Classifications

• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00009—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with a sample supporting tape, e.g. with absorbent zones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N2035/00465—Separating and mixing arrangements
  • G01N2035/00475—Filters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N2035/00465—Separating and mixing arrangements
  • G01N2035/00534—Mixing by a special element, e.g. stirrer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
  • G01N35/04—Details of the conveyor system
  • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
  • G01N2035/0403—Sample carriers with closing or sealing means
  • G01N2035/0405—Sample carriers with closing or sealing means manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
  • G01N35/04—Details of the conveyor system
  • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
  • G01N2035/0429—Sample carriers adapted for special purposes
  • G01N2035/0436—Sample carriers adapted for special purposes with pre-packaged reagents, i.e. test-packs
• • 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
  • G01N21/75—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

Definitions

• the invention relates to a method for assaying a biological sample liquid, in particular for the determination of microorganisms such as coliform bacteria, such as Escherichia coli, wherein the sample liquid is passed through a particular photo ⁇ metric measuring device with an excitation means and a detector means.
• the invention relates to an apparatus for analyzing a biological sample liquid, in particular for Bestim ⁇ mung of microorganisms such as coliform bacteria, such as
• E. coli with a measuring device having an excitation device and a detector device, and with a sample supply device for supplying sample liquid to the measuring device.
• the object of the present invention is a method and an apparatus of the type mentioned to provide, with which or with which reliable and accurate investigations of a sample liquid is made possible even at low levels of the biological material contained therein.
• sample volumes are generated which are examined individually in the measuring chamber of the measuring device, ie independently of the remaining sample volumes. Due to the on ⁇ distribution of the sample liquid into separate sample volumes can advantageously have a higher resolution in the evaluation of the measurement signals from the measuring chamber can be achieved. In comparison to the investigation of a continuous liquid stream can be obtained comparatively sharp measurement signals for the metrological analysis of the separated sample volumes, which in some way legally facilitate the determination of the organic content of the Pro ⁇ benfactkeit before ⁇ .
• the examination of individual sample volumes also advantageously allows the use of statistical evaluation ⁇ shear, which are based on the evaluation of the measurement signals from the discrete sample volumes.
• an interruption-free process for the Availability checked ⁇ supply is provided, wherein the sample volumes gradually separates ask ⁇ and examined individually.
• the sample liquid For dividing the sample liquid into defined sample volumes, it is favorable when the sample liquid is filled into each other ge ⁇ separated receiving chambers of a sample-receiving device. Accordingly, the receiving chambers of the sample-receiving device with the sample volumes of the sample liquid are ⁇ be filled, which are then tested individually in the measuring chamber of the measuring device.
• the measuring chamber of the measuring device is in this case preferably dimensioned and designed to accommodate only a single receiving chamber. Due to the sequential examination of the sample volumes, the measuring device can advantageously be comparatively simple and small in size. lumig be designed.
• the sample receiving device is in this case connected to a conveying device with which the sample receiving device is conveyed through the measuring device.
• an elongated sample receiving device which has longitudinally successive receiving chambers, which are guided successively through the measuring chamber of the measuring device.
• the elongated sample-receiving device is in this case preferably gradually moved such in the longitudinal direction of the sample ⁇ cradle that the receiving chambers can be individually analyzed in the measuring chamber of the measuring device.
• the receiving chamber of the sample-receiving device has a filling opening which is closed after filling with the Probenflüs ⁇ stechnik, preferably by means of a film or a lid.
• the thus-closed receiving chambers of the sample receiving device can subsequently be conveyed through the measuring chamber of the measuring device in which the examination of the biological sample volumes takes place.
• the receiving chamber of the sample receiving device is provided with a preferably dry-chemical reagent, in particular with a substrate for catalysis ei ⁇ ner biochemical reaction.
• Particularly preferred is an embodiment in which at least two capsules are arranged with different dry chemical reagents in the receiving chamber.
• the capsules have the dry ⁇ chemical reagents enclosing sheaths, which dissolve at predetermined, different times and / or at different temperatures.
• the reagent of the sample liquid can be added during the filling of the receiving chambers of the sample receiving device of the measuring liquid.
• the reagent-containing storage chamber of the sample-receiving device is preferably sealed by a protective foil which is removed with the sample liquid prior to filling.
• the administration with a liquid, optionally with the To ⁇ of a reagent.
• the sample fluid is through the filter, in particular a disposable filter ⁇ , filtered.
• the contaminated filter can then be further subjected to a cleaning step in which a cleaning solution is passed through the filter before the filter is placed in the corresponding receiving chamber of the sample receiving device.
• the sample prior to the expiry of incubation is investigated in the measuring device for the filling of the receiving chamber of the sample-receiving device, preferably being a further measurement at the beginning of the in ⁇ incubation time after filling of the Recording chamber is made.
• the conveyor can in this case controlled in such a ⁇ to that in the promotion of the sample-receiving device between the filling station, in which the receiving chambers of the sample-receiving device are filled with the sample liquid, and the inspection station in which the sample volumes to be analyzed in the measuring chamber of the measuring device, the predetermined Incubation period elapses.
• each receiving chamber is measured a first time after filling and thorough mixing, whereby can be made whether the reagents are in the proper condition. During the incubation period you can then measure several times. In any case, at least one measurement is taken at the end of the incubation period.
• individual receiving chambers can also be filled with a calibration solution in order to check or calibrate the function and setting of the measuring unit.
• the "incubation time” is the period between mixing the sample with the reagent (s) and measuring, during which time the reagent can react with any reactants (e.g., enzymes) present in the sample.
• bacteriological tests are carried out in addition to chemical analyzes.
• the latter studies include in particular the detection of the presence of bacteria of faecal origin, such as E. coli and enterococci, as the non ⁇ presence of these bacteria in a given volume of water indicates that the water is clean.
• CFU colony forming units
• microorganisms particularly coliforms and enterococci
• substances and materials in particular meta ⁇ metabolites, secondary metabolites, enzymes or proteins which are released by the microorganisms, for example, to the nutrient medium or water.
• substances and substances can either be used directly in the nutrient medium or water (eg by means of fluorescence, sorption at defined wavelengths) or indirectly, in that the substances or substances serve as substrate for further chemical or biochemical reactions.
• a method based on this principle and detection of colifor ⁇ men bacteria such as E.
• coli may be used, based on the reaction of 4-methylumbelliferyl-ß-D-galactopyranoside (MUG) by coliforms and enterococci into the water or nutrient medium delivered ß-galactosidases.
• MUG 4-methylumbelliferyl-ß-D-galactopyranoside
• coliforms and enterococci into the water or nutrient medium delivered ß-galactosidases.
• These enzymes cleave MUG, so that a fluorescent dye, wel ⁇ cher it allows deduce whether, and in what quantities coliforms and enterococci are present in an aqueous sample.
• ⁇ serlösliche coumarins, resorufin-releasing substances, Flu- can be determined depending on the microorganism as substrates, which are implemented by micro-organisms, and oresceine Naphthalene be used.
• Particularly preferred coumarins are selected from the group ⁇ be detached from 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, 4-methylumbelliferyl caprylate, 4-methylumbelliferyl-beta-D-cellobioside, 4-methylumbelliferyl-beta-D-galactopyranoside , 4- methylumbelliferyl-beta-D-glucuronic acid trihydrate, 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-alpha-L-rhamnopyranoside, 4-methylumbelliferylsulfate, 4-methylumbelliferyl-alpha-L-rhamnopyranoside, 4-
• Methylumbelliferyl-beta-D-ribofuranoside 4-trifluoromethyl-7-hydroxycoumarine, L-alanine 7-amido-4- (trifluoromethyl) coumarin, N-alpha-CBZ-L-arginine 7-amido-4-methylcoumarin, L-citrulline 7- amido-4-methylcoumarin, glutaryl-glycyl-L-arginine 7-amido-4-methylcoumarin, L-histidine 7-amido-4-methylcoumarin, L-hydroxyproline 7-amido-4-methylcoumarin, L-lysyl-L-alanine 7-amido-4-methylcoumarin, L-ornithine 7-amido-4-methylcoumarin, L-pyroglutamic acid 7-amido-4-methylcoumarin L-tryptophan 7-amido-4-methylcoumarin and 6, 8-difluoro-7-hydroxy 4-methyl coumarin (DiFMU).
• Particularly preferred resorufin-releasing substances are made ⁇ selected from the group consisting of 10-acetyl-3, 7- dihydroxyphenoxazine, 7-ethoxyresorufin, pentoxyresorufin, resorufin beta-D-galactopyranoside and resazurin.
• fluoresceins selected from the group consisting of 6-carboxyfluorescein, fluorescein iso ⁇ mer I, fluoresceinamine isomer II acetate, 9-deoxy-9-N- (fluorescein-5-aminothiocarbonyl) amino-N-acetylneuraminic acid, Fluoresceindi-, fluorescein isothiocyanate Isomer I and Rose Bengal Dinarium salt.
• naphthalenes are selected from the group consisting of 6-bromo-2-naphthyl-alpha-D-galactopyranoside, 2-naphthyl-beta-D-galactopyranoside, 1-naphthyl-beta-D-glucopyranoside, L-alanine 2-naphthylamide , L-leucine beta-naphthylamide hydrochloride, L-proline beta-naphthylamide hydrochloride and L-pyroglutamic acid beta-naphthylamide.
• the receiving chambers of the sample receiving device and the measuring chamber of the measuring device at least partially have corresponding cross-sectional ⁇ shapes.
• the sample receiving ⁇ device polygonal in longitudinal section, in particular in Wesent ⁇ union square or substantially rectangular, Low ⁇ chambers and / or in longitudinal section round, in particular in Wesent ⁇ union circular , Receiving chambers.
• the receiving chamber has a support surface for supporting a filter. Accordingly, the filter is placed on the support surface of the receiving chamber, so that the filter is placed in a stable position during the incubation or examination phase.
• a substantially horizontal support surface ⁇ is provided so that the filter is held under the action of gravity in the predetermined position when the sample receiving device is arranged in the horizontal direction.
• the receiving chamber for forming the support surface for the filter has a sieve-like or grid-shaped support element.
• the filter can be reliably held in the predetermined position.
• the sieve or lattice-shaped support element for the filter membrane can already be mounted in the receiving chamber or alternatively be inserted before or with the filter membrane in the receiving chamber.
• the support surface is formed by a gradation of the receiving chamber.
• a trough-shaped receiving chamber is provided ⁇ see, which has a circumferential gradation on a side wall of the receiving chamber.
• the sample receiving device has receiving chambers of a first type with a first receiving volume for receiving a first defined sample volume and receiving chambers of a second type with a second defined receiving volume for receiving a second defined sample volume.
• the sample receiving ⁇ device may have a fixed sequence of receiving chambers of the first and the second type.
• the MPN method most probable number
• the receiving chamber of the sample-receiving device has an elastically deformable to mix the sample liquid portion.
• the excitation device has a radiation source for irradiating the sample liquid and the detector device has a radiation detector for detecting radiation interacting with the sample liquid.
• a radiation source for irradiating the sample liquid
• the detector device has a radiation detector for detecting radiation interacting with the sample liquid.
• a wide variety of types of radiation sources can be used.
• light emitting diodes or working with a continuous spectrum radiation sources may be provided. If a high intensity is required, a La ⁇ serttle may in particular be used.
• the use of light-emitting diodes is often preferred, as this represents a very cost-effective design, which are also generally available for most wavelength ranges.
• the radiation detector photodiodes are preferably used.
• photomultipliers can also be used.
• a spectrometer in particular a CCD ("charge coupled device") sensor, if not only the light intensity of one or more wavelengths but the spectral characteristic is to be investigated.
• the radiation detector preferably to be arranged to measure in the sample liquid excited fluorescence ⁇ radiation.
• the radiation detector can detect the transmitted radiation, which contains information about the absorption capacity of the sample liquid.
• the receiving chambers of the sample receiving device are releasably connected to each other.
• the receiving chambers advantageously in particular a ⁇ individually can be separated from the sample collection device.
• the sample-receiving device may, for example, attenuations, in particular Perforatio ⁇ NEN, have between the receiving chambers.
• the individual receiving chambers are arranged in a conveyor belt or a transport device from which or which they can be removed for further processing.
• FIG. 1 shows a device according to the invention for the examination of a biological sample liquid, in which the sample liquid is divided into defined sample volumes into one another for division filling separate receiving chambers of a sample receiving device, wherein the sample volumes are examined individually in a measuring chamber;
• FIG. 2 shows an embodiment of the device in which the receiving chambers have bearing surfaces for arranging filters with the concentrated sample
• Figure 3 is a disclosed embodiment of the apparatus in which the sample liquid without filtration is ⁇ into the receiving chambers.
• FIG. 4a is a plan view of one disclosed embodiment, the sample-receiving device which has square in longitudinal sectioncard ⁇ chambers a first and a second type;
• FIG. 4b includes a plan view of another disclosed embodiment, the sample-receiving device, which is circular in longitudinal section on ⁇ acceptance chambers a first and a second type;
• Fig. 5 is a section along the line V-V in Fig. 4b;
• FIG. 6a shows a sectional view of a further embodiment of the sample receiving device, in which the receiving chamber has an elastically deformable section;
• Fig. 6c is a sectional view of another embodiment of the sample receiving apparatus in which the elastically deformable portion is provided annularly on the side wall of the receiving chamber;
• FIG. 7 shows a view of the sample receiving device according to FIG. 6 as it passes through the correspondingly shaped measuring chamber of the device according to the invention
• FIG. 8 shows a view of an embodiment of the measuring chamber of the invention. inventive device, and
• FIG. 9 shows a view of a further embodiment of the device, in which the measuring device has two measuring chambers for the simultaneous measurement of two sample volumes.
• FIG. 1 there is shown an apparatus 1 for assaying a biological sample liquid, which according to a preferred embodiment comprises coliform bacteria, e.g. E. coli.
• a biological fluid for the purposes of this disclosure, a liquid having a living or viable material is generally considered.
• the device 1 has a measuring device 2, in which the sample liquid is examined.
• the measuring device 2 comprises a measuring chamber 3, which with an excitation means (not shown) for excitation of the sample liquid and with a detector device (not ge ⁇ shows) for detecting an originating from the excited sample fluid measurement signal in communication.
• a radiation source for example a light- emitting diode, and a radiation detector, for example a CCD sensor, may be provided as the excitation device.
• the measuring device 2 is connected to a sample supply device 4, with which the sample liquid is passed through the measuring device 3.
• the sample liquid is divided in the embodiment shown in defined sample volumes, which nacheinan ⁇ are guided by the measuring chamber 3 of the measuring device 2.
• the discrete sample volumes are then tested individually in the Messkam ⁇ mer 3 of the measuring device. 2
• the sample supply device 4 has a sample receiving device 5 with separate receiving chambers 6, in which the sample volumes are accommodated.
• an elongated sample receiving device 5 is provided, which in
• the sample receiving device 5 is moved by means of a (not shown) conveying or transporting device in the direction of arrow 4 ' moved so that the receiving chambers 6 of the sample receiving device 4 spatially and temporally successive in the measuring chamber 3 of the measuring device 2 arrive, in which the sample volumes are examined individually.
• the receiving chambers 6 of the sample receiving device 4 are provided in the embodiment shown with a preferably dry chemical reagent 7, which forms a substrate for the biochemical reaction with the biological sample liquid.
• the receiving chambers 6 with the reagent 7 are closed by means of a protective film 8 which is unwound from a roll 9 (shown schematically in the drawing).
• a protective film 8 which is unwound from a roll 9 (shown schematically in the drawing).
• an adhesive or welded connection is preferably provided for sealing the receiving chambers 6 with the protective film 15.
• the protective film 8 is removed before filling the receiving chambers 6 with the sample liquid.
• the protective film 8 is wound in the embodiment shown via a deflection roller 10 on a take-up reel 10 '. By removing the protective film 8 top filling openings 11 of the receiving chambers 6 are released.
• the receiving chambers 6 are filled by means of a filling device 12 via the exposed filling openings 11 with a measuring liquid.
• the dry chemical reagent 7 is previously enclosed in the receiving chambers 6, so that the addition of a reagent via the measuring liquid can be dispensed with.
• the measuring fluid may contain the reagent.
• the next step is inserted by means of a Filteranord ⁇ voltage 13, a sample contaminated with the filter 14 into the corresponding receiving chamber 6 of the sample-receiving device. 5 Accordingly, the bacteria are filtered out of the sample volume, so that the bacteria present in the filtered sample volume are available for the enzymatic reaction in the measurement volume.
• the filling openings 11 of the sample receiving device 5 are closed by means of a foil 15 after the filling of the receiving chambers 6.
• the film 15 is unwound in the embodiment shown by a take-off roll 16 (shown schematically) and fed to the sample receiving device 5 via a deflection roller 17.
• the film 15 is preferably attached via a Kle ⁇ welded or welded connection to the sample receiving device 5.
• the receiving chambers 6 of the sample receiving device 5 each have a circumferential, substantially horizontal support surface 18 for supporting the filter 14.
• the Auflageflä ⁇ che 18 may also be provided with a screen or lattice-shaped support element (not shown) on which the Fil ⁇ ter 14 can be placed.
• the support surface 17 is formed by a step 19 of the trough-shaped receiving chamber 6.
• the sample is introduced together with the measuring liquid into the receiving chambers 6 of the sample receiving device 5, wherein the use of the filter 14 is dispensed with.
• the sample volume received therein is mixed before the filling opening 11 is closed with the film 15.
• the sample receiving device 5 preferably has receiving chambers of a first type 6 'having a first receiving volume for receiving a first defined sample volume and receiving chambers of a second type 6 "having a second defined receiving volume for receiving a second defined sample volume.
• This design facilitates the statistical evaluation of the measurement results.
• the MPV method can be used in this case.
• the sample receiving device 5 in plan view substantially square receiving chambers 6, wherein a fixed sequence of receiving chambers of the first type 6 'and receiving chambers of the second type 6' 'is provided.
• substantially circular receiving chambers are shown. 6, wherein also in this embodiment, a fixed sequence of receiving chambers of the first type 6 'and receiving chambers of the second type 6''are provided.
• the receiving chamber 6 of the sample receiving device 5 has an elastically deformable section 20.
• the portion 20 of the receiving chamber 6 can be elastically deformed with jaws 21 in the direction of arrow 22, whereby the sample liquid contained therein in the remaining portions of the receiving chamber ⁇ 6 is displaced (see arrows 23 in Fig. 6b).
• the elastically deformable section 20 is formed at the bottom of the receiving chamber 6.
• Fig. 6c of the elastically deformable portion 20 is formed ring ⁇ shaped circumferentially on the side wall of the receiving chamber 6.
• the receiving chamber 6 have the sample-receiving device 5 and the measuring chamber 3 of the measurement device 2 at least in sections mutually corresponding cross sectional shapes ⁇ .
• the elastically deformable portion 20 of the receiving chamber 6 in Wesentli ⁇ surfaces with an accurate fit in the measuring chamber 3 of the measuring device. 2
• FIG. 8 shows in cross-section an alternative embodiment of the measuring chamber 3, which is shaped in sections corresponding to the receiving chamber 6 of the first type 6 'according to FIGS. 4, 5.
• the measuring device 2 can contain at least two measuring chambers 3, so that a plurality of sample volumes can be measured simultaneously.
• two sample receiving devices 5 are provided, which are supplied to the separate measuring chambers 3.
• the receiving chambers 6 of the sample receiving device 5 in this case have different receiving volumes.

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
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• Wood Science & Technology (AREA)
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• Organic Chemistry (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Sustainable Development (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2012
  • 2012-07-04 AT ATA50268/2012A patent/AT513085B1/de not_active IP Right Cessation
• 2013
  • 2013-07-04 DE DE212013000143.6U patent/DE212013000143U1/de not_active Expired - Lifetime
  • 2013-07-04 WO PCT/AT2013/050132 patent/WO2014005168A2/fr not_active Ceased

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