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WO1999025465A1 - Filtres a diaphragme en gelatine et leur procede de production - Google Patents

Filtres a diaphragme en gelatine et leur procede de production Download PDF

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Publication number
WO1999025465A1
WO1999025465A1 PCT/EP1998/007118 EP9807118W WO9925465A1 WO 1999025465 A1 WO1999025465 A1 WO 1999025465A1 EP 9807118 W EP9807118 W EP 9807118W WO 9925465 A1 WO9925465 A1 WO 9925465A1
Authority
WO
WIPO (PCT)
Prior art keywords
gelatin
membrane
osmoprotective
drawing solution
membrane filters
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.)
Ceased
Application number
PCT/EP1998/007118
Other languages
German (de)
English (en)
Inventor
Elmar Herbig
Helmut Jaschhof
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
Priority to EP98955551A priority Critical patent/EP1019179A1/fr
Priority to JP2000520893A priority patent/JP2001523549A/ja
Publication of WO1999025465A1 publication Critical patent/WO1999025465A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2202—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling
    • G01N1/2205—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling with filters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/1411—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes containing dispersed material in a continuous matrix
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/74—Natural macromolecular material or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2202—Devices for withdrawing samples in the gaseous state involving separation of sample components during sampling
    • G01N2001/222—Other features
    • G01N2001/2223—Other features aerosol sampling devices

Definitions

  • the invention relates to gelatin membrane filters for collecting microorganisms from gases while maintaining an increased number of viable microorganisms, and to processes for their production.
  • the gelatin membrane filters according to the invention can be used to collect 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 number of bacteria in the media. For example, they are used in combination with an air sampler to collect bacteria, viruses, yeasts and fungi in order to determine their concentration in rooms. Such monitoring is a prerequisite for the timely initiation of measures to protect people and products from damage caused by excessive microorganism concentrations, for example in the ambient air.
  • the air is regularly checked for its germ content. Since it is usually filtered air, which naturally has a low microorganism content, large volumes usually have to be examined in order to collect sufficient germs for meaningful results. To do this, an air sample is filtered through a suitable filter.
  • a suitable filter For this purpose, sterile membrane filters with pore sizes in the microfiltration range, predominantly based on cellulose nitrate, cellulose acetate and gelatin, are used as filters, as are described, for example, in DE-PS 11 73 640. Gelatin membrane filters on which the retained microorganisms are to be kept moist and capable of reproduction are particularly suitable.
  • the gelatin membrane filters can either be placed on an agar medium are incubated, with microorganism colonies growing out of the individual germ aggregates collected (the gelatin membrane filter liquefies and disappears and the microorganism colonies can be counted directly on the agar), or dissolved in a sterile solution such as a peptone water or a physiological saline solution, so that portions can be incubated on different nutrient media.
  • a sterile solution such as a peptone water or a physiological saline solution
  • DE-PS 11 73 640 also generally suggests that, in the manufacture of a gelatin membrane, nutrients for the cultivation of microorganisms, as well as buffering substances, dyes or chemicals, which are suitable for containing biologically toxic substances in the air such as traces of heavy metals or harmful gases.
  • the disadvantage is that, despite the addition of nutrient media or absorbent substances to the gelatin membranes, significantly fewer colonies can grow and be determined from the microorganisms collected on them than was actually present in the contaminated medium.
  • the invention is therefore based on the object of creating gelatin membrane filters for collecting microorganisms from gases in which the number of detectable microorganisms is significantly increased than in the gelatin membrane filters of the prior art, and to find a method for producing these gelatin membrane filters.
  • gelatin membrane filters which are characterized by the features of the independent claims.
  • Advantageous developments of the invention are identified by the features of the dependent claims.
  • osmoprotective substances include, for example, inositol, betaine, lycin, oxyneurin and others. It is known that these substances counteract the dehydration of the microorganisms, an osmotic stress. Obviously, some of the microorganisms lose on collection Gelatin membrane filters so much of cellular water that it is no longer viable and is no longer detectable through colony formation through incubation.
  • the gelatin membrane filters contain the osmoprotective substances in an amount which leads to at least a doubling of the number of viable microorganisms in comparison to gelatin membrane filters without the osmoprotective substances if the gelatin membrane filters are flowed through for one minute and a further 8 minutes with an air flow of 7.5 m 3 / h found that equal proportions of osmoprotective substances in the gelatin membrane filters with different amounts of gases flowing through the gelatin membrane filters with the same number of collected microorganisms, to different proportions of viable Mi. lead to microorganisms.
  • a content of trimethylammonioacetate has proven to be particularly suitable as an osmoprotective substance in the gelatin membrane filters, in particular if this substance is present in the membrane drawing solution from which the membrane is obtainable in a proportion of 0.005 to 0.75% based on the content of the gelatin is.
  • the membrane drawing solution is an aqueous homogeneous solution which contains gelatin with a share of 4.6 to 5.6% in the total membrane drawing solution and ethanol with a share of 38 to 46% in the total membrane drawing solution. It is advantageous for the stability of the membrane if the gelatin is additionally stabilized by a binder.
  • the binder can be, for example, polyvinyl alcohol or starch.
  • the osmoprotective substances penetrate the gelatin matrix as evenly as possible. If these substances are only on the outer surface of the gelatin membrane matrix, there is a risk that part of the osmoprotective substances in the inner matrix during long-term storage migrates and is only limited or no longer effective for the collected microorganisms. Therefore, in a preferred method according to the invention, the osmoprotective substances are incorporated into the membrane matrix in a homogeneous distribution via a phase inversion process, by means of which the gelatin membrane filters are produced. For this purpose, the osmoprotective substances are introduced into the membrane drawing solution.
  • the osmoprotective substances are introduced into the solidification bath during the formation of the gelatin membrane filter.
  • the gelatin membrane filters are not yet fully formed in this first solidification bath, so that the osmoprotective substances can penetrate into the entire membrane matrix.
  • the gelatin membrane filters according to the invention Compared to the gelatin membrane filters of the prior art, including those available according to DE-PS 11 73 640, the gelatin membrane filters according to the invention not only have increased mechanical stability, which significantly improves their handling, but they also have a significantly higher air flow rate is at least a factor of 2 larger. They are therefore particularly suitable for high air throughputs for germ collection. This makes it possible to use the gelatin membrane filters according to the invention to reduce the time for the collection of microorganisms or to test extremely lightly contaminated gases because larger volumes can be filtered without the gelatin membrane filters being impaired or destroyed in their action.
  • the process for producing the gelatin membrane filter according to the invention is carried out by a) preparing an aqueous homogeneous membrane drawing solution which contains at least gelatin and ethanol, b) spreading a thin film from the membrane drawing solution on a base, c) the thin film to form a gelled one Phase exposed to air, d) the gelled phase is placed in a precipitation bath for post-treatment and e) the gelatin membrane filter is dried, and is characterized in that f) at least one osmoprotective substance is added to the membrane drawing solution.
  • the membrane drawing solution contains between 0.005 and 0.75% trimethylammonioacetate, based on the gelatin.
  • a binder is introduced over the membrane drawing solution in a proportion of 0.02 to 0.1% of the total membrane drawing solution.
  • methyl acetate with an alcohol, in particular methanol 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 solidification bath for a period of one to three hours at room temperature before it is transferred to a second solidification bath made of pure methyl acetate. Drying and sterilization, preferably with gamma rays, follow.
  • gelatin membrane filter To produce a gelatin membrane filter, 200g gelatin (Gelita powder gelatin 250 Bloom, DGF Deutsche Gelatine-Fabriken Stoess AG) and 2g polyvinyl alcohol (Mowiol type 18-88, Hoechst AG) are dissolved in 2000g water with stirring at 60 ° C for 1 hour and then with 1645g Ethanol and 0.02g trimethylammonioacetate dissolved in 10g water.
  • the membrane drawing solution heated to 33 ° C., is spread out on a base to form a 350 ⁇ m thick film and exposed to air with a relative humidity of 45% at room temperature for 5 minutes.
  • the gelled film, together with the support, is placed in a first solidifying bath consisting of methyl acetate with a content of 14% methanol for 3 hours and then in a second solidifying bath made of pure Methyl acetate introduced for a period of 3 hours.
  • the gelatin membrane filter is removed from the base and dried.
  • the gelatin membrane filters obtained in this way have a pore diameter of about 3 ⁇ m and an air flow of 138 1 / min cm 2 bar.
  • the gelatin membrane filters obtained according to Example 1 are designated Sample A.
  • Samples B, C, D and E are prepared analogously to Example 1, only the mass of trimethylammonioacetate in the membrane drawing solution being varied (table, line 2).
  • a comparative sample F is produced analogously to Example 1, but the addition of trimethylammonioacetate is dispensed with.
  • the gelatin membrane filters A to F produced according to Examples 1 to 6 were exposed to the same amount of Escherichia coli ATCC 8739 germs for one minute and air for a further period of 8 minutes with a flow of 7.5 m 3 / h sucked through the loaded gelatin membrane filter.
  • the gelatin membrane filters were applied to agar nutrient cardboard slices containing tryptone soy broth agar (TSBA nutrient broth), incubated overnight at 37 ° C. and the colony number was determined microscopically. The results are shown in rows 3 and 4 of the table.
  • Escherichia coli ATTCC 8739 was used in the tests. This strain is widely used in microbiological QA departments for pharmaceutical validation testing. A frozen vial with a concentration of approximately 10 3 colony-forming units per milliliter [CFU / ml] was used. In each case 0.1 ml of the thawed suspension was spread on plates with tryptone-soy broth agar (TSBA nutrient broth) in two identical parallels. The agar plates were incubated overnight at 37 ° C. ⁇ 2 ° C., and the bacteria were determined microscopically after the Gram staining. The slides were archived as reserve samples.
  • TSBA nutrient broth tryptone-soy broth agar
  • a culture suspension was prepared by inoculating 50 ml of the above nutrient broth with a colony previously grown on a plate in a 250 ml vessel. The vessel was left at 37 ° C overnight Incubated ⁇ 2 ° C with shaking in a water bath. The suspension was examined using cultivation techniques, transferred to sterile bottles and stored at 4 ° C ⁇ 2 ° C until further use.
  • Bacillus subtilis var niger spores (NCTC 10073) were used as microbiological tracers. These spores were washed 3 times by centrifugation and resuspended with sterile, distilled water. The concentration of the B. subtilis stock solution was approx. 2 x 10 u CFU / ml. A 1 liter suspension of Bacillus subtilis was prepared for use in these tests by diluting the stock solution to approximately 2.5 x 10 9 CFU / ml with sterile, distilled water and 40 minutes at 60 ° C to kill any that were present vegetative organisms was heated. The determination of the spore suspension used for the tests gave a final concentration of B. subtilis spores of 2.26 x 10 9 CFU / ml.
  • the suspension of E. coli ATCC 8739 was kept at 4 ° C + 2 ° C for two weeks before use. A subsequent determination using cultivation techniques showed that the E. coli concentration remained constant during this time. Preliminary tests were carried out with different concentrations of the test and tracer germs in order to determine the suitable concentrations for the final tests.
  • the concentration of the stock suspension of E. coli ATCC 8739 was 2.1 x 10 10 CFU / ml.
  • a nebulizing suspension of 10 ml was prepared by mixing 5 ml suspensions of E. coli (diluted with distilled water, giving 7.6 x 10 3 to 2.8 x 10 4 CFU / ml) and a 5 ml Suspension of B. subtilis (diluted with distilled water, gives 4.0 x 10 3 CFU / ml). The final suspension was aerosolized within 30 minutes.
  • the test bench consists of a nebulizer of the Collison 3-jet nebulizer type for the generation of bacterial aerosols in a closed and sealed
  • the aerosol chamber is connected to a 1 meter long stainless steel pipe system with an inner diameter of 45 mm.
  • the Sartorius filter head Air sampler is connected to this pipeline using an appropriate adapter.
  • the entrance to the chamber is protected by a powerful air filter.
  • the Sartorius MD 8 airscan air sampler (serial no. 9607001) with calibration certificate, hose and filter head was operated in accordance with the operating instructions.
  • the MD8 airscan was set to an air flow of 7.5 m 3 / h.
  • the MD8 airscan was turned on and after 15 seconds the Collison nebulizer was activated by connecting to a pressurized gas line at 180 kPa. The nebulizer was activated for 1 minute and the MD8 was then switched off.
  • gelatin membrane filters were placed on the TSBA plates either directly after this 1-minute exposure to germ-containing aerosol (sucked in with the aid of the air germ collector MD8) or after a further 8-minute exposure to filtered air and incubated and measured as described above.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne des filtres à diaphragme destinés à piéger des micro-organismes contenus dans des gaz pour obtenir une plus grande proportion de micro-organismes capables de vivre sur ces filtres. L'invention concerne également leur procédé de production. Pour ce faire, les filtres à diaphragme en gélatine contiennent des substances osmoprotectives qui sont ajoutées à la solution d'attraction constituant la membrane ou bien dans un premier bain de solidification. Les filtres à diaphragme en gélatine selon l'invention, sont utilisés dans les industries pharmaceutique, biotechnologique et alimentaire, dans la protection de l'environnement, la gestion des déchets et les installations médicales afin de déterminer le nombre de bactéries des différents milieux. Par exemple en association avec un appareil servant à piéger les germes contenus dans l'air, ces filtres permettent de piéger des bactéries, des virus, des levures et des champignons en vue de déterminer leur concentration dans les pièces.
PCT/EP1998/007118 1997-11-13 1998-11-07 Filtres a diaphragme en gelatine et leur procede de production Ceased WO1999025465A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98955551A EP1019179A1 (fr) 1997-11-13 1998-11-07 Filtres a diaphragme en gelatine et leur procede de production
JP2000520893A JP2001523549A (ja) 1997-11-13 1998-11-07 ゼラチン膜フィルタおよびその製造方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19750215A DE19750215C1 (de) 1997-11-13 1997-11-13 Gelatinemembranfilter und Verfahren zu ihrer Herstellung
DE19750215.6 1997-11-13

Publications (1)

Publication Number Publication Date
WO1999025465A1 true WO1999025465A1 (fr) 1999-05-27

Family

ID=7848554

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/007118 Ceased WO1999025465A1 (fr) 1997-11-13 1998-11-07 Filtres a diaphragme en gelatine et leur procede de production

Country Status (4)

Country Link
EP (1) EP1019179A1 (fr)
JP (1) JP2001523549A (fr)
DE (1) DE19750215C1 (fr)
WO (1) WO1999025465A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9834806B2 (en) 2008-06-27 2017-12-05 Hitachi Plant Services Co., Ltd. Microbe-collecting carrier cartridge, carrier treating apparatus, and method of measuring microbes
EP3336515A1 (fr) 2016-12-14 2018-06-20 Airbus Defence and Space GmbH Échantillonneur d'air portatif comprenant un magasin de filtres pour contenir et positioner lesdits filtres
CN118558067A (zh) * 2024-04-30 2024-08-30 青岛大学 空气微生物绿色捕获膜材料及其制备方法和应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19814715A1 (de) * 1998-04-02 1999-10-07 Sartorius Gmbh Gelatinemembranfilter, Verfahren zu ihrer Herstellung und ihre Verwendung
JP4266637B2 (ja) * 2001-02-05 2009-05-20 ミリポア・コーポレイション 微生物の検出
GB2387130A (en) 2002-04-04 2003-10-08 Fluid Technologies Plc Hollow fibre filter membrane unit with microorganism detector, and associated usage
FR2917095B1 (fr) * 2007-06-07 2009-07-17 Biomerieux Sa Dispositif de lyse de microorganismes presents dans un echantillon environnemental ou clinique et d'extraction des acides nucleiques desdits microorganismes aux fins d'analyse.
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
DE102014203855A1 (de) * 2014-03-03 2015-09-03 Biotec Gmbh, Umwelt-Analytik-Beratung-Service Applikation zur PCR-Analytik von luftgetragenen Nukleinsäuren

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1173640B (de) * 1959-05-21 1964-07-09 Membranfiltergesellschaft G M Verfahren zur Herstellung von Membranfiltern
WO1992008355A1 (fr) * 1990-11-13 1992-05-29 Liphatech, Inc. Procede pour la preparation de produits agricoles bacteriens
WO1996036693A1 (fr) * 1995-05-19 1996-11-21 Phytera, Inc. Manipulation de cultures de cellules et de tissus vegetaux
WO1997026365A2 (fr) * 1996-01-19 1997-07-24 Dekalb Genetics Corporation Mais transgenique a teneur accrue en mannitol

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1173640B (de) * 1959-05-21 1964-07-09 Membranfiltergesellschaft G M Verfahren zur Herstellung von Membranfiltern
WO1992008355A1 (fr) * 1990-11-13 1992-05-29 Liphatech, Inc. Procede pour la preparation de produits agricoles bacteriens
WO1996036693A1 (fr) * 1995-05-19 1996-11-21 Phytera, Inc. Manipulation de cultures de cellules et de tissus vegetaux
WO1997026365A2 (fr) * 1996-01-19 1997-07-24 Dekalb Genetics Corporation Mais transgenique a teneur accrue en mannitol

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9834806B2 (en) 2008-06-27 2017-12-05 Hitachi Plant Services Co., Ltd. Microbe-collecting carrier cartridge, carrier treating apparatus, and method of measuring microbes
EP3336515A1 (fr) 2016-12-14 2018-06-20 Airbus Defence and Space GmbH Échantillonneur d'air portatif comprenant un magasin de filtres pour contenir et positioner lesdits filtres
CN118558067A (zh) * 2024-04-30 2024-08-30 青岛大学 空气微生物绿色捕获膜材料及其制备方法和应用

Also Published As

Publication number Publication date
EP1019179A1 (fr) 2000-07-19
JP2001523549A (ja) 2001-11-27
DE19750215C1 (de) 1999-02-04

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