WO2008156462A1 - Dispositif et procédé de détection et de numération de multiples groupes de microorganismes - Google Patents

Dispositif et procédé de détection et de numération de multiples groupes de microorganismes Download PDF

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Publication number
WO2008156462A1
WO2008156462A1 PCT/US2007/015204 US2007015204W WO2008156462A1 WO 2008156462 A1 WO2008156462 A1 WO 2008156462A1 US 2007015204 W US2007015204 W US 2007015204W WO 2008156462 A1 WO2008156462 A1 WO 2008156462A1
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WO
WIPO (PCT)
Prior art keywords
microorganisms
light source
primary
growth
interaction
Prior art date
Application number
PCT/US2007/015204
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English (en)
Inventor
Gideon Eden
Ruth Eden
Original Assignee
Gideon Eden
Ruth Eden
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/766,208 external-priority patent/US7745169B2/en
Application filed by Gideon Eden, Ruth Eden filed Critical Gideon Eden
Publication of WO2008156462A1 publication Critical patent/WO2008156462A1/fr

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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
    • 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

Definitions

  • Boyd U.S. Pat. 5,510,243
  • the new device and method simultaneously detects and enumerates two groups of microorganisms in a test sample, utilizing a single test container.
  • a chromogenic substrate and a fluorogenic substrate are mixed with the test sample.
  • the test container is incubated to allow bacterial growth and metabolism.
  • Spectral changes of the substrates are dynamically detected using two external light sources aimed at a transparent section of the test container, and a single external photo detector.
  • One light source operates in the visible band and the second in the long ultraviolet band.
  • a single light source generating both bands may be employed.
  • the two dynamic time patterns generated by the two substrates are analyzed in real time to determine the presence or absence of each microorganism group and to enumerate their original concentrations in the test sample.
  • FIG. 1 is a diagram of a preferred embodiment of the device
  • FIG. 2 is a diagram of an alternative embodiment of the device
  • FIG. 3 is a chart of a plot of the experimental data for water inoculated with E. coli
  • FIG. 4 is a chart of a plot of the experimental data for water inoculated with Enterobacter aerogenes.
  • FIG. 5 is a chart of a plot of the experimental data for water inoculated with E. coli using the device of FIG. 2.
  • a chromogen (or chromogenic substrate) is a substance (usually colorless) that, when cleaved by a specific enzyme produced by microorganisms, produces a pigment or dye.
  • a chromophore is a group on, or part of, a chromogen that produces a color when the chromogen is cleaved by an enzyme.
  • a fluorogen (or fluorogenic substrate) is a non-fluorescent material that, when cleaved by a specific enzyme produced by microorganisms, produces a fluorescent compound.
  • a fluorophore is a group on, or part of, a fluorogen that is responsible for the fluorescence when a fluorogen is cleaved by an enzyme.
  • Primary group of organisms can refer to a single microbe, a related species of microbes, or a large genus of microbes possessing a common taxonomic characteristic.
  • Secondary group of organisms can refer to a single microbe, a related species of microbes, or a large genus of microbes possessing a common taxonomic characteristic.
  • FIG. 1 illustrates the device for monitoring the growth of two groups of microorganisms in a sample.
  • the device 1 comprises a container 2 which is transparent to light at least in a window section 8.
  • the container can be made of glass or polymer, transparent to visible light and at least a limited band of the ultraviolet spectrum range.
  • polystyrene can be employed which is transparent to the whole visible spectrum and to the long ultraviolet segment residing above 350 nanometers wavelength.
  • the tested sample 6 is introduced to a liquid mixture 5 comprising media capable of growing the target microorganisms and two substrate indicators, each capable of indicating growth of one of the target groups.
  • one substrate indicator is chromogenic and therefore can change its color due to growth of the primary group
  • the other substrate indicator is fluorogenic and therefore can change its fluorescence characteristic due to growth of the secondary group.
  • a visible light source 11 and ultraviolet light source 12 are placed interfacing the transparent window 8 filled with the liquid 5.
  • a single photo detector 15 is also placed interfacing the window 8.
  • the photo detector 15 detects the transmission of light from the visible light source 11 through the liquid solution 5, and at 90° therefrom the fluorescent light excited by the ultraviolet source 12.
  • the light sources 11 and 12 are controlled by the electronic controllers 13 and 14, respectively.
  • the light controllers can switch on and off the light sources, determine their intensity and modulate them in predetermined frequencies.
  • a wideband visible light source and optical filter may also be used.
  • the signals detected by the photo detector 15 are amplified and processed by the photo detector processor 16.
  • a central processor 17 controls and synchronizes the operation of the light sources and receives the processed signal from the processor 16. The whole operation can be monitored by a computer 18 that stores the data and provides a user interface, real time data analysis and reports.
  • the advantage of this embodiment over the prior art is that visible and fluorescence light generated by the system can now be recorded and analyzed dynamically. Instead of observing the changes when the test is over, the system can record the signals repeatedly (e.g., every 10 minutes) and detect immediate changes occurring in the substrates. For larger concentrations of microorganisms, these changes take place faster than those of lower concentrations. Since the processor 17 and the computer 18 analyze these changes in real time, the duration of the tests are shorter than the prior art tests in which human observation is required at specific times (18-24 hours) determined by the maximal incubation time that ensures the detection of the lowest possible bacterial concentration.
  • FIG. 2 illustrates another embodiment for monitoring the growth of two groups of microorganisms in a sample.
  • the device 1 comprises a container 2 which is transparent to light, at least in a window section 8. Similar to the first embodiment, the container can be made of glass or polymer, transparent to visible light and at least a limited band of the ultraviolet spectrum range. For example, polystyrene can be employed which is transparent to the whole visible spectrum and to long ultraviolet segment residing above 350 nanometers.
  • the tested sample 6 is introduced to a liquid mixture 5 comprising media capable of growing the target microorganisms and two substrate indicators, each capable of indicating growth of one of the target groups. Similar to the previous embodiment, one substrate indicator is chromogenic and therefore can change its color due to growth of the primary group, and the other substrate indicator is fluorogenic and therefore can change its fluorescence characteristic due to growth of the secondary group.
  • a single light source 11 is placed interfacing the transparent window 8 filled with the liquid 5.
  • a single photo detector 15 is also placed interfacing the window 8.
  • the light source 11 is a wide spectral band source covering both ultraviolet and a portion of the visible spectrum.
  • a gas discharge tube can cover the long UV range, but also a limited visible range that appears in the violet-blue range.
  • the photo detector 15 detects the transmission of visible light and the fluorescent light excited by the combined source 11.
  • the light source 11 is controlled by the electronic controller 13.
  • the electronic controller 13 can switch on and off the light source, determine its intensity and modulate it in predetermined frequencies.
  • the signal detected by the photo detector 15 is amplified and processed by the photo detector processor 16.
  • the central processor 17 controls the operation of the light source and receives the processed signal from the processor 16.
  • the whole operation can be monitored by a computer 18 that stores the data and provides user interface, real time data analysis and reports.
  • a computer 18 that stores the data and provides user interface, real time data analysis and reports.
  • this embodiment can be used for a combined coliform/E.
  • the color change results in a decrease of the signal detected by the photo detector 15, while the increase in fluorescence results in the increase of the detected signal. Consequently, the combined curve is capable of indicating individual growth of the two groups, avoiding the necessity for two light sources.
  • GNB gram negative bacteria
  • E. coli can be monitored by the inclusion of a dye indicator in the medium of an L-alanine aminopeptidase for GNB and ⁇ glucuronidase dye indicator for the detection of E. coli.
  • a color indicator such as ⁇ -napthalamide- ⁇ -L-alanine (color change from colorless to purple)
  • a fluorescent dye such as 4-methylumbelliferyl- ⁇ -L-alanine
  • a color indicator such as ⁇ -D- glucuronide (color change from colorless to purple)
  • a fluorescent dye such as 4-methylumbelliferyl- ⁇ -D- glucuronide
  • a method for the detection of all gram positive bacteria and Staphylococcus aureus can be developed by the utilization of 4-methylumbelliferyl phosphate in conjunction with GNB inhibitors and/or antibioitics. S. aureus can be detected by the use of dye indicator orthonitrophenyl phosphate.
  • the growth pattern of the corresponding microorganisms is determined. Then the number of
  • CFU is the colony forming units; log denotes the 10 base logarithmic function;
  • C s i is the initial concentration of the corresponding indicator substrate modifying reagents
  • K B is the bacterial activity
  • t g is the bacterial generation time
  • to is said Detection Time
  • t L is the time duration of the lag phase.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • glucoronidase (MUG) 0.1 g/1 was used for the simultaneous detection of coliform and
  • FIG. 3 shows the curve obtained with water inoculated with E. coli.
  • the color signal starts going down in a rapid rate due to the creation of the yellow color after around 6 hours.
  • the fluorescent signal starts a sharp upward trend around 7 hours due to the formation of the UV signal.
  • FIG. 4 shows the curves obtained with the coliform Enterobacter aerogenes. There is only a decrease in the curves due to the color formation, and no fluorescence is observed.
  • FIG. 5 shows the curve obtained for E. coli with the single light source associated with the embodiment of FIG. 2.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un dispositif et un procédé qui permettent la détection et la numération simultanées de deux groupes de microorganismes dans un échantillon d'analyse à l'aide d'un seul et unique contenant d'analyse. Dans le milieu de croissance liquide du contenant, on mélange à l'échantillon d'analyse un substrat chromogène et un substrat fluorogène. On fait incuber le contenant d'analyse afin de donner libre cours à la croissance et au métabolisme bactériens. On détecte de manière dynamique les changements spectraux des substrats au moyen de deux sources lumineuses externes ciblées sur une section transparente du contenant d'analyse et d'un seul et unique photodétecteur. Une source lumineuse fonctionne dans la bande visible et la seconde, dans la bande des ultraviolets longs. On analyse en temps réel les deux modèles temporels dynamiques produits par les deux substrats afin de déterminer la présence ou l'absence de chaque groupe de microorganismes et de dénombrer leurs concentrations originales dans l'échantillon d'analyse.
PCT/US2007/015204 2007-06-21 2007-06-29 Dispositif et procédé de détection et de numération de multiples groupes de microorganismes WO2008156462A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11/766,208 US7745169B2 (en) 2006-11-10 2007-06-21 Device and method for the detection and enumeration of multiple groups of microorganisms
US11/766,208 2007-06-21

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WO2008156462A1 true WO2008156462A1 (fr) 2008-12-24

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2343881A1 (es) * 2010-03-01 2010-08-11 Adasa Sistemas S.A.U Cubeta, instalacion y procedimiento para la medida de coliformes totales y escherichia coli basado en deteccion optica para aplicaciones de control de calidad de aguas.
US10180248B2 (en) 2015-09-02 2019-01-15 ProPhotonix Limited LED lamp with sensing capabilities
CN109580626A (zh) * 2018-07-30 2019-04-05 海南微氪生物科技股份有限公司 一种基于光电传感器的微生物快速检测仪

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773426A (en) * 1972-02-22 1973-11-20 Department Of Health Educ Welf Bacterial growth detector
US5266486A (en) * 1989-05-12 1993-11-30 Nvl Photronics Corporation Method and apparatus for detecting biological activities in a specimen
US5427920A (en) * 1992-04-24 1995-06-27 Becton Dickinson And Company Methods and apparatus for detecting biological activities in a specimen
US6020150A (en) * 1995-03-20 2000-02-01 Diffusion Bacteriologie Du Var Gelled system and method for detecting microorganisms by separation and culture on gelled system
US6077673A (en) * 1998-03-31 2000-06-20 Clontech Laboratories, Inc. Mouse arrays and kits comprising the same
US6197576B1 (en) * 1998-05-22 2001-03-06 Gideon Eden Instrument for detection of microorganisms

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773426A (en) * 1972-02-22 1973-11-20 Department Of Health Educ Welf Bacterial growth detector
US5266486A (en) * 1989-05-12 1993-11-30 Nvl Photronics Corporation Method and apparatus for detecting biological activities in a specimen
US5427920A (en) * 1992-04-24 1995-06-27 Becton Dickinson And Company Methods and apparatus for detecting biological activities in a specimen
US6020150A (en) * 1995-03-20 2000-02-01 Diffusion Bacteriologie Du Var Gelled system and method for detecting microorganisms by separation and culture on gelled system
US6077673A (en) * 1998-03-31 2000-06-20 Clontech Laboratories, Inc. Mouse arrays and kits comprising the same
US6197576B1 (en) * 1998-05-22 2001-03-06 Gideon Eden Instrument for detection of microorganisms

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2343881A1 (es) * 2010-03-01 2010-08-11 Adasa Sistemas S.A.U Cubeta, instalacion y procedimiento para la medida de coliformes totales y escherichia coli basado en deteccion optica para aplicaciones de control de calidad de aguas.
US10180248B2 (en) 2015-09-02 2019-01-15 ProPhotonix Limited LED lamp with sensing capabilities
CN109580626A (zh) * 2018-07-30 2019-04-05 海南微氪生物科技股份有限公司 一种基于光电传感器的微生物快速检测仪

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