an incubating system mounted within the housing for incubating microbiological materials within the liquid sample, and a spectrophotometer system mounted within the housing for measuring light absorbed, emitted or scattered by the liquid samples as the microbiological materials are incubated by the incubating system over time. The specimen container is filled with a liquid sample to be tested and mixed with a reagent that provides a detectable parameter, and placed inside the apparatus. The incubation system heats and maintains the temperature of the liquid sample within a preset range while the spectrophotometer system propagates light within the specimen container, and monitors and records changes in the light as the light propagates through the container. A continuous non-intrusive monitoring and recording of the test parameter is achieved as the incubation progresses. Any significant deviation of the signal output is an indication of presence of the detectable parameter, while the time taken to reach the significant deviation provides quantification of the microbiological parameter under investigation.
Description
Technical field
The present invention relates to be used for to survey the existence of microbiological materials of liquid sample and the method and apparatus of counting (enumeration), in particular to the method and apparatus of the pathogenic bacteria that is used for the quantitative test water sample.
Background technology
Potable water and recreational water (water at sandy beach place and other swimming facility places) should be regularly tested, and this test result should obtain in short time period, so that the protection public is away from harmful and infectious disease.
At present, most water sample tests are to carry out under away from the laboratory environment in drainage facility or place.Be used for a lot of methods and program exploitation before surpassing 100 years of conventional microbiological analysis at present.Operation and data aggregation this aspect two, they all are labor capacity programs big and consuming time.The operator of these facilities generally can not obtain the result of such test in about 36 to 72 hours.Therefore, after contaminated water had been consumed or has used for a long time, the operating personnel of drainage facility just can take action and proofread and correct this contaminated water usually.
In addition, aquatic transmission of disease still is a serious problem, although this problem is all being attempted to control in the whole world.This problem is not only limited to developing country and less developed country, but is global in essence.Its some main reasons are:
(1) present test frequency also is not enough to provide early warning to make can to take corrective action to stop illness outbreak; And
(2) present method of testing effort and consuming time, thereby hindered regular test;
1988, people such as Edberg S.C have developed a kind of new technology based on chemically defined substrate MTF method (chemically defined subs trate MTF method), be called that " (Autoanalysis Colilert; AC): " this makes ' National filed evaluation of a defined substrate methodfor the simultaneous evaluation of total coliforms andEscherichia coli from drinking water:comparison with standardmultiple tube technique ', Appl.Environ.Microbiol.54 1595 (1988). " and can be in less than 24 hours time to survey and to discern total coli group and Escherichia coli (E.coli) in the water simultaneously with the detectability of the every 100ml of 1CFU coliform bacteria film by fluorimetry on-the-spot fast quantification detection method.Colilert , a kind of colour developing-fluorogenic reagent medium is for specific nutrient and zymolyte are provided for surveying simultaneously total coli group and colibacillary chromophore and fluorophore.In 1989, U.S. EPA was ratified the mode that this method is the total coli group in a kind of qualitative test potable water.
Colour developing/fluorescent reagent has been opened and has been led to better and the gate of testing scheme faster carrying out development aspect the microorganism testing.In addition, these products provide the technology of utilizing such as optical spectroscopy to carry out biology, microorganism and chemico-analytic additional possibility.Spectrophotometric analysis is very sensitive, therefore can detect the low-down color of being concerned about of the concentration that exists in the liquid sample and produce composition (parts per million (ppm)).Visually, human eyes only just can detect color when mentioned component exists with very high concentration, are in 18 to 72 hours these scopes to the requirement of incubation time section therefore.When cultivating when combining, can significantly reduce identification or estimate required time that exists of microorganism indicator in water, food and the environmental samples with photometric analysis.
Use and advantage that spectrophotometric is applied in the microbiological analysis aspect of carrying out in the liquid sample have been quoted from the document.Yet, these tests be by with different time at interval with the part sample from cultivating vessel and extract the photometer test tube and using the standard light spectrometer to measure, finish in the culture chamber outdoor.This is not only consuming time, but also needs separative incubator, spectrometer and need the technician to test, and is also needing the robot sampling system in some cases.If when analyzing, do not give suitable attention, also have the potential risk that produces cross pollution and personal error.
Therefore, be badly in need of improved method and the device that is used for testing the microbiological materials of potable water, recreational water and waste water, so that better management and protection public health and the environment to drainage facility to be provided.
Summary of the invention
The present invention relates to a kind of system that is used for the bacterium of the fluid sample of fast quantitative analysis such as water.An aspect of of the present present invention is an a kind of sampling receptacle and system with device of spectrophotometer system that is used to hold test sample book that comprises, this spectrophotometer system comprises a suitable light emitting source and a detector close with the sampling receptacle of the shell that is positioned at described device.Add a kind of reagent that detectable parameter (as color, fluorescence etc.) is provided in the test sample book in sampling receptacle.When sample when being cultivated, detector monitors is from the light that passes sample and sampling receptacle in described source.This detector is connected to the spectrophotometer processor of measurement, processing, record and canned data.This processor can also be connected to such as computing machine, multimeter or any other and can measure and write down from suitable measurement and recording unit the equipment of the output signal of detector.This provides the non-intrusion type Continuous Cultivation and the signal growth (growth) of examination parameter has been measured.
Another aspect of the present invention is a kind of system that is used for the microbiological parameters of express-analysis liquid sample.This system comprises: a sampling receptacle that is used for the receiving fluids sample, this sampling receptacle are by the made that allows light to propagate; But one limits a shell that is used to keep the closed chamber of sampling receptacle; Installation culture systems in the enclosure is used for the microbiological materials of nutrient solution sample body; And installation spectrophotometer system in the enclosure, be used in sampling receptacle propagates light and when cultivating microbiological materials, measure light by this liquid sample absorption, emission or scattering along with the past culture systems of time.
Of the present invention on the one hand is a kind of device that is used for surveying the microbiological materials of liquid sample again.This device comprises: the shell with closed chamber, this chamber are shaped as and are suitable for transparent (clear) plastic containers of keeping one to be used for the receiving fluids sample; Installation culture apparatus in the enclosure is used for any microbiological materials of nutrient solution sample body; And installation spectrophotometric counter device in the enclosure, be used for when cultivating microbiological materials, measuring light by this liquid sample absorption, emission or scattering along with the past culture apparatus of time.
The invention still further relates to a kind of method that is used for the microbiological materials of express-analysis liquid sample, this method may further comprise the steps:
(a) will have the liquid sample and a reagent mix of the unknown microbiological materials of initial number (population) in sampling receptacle, thereby produce a sample/reagent mixture, this reagent provides the detectable parameter of indicator microoraganism material;
(b) sampling receptacle is put into a closed shell and seal this shell;
(c) in a preselected temperature scope, sample/reagent mixture is being cultivated a period of time in the shell of sealing under the temperature; And
(d) when in this section culture sample in the time/reagent mixture, the variation of measuring detectable parameter.
The invention still further relates to a kind of method that is used for the microbiological materials of fast quantitative analysis liquid sample.This method may further comprise the steps:
(a) liquid sample that will have the microbiological materials of initial number the unknown is put into a sampling receptacle, and this sampling receptacle is made by the material that allows light to propagate;
(b) produce a sample/reagent mixture by liquid sample and a reagent are mixed mutually, this reagent provides the detectable parameter of an indicator microoraganism material once exposing;
(c) in a preselected temperature scope, sample/reagent mixture is being cultivated a period of time in the shell of a sealing under the temperature;
(d) light by in sample/reagent mixture, propagating a known strength and the time-dependent variation in intensity of measuring light, the variation of when culture sample/reagent mixture, measuring detectable parameter;
(e) change records with light intensity is the function of time;
(f) the remarkable inconsistent time takes place in record, in this time detectable parameter generation index variation;
(g) relevant by the known time that makes the detectable parameter generation exponential growth that the known microbiological materials of remarkable inconsistent time and initial number takes place, determine initial number.
Description of drawings
Referring now to accompanying drawing, only the present invention is described in the mode of example, wherein:
Fig. 1 is the synoptic diagram of system of the present invention;
Fig. 2 is the skeleton view according to a device of a preferred embodiment manufacturing of theme invention;
Fig. 3 is the decomposition diagram of subject apparatus, shows from the lid of base unit disengaging and the sampling receptacle of removing;
Fig. 4 is the sectional view of subject apparatus along the line 4-4 among Fig. 2;
Fig. 5 is the sectional view of subject apparatus along the line 5-5 among Fig. 4;
Fig. 6 is the sectional top view of subject apparatus along the line 6-6 among Fig. 4;
Fig. 7 is the cross sectional elevation of a device making according to an alternative embodiment of the present invention;
Fig. 8 is the process flow diagram of the method for diagram theme invention;
Fig. 9 is diagram one a typical growth time curve map;
Figure 10 is the data plot that shows about the exemplary growth curve of two kinds of different microbiological parameters;
Figure 11 is the tables of data that the result who is generated by method of the present invention is shown;
Figure 12 is an exemplary linear correlation curve used in the method for subject apparatus;
Figure 13 is the test report that is generated by subject methods;
Figure 14 a is the process flow diagram of diagram heating control algolithm of the present invention; And
Figure 14 b is the process flow diagram of diagram temperature control of the present invention and data collection algorithm;
Embodiment
With reference to figure 1, wherein illustrate a kind of that make according to theme invention, utilize the system of cultivating and surveying the express-analysis microbiological parameters in the non-intrusion type vessel.This system 10 comprises an incubator-detector assembly 12, a sampling receptacle 14 and an external data recorder 80 that is used to hold the liquid sample 11 that mixes with reagent 20.Incubator-detector assembly 12 comprises: one has the shell 15 of surveying chamber 65, and this detection chamber is shaped to and is suitable for receiving sampling receptacle 14; A culture systems 60 that is installed in the microbiological materials that is used for nutrient solution sample body 11 in the shell 15; And spectrophotometer system 62 that is installed in the shell 15.This spectrophotometer system 62 is measured by the amount of the light of 11 absorptions of the liquid sample in the sampling receptacle 14, emission or scattering when culture systems 60 is cultivated microbiological materials.
Culture systems 60 comprises heating controller 92, and spectrophotometer system 62 comprises spectrophotometer controller 94.Power supply 90 is to culture systems 60 and spectrophotometer system 62 power supplies.Preferably, external data recorder 80 comprises a computing machine 85 and the output device such as printer 82 that is connected to computing machine 85 with microprocessor 86 and memory device 88.
With reference now to Fig. 2-6,, wherein illustrates a preferred embodiment of incubator-detector assembly 12.The shell 15 of device 12 is the columned shells of cardinal principle, and this shell comprises a base 16, is shaped as the 18 and lids that can remove 50 of container holder (holder) that keep sampling receptacle 14.Base 16 comprises that one is shaped as to receive and covers 50 upwardly extending cylindrical lip 55.Base 16 accommodates power supply 90, heating controller 92 and spectrophotometer controller 94.Power supply 90 can be any suitable power supply known in the art, and such as a rechargeable battery that is positioned at base 16, this rechargeable battery has the supply socket 99 that is used to be connected to outside 120 or 220 volts of AC (interchange) power supplys or DC (direct current) power supply.Be installed on the outside of base 16 is power switch 13, state LED (light emitting diode) 97 and FPDP 98.
The lid 50 that can remove is shaped as around the wall 19 of container holder 18 and fits tightly.Preferably, lid 50 comprises the double-walled cylindrical shell of a high thermal efficiency, and this shell has the top 52 and the open-ended bottom flange 53 of outer wall 51, inwall 59, sealing.The outside surface of bottom flange 53 is provided with sealing strip (bead) 66, and the sealing bar is shaped as in the groove 67 on the inside surface on the limit 55 that is encased in base 16.The inside surface of inwall 59 comprises being shaped as and engages outstanding 68 of ring 49 that the base 21 around container holder 18 extends hermetically.Alternatively, lid 50 can be equipped with vacuum or inert gas between wall 51 and 59.
When lid 50 was placed on the container holder 18, lid 50 and container holder 18 limited a very effective adiabatic cultivation-detection chamber 65.Preferably, make the inside surface blackening of the wall 19 of sample fixer 18, so that chamber 65 becomes an effective black box (darkroom) that is used for photodetection and measurement.
The culture systems 60 of device 12 comprises heating element 24, temperature sensor 25 and cultivates controller 92.Heating element 24 is installed in the heating finger piece 57, and this heating finger piece extends up through a hole in the base 21 of container holder 18.This temperature sensor 25 is installed in the base 21 upwardly extending temperature finger 56 of container holder 18.This temperature sensor 25 can comprise that is placed near the thermal resistor 26 in finger piece 56 tops.
Spectrophotometer system 62 is to measure by the system of the light of liquid sample absorption, emission or scattering when cultivating microbiological materials along with the past of time a kind of being used for.As best image in Fig. 4, spectrophotometer system 62 preferably includes three spectrophotometers: one comprises first spectrophotometer of light emitting source 30a and detector 35a, second spectrophotometer that comprises light emitting source 30b and detector 35b and the 3rd spectrophotometer that comprises light emitting source 30c and detector 35c.Light emitting source 30a, b, c propagate the light beam of given intensity along selected light path in sampling receptacle 14.Photo-detector 35a, b, c are placed with the variation of surveying the beam intensity in the selected visual field relevant with light path, and this variation is to be caused by liquid sample 11 absorptions, emission or scattering in the culture systems cultivation microbiological materials time in past along with the time.
Preferably, each light source 30a, b, c comprise the light emitting diode (LED) of a specific wavelength maximum, and preferably, each detector 35a, b, c comprise a photo transistor detector.Light source 30a, b, c and detector 35a, b, c are installed on the printed circuit board (PCB) 33a, the b that are electrically connected to spectrophotometer controller 94, the c.
The operation of spectrophotometer controller 94 control spectrophotometer system.Spectrophotometer controller 94 is enabled and is stopped using, and can drive signal emitting-source 30a, b, c pulsedly.The output signal that is generated by detector 35a, 35b and 35c is also measured and handled to spectrophotometer controller 94.Spectrophotometer controller 94 comprises the microprocessor 95 with an onboard clock, and this onboard clock plays data recorder and stores the detector signal value surveyed in the microprocessor 95 particular memory location with the relevant temperature of liquid sample 11 and Measuring Time.Spectrophotometer controller 94 can be by enabling one or more state LED 97, or sound signal is sent the end that the equipment (not shown) is indicated test.Spectrophotometer controller 94 is also communicated by letter with external data record equipment 80, multimeter or other external signal executors such as computing machine 85 by FPDP 98.
The microprocessor 95 of spectrophotometer controller 94 comprises storer, and this storer is used to store the software of the method for testing of carrying out the theme invention.This method of testing regulation is implemented the standard of test process and required condition.By the software of customization, this method can be programmed and be downloaded to by FPDP 98 in the storer of microprocessor 95.This software can also erasure controller all memory locations in 94.
In order to utilize device 12 test liquid samples of the present invention, liquid sample 11 is placed in the sample container 14.Liquid sample 11 is not limited to water, and can comprise that other liquid or other contain the fluid nutrient medium of suspension, filter paper and other solids such as food particle.Liquid sample in sampling receptacle 14 11 adds suitable reagent 20 then.Reagent 20 can be chemistry or biological in essence, can provide indication to exist or do not exist the microbiological materials examined or check such as detectable parameter such as color, fluorescence, turbidity.
The detection of color, fluorescence or turbidity signal depends on the time, and detection time is relevant with the number of bacteria that exists when test begins.Thereby, but be detected time, the total coli group in water sample that can obtain to be surveyed and the quantification of the microbiological parameters the Escherichia coli with evaluation quantity by the total coli group and the Escherichia coli of measuring in the caused signal of change color or fluorescence signal and the water sample.
The spectrophotometer system 62 of operative installations 12 is measured the detection of color or the detection of fluorescence signal.In preferred embodiments, spectrophotometer system 62 comprises three spectrophotometers, they provide respectively to the colourity of total coli group survey, to colibacillary fluorescence detection and the growth of microorganism turbidity that obtains by nephelometry.
The onboard clock of the microprocessor 95 of spectrophotometer controller 94 provides growth time, and the steady temperature of culture systems 60 provides growth of microorganism and optics repeatability.
In a preferred embodiment of system 10 of the present invention, spectrophotometer system 62 comprises three " growth time-spectrophotometer (time-of-growth-spectrophotometer) " that are positioned at single steady temperature incubator, and the growth that is about to the prescribed microorganism parameter is recorded as the spectrophotometer of the function of time.The type of the spectrophotometric analysis of being finished by each spectrophotometer depends on the configuration and the technical requirement of this spectrophotometric source and detector.Source-detector provides colourity or nephelometric analysis to the 180 degree configurations of 30c and 35c, and source-detector provides fluorescence or nephelometric analysis to the 90 degree configurations of 30a, 35a and 30b, 35b.
Preferably, the spectrophotometric source 30c of colorimeter comprises that maximum wavelength is the LED of 620nm, and preferably, detector 35c is the photo transistor detector that has signal response in comprising the whole visible-range of 620nm.Preferably, the spectrophotometric source 30a of photofluorometer is that maximum wavelength is the UVLED (ultraviolet LED) of 380nm, and preferably, the detector 35a that is become 90 degree to place with source 30b by strategy ground is the photo transistor detector that has signal response in the visible region of the scope that comprises 400-500nm.Turbidimetric configuration is similar to the configuration of photofluorometer, just source 30b preferably maximum wavelength be the LED of 400nm.
After reagent 20 is added in the sample under aseptic condition, sample lid 42 is fixed on the sample bottle 44, and rocks sampling receptacle 14 lightly, with solubilising reagent, form sample/reagent mixture.For total coli group and the Escherichia coli in the while test water sample, typical reagent not only provides best growth nutrient, but also provide about the change color of total coli group and about colibacillary fluorescence signal, if they are present in the sample with any amount.The example of typical colour developing/fluorescent reagent is:
Merck?KGaA-Readycult
IDEXX-Colilert
Then sampling receptacle 14 is put into container holder 18, as best illustrating among Fig. 2.In case the lid 50 that can remove is placed on the base 16, cultivation-detection chamber 65 just provides growth of microorganism and desirable black box (darkroom) condition of spectrophotometric detection.
Press the start button 13 on the base 16 of lower device 12, enable and cultivate circulation and detection process.Alternatively, can utilize an independent button of enabling, enable detection process with the schedule time after cultivating beginning.
Enable the power supply that detection process can comprise opening signal emissive source 30a, b, c and detector 35a, b, c, pulsedly the signal output of drive signal emission and supervision detector 35a, b, c.
Preferably, the colourity TDGSP of total coli group and colibacillary fluorescence TDGSP are recorded simultaneously with the turbidity TDGSP of the turbidity of the increase that is caused by the bacterial growth in the water.
There is the parameter of being examined or check in the remarkable inconsistent indication of output signal and initial baseline, and significantly inconsistent required time provides indication to the test parameter original vol from beginning to reach this.
The clock of microprocessor 95 provides the date and time that begins to test, the time of each measured signal and the time that temperature, test accordingly finished or stopped.The end of test can be indicated by state LED 97 and/or sound signal, perhaps can control by software program.
With reference now to Fig. 7,, wherein illustrates the synoptic diagram of a device 112 of making according to an alternative embodiment of the present invention.Except changed at several places, device 112 was similar to the device 12 of the preferred embodiment shown in Fig. 2-6 substantially.
Heating element 124 is installed in the open-ended circular cylindrical wall 160 of sample holder 150.As directed, heating element 124 comprises resistance wire 125.Alternatively, heating element 124 can be resistance coil, resistor foil etc.The length of resistance wire is by rated resistance temperature and every foot design ohmic value regulation of tinsel 125.
Sampling receptacle 114 comprises sample lid 142 and sample bottle 144.This sample bottle is columned substantially, has one and is shaped as the bottom cavity 145. of holding temperature controller finger piece 156
Heating controller 192 is kept the constant preset temperature range in the sample.Alternatively, it can comprise a timer (not shown), is used to measure incubation time from the outset, and stops heating when Preset Time finishes.
The spectrophotometer system of device 112 is similar to the spectrophotometer system of the device 12 of preferred embodiment.Light emitting source 130a is along the direction of arrow 1 emission light, and detector 135a surveys and is launched or light that the direction along arrow 2 of scattering is advanced.Light emitting source 130b is along the direction of arrow 4 emission light, and detector 135b surveys and is launched or light that the direction along arrow 5 of scattering is advanced.Light emitting source 130c is along the direction emission light of arrow 3, and detector 135 is surveyed the light of advancing along the direction of arrow 3.This spectrophotometer system also comprises spectrophotometer controller 194, and this spectrophotometer controller is used to control the operation of light source 130a, b, c and detector 35a, b, c and power supply 190.
With reference now to Fig. 8-13,, wherein illustrates a preferred embodiment of quantitative analysis method of the present invention.
Quantitative analysis method of the present invention is based on and has this understanding of contact between initial number and the growth population in time.The time interval between the beginning (initial number) of test and the growth population of fixing is the function of initial number, cultivation temperature and growth medium.Therefore, cultivation temperature and growth medium are kept constant, reaching the fixing required time of growth population is the direct function (direct function) of initial number.
Colour developing/fluorescent reagent such as Readycult (Merck KgaA) or Colilert (IDEXX) provides a kind of like this mechanism, promptly, can come monitoring and measuring growth of microorganism quantity (total coli group and Escherichia coli) by the luminosity detection process in the present invention by means of this mechanism.The certain enzyme (for example galactosidase (total coli group) and glucuronidase (Escherichia coli are distinctive)) that is produced by these organisms will make the metabolism of nutrient indicator and chromophore or fluorophore will be discharged in the fluid nutrient medium.The concentration of surveying chromophore in the nutrient culture media or fluorophore at a given time is proportional with this growth population constantly, so is measuring time-based growth population by the change in signal strength that the concentration increase of color development composition causes.
Quantity detection time (t
Pop) being defined as reaching the time that detectable population size spends, it has been used to estimate bacterial growth.Show that the logarithm of this detection time and inoculum (inoculum) (initial number of microorganism) level is inversely proportional to:
t
pop∝1/log?X
o {1}
X wherein
o=initial bacterial number.
It is that the photometric signal amount of being surveyed that surpasses background signal is the statistically evident time that the remarkable inconsistent time (TSD) takes place.TSD also depends on the initial concentration of the bacterium in the sample, and initial total number of bacteria is high more, and TSD is short more.Because the increase that can use the increase of signal output to measure population size, thus required time of picked up signal output and significantly inconsistent (TSD) of baseline should and t
PopCorrespondence is if measure the words of this time at growth phase.This method is
t
pop＝TSD {2}
Therefore
TSD∝1/log?X
o {3}
At the initial number (X of TSD with the microorganism of being examined or check
o) between this linear correlation curve equation---LCCE, except detect existing, also provide the quantitative information of bacterial number.
Fig. 8 is a process flow diagram of describing the step of subject methods.At square frame 200, in sampling receptacle 14, reagent 20 is mixed with the liquid sample 11 of the microbiological materials with initial number the unknown, thereby produce a sample/reagent mixture.Reagent 20 provides the detectable parameter such as color or fluorescence of indicator microoraganism material.At square frame 202, sampling receptacle 14 put into shell 15 and will cover 50 be placed on the shell 15.At square frame 204, start incubation, under the steady temperature sample/reagent mixture is being cultivated a period of time in the shell of sealing.
At square frame 206, log-on data is collected, the variation of measuring detectable parameter along with the past culture sample of time/reagent mixture the time.Also survey the variation of light intensity by the light of propagating a known strength in the sample/reagent mixture in sampling receptacle 14 in the training period and measure these variations.
In the training period, collect by spectrophotometer controller 94 about the data of the photometric signal of time, temperature and pilot light Strength Changes.Micro organism quantity increase in time is attended by the increase of photometric signal.This produces a real-time microbial growth curve, all curves as shown in Figure 9.At square frame 208, stop test, and with the data storage of collecting in the storer of the microprocessor 95 of spectrophotometer controller 94.
At square frame 210, above-mentioned data are downloaded in the external data recorder 80, preferably download in the computing machine 85 that customized software is installed.At square frame 212, computing machine 80 deal with data and with graphical format and table format display result.Figure 10 illustrates the growth curve of all selected parameters of a typical sample---this curve be real time record or download from installing 12, also illustrate the cultivation temperature sketch plan.The vertical value representation signal intensity (Any Digit) of left-hand side.Right-hand side be with ℃ (degree centigrade) be the temperature of unit.The horizontal scale of bottom is represented hour being the time of unit.Figure 11 illustrates the tables of data of the value of the parameter signal that comprises a typical sample and time and temperature.
At square frame 214, the software of computing machine 85 is automatically based on the photometric signal preset value calculating TSD that surpasses by the background signal value of analyst's definition.At square frame 216, utilize built-in predefine linear correlation curve equation to calculate the initial number of the microbiological parameters of being examined or check (with the expression of the colony forming unit (CFU) in the given sample volume).In order to obtain this linear correlation curve equation, both handle a series of samples with different Initial microorganisms quantity (as Escherichia coli) that separate to use method of the present invention and standard method (membrane filtration).This predefined linear correlation curve equation (LCCE) is by the TSD value that will obtain from the present invention and the corresponding initial population values (X that obtains from membrane filtering method
0) section out generation with coordinate.Figure 12 shows the linear correlation curve of sample.
At square frame 218, initial population values is presented on the computer screen, shown in Figure 13.
Therefore, method of the present invention is provided at incubation when carrying out, to the supervision continuous, non-intrusion type and the record of one or more detectable parameter.There is detectable parameter in the remarkable inconsistent indication of output signal, and the remarkable inconsistent time that spends of reaching provides the quantitative test to parameter.
With reference now to Figure 14 a and 14b,, wherein illustrate heating and the temperature control and the data collection algorithm of controller 92 and 94.Press the program of power switch 13 start-up control devices 92 in command block 300.
In command block 305, controller 92 and 94 implementation quality inspections are to examine cultivation and detection system and parts in operation normally.
In command block 310, if logic is a "Yes", controller 92 and 94 will advance to piece 320.If logic is a "No", these two controllers will start suitable LED to send signal report " cell failure ".
In command block 320, controller 92 is by the sample in heating element 24 heating containers 14 11 and by the temperature of temperature sensor 25 with predetermined time interval monitoring sample 11.
If the logic at command block 330 places is a "No", then controller 92 continues the sample 11 in the heating container 14 and monitors its temperature.
If the logic at command block 330 places is a "Yes", then the temperature of the sample of being surveyed as temperature sensor 25 11 has reached predetermined temperature value.Then, controller 92 will be provided with zero point test duration, and continue the temperature of the sample 11 in the monitoring of containers 14.Controller 92 also begins monitoring period.
If the logic at command block 350 places is a "No", then controller 95 will continue heating sample 11 and monitor its temperature.
If the logic at command block 350 places is a "Yes", then controller 92 enters square frame 355, stops the sample 11 in the heating container 14, and enters command block 365.
In command block 365, controller 92 begins to collect temperature data and time data, and controller 94 begins to collect signal data.Controller 92 is start-up temperature Control Circulation 1 also, to keep cultivation temperature in preset range.If the logic at square frame 400 places in circulation 1 is "Yes", then this controller turns back to square frame 355 and continues this circulation.
If the logic at square frame 400 places is a "No", then this controller will forward command block 410 to, the sample 11 in the beginning heating container 14.Circulation 2 logics that will always proceed at square frame 400 places become "Yes", and turn back to circulation 1.
No matter be that logic loops 1 or 2, two controllers 92 and 94 all will be collected data separately at default time interval place, and with data storage in the storer of processor.
At square frame 370, state LED 97 is updated to indicate ongoing test.
If the logic at command block 375 places is a "No", then controller 92 and 94 will turn back to command block 365, and continue to collect data, thereby log-on data is collected circulation---circulation 3.If the logic at command block 375 places is a "Yes", think then to test and finish that controller 92 and 94 stops to monitor and collecting data, cuts out and cultivate and detection system, in command block 385 (stand-by) pattern that enters the free time, wait for further indication from user or analyst.
With respect to the membrane filtering method of standard, the method and apparatus of this theme invention provides a plurality of advantages.This subject methods and device are for fast but simply, reliably, accurately the microbiological materials in the various types of liquid samples of on-the-spot test is got ready, and these liquid samples comprise potable water and recreational water.Other advantages comprise be subjected to turbidity interference still less because the performance analysis scope needs dilution greatly and not, simplified operation, and built-in quality control (QC) provides automatic QC for each test by full-automatic.
Should be understood that and to carry out various changes to the embodiment of method and apparatus described here.Though the spectrophotometer system of preferred embodiment comprises three spectrophotometers, should be understood that this device can comprise the spectrophotometer of different numbers.Equally, the spatial configuration of source-detector can have significant change, and does not deviate from the present invention.And each spectrophotometer can be configured to be used to survey different test parameters, and can be independently operated or operation simultaneously.
Should also be understood that light emitting source is not limited to LED (can be laser instrument or laser diode as them), and detector is not limited to phototransistor (can be photodiode, photo-resistor, CCD etc. as them).
In addition, method of the present invention is not limited to the detectable parameter in the preferred embodiment, can be used for surveying the light emission that the bioluminescence that caused by the biological or chemical composition in the reagent in the sample container 14 20 or chemiluminescence process are caused as this method.This will make method and apparatus of the present invention can be used to utilize the toxicity research of bioluminescence bacterium.
And it is outside but be positioned at device 10 that any or all spectrophotometric light emitting source and detector can be placed on cultivation-detection chamber 65, and can be used for the supervisory signal growth by be placed on optical fiber in the chamber 65 tactfully.
Therefore, without departing from the invention, can be to describing at this and graphic embodiment of the present invention are carried out various changes, scope of the present invention is limited by the accompanying claims.
Claims (28)
1. system that is used for the microbiological materials of express-analysis liquid sample comprises:
(a) sampling receptacle that is used for the receiving fluids sample, this sampling receptacle are to be made by the material that allows light to propagate;
(b) shell with closed chamber, this chamber is shaped to and is suitable for keeping described sampling receptacle;
(c) installation culture systems in the enclosure is used for any microbiological materials of nutrient solution sample body; And
(d) installation spectrophotometer system in the enclosure is used in sampling receptacle propagates light and measures light by this liquid sample absorption, emission or scattering when cultivating microbiological materials along with the past culture systems of time.
2. according to the system of claim 1, wherein spectrophotometer system comprises at least one spectrophotometer, and this spectrophotometer comprises: a light emitting source, this light emitting source are positioned at propagates light in the sampling receptacle; A detector, this detector are oriented to be used to survey the variation of light light quantity when propagating by microbiological materials and the detector signal that is used to produce this variation of indication; And a spectrophotometer controller, be used to control light emitting source and detector.
3. according to the system of claim 2, wherein the spectrophotometer controller comprises a microprocessor, and this microprocessor is used to handle detector signal and is used to be generated as the record that the light of the function of time changes.
4. according to the system of claim 2, wherein light emitting source is propagated the light with known strength along first light path in sampling receptacle, and detector is surveyed the variation of the light intensity in the visual field relevant with first light path.
5. according to the system of claim 3, wherein the visual field is oriented to become with first light path 180 ° of angles.
6. according to the system of claim 3, wherein the visual field be oriented to first light path at an angle of 90.
7. according to the system of claim 2, wherein light emitting source comprises a light emitting diode, and detector comprises a phototransistor.
8. according to the system of claim 1, wherein spectrophotometer system comprises one first spectrophotometer and one second spectrophotometer, wherein first spectrophotometer comprises that first light emitting source that is used in sampling receptacle propagating first light beam and one are used to survey first detector of the variation of first light beam, and second spectrophotometer comprises that second light emitting source that is used for propagating second light beam in sampling receptacle and one are used to survey second detector of the variation of second light beam.
9. system according to Claim 8, wherein spectrophotometer system comprises one the 3rd spectrophotometer, and the 3rd spectrophotometer comprises that the 3rd light emitting source that is used in sampling receptacle propagating the 3rd light beam and one are used to survey the 3rd detector of the variation of the 3rd light beam.
10. according to the system of claim 2, wherein shell comprises upwardly extending, as to have an an open top end columned container holder of cardinal principle, and a lid of removing that is used to seal this shell, this lid that can remove is shaped as around container holder.
11. according to the system of claim 10, wherein light emitting source and detector are installed in the container holder position of close sampling receptacle when sampling receptacle is placed in the enclosure.
12. according to the system of claim 10, wherein container holder comprises a groove that extends internally, sampling receptacle comprises a side recess, and this side recess is shaped to when sampling receptacle is placed in the container holder and this groove registration.
13. according to the system of claim 10, wherein container holder comprise a pair of over against groove, sampling receptacle comprise a pair of over against recess, this to recess be shaped as with this to the groove registration.
14. according to the system of claim 1, wherein culture systems comprises that a heating element, temperature sensor and response temperature sensor are used to control the heating controller of heating element.
15. according to the system of claim 14, wherein temperature sensor extends up in the chamber, sampling receptacle has a bottom cavity, and this bottom cavity is shaped as holds this temperature sensor.
16. according to the system of claim 14, wherein heating element extends up in the chamber, sampling receptacle has a bottom cavity, and this bottom cavity is shaped as holds this heating element.
17. a device that is used for cultivating and survey the microbiological materials of liquid sample comprises:
(a) shell with closed chamber, this chamber are shaped to and are suitable for the transparent plastic sampling receptacle that keeps one to be used for the receiving fluids sample;
(b) in the enclosure culture apparatus is installed, is used for any microbiological materials of nutrient solution sample body;
(c) in the enclosure spectrophotometric counter device is installed, is used in sampling receptacle propagates light and when cultivating microbiological materials, measures light by this liquid sample absorption, emission or scattering along with the past culture apparatus of time.
18. according to the device of claim 17, wherein the spectrophotometric counter device comprises: a light emitting source is used for propagating the light beam with known strength along first light path in sampling receptacle; And a detector, be used to survey variation along the light intensity of second light path relevant with first light path.
19. according to the device of claim 18, wherein this shell comprises: a base; One is installed in the container holder that is used to keep sampling receptacle on the base, and this container holder has the top of an opening; And the lid that can remove, this lid is shaped as the top of this sample holder of sealing.
20. according to the device of claim 19, wherein culture apparatus comprises that a heating element, temperature sensor and one is coupled to heating and the temperature controller that temperature sensor is used to control heating element.
21. a method that is used for the microbiological materials of express-analysis liquid sample, this method may further comprise the steps:
(a) will have the liquid sample and a reagent mix of the microbiological materials of initial number the unknown in sampling receptacle, thereby produce a sample/reagent mixture, this reagent provides the detectable parameter of indicator microoraganism material;
(b) sampling receptacle is put into a closed shell and seal this shell;
(c) in a preselected temperature scope, sample/reagent mixture is being cultivated a period of time in the shell of sealing under the temperature; And
(d) when in this section culture sample in the time/reagent mixture, the variation of measuring detectable parameter.
22., comprise that also the change records with detectable parameter is the step of the function of time according to the method for claim 21.
23. according to the method for claim 21, the step of wherein measuring the variation of detectable parameter is included in interior propagates light of sample/reagent mixture in the sampling receptacle and the variation of surveying light.
24., further comprising the steps of according to the method for claim 22:
(a) the remarkable inconsistent time takes place in record, in this time detectable parameter generation index variation; And
(b) relevant by the known time that makes the known microbiological materials generation exponential growth of remarkable inconsistent time of generation and initial concentration, determine initial number.
25. according to the method for claim 21, wherein the preselected temperature scope is ± 1 ℃.
26. according to the method for claim 21, wherein detectable parameter is selected from color, fluorescence and turbidity.
27. according to the method for claim 21, wherein detectable parameter comprises bioluminescence or the chemiluminescence that is caused by the biological or chemical composition in the reagent.
28. a method that is used for the microbiological materials of fast quantitative analysis liquid sample, this method may further comprise the steps:
(a) liquid sample that will have the microbiological materials of initial number the unknown is put into a sampling receptacle, and this sampling receptacle is made by the material that allows light to propagate;
(b) produce a sample/reagent mixture by liquid sample and a reagent are mixed mutually, this reagent provides the detectable parameter of indicator microoraganism material once exposing;
(c) in a preselected temperature scope, sample/reagent mixture is being cultivated a period of time in the shell of a sealing under the temperature;
(d) by propagates light in the sample/reagent mixture in sampling receptacle and survey the variation of light intensity, the variation of measuring detectable parameter when being culture sample in this section period/reagent mixture;
(e) change records with light intensity is the function of time;
(f) the remarkable inconsistent time takes place in record, in this time light intensity generation index variation;
(g) relevant by the known time that makes the known microbiological materials generation exponential growth of remarkable inconsistent time of generation and initial concentration, determine initial number.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CA2005/000686 WO2006116835A1 (en) | 2005-05-05 | 2005-05-05 | System for rapid analysis of microbiological materials in liquid samples |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101218507A true CN101218507A (en) | 2008-07-09 |
Family
ID=37307544
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| CNA2005800510056A Pending CN101218507A (en) | 2005-05-05 | 2005-05-05 | System for rapid analysis of microbiological materials in liquid samples |
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| Country | Link |
|---|---|
| EP (1) | EP1886136A4 (en) |
| CN (1) | CN101218507A (en) |
| AU (1) | AU2005331515A1 (en) |
| CA (1) | CA2607086C (en) |
| EA (1) | EA012956B1 (en) |
| IL (1) | IL187064A0 (en) |
| MX (1) | MX2007013637A (en) |
| WO (1) | WO2006116835A1 (en) |
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| CN102175661A (en) * | 2011-01-25 | 2011-09-07 | 宇星科技发展(深圳)有限公司 | Online analyzer of Escherichia coli |
| CN101701184B (en) * | 2009-11-12 | 2012-07-11 | 何宗彦 | Microbe rapid detection device and detection method thereof |
| CN101598662B (en) * | 2008-04-03 | 2012-12-19 | 克洛纳光学传感器公司 | Device for measuring the dispersion and/or absorption and/or refraction of a sample |
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-
2005
- 2005-05-05 AU AU2005331515A patent/AU2005331515A1/en not_active Abandoned
- 2005-05-05 CN CNA2005800510056A patent/CN101218507A/en active Pending
- 2005-05-05 WO PCT/CA2005/000686 patent/WO2006116835A1/en active Application Filing
- 2005-05-05 MX MX2007013637A patent/MX2007013637A/en not_active Application Discontinuation
- 2005-05-05 EP EP05741372A patent/EP1886136A4/en not_active Withdrawn
- 2005-05-05 EA EA200702418A patent/EA012956B1/en not_active IP Right Cessation
- 2005-05-05 CA CA2607086A patent/CA2607086C/en not_active Expired - Fee Related
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2007
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101598662B (en) * | 2008-04-03 | 2012-12-19 | 克洛纳光学传感器公司 | Device for measuring the dispersion and/or absorption and/or refraction of a sample |
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| CN113504250A (en) * | 2021-05-26 | 2021-10-15 | 杭州电子科技大学 | Peanut aflatoxin detection device and method based on prism type RGB color extraction |
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2006116835A8 (en) | 2007-12-13 |
| EA200702418A1 (en) | 2008-06-30 |
| EP1886136A1 (en) | 2008-02-13 |
| CA2607086C (en) | 2015-11-17 |
| CA2607086A1 (en) | 2006-11-09 |
| IL187064A0 (en) | 2008-02-09 |
| AU2005331515A1 (en) | 2006-11-09 |
| MX2007013637A (en) | 2008-04-08 |
| WO2006116835A1 (en) | 2006-11-09 |
| EA012956B1 (en) | 2010-02-26 |
| EP1886136A4 (en) | 2010-02-03 |
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