EP1066403A1 - Procede pour deceler des micro-organismes dans des gaz - Google Patents

Procede pour deceler des micro-organismes dans des gaz

Info

Publication number
EP1066403A1
EP1066403A1 EP99917928A EP99917928A EP1066403A1 EP 1066403 A1 EP1066403 A1 EP 1066403A1 EP 99917928 A EP99917928 A EP 99917928A EP 99917928 A EP99917928 A EP 99917928A EP 1066403 A1 EP1066403 A1 EP 1066403A1
Authority
EP
European Patent Office
Prior art keywords
membrane
gelatin
microorganisms
organisms
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Withdrawn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
EP99917928A
Other languages
German (de)
English (en)
Inventor
Elmar Herbig
Dieter Nussbaumer
Khuong To Vinh
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.)
Sartorius AG
Original Assignee
Sartorius AG
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 Sartorius AG filed Critical Sartorius AG
Publication of EP1066403A1 publication Critical patent/EP1066403A1/fr
Withdrawn legal-status Critical Current

Links

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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus
    • Y10S435/836—Bacillus licheniformis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Definitions

  • the invention relates to a method for the detection of microorganisms in gases, in particular air and gelatin membrane filters for carrying out the method, which are characterized by a particular purity
  • the method can be used for the detection of microorganisms from gaseous media, in particular from air, in the pharmaceutical, biotechnological and food industries, in environmental protection, in waste management and in medical facilities for determining the bacterial count of the media for the collection of bacteria, spores, viruses, yeasts and fungi (microorganisms) in order to be able to determine their concentration in rooms by means of the method according to the invention.
  • Such surveillance is a prerequisite for the timely initiation of measures to prevent people and products from being damaged by excessive concentrations of microorganisms Preserve example in indoor air
  • the air is regularly checked for its microbial content. As a rule, it is filtered air, which naturally has a low content of microorganisms. Large volumes usually have to be examined in order to collect sufficient germs for meaningful results
  • Air germ collecting device as it is sold by the company Sartorius AG under the name "MD8 airscan" is filtered through a suitable filter.
  • MD8 airscan sterile membrane filters with pore sizes in the microfiltration range, mainly based on gelatin, are used, as described, for example, in DE -PS 11 73 640 are described
  • Gelatin membrane filters on which the retained microorganisms are to be kept moist and capable of reproduction are particularly suitable.
  • Gelatin membranes on which the collected microorganisms have a particularly high viability due to the addition of osmoprotective substances are described in DE-PS 197 50 215.
  • the gelatin membrane filters can either incubated on an agar floor, whereby microorganism colonies grow out of the individual collected germ aggregates (the gelatin membrane filter liquefies and disappears and the microorganism colonies can be counted directly on the agar), or in a sterile solution, such as a peptone water or a physiological saline solution be dissolved so that subsets can be incubated on different nutrient media.
  • the analysis results are only available relatively late because the colony growth is usually takes up to 7 days
  • a rapid microbiological analysis process rapid test system for the detection of microorganisms
  • ChemScan the results are available after only 30 to 90 minutes, because this process detects the individual microorganism and the otherwise time-consuming process Microorganism multiplication in the incubator no longer required
  • an aqueous sample containing the living microorganisms is filtered through a 0.22 ⁇ m or 0.45 ⁇ m analysis membrane in order to retain the microorganisms contained in the sample on the analysis membrane.
  • the analysis membrane is kept at 30 ° for 30 minutes C placed on an absorption pad which is soaked with a labeling liquid
  • the labeling liquid has the ability to interact with the cell cytoplasm of living microorganisms via an enzyme-controlled reaction under yellowish fluorescence Microscope, preferably with a laser scan system, every single microorganism can be recognized on the analysis membrane.
  • the gelatin membrane filters, on which the microorganisms were collected from the air only by dissolving the Gelatin membrane filter in a water solution, such as a peptone-water solution, is to be transferred to a sample and this sample is to be analyzed as described above.
  • a water solution such as a peptone-water solution
  • the invention is therefore based on the object of proposing a method for the rapid detection of microorganisms and gelatin membrane filters suitable for this method for collecting these microorganisms from gases
  • the object is achieved by a method for the rapid detection of microorganisms in gases, in which a) the gas for collecting the microorganisms is passed through a gelatin membrane filter free of particles with a diameter of large 0.45 ⁇ m, preferably 0.1 ⁇ m, b) the gelatin membrane filter with the collected microorganisms is dissolved in an aqueous solution which contains an enzymatic marker which produces fluorescence with the microorganisms, c) the solution obtained with the microorganisms through an analysis membrane of a pore size in the range from about 0.2 to about 0.45 ⁇ m is filtered and d) the fluorescent microorganisms remaining on the analysis membrane are paid out
  • gelatin membrane filters are suitable for carrying out the process which are free from particles with sizes which are retained by microfiltration membranes with a pore diameter of at most 0.45 ⁇ m, preferably of at most 0.2 ⁇ m. That is, such Gelatin membrane filters are free of microorganisms, including dead microorganisms.
  • the gelatin membrane filters in a preferred embodiment contain osmoprotective substances, for example methyl methylammonioacetate (betaine), in order to significantly increase the viability of the living microorganisms collected on the gelatin membrane filters.
  • a gelatin freed from such particles as the starting material for the production of the gelatin membrane filter.
  • a solution of commercially available gelatin is filtered through a microfiltration membrane with a pore size of up to approximately 0.45 ⁇ m, preferably of up to about 0.2 ⁇ m and particularly preferably up to 0.1 ⁇ m. It has proven to be expedient to filter the already formulated membrane drawing solution under pressure through a microfiltration membrane of the aforementioned pore size. This ensures that in one work step each component, from which the membrane drawing solution was produced, all particles which have a size are removed so that they are retained by microfiltration membranes of the pore size mentioned.
  • Pressure filtration is to be preferred because, compared to vacuum filtration, volatile solution is used can not evaporate and the quantitative composition of the membrane drawing solution is not changed
  • a hydrophilic, cross-linked 0.2 ⁇ m cellulose hydrate membrane which is marketed under the name Hydrosart by Sartonus AG, Gottingen, has proven to be particularly suitable for filtration.
  • the gelatin membrane filters according to the invention are particularly suitable for rapid microbiological analysis methods for the direct determination of individual microorganisms, in particular living microorganisms, which show fluorescence signals with marking agents.
  • the gelatin membrane filter on which the microorganisms have been collected from gases is brought into a watery solution for dissolution an analysis membrane with pore sizes in the range from about 0.2 to at most 0.45 ⁇ m is filtered in order to retain the microorganisms collected on the gelatin membrane filter on the analysis membrane.
  • the use of microsieve membrane types, as described, for example, in WO 95/13860 AI are preferred, in particular nuclear track membranes with pore sizes of about 0.2 ⁇ m.
  • the use of such analysis membranes is advantageous because they are characterized by a narrow pore size distribution and by the straight pore profile across the membrane surface are characterized by a high filtration rate and because microorganisms remain completely on the surface of these membranes and cannot penetrate into the pore structure as with conventional microfiltration membranes
  • the labeling fluid is added directly to the aqueous solution used to dissolve the gelatin membrane filter, and after a labeling time to be tested, the labeled microorganisms are collected on the analysis membrane.
  • an aqueous homogeneous membrane drawing solution consisting at least of gelatin with a share of 4.5 to 5.6% of the total membrane drawing solution, made of ethanol with a share of 38 to 46 % of the total membrane drawing solution and optionally made from a binder with a content of 0.02 to 0.1% of the total membrane drawing solution.
  • Polyvinyl alcohol for example, can be used as the binding agent.
  • the membrane drawing solution is pressed under pressure through a microfiltration membrane with a maximum pore size of 0.45 ⁇ , preferably filtered to 0.2 ⁇ m, then a thin film of the membrane drawing solution is spread out on a support and the thin film is exposed to air to form a gelled phase.
  • the gelled phase is then introduced into a precipitation bath consisting of methyl acetate for post-treatment
  • the membrane drawing solution additionally contains at least one osmoprotective substance with a proportion of 0.005 to 0.75% based on the gelatin content.
  • trimethylammonioacetate can be used as the osmoprotective substance
  • methyl acetate with an alcohol in particular methanol
  • a proportion of 10 to 20% of the total first hardening bath is used as the first hardening bath with a proportion of 10 to 20% of the total first hardening bath.
  • the membrane remains in this first consolidation bath for a period of one to three hours at room temperature before it is transferred to a second consolidation bath made of pure methyl acetate. Drying and sterilization, preferably with gamma rays, follow. The invention is explained using the following exemplary embodiment
  • Example 1 To produce a gelatin membrane filter, 200 g of commercially available gelatin powder and 2 g of polyvinyl alcohol as a binder (Mowiol type 18-88, Hoechst AG) are dissolved in 2000 g of water with stirring at 60 ° C. for one hour and then with 1645 g ethanol and 0.02 g T ⁇ methylammonioacetat as an osmoprotective substance dissolved in 10 g water.
  • This membrane drawing solution is in dead-end mode at a pressure difference of 3 bar and a temperature of about 40 ° C through a cross-linked cellulose hydrate membrane with a pore size of 0.2 ⁇ m filtered
  • the filtered membrane solution heated to 21 ° C., is spread out on a base to form a film of thickness 350 ⁇ m and exposed to air with a relative humidity of about 45% at room temperature for about 5 minutes.
  • the gelled film is mixed with the Underlay in a first solidifying bath, which consists of methyl acetate with a content of 14% methanol, for 3 hours and then placed in a second solidifying bath made of pure methyl acetate for 3 hours.
  • the gelatin membrane filter is removed from the base, dried and sterilized with gamma rays
  • the gelatin membrane filter thus obtained has an air flow of 140 1 / min cm 2 bar.
  • a gelatin membrane filter produced according to this example and containing 150 mg gelatin was dissolved in 50 ml Peptone water and mixed with 100 ⁇ l delvolase enzyme and fluorescent dye. After an incubation period of The solution was filtered from cellulose nitrate over a 0.4 ⁇ m analysis membrane for 5 minutes at 37 ° C. and the analysis membrane was evaluated under the microscope. No microorganisms were found
  • gelatin membrane filters according to the invention for collecting airborne germs together with a rapid test system for the detection of microorganisms, such as the ChemScan system, represent an extremely precise detection method for very low bacterial counts, which makes them particularly suitable for quality control questions in the pharmaceutical industry and biotechnology by preserving the microbiological Analysis results in just a few minutes support a just-in-time production. There are practically no waiting times for the release of production rooms, since microbiological contamination can be detected in a very short time and, as with conventional methods, incubation times are no longer required for up to a week to arrive at the microbiological finding. This increases the production security significantly

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé permettant de déceler rapidement des micro-organismes dans des gaz, en particulier dans l'air, ainsi qu'un filtre à membrane pour la mise en oeuvre de ce procédé, remarquable en ce qu'il présente une pureté particulière. A cet effet, le gaz destiné à collecter les micro-organismes est envoyé à travers un filtre à membrane en gélatine, exempt de particules de diamètre supérieur à 0,45 mu m, de préférence égal à 0,1 mu m, le filtre à membrane en gélatine est dissous, avec les micro-organismes collectés, dans une solution aqueuse renfermant un agent de marquage enzymatique générant une fluorescence avec les micro-organismes, la solution obtenue, avec les micro-organismes, est filtrée sur membrane pour analyse d'une porosité comprise entre environ 0,2 et environ 0,45 mu m, et les micro-organismes fluorescents demeurant sur la membrane pour analyse sont dénombrés. L'invention est utilisée dans l'industrie pharmaceutique, en biotechnologie et dans l'industrie alimentaire, pour la protection de l'environnement, dans le secteur du traitement des résidus et dans des dispositifs médicaux pour la détermination de la charge de germes bactériens dans des milieux.
EP99917928A 1998-04-02 1999-03-30 Procede pour deceler des micro-organismes dans des gaz Withdrawn EP1066403A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19814715 1998-04-02
DE19814715A DE19814715A1 (de) 1998-04-02 1998-04-02 Gelatinemembranfilter, Verfahren zu ihrer Herstellung und ihre Verwendung
PCT/EP1999/002194 WO1999051765A1 (fr) 1998-04-02 1999-03-30 Procede pour deceler des micro-organismes dans des gaz

Publications (1)

Publication Number Publication Date
EP1066403A1 true EP1066403A1 (fr) 2001-01-10

Family

ID=7863310

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99917928A Withdrawn EP1066403A1 (fr) 1998-04-02 1999-03-30 Procede pour deceler des micro-organismes dans des gaz

Country Status (5)

Country Link
US (1) US6562583B1 (fr)
EP (1) EP1066403A1 (fr)
JP (1) JP2002510501A (fr)
DE (1) DE19814715A1 (fr)
WO (1) WO1999051765A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062941A1 (fr) * 2001-02-05 2002-08-15 Micap Plc Detection de micro-organismes
GB2387130A (en) 2002-04-04 2003-10-08 Fluid Technologies Plc Hollow fibre filter membrane unit with microorganism detector, and associated usage
US20040002126A1 (en) * 2002-06-28 2004-01-01 Michel Houde Method, device and system for detecting the presence of microorganisms
WO2007035763A2 (fr) * 2005-09-20 2007-03-29 The Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University Méthodes pour détecter et caractériser des micro-organismes
JP5245379B2 (ja) * 2007-12-04 2013-07-24 株式会社日立プラントテクノロジー 捕集デバイス、及びそれを用いる分析システム
US8628953B2 (en) 2007-11-29 2014-01-14 Hitachi Plant Technologies, Ltd. Capturing carrier, capturing device, analysis system using the same, and method for capturing and testing microorganisms
JP5252176B2 (ja) * 2007-11-29 2013-07-31 株式会社日立プラントテクノロジー 捕集担体、捕集ユニット、捕集装置及び捕集・検査方法
EP2241875A1 (fr) * 2009-04-14 2010-10-20 Koninklijke Philips Electronics N.V. Concentration élevée de micro-objets organiques pour l'imagerie microscopique
JP5738244B2 (ja) * 2012-08-01 2015-06-17 株式会社日立製作所 検査方法
CN103323590B (zh) * 2013-06-08 2015-05-06 上海云泽生物科技有限公司 一种基于纤维膜捕集分离的定量检测装置及其检测方法
DE102014203855A1 (de) * 2014-03-03 2015-09-03 Biotec Gmbh, Umwelt-Analytik-Beratung-Service Applikation zur PCR-Analytik von luftgetragenen Nukleinsäuren
WO2016078667A1 (fr) * 2014-11-17 2016-05-26 Syddansk Universitet Ensemble d'évaluation de la perméabilité d'un médicament présentant des propriétés biomimétiques modulables
US10876949B2 (en) 2019-04-26 2020-12-29 Honeywell International Inc. Flow device and associated method and system
EP3865583A1 (fr) * 2020-02-14 2021-08-18 Testo bioAnalytics GmbH Procédé de détection des microorganismes et porte-échantillons en forme de disque
US11835432B2 (en) 2020-10-26 2023-12-05 Honeywell International Inc. Fluid composition sensor device and method of using the same
US20220364973A1 (en) * 2021-05-13 2022-11-17 Honeywell International Inc. In situ fluid sampling device and method of using the same

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Publication number Priority date Publication date Assignee Title
DE1173640B (de) * 1959-05-21 1964-07-09 Membranfiltergesellschaft G M Verfahren zur Herstellung von Membranfiltern
FR2628530B1 (fr) * 1988-03-08 1994-01-28 Chemunex Sa Appareil et procede de detection et de numeration de particules fluorescentes, portees par un support solide
US5739004A (en) * 1993-05-20 1998-04-14 Minnesota Mining And Manufacturing Company Biological sterilization indication for use with or without test pack materials or devices
US5498526A (en) * 1993-08-25 1996-03-12 Abtox, Inc. Bacillus circulans based biological indicator for gaseous sterilants
DE19750215C1 (de) * 1997-11-13 1999-02-04 Sartorius Gmbh Gelatinemembranfilter und Verfahren zu ihrer Herstellung

Non-Patent Citations (1)

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Title
See references of WO9951765A1 *

Also Published As

Publication number Publication date
DE19814715A1 (de) 1999-10-07
US6562583B1 (en) 2003-05-13
JP2002510501A (ja) 2002-04-09
WO1999051765A1 (fr) 1999-10-14

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