EP1573051A1 - Procede et dispositif pour determiner des germes - Google Patents

Procede et dispositif pour determiner des germes

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Publication number
EP1573051A1
EP1573051A1 EP03767723A EP03767723A EP1573051A1 EP 1573051 A1 EP1573051 A1 EP 1573051A1 EP 03767723 A EP03767723 A EP 03767723A EP 03767723 A EP03767723 A EP 03767723A EP 1573051 A1 EP1573051 A1 EP 1573051A1
Authority
EP
European Patent Office
Prior art keywords
germs
sample
fluorescent
fluorescence
membrane filter
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP03767723A
Other languages
German (de)
English (en)
Inventor
Tilo Weiss
Stefan Stumpe
Friedhelm Siepmann
Andreas Thunchen
Frank Wienhausen
Andreas Katerkamp
Michael Heinzel
Roland Breves
Mirko Weide
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
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
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP1573051A1 publication Critical patent/EP1573051A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Definitions

  • the present invention relates to a method for the quantitative and / or qualitative determination of germs according to the preamble of claim 1. Furthermore, the present invention relates to a device for the quantitative and / or qualitative determination of germs according to the preamble of claim 27, in particular for carrying out the aforementioned method, and the use of this device, in particular for the preferably automated production and / or quality control.
  • Microbiological safety must be guaranteed for a large number of substances, raw materials and products from the various areas of industry, handicraft, household, health, catering, etc.
  • Quantitative microbiological analysis techniques have been used for quality assurance for a long time.
  • the basis of these methods is to multiply individual germs that occur in the material to be examined so that they become visible as smears or colonies with the naked eye.
  • cultivation methods are used for this, which multiply these germs either on solid nutrient media or in liquid nutrient solutions or media. Depending on the method and type of germ, it takes up to several days to carry out conventional cultivation methods for the detection of bacteria and fungi.
  • the cultivation processes are generally carried out in such a way that culture media (typically culture dishes with culture media based on agar-agar) are inoculated with the sample and, at the respective germs, adapted, generally elevated temperatures for up to a week can be cultivated (e.g. in an incubator). From growth and the form of the resulting cultures, a person skilled in the art can then deduce the type and extent of the microbial load on the sample.
  • culture media typically culture dishes with culture media based on agar-agar
  • adapted, generally elevated temperatures for up to a week can be cultivated (e.g. in an incubator). From growth and the form of the resulting cultures, a person skilled in the art can then deduce the type and extent of the microbial load on the sample.
  • This technology has the decisive disadvantage that only an undetermined fraction of the germs contained in the sample can be cultivated and the information is only available after a week.
  • fluorescence-optical methods have increasingly replaced the conventional "rapid detection methods" and cultivation methods.
  • the direct epifluorescence filter technique (DEFT) is the first direct method available that also allows quantitative "live / dead” germ detection in less than an hour.
  • This non-specific, fluorescence-optical method has been known as a qualitative method for over 25 years in basic university research (see, for example, Pettipher et al., Appl. Environ. Microbiol. 44 (4): 809-13, 1982) and has been used since In the early 1990s increasingly in industrial applications (e.g. breweries, dairies, food industry etc.) as quantitative Test method established (Hermida et al., J. AOAC Int.
  • MMCF method membrane filter microcolony fluorescence method
  • MMCF method see e.g. Baumgart, microbiological analysis of food, Behr's ... Verlag 1993, 3rd edition, p. 98 ff.
  • the disadvantages here are that a time-consuming pre-enrichment of the germs must precede, the membrane filter must be pretreated for the subsequent epifluorescence microscopy (moistening with special media, dimensioning and drying) and the number of germs by counting the fluorescence-marked colonies under an epifluorescence microscope or a UV lamp must be done.
  • the significance of the analysis is limited by the time available. At the end of the enrichment period, all germs must have multiplied to such an extent that they have become visible. Delayed growth due to unfavorable sampling or growing conditions can lead to erroneously negative results. Therefore, the implementation of the conventional cultivation methods for the detection of bacteria and fungi often requires several days, so that the microbiological results are often too late to allow a regulatory intervention in the production process.
  • the present invention is therefore based on the object of providing a method of the type mentioned at the outset which is suitable for the quantitative or qualitative determination of germs and in particular at least partially avoids the disadvantages described above, and a corresponding device for carrying out such a method.
  • the subject of the present invention is therefore a method for the quantitative and / or qualitative determination of germs in a sample, the method comprising a method step (a) of sample preparation and a method step (b) of detection and / or evaluation comprises, in method step (a) marking at least some of the germs present in the sample by means of at least one fluorescence marker and in method step (b) quantitative and / or qualitative detection and / or evaluation taking place, the method step (b) Detection and / or evaluation carried out is carried out by fluorescence reflection spectrometry or fluorescence reflection photometry (the two terms "fluorescence reflection spectrometry” and “fluorescence reflection photometry” are used synonymously in the following, and the basic measurement method is explained in more detail below).
  • a special feature of the method according to the invention is thus the combination of fluorescent labeling of germs, in particular microorganisms, with suitable fluorescent markers and the subsequent detection of the fluorescence-labeled germs with a simplified, namely fluorescence reflection photometric detection or evaluation method and a corresponding device. Since the sample is only exposed to radiation for a short time in the method of fluorescence reflection photometry, the fluorescent markers fade and thus falsify the measurement result is efficiently avoided.
  • the fluorescence reflection spectrometric or fluorescence reflection photometric detection or evaluation detects the radiation emitted by the fluorescence-marked nuclei upon irradiation of the corresponding wavelength in reflection or their intensity, which correlates with the number of bacteria present in the sample (for further details on reflection photometric or reflection photometric methods, for example the relevant explanations in Römpp, Chemielexikon, Thieme Verlag, 10th edition, Vol. 5, pages 3756/3757, keywords “reflection” and “reflection spectroscopy” as well as Vol. 4, pages 3312 to 3314, keyword “photometry”, each including the referenced literature, the entire content of which is hereby incorporated by reference).
  • the z. B. applied in the context of the MMCF method (membrane filter microcolony fluorescence method)
  • Fluorescence reflection photometers or spectrometers which can be used in the context of the method according to the invention can be readily designed for the person skilled in the art on the basis of commercially available components which are usually available for this purpose.
  • the phrase "marking at least part of the germs present in the sample” means in particular the following: depending on whether only special ones germs or types of germs present in the sample or all the germs present in the sample are to be determined, in the former case only a specific part of the germs present in the sample (generally with germ-specific fluorescence markers) is marked, while in the latter case all Marked germs present in the sample (generally with non-germ-specific fluorescent markers or a mixture of different germ-specific fluorescent markers).
  • the number of germs present in the sample can be determined by means of suitable calibration on the basis of the measurement value determined by fluorescence reflection photometry (in the case of fluorescent labeling of all germs present in the sample, the total number of all germs present in the sample and only in the case of fluorescent labeling) special germs their total number).
  • fluorescence reflection photometry in the case of fluorescent labeling of all germs present in the sample, the total number of all germs present in the sample and only in the case of fluorescent labeling
  • special germs their total number special germs their total number.
  • the fluorescence-labeled nuclei are applied to a membrane filter (for example a polycarbonate membrane filter), which is preferably porous, in the sample preparation carried out in process step (a).
  • a membrane filter for example a polycarbonate membrane filter
  • the membrane filter should generally be designed such that it retains the germs or is impermeable to the germs.
  • the size of the pores of the membrane filter should be chosen so that the pore size is smaller than the germs present in the sample.
  • part or area of the membrane filter generally the edge, should not be provided with germs, because this ensures referencing or internal calibration or standardization for each sample.
  • membrane filter materials suitable according to the invention are also PTFE, polyester and cellulose and cellulose derivatives, such as cellulose acetate, regenerated cellulose, nitrocellulose or mixed cellulose esters.
  • Membrane filters suitable according to the invention are sold, for example, by Macherey-Nagel (for example the "PORAFIL ® " series).
  • Macherey-Nagel for example the "PORAFIL ® " series.
  • the use of a membrane filter offers the great advantage that the fluorescence reflection photometric detection or evaluation, in particular without further sample treatment, preparation, transfer or the like (ie in particular without pre-enrichment), can be carried out directly on the membrane filter.
  • the fluorescence-labeled germs are applied to a silicon microsieve in the sample preparation carried out in method step (a).
  • silicon microsieves have a particularly smooth and even surface and therefore germs located thereon can be better detected.
  • the cleaning of such screens is relatively easy to do and they can be used several times.
  • the silicon microsieves also have good biocompatibility and good reflectivity. Another advantage of microsieves can be seen in their rigid structure, which have considerable advantages in their handling.
  • the particularly uniform pore size is another advantage that further increases the accuracy of selective filtrations.
  • the pore sizes of the microsieves to be used for the method according to the invention should advantageously be between approximately 0.1 and 2 ⁇ m.
  • Microsieves with pore sizes of 0.45 ⁇ m and 1.2 ⁇ m are particularly preferred.
  • sieves with different pore sizes can also be used, i.e. also with pores smaller than 0.1 ⁇ m or larger than 2 ⁇ m.
  • the fluorescent marker used in the method according to the invention is advantageously selected such that it is membrane-permeable with respect to the membrane filter used in the sample preparation carried out in method step (a). This has the advantage that the fluorescence reflection photometric detection or evaluation no interference signals caused by excess fluorescence markers or no background noise occurs and consequently a favorable signal / background ratio or signal / noise ratio is achieved.
  • the fluorescent labeling of the germs present in the sample in method step (a) of the method according to the invention is carried out in a manner known per se.
  • the germs to be labeled can be brought into contact with a solution or dispersion of the fluorescent markers present in excess with respect to the existing germs, the contact time having to be sufficient to ensure complete fluorescence labeling of all the germs involved in this process step should be marked (depending on the selection of the fluorescent marker, for example, all germs present in the sample or all germs of only one or more types of germs).
  • the excess fluorescence markers can then be removed or separated from the fluorescence-marked germs.
  • method step (a) of sample preparation can also include inactivating and / or removing any germ-inhibiting and / or germicidal substances or components (eg preservatives, surfactants etc.) present in the sample.
  • germ-inhibiting and / or germicidal substances or components eg preservatives, surfactants etc.
  • method step (a) of sample preparation can also include inactivating and / or removing any germ-inhibiting and / or germicidal substances or components (eg preservatives, surfactants etc.) present in the sample.
  • germ-inhibiting and / or germicidal substances or components e.g. preservatives, surfactants, etc.
  • the method step of inactivating or removing any germ-inhibiting or germ-killing substances or constituents that may be present in the sample is advantageously carried out before the fluorescence marking, preferably immediately after sampling or immediately at the beginning of the method step (a) performed sample preparation performed by the method according to the invention; In this way it is ensured that the germ-inhibiting or germ-killing substances, constituents, ingredients and the like can essentially have not yet brought about a change in the number of germs present in the original sample.
  • the process step of inactivating or removing any germ-inhibiting or germ-killing substances or constituents which may be present in the sample, depending on the type of sample, is carried out in a manner known per se.
  • Stumpe et al. “Chemiluminescence-based direct detection of microorganisms - An experience report from the food and cosmetics industry” on pages 317 to 323 of the conference proceedings "HY-PRO 2001, Hygienic Production Technology / Hygienic Production Technology", 2nd international Specialist congress and exhibition, Wiesbaden May 15-17, 2001 and the literature cited in this article; the entire content of this article, including the content of the literature cited therein, is hereby incorporated by reference.
  • An inactivation or conditioning solution which is particularly suitable according to the invention has the following composition:
  • the TLH water which can be used according to the invention has the following composition in particular:
  • Polysorbate 80 (Tween 80) 30.0 g
  • the phosphate buffer solution which can be used according to the invention has the following composition in particular:
  • fluorescent marker is understood very broadly in the context of the present invention and means in particular any fluorescent marker which is designed such that it interacts with the germs, for example on the germs, in particular on their cell wall (shell) and / or nucleic acid , binds and / or is taken up by the germs, in particular metabolized and / or implemented enzymatically.
  • the fluorescent marker used according to the invention can be, for example, a non-germ-specific fluorescent marker or a mixture of non-germ-specific fluorescent markers. This enables a relatively inexpensive fluorescence labeling of all germs present in the sample and thus a relatively quick determination of the total number of germs in the sample.
  • a germ-specific fluorescence marker or a mixture of different germ-specific fluorescence markers can be used as the fluorescence marker.
  • a mixture of germ-nonspecific and germ-specific fluorescence markers can be used as the fluorescence marker.
  • a fluorescent marker which interacts with living germs can also be used as the fluorescent marker.
  • a fluorescent marker which interacts with "dead” germs can also be used as the fluorescent marker.
  • Such mixtures are known per se from the prior art (see, for example, Stumpe et al., Loc. Cit. And the system from EasyProof Labor case GmbH, Voerde).
  • a non-fluorescent precursor i.e. a precursor or precursor
  • esterase enzymatic activity
  • green color living proof
  • an intact cell membrane with membrane potential must be present.
  • the fluorescent markers commonly used for seed labeling in epifluorescence microscopy or in DEFT (direct epifluorescence filter technology) or in MMCF (membrane filter microcolony fluorescence method) can also be used as fluorescent markers.
  • a fluorescent dye or a precursor of such a fluorescent dye can be used as the fluorescent marker, from which interaction with the germs, in particular through metabolism and / or enzymatic conversion, the fluorescent dye is generated, are used.
  • Examples of such precursors of fluorescent dyes are e.g. B. is described in WO 86/05206 A1 and in EP 0 443 700 A2, the entire respective disclosure content of which is hereby incorporated by reference (eg non-fluorescent diacetyl fluorescein which can be converted enzymatically to fluorescein).
  • fluorescent dyes which can be used according to the invention as fluorescent markers are, without limitation, e.g. B. 3,6-bis [dimethylamino] acridine (acridine orange), 4 ', 6-diamido-2-phenylindole (DAPI), 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (ethidium bromide), 3,8-diamino-5- [3- (diethylmethylammonio) propyl] -6-phenylphenanthridinium diiodide (propidium iodide),
  • Rhodamines such as Rhodamine B and Sulforhodamine B as well as fluorescein isothiocyanate.
  • EP 0 940 472 A1 or to Molecular Probes' Handbook of Fluorescent Probes and Research Chemicals, 5th edition, Molecular Probes Inc., Eugene, Oregon (PR Haugland, Editor, 1992), the the entire respective disclosure content is hereby incorporated by reference.
  • nucleic acid probes for example, germ-specific nucleic acid probes
  • fluorescent markers which in turn are fluorescence-labeled, in particular with a fluorescent group or a fluorescent molecule.
  • the fluorescent group or the fluorescent molecule can, for example, be covalently or otherwise bound to the nucleic acid probe.
  • the nucleic acid probe used according to the invention as a fluorescence marker can be, for example, a fluorescence-labeled oligo or polynucleotide or a fluorescence-labeled DNA or RNA probe.
  • DNA probes are preferred according to the invention for reasons of stability.
  • nucleic acid probes which can be used according to the invention as fluorescent markers are, for example, “the probes mentioned in WO 01/85340 A2, WO 01/07649 A2 and WO 97/14816 A1, the entire respective disclosure content of which is hereby incorporated by reference.
  • nucleic acid probes that are typically used for fluorescence jn situ hybridization (FISH) for labeling (DNA or RNA labeling) used nucleic acid probes are used.
  • FISH fluorescence jn situ hybridization
  • DNA or RNA labeling used nucleic acid probes.
  • a particularly germ-specific antibody can also be used as the fluorescence marker, which in turn is fluorescence-labeled, in particular with a fluorescent group or a fluorescent molecule, it being possible for the fluorescent group or the fluorescent molecule to be covalently or otherwise bound to the antibody.
  • the detection limit in the method according to the invention with regard to the germs to be determined is ⁇ 100 colony-forming units (CFU) per milliliter of sample volume, preferably ⁇ 10 colony-forming units (CFU) per milliliter of sample volume.
  • the method according to the invention therefore does not require any prior enrichment.
  • the low detection limit is of crucial importance for the fulfillment of certain guidelines or regulations, for example.
  • a significantly higher bacterial count limit e.g. 10 2 to 10 3 CFU / ml
  • a time-consuming and cost-intensive absence check of certain problem germs, ie pathogenic germs must be carried out.
  • the number of bacteria in the range from about 10 CFU per milliliter of sample volume or even less to about 10 8 CFU per milliliter of sample volume can be determined with the method according to the invention.
  • the sample should be diluted in advance accordingly, ie in a suitable manner, for the purpose of quantitative evaluations from a certain number of bacteria (generally from about 10 2 CFU per milliliter sample volume).
  • the method according to the invention is suitable in principle for the determination of any germs, in particular pathogenic germs of all kinds (e.g. microorganisms of all kinds, in particular unicellular microorganisms such as bacteria and fungi, e.g. yeasts or molds).
  • pathogenic germs of all kinds e.g. microorganisms of all kinds, in particular unicellular microorganisms such as bacteria and fungi, e.g. yeasts or molds.
  • the method according to the invention is suitable in principle for the quantitative and / or qualitative determination of germs in any products (i.e. media, matrices, solutions etc.), preferably filterable, in particular liquid and / or flowable products.
  • any products i.e. media, matrices, solutions etc.
  • filterable in particular liquid and / or flowable products.
  • solid products or products which cannot be filtered as such these have to be converted into a form accessible to the method according to the invention during sample preparation; this is done using methods known per se, for example by transferring to a solution or dispersion, comminution, extraction, etc.
  • the method according to the invention is suitable for the quantitative and / or qualitative determination of germs in foods, surfactant-containing products such as detergents and cleaning agents, agents for surface treatment, dispersion products, cosmetics, hygiene products and personal care products, pharmaceuticals, adhesives, cooling lubricants, lacquers and (lacquer -) Coagulation as well as raw materials and raw materials for the aforementioned products.
  • surfactant-containing products such as detergents and cleaning agents, agents for surface treatment, dispersion products, cosmetics, hygiene products and personal care products, pharmaceuticals, adhesives, cooling lubricants, lacquers and (lacquer -) Coagulation as well as raw materials and raw materials for the aforementioned products.
  • the method according to the invention is therefore suitable for all types of possible raw materials, intermediate and end products from the various fields, such as, for example, food, branded articles, cosmetics, adhesives, cooling lubricants (for example oily cooling lubricant emulsions); Process fluids from plants etc., with the restriction that the germs to be detected can be separated using a separation process, such as filtration or sedimentation. It does not matter whether the products are in solid or liquid form.
  • the process according to the invention is particularly suitable for automated implementation (for example in the context of production and / or quality control).
  • the method according to the invention is usually carried out as follows:
  • the sample with the germs to be determined quantitatively and / or qualitatively is introduced into a suitable sample vessel, the bottom of which is provided with a generally round membrane filter and which should be closable without germs.
  • the outer edge of the membrane filter lies on the sample vessel, so that the outer, concentric edge is not covered with germs.
  • germ-inhibiting or germ-killing substances or components e.g. preservatives or surfactants
  • these substances or components are first inactivated and / or removed by removing the sample with a suitable inactivation and / or conditioning solution is brought into contact for a period of time sufficient to enable the inactivation and / or removal of these substances or components.
  • the inactivation and / or conditioning solution is removed via the membrane filter by means of positive or negative pressure. Subsequently, excess or remaining inactivating and / or conditioning solution is removed via the membrane filter, if necessary by simple or multiple washing with water, usually by applying an overpressure or underpressure, so that the washing water is also on simple way is removed. This is followed by a marking of at least part of the germs present in the sample by means of at least one fluorescent marker.
  • the nuclei can, for example, be brought into contact with a solution or dispersion of the fluorescent marker for a time sufficient to mark the nuclei. The excess solution or dispersion of the fluorescent marker is then removed by applying an overpressure or underpressure again via the membrane filter.
  • the sample can be subjected to a single or multiple washing with water, buffer solutions or other liquids.
  • the membrane filter can then finally be separated from the sample vessel, so that a membrane filter coated with fluorescence-labeled germs, the outer edge of which is free of germs, results.
  • This can be fed directly to the fluorescence reflection photometry, ie generally without further preparation of the sample or treatment of the sample or filter.
  • the membrane filter covered with fluorescence-labeled nuclei is then irradiated with light of a suitable wavelength and, as it were, scanned or scanned.
  • the measured value determined correlates with the number of bacteria on the membrane filter or in the sample.
  • the following detergent products of different viscosities were examined using the device according to the invention:
  • a typical process sequence of the method according to the invention in the case of automated implementation includes, for example, that the user of a sample in a defined quantity (for example 1 ml) in a predetermined sample vessel is sealed germ-free and contains a conditioning or inactivation medium and a membrane filter at the outlet.
  • a conditioning or inactivation medium for example 1 ml
  • a membrane filter at the outlet.
  • the sample is shaken and thermostatted at a suitable temperature (for example 37 ° C.) depending on the type of germs.
  • the medium is filtered off through the membrane filter.
  • One or more washing steps with aseptic water, buffer solutions or other liquids follow automatically in order to wash out substances contained in the sample and then the marking with a fluorescent marker.
  • the labeling can be done with an unspecific fluorescent marker that attaches to all germs present in the sample (e.g. nucleotide fragments), or with a marker that uses the DEFT method to distinguish all living and "dead” microorganisms, or in the third case with a fluorescence marker based on FISH technology, which enables the selective staining of individual microorganisms by means of gene-labeled fluorescence probes.
  • excess marking solution is in turn automatically washed off with water, so that after completion of the automatic protocol, fluorescence-marked microorganisms are present on a membrane filter.
  • the intensity of the respective fluorescence and thus the number of microorganisms is then automatically determined with a fluorescence reflection photometer or spectrometer.
  • the filter membrane is irradiated with light of a suitable wavelength and the fluorescent light emitted by the marked microorganisms is detected in a correspondingly wavelength-resolved manner.
  • the subsequent evaluation compares the intensity in the edge area of the membrane filter, which should not be covered with germs, with the intensity in the area with marked microorganisms and calculates the number of germs present in the sample on the basis of a stored calibration.
  • An advantage of the method according to the invention is that it can be carried out automatically. By fully automating the entire process, the process can be carried out more easily, quickly and reproducibly. This has advantages in terms of costs, personnel expenditure and sensitivity. The high reproducibility when carrying out the examinations is also of great advantage.
  • Another advantage of the method according to the invention is that it does not require an epifluorescence microscope.
  • the epifluorescence microscope used according to the prior art by the simplified, namely fluorescence reflection photometric evaluation or detection method or system, the work and investment required for the user is reduced, and the evaluation of the samples can be carried out fully automatically (e.g. with an appropriate algorithm). Furthermore, the radiation exposure is reduced.
  • Yet another advantage of the method according to the invention is the use of standardized or conventional components: the overall system required for carrying out the method according to the invention is integrated in such a way that a large number of standardized or conventional components (vessels, media, filters etc.) are used can be reduced, thereby reducing operator effort and increasing the security of the process.
  • Another advantage of the method according to the invention is the simple detection and evaluation: the method according to the invention can be carried out, for example, in a suitable sample vessel, so that the marked germs are prepared on a suitable filter membrane.
  • a suitable sample vessel e.g. 8 mm diameter
  • a concentric, non-seeded edge enables referencing and internal standardization for each sample.
  • Another advantage of the method according to the invention is the speed with which the method according to the invention is carried out: the method according to the invention already allows the number of bacteria to be determined a time between a few (approx. 3-5) minutes, depending on the type of germs and their number. Conventional culture methods, on the other hand, take up to several days.
  • the high sensitivity of the method according to the invention is also of particular advantage: the method according to the invention allows the determination of germs even in high dilution. Accelerated culture methods are not sufficiently sensitive for a detection limit of 10 CFU per milliliter sample volume.
  • Another advantage of the method according to the invention is the fact that the fluorescence-labeled germs are only exposed to radiation for a relatively short time, since the measured values are acquired relatively quickly by fluorescence reflection photometry. This largely prevents the fluorescence marker from bleaching and thus falsifying the measurement result. This also avoids the killing of living germs, which is particularly important in the case of a live / dead differentiation.
  • “Scanning” or “scanning” the sample (more precisely the membrane filter coated with fluorescence-marked nuclei) in the context of the method according to the invention or in the context of fluorescence reflection photometric evaluation has further advantages: On the one hand, the Scanning large areas can be stimulated. On the other hand - in particular in comparison to a conventional fluorescence microscope with a limited detected area - a high excitation intensity is achieved with a brief comparison. The homogeneous illumination of the samples by scanning is particularly important for intensity measurements.
  • the entire process of determining the microbial load of a sample in the case of automated execution merely consists of filling the sample and starting the process. Subsequently, the User the numerical value for the germ load. The effort for sample preparation and measurement is minimal.
  • the system can thus also be ideally integrated into process systems for quality monitoring, assurance and documentation.
  • the present invention also relates to a device as described in claim 28 for the quantitative and / or qualitative determination of germs in a sample by a method with sample preparation and subsequent detection and / or evaluation, by means of the device in the course of sample preparation, at least a portion of the germs present in the sample can be marked by means of at least one fluorescence marker and the detection and / or evaluation can be carried out using the fluorescence marker, in particular for carrying out a method as described above.
  • sample container 1 shows the schematic representation of a device for carrying out a method for the quantitative and / or qualitative determination of germs in a sample.
  • the heart of this device is a sample container 1.
  • This sample container 1 is, as indicated schematically, in the device via various lines 2 to control valves 3 for ventilation 4 and compressed air 5 and via pumps 6 to connections for dye 7 ("fluorescent marker") and for Rinsing solution 8 included.
  • dye 7 dye 7
  • Rinsing solution 8 included.
  • Sterilfilter_2a integrated.
  • Fig. 1 shows the relationships that have been explained above in a schematic representation. The method of operation of such a device logically follows the procedure explained for the method claims.
  • a membrane filter 9 is arranged, which is indicated in the illustrated embodiment as a simple circular disk. This is designed so that it retains the germs to be detected and / or is impermeable to the germs to be detected.
  • a detection system 10 which is designed to carry out a fluorescence reflection photometric measurement and on which the sample receptacle 1 with the membrane filter 9 or preferably the membrane filter 9 removed from the sample receptacle 1 can be positioned for the purposes of detection and / or evaluation.
  • FIG. 1 shows the membrane filter 9 indicated at the bottom of the sample receptacle 1. This means that the membrane filter 9 in the embodiment shown in FIG. 1 can be removed downwards from the sample receptacle 1 in order to be fed to the detection system 10. How the arrangement looks in detail here is left to the constructive considerations of the expert.
  • the membrane filter 9 is a membrane filter having pores, in particular a polycarbonate membrane filter, and if, in particular, the size of the pores of the membrane filter 9 is smaller than the size of the germs present or to be determined in the sample to be determined.
  • a silicon microsieve is used instead of the membrane filter 9.
  • the advantages of using silicon microsieves have already been discussed in detail above.
  • a silicon microsieve can generally be used in the device according to the invention instead of a membrane filter. Both when arranged within the sample container 1 and when the membrane filter 9 is separated from the sample container 1 for the purpose of detecting the marked germs, it is advisable to work with a reference surface in order to be able to standardize the respective sample independently.
  • the membrane filter 9 arranged in the sample receptacle 1 has an area 12, in particular edge, which cannot or cannot be practically occupied during the sample preparation of germs and which serves as a reference surface when carrying out the detection and / or evaluation.
  • the area or edge 12 not covered with markings enables the internal standardization of the sample itself on the membrane filter 9.
  • the membrane filter 9 has an effective diameter for the filtration of approximately 5 to approximately 25 mm, in particular approximately 6 mm to approximately 12 mm, preferably approximately 8 mm to about 10 mm.
  • the membrane filter 9 retains the germs, that is to say the germs marked with the fluorescence marker remain on the top of the membrane filter 9. It is advisable to select the fluorescence marker in such a way that it is membrane-permeable with respect to the membrane filter 9, so that a favorable signal / noise ratio is achieved in the detection system 10. To handle the sample in the sample receptacle 1, one has to isolate the fluorescence-marked ("marked") germs on the membrane filter 9.
  • FIG. 1 shows a variant in which pressure is taken with compressed air 5.
  • FIG. 1 also shows that the device has a thermostat 13 for thermostatting the sample receptacle 1.
  • the thermostatic device 13 with a total of four receiving openings can be seen here, so that a total of four sample receiving containers 1 can be thermostatted simultaneously to the normally desired temperature, which is dependent on the target germs (e.g. 37 ° C.).
  • the evaluation device 11 controls a central controller 14, which forwards the measurement parameters to control electronics 15 for the excitation optics 16 and a positioning table 17.
  • the measuring object is located on the positioning table 17, that is to say here the membrane filter 9 is populated with marked germs.
  • Excitation light emitted by the excitation optics 16 onto the measurement object 18 is reflected as fluorescence light to a detection optics 19.
  • the detected signal is fed to measurement electronics 20, which in turn feeds the controller 14.
  • the present invention relates to the use according to the invention of the device according to the invention, as described above, as is the subject of the claims for use (claims 37 to 39).

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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
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Abstract

La présente invention concerne un procédé de détermination quantitative et/ou qualitative de germes dans un échantillon. Ce procédé consiste (a) à préparer l'échantillon et (b) à effectuer une détection et/ou une analyse. Dans l'étape (a),au moins une partie des germes présents dans l'échantillon est marquée au moyen d'au moins un marqueur fluorescent. La détection et/ou l'analyse de l'étape (b) sont effectuées par photométrie à réflexion et fluorescence. La présente invention concerne également un dispositif correspondant, conçu pour mettre en oeuvre ledit procédé.
EP03767723A 2002-12-17 2003-12-02 Procede et dispositif pour determiner des germes Withdrawn EP1573051A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10259302 2002-12-17
DE10259302A DE10259302A1 (de) 2002-12-17 2002-12-17 Verfahren und Vorrichtung zur Bestimmung von Keimen
PCT/EP2003/013567 WO2004055203A1 (fr) 2002-12-17 2003-12-02 Procede et dispositif pour determiner des germes

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EP1573051A1 true EP1573051A1 (fr) 2005-09-14

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US (1) US20060024710A1 (fr)
EP (1) EP1573051A1 (fr)
JP (1) JP2006509514A (fr)
CN (1) CN1726286A (fr)
AU (1) AU2003292168A1 (fr)
DE (1) DE10259302A1 (fr)
RU (1) RU2005122441A (fr)
WO (1) WO2004055203A1 (fr)

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JP4646716B2 (ja) * 2005-02-03 2011-03-09 三洋電機株式会社 微生物検出装置及び微生物検出用カセット
DE102005006237A1 (de) * 2005-02-10 2006-08-24 Henkel Kgaa Verfahren zur Bestimmung von Keimen
CN101720346B (zh) * 2007-05-29 2012-02-29 汉高有限公司 胶粘剂检测方法
US20080305514A1 (en) * 2007-06-06 2008-12-11 Alcon Research, Ltd. Method for detecting microbes
US20110263044A1 (en) * 2008-07-31 2011-10-27 Eads Deutschland Gmbh Device and method for the automatic detection of biological particles
JP5030897B2 (ja) * 2008-08-28 2012-09-19 メタウォーター株式会社 微生物計測方法
JP5779837B2 (ja) * 2010-02-24 2015-09-16 株式会社Ihi 微生物検出方法
CN103063633A (zh) * 2012-12-25 2013-04-24 南昌大学 一种快速自动检测水中细菌的系统
CN104117077A (zh) * 2013-04-27 2014-10-29 昆山研达电脑科技有限公司 电子产品消毒系统
DK3108249T3 (en) 2014-02-18 2019-03-18 Laboratory Corp America Holdings METHOD AND SYSTEMS FOR QUICK DETECTION OF MICROORGANISMS USING FREE ANTIBODIES
CN104297038B (zh) * 2014-10-10 2017-05-31 丁昊 结核分枝杆菌荧光抗酸染色染液
US11248993B2 (en) * 2018-02-15 2022-02-15 Colorado State University Research Foundation Systems and methods for direct particle sampling
CN109082456B (zh) * 2018-08-24 2021-07-23 张家口健垣科技有限公司 一种自动化食品微生物检测前处理方法及装置
FR3086951B1 (fr) * 2018-10-05 2021-02-26 Redberry Methode et dispositif pour la detection d'au moins un microorganisme selon sa cinetique de marquage, et support de detection
CN117368470A (zh) * 2023-10-09 2024-01-09 南通如日纺织有限公司 一种纺织品抗菌检测与质量评估系统

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Also Published As

Publication number Publication date
DE10259302A1 (de) 2004-07-08
CN1726286A (zh) 2006-01-25
WO2004055203A1 (fr) 2004-07-01
AU2003292168A1 (en) 2004-07-09
JP2006509514A (ja) 2006-03-23
US20060024710A1 (en) 2006-02-02
RU2005122441A (ru) 2006-02-20

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