EP1019179A1 - Gelatine membrane filters and method for producing the same - Google Patents

Gelatine membrane filters and method for producing the same

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Publication number
EP1019179A1
EP1019179A1 EP98955551A EP98955551A EP1019179A1 EP 1019179 A1 EP1019179 A1 EP 1019179A1 EP 98955551 A EP98955551 A EP 98955551A EP 98955551 A EP98955551 A EP 98955551A EP 1019179 A1 EP1019179 A1 EP 1019179A1
Authority
EP
European Patent Office
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.)
Withdrawn
Application number
EP98955551A
Other languages
German (de)
French (fr)
Inventor
Helmar 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
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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 EP1019179A1 publication Critical patent/EP1019179A1/en
Withdrawn legal-status Critical Current

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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.

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  • 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

The invention relates to gelatine membrane filters for collecting micro-organisms from gases and obtaining a higher proportion of micro-organisms capable of living on said filters, and to a method for producing the same. To this end, the inventive gelatine membrane filters contain osmoprotective substances which are added either to the membrane attraction solution or to a first setting bath. The inventive gelatine membrane filters are used in the pharmaceutical, biotechnology and food industries, in environmental protection, waste management and in medical devices for determining the bacterial count in media. They can be used in conjunction with an apparatus for collecting airborne bacteria for example, for collecting bacteria, viruses, yeast and fungi in order to determine the concentration of these micro-organisms in rooms.

Description

Gelatinemembranfilter und Verfahren zu ihrer Herstellung Gelatin membrane filters and process for their manufacture
Die Erfindung betrifft Gelatinemembranfilter zur Sammlung von Mikroorganismen aus Gasen unter Erhalt einer erhöhten Anzahl lebensfähiger Mikroorganismen sowie Verfahren zu ihrer Herstellung.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.
Die erfindungsgemäßen Gelatinemembranfilter sind zur Sammlung von Mikroorganismen aus gasförmigen Medien, insbesondere aus Luft, in der pharmazeutischen, biotechnologischen und Lebensmittelindustrie, im Umweltschutz, in der Abfallwirtschaft und in medizinischen Einrichtungen zur Bestimmung der Keimzahlbelastung der Medien einsetzbar. Sie dienen beispielsweise in Kombination mit einem Luftkeimsarnmelgerät zur Sammlung von Bakterien, Viren, Hefen und Pilzen, um deren Konzentration in Räumen bestimmen zu können. Derartige Überwachungen sind Voraussetzung für die rechtzeitige Einleitung von Maßnahmen, um Personen und Produkte vor Schädigungen durch zu hohe Mkroorganismenkonzentrationen zum Beispiel in der Raumluft zu bewahren.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.
In bestimmten Räumen mit besonderen Anforderungen an die Beschaffenheit der Luft, wie in klimatisierten Räumen, Reinräumen und Isolatoren, wird die Luft regelmäßig auf ihren Keimgehalt hin untersucht. Da es sich in der Regel um gefilterte Luft handelt, die naturgemäß einen geringen Gehalt an Mikroorganismen aufweist, müssen gewöhnlich große Volumina untersucht werden, um ausreichend Keime für aussagefähige Ergebnisse zu sammeln. Dazu wird eine Luftprobe durch ein geeignetes Filter filtriert. Als Filter werden für diesen Zweck sterile Membranfilter mit Porengrößen im Mikrofiltrationsbereich vorwiegend auf der Basis von Cellulosenitrat, Celluloseacetat und Gelatine eingesetzt, wie sie beispielsweise in der DE-PS 11 73 640 beschrieben sind. Besonders geeignet sind Gelatinemembranfilter, auf denen die zurückgehaltenen Mikroorganismen feucht und vermehrungsfähig gehalten werden sollen. Nach der Probenahme können die Gelatinemembranfilter entweder auf einem Agarnährboden bebrütet werden, wobei aus den einzelnen gesammelten Keimaggregaten Mikroorganismenkolonien heranwachsen (der Gelatinemembranfilter verflüssigt sich und verschwindet und die Mikroorganismenkolonien können direkt auf dem Agar gezählt werden), oder in einer sterilen Lösung, wie einer Pepton-Wasser- oder einer physiologischen Kochsalzlösung gelöst werden, so daß Teilmengen auf verschiedenen Nährmedien bebrütet werden können.In certain rooms with special requirements for the nature of the air, such as in air-conditioned rooms, clean rooms and isolators, 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. 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. After sampling, 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.
Die DE-PS 11 73 640 schlägt auch allgemein vor, bei der Herstellung in das Gefüge einer Gelatinemembran Nährstoffe für die Anzüchtung von Mikroorganismen, ferner puffernd wirkende Substanzen, Farbstoffe oder Chemikalien einzulagern, die geeignet sind, in der Luft enthaltene, biologisch giftig wirkende Stoffe, wie Spuren von Schwermetallen oder schädliche Gase, zu absorbieren. Nachteilig ist, daß trotz Zugabe von Nährmedien oder von absorbierenden Stoffen zu den Gelatinemembranen, aus den auf ihnen gesammelten Mikororganismen deutlich weniger Kolonien heranwachsen und bestimmt werden können als tatsächlich in dem kontaminierten Medium ursprünglich vorhanden waren.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.
Der Erfindung liegt daher die Aufgabe zugrunde, Gelatinemembranfilter zur Sammlung von Mikroorganismen aus Gasen zu schaffen, bei denen die Anzahl der nachweisbaren Mikroorganismen deutlich erhöht ist als bei den Gelatinemembranfiltern des Standes der Technik sowie ein Verfahren zur Herstellung dieser Gelatinemembranfilter zu finden.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.
Die Aufgabe wird gelöst durch Gelatinemembranfilter, die durch die Merkmale der unabhängigen Patentansprüche charakterisiert sind. Vorteilhafte Weiterbildungen der Erfindung sind durch die Merkmale der abhängigen Patentansprüche benannt.The object is achieved by 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.
Überraschenderweise wurde gefunden, daß die Anzahl der nachweisbaren Mikroorganismen, die durch Filtration aus Gasen auf Gelatinemembranfilter gesammelt wurden, deutlich ansteigt, wenn die Gelatinemembranfilter sogenannte osmoprotektive Substanzen enthalten. Zu den osmoprotektiven Substanzen gehören beispielsweise Inositol, Betain, Lycin, Oxyneurin und andere. Es ist bekannt, daß diese Substanzen der Dehydratation der Mikroorganismen, einem osmotischen Streß, entgegenwirken. Offensichtlich verliert ein Teil der Mikroorganismen bei der Sammlung auf Gelatinemembranfiltern soviel an zellulärem Wasser, daß er nicht mehr lebensfähig ist und durch Bebrüten nicht mehr über Koloniebildungen nachweisbar ist. Darauf deutet auch die Tatsache hin, daß nurmehr etwa die Hälfte an Bakterien nachgewiesen werden konnte, wenn mit der gleichen Anzahl Bakterien beaufschlagte Gelatinemembranfilter beispielsweise statt eine Minute weitere 8 Minuten lang mit einem Luftstrom von 7,5 m3/h durchströmt wurden. Dieser Effekt war bei Gelatinemembranfiltern nicht zu erwarten, weil Gelatinemembranfilter Mikroorganismen ausdrücklich feucht und vermehrungsfähig halten sollten (Prospekt „Laborfiltration, Mikrobiologie, Elektrophorese" Seite 14, Sartorius GmbH 1984). In einer bevorzugten Ausfuhrungsform der Erfindung enthalten die Gelatinemembranfilter die osmoprotektiven Substanzen in einer Menge, die wenigstens zu einer Verdopplung der Anzahl lebensfähiger Mikroorganismen führt im Vergleich zu Gelatinemembranfiltern ohne die osmoprotektiven Substanzen, wenn die Gelatinemembranfilter zur Sammlung der Mikroorganismen eine Minute und weitere 8 Minuten mit einem Luftstrom von 7,5 m3/h durchströmt werden. Es wurde nämlich gefunden, daß gleiche Anteile an osmoprotektiven Substanzen in den Gelatinemembranfiltern bei unterschiedlichen Mengen an Gasen, die die Gelatinemembranfilter mit der gleichen Anzahl gesammelter Mikroorganismen durchströmen, zu unterschiedlich Anteilen an lebensfähigen Mikroorganismen führen. Als besonders geeignet hat sich ein Gehalt an Trimethylammonioacetat als osmoprotektiver Substanz in den Gelatinemembranfiltern erwiesen, insbesondere wenn diese Substanz in der Membranziehlösung, aus der die Membran erhältlich ist, mit einem Anteil von 0,005 bis 0,75 % bezogen auf den Gehalt an der Gelatine vorhanden ist. Die Membranziehlösung stellt eine wässrige homogene Lösung dar, die Gelatine mit einem Anteil von 4,6 bis 5,6 % an der gesamten Membranziehlösung und Ethanol mit einem Anteil von 38 bis 46 % an der gesamten Membranziehlösung enthält. Dabei ist es für die Stabilität der Membran von Vorteil, wenn die Gelatine zusätzlich durch ein Bindemittel stabilisiert ist. Das Bindemittel kann beispielsweise Polyvinylalkohol oder Stärke sein. Aufgrund der Gelatinestruktur ist es von Vorteil, wenn die osmoprotektiven Substanzen die Gelatinematrix möglichst gleichmäßig durchsetzen. Befinden sich diese Substanzen nur auf der äußeren Oberfläche der Gelatinemembranmatrix, besteht die Gefahr, daß bei Langzeitlagerungen ein Teil der osmoprotektiven Substanzen in die innere Matrix abwandert und für die gesammelten Mikroorganismen nur noch eingeschränkt oder gar nicht mehr wirksam ist. Deshalb werden die osmoprotektiven Substanzen in einem bevorzugten erfindungsgemäßen Verfahren über einen Phaseninversionsprozeß, durch welchen die Gelatinemembranfilter erzeugt werden, in homogener Verteilung in die Membranmatrix eingebaut. Dazu werden die osmoprotektiven Substanzen in die Membranziehlösung eingebracht. Alternativ werden die osmoprotektiven Substanzen während der Ausbildung der Gelatinemembranfilter im Verfestigungsbad eingebracht. Das geschieht in einem ersten Verfestigungsbad, das aus Methylacetat mit einem Alkohol, vorzugsweise Methanol, mit einem Anteil von 10 bis 20 % an der gesamten Lösung besteht, sowie aus den osmoprotektiven Substanzen in einer Konzentration von bis zu etwa 2 % an der gesamten Lösung. In diesem ersten Verfestigungsbad sind die Gelatinemembranfilter noch nicht vollständig ausgebildet, so daß die osmoprotektiven Substanzen in die gesamte Membranmatrix eindringen können. Die erfindungsgemäßen Gelatinemembranfilter weisen gegenüber den Gelatinemembranfiltern des Standes der Technik, einschließlich den nach der DE-PS 11 73 640 erhältlichen, nicht nur eine erhöhte mechanische Stabilität auf, wodurch ihre Handhabung deutlich verbessert wird, sondern sie verfügen auch über einen deutlich höheren Luftdurchfluß, der mindestens um den Faktor 2 größer ist. Sie sind damit für hohe Luftdurchsätze zur Keimsammlung besonders geeignet. Dadurch ist es möglich, mit den erfindungsgemäßen Gelatinemembranfiltern die Zeit für die Sammlung von Mikroorganismen zu reduzieren oder äußerst gering belastete Gase zu testen, weil größere Volumina filtriert werden können, ohne daß die Gelatinemembranfilter in ihrer Wirkung beeinträchtigt oder zerstört werden.Surprisingly, it was found that the number of detectable microorganisms, which were collected by filtration from gases on gelatin membrane filters, increases significantly if the gelatin membrane filters contain so-called osmoprotective substances. The 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. This is also indicated by the fact that only about half of the bacteria could be detected if the same number of bacteria was applied to the gelatin membrane filters with the same number of bacteria, for example, instead of a minute, with an air flow of 7.5 m 3 / h for a further 8 minutes. This effect was not to be expected with gelatin membrane filters, because gelatin membrane filters should keep microorganisms expressly moist and capable of reproduction (prospectus "Laboratory Filtration, Microbiology, Electrophoresis" page 14, Sartorius GmbH 1984). In a preferred embodiment of the invention, 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. Due to the gelatin structure, it is advantageous if 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. Alternatively, the osmoprotective substances are introduced into the solidification bath during the formation of the gelatin membrane filter. This takes place in a first solidification bath, which consists of methyl acetate with an alcohol, preferably methanol, in a proportion of 10 to 20% of the total solution, and of the osmoprotective substances in a concentration of up to about 2% of the total solution. 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. 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.
Das Verfahren zur Herstellung der erfindungsgemäßen Gelatinemembranfilter wird durchgeführt, in dem a) eine wässrige homogene Membranziehlösung, die mindestens Gelatine und Ethanol enthält, hergestellt, b) ein dünner Film aus der Membranziehlösung auf einer Unterlage ausgebreitet, c) der dünne Film zur Ausbildung einer gelierten Phase Luft ausgesetzt, d) die gelierte Phase zur Nachbehandlung in ein Fällbad eingebracht und e) der Gelatinemembranfilter getrocknet wird, und ist gekennzeichnet, dadurch, daß f) der Membranziehlösung wenigstens eine osmoprotektive Substanz hinzugefügt wird.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.
In einer bevorzugten Ausführungsform des Verfahrens enthält die Membranziehlösung zwischen 0,005 und 0,75 % Trimethylammonioacetat bezogen auf die Gelatine.In a preferred embodiment of the method, the membrane drawing solution contains between 0.005 and 0.75% trimethylammonioacetate, based on the gelatin.
In einer weiteren Ausführungsform des Verfahrens, wird zur Erhöhung der mechanischen Stabilität der Gelatinemembranfilter ein Bindemittel über die Membranziehlösung mit einem Anteil von 0,02 bis 0,1 % an der gesamten Membranziehlösung eingebracht. In einer weiteren bevorzugten Ausführungsform der Erfindung wird als erstes Verfestigungsbad Methylacetat mit einem Alkohol, insbesondere Methanol, mit einem Anteil von 10 bis 20 % am gesamten ersten Verfestigungsbad verwendet. Die Membran verbleibt in diesem ersten Verfestigungsbad über eine Zeitdauer von ein bis drei Stunden bei Raumtemperatur bevor sie in ein zweites Verfestigungsbad aus reinem Methylacetat überführt wird. Trocknung und Sterilisierung, vorzugsweise mit Gammastrahlen, schließen sich an.In a further embodiment of the method, in order to increase the mechanical stability of the gelatin membrane filter, a binder is introduced over the membrane drawing solution in a proportion of 0.02 to 0.1% of the total membrane drawing solution. In a further preferred embodiment of the invention, 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.
Die Erfindung wird nun anhand der nachstehenden Ausführungsbeispiele erläutert.The invention will now be explained on the basis of the exemplary embodiments below.
Beispiel 1example 1
Zur Herstellung eines Gelatinemembranfilters werden 200g Gelatine (Gelita- Pulvergelatine 250 Bloom, DGF Deutsche Gelatine-Fabriken Stoess AG) und 2g Polyvinylalkohol (Mowiol Typ 18-88, Hoechst AG) unter einstündigem Rühren bei 60°C in 2000g Wasser gelöst und anschließend mit 1645g Ethanol und 0,02g Trimethylammonioacetat gelöst in 10g Wasser versetzt. Die auf 33°C temperierte Membranziehlösung wird zu einem Film der Stärke 350μm auf eine Unterlage ausgebreitet und Luft mit einer relativen Feuchte von 45 % bei Raumtemperatur 5 Minuten ausgesetzt. Der gelierte Film wird zusammen mit der Unterlage in ein erstes Verfestigungsbad, daß aus Methylacetat mit einem Anteil von 14 % Methanol besteht, für die Dauer von 3 Stunden und danach in ein zweites Verfestigungsbad aus reinem Methylacetat für die Dauer von 3 Stunden eingebracht. Der Gelatinemembranfilter wird von der Unterlage abgezogen und getrocknet.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 μm 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.
Die so erhaltenen Gelatinemembranfilter weisen einen Porendurchmesser von etwa 3 μm und einen Luftdurchfluß von 138 1/min cm2 bar auf. Die nach Beispiel 1 erhaltenen Gelatinemembranfilter werden mit Probe A bezeichnet.The gelatin membrane filters obtained in this way have a pore diameter of about 3 μm and an air flow of 138 1 / min cm 2 bar. The gelatin membrane filters obtained according to Example 1 are designated Sample A.
Beispiel 2-5Example 2-5
Analog Beispiel 1 werden Proben B, C, D und E hergestellt, wobei lediglich die Masse an Trimethylammonioacetat in der Membranziehlösung variiert wird (Tabelle, Zeile 2).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).
Beispiel 6Example 6
Analog Beispiel 1 wird eine Vergleichsprobe F hergestellt, wobei aber auf den Zusatz von Trimethylammonioacetat verzichtet wird.A comparative sample F is produced analogously to Example 1, but the addition of trimethylammonioacetate is dispensed with.
Zur Überprüfung der Wirksamkeit der osmoprotektiven Substanzen wurden die nach den Beispielen 1 bis 6 hergestellten Gelatinemembranfilter A bis F mit der gleichen Menge Keime von Escherichia coli ATCC 8739 während einer Minute beaufschlagt und über einen weiteren Zeitraum 8 Minuten Luft mit einem Fluß von 7,5 m3/h durch die beaufschlagten Gelatinemembranfilter hindurchgesaugt. Die Gelatinemembranfilter wurden unmittelbar danach auf Agar-Nährkartonscheiben, die Trypton-Soja-Bouillon- Agar (TSBA-Nährbouillon) enthalten, aufgebracht, über Nacht bei 37°C inkubiert und die Koloniezahl mikroskopisch bestimmt. Die Ergebnisse sind in den Zeilen 3 und 4 der Tabelle enthalten. Sie stellen Mittelwerte aus jeweils 5 Messungen dar und zeigen, daß Trimethylammonioacetat als osmoprotektive Substanz wenigstens zu einer Verdopplung der Anzahl lebensfähiger Mikroorganismen führt (Proben A, B und C) im Vergleich zu Gelatinemembranfiltern ohne diese osmoprotektive Substanz (Probe F), wenn es mit einem Anteil von 0,01 bis 0,25 % bezogen auf den Gelatinegehalt in der Membranziehlösung vorhanden ist und wenn die Gelatinemembranfilter zur Sar mlung der Mikroorganismen eine Minute und weitere 8 Minuten mit einem Luftstrom von 7,5 m3/h durchströmt werden. TabelleTo check the effectiveness of the osmoprotective substances, 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. Immediately afterwards, 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. They represent mean values from 5 measurements each and show that trimethylammonioacetate as an osmoprotective substance leads to at least a doubling of the number of viable microorganisms (samples A, B and C) compared to gelatin membrane filters without this osmoprotective substance (sample F), if it is used with a A proportion of 0.01 to 0.25%, based on the gelatin content, is present in the membrane drawing solution and when the gelatin membrane filters for the collection of the microorganisms are flowed through for one minute and a further 8 minutes with an air flow of 7.5 m 3 / h. table
* Durchschnittliche Abweichung ± 8,0* Average deviation ± 8.0
Testmaterialien und TestmethodenTest materials and test methods
Testkeime:Test germs:
In den Tests wurde Escherichia coli ATTCC 8739 verwendet. Dieser Stamm findet breite Anwendung in mikrobiologischen QS-Abteilungen für pharmazeutische Validierungsprüfungen. Es wurde ein eingefrorenes Fläschchen mit einer Konzentration ca. 103 koloniebildende Einheiten pro Milliliter [KBE/ml] verwendet. Jeweils 0, 1 ml der aufgetauten Suspension wurde auf Platten mit Trypton-Soja-Bouillon-Agar (TSBA- Nährbouillon) in zwei identischen Parallelen ausgestrichen. Die Agarplatten wurden über Nacht bei 37 °C ± 2 °C bebrütet, und die Bakterien nach der Gram-Färbung mikroskopisch bestimmt. Die Objektträger wurden als Rückstellmuster archiviert.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.
Eine Kultur-Suspension wurde durch Beimpfen von 50 ml der oben genannten Nährbouillon mit einer vorher auf einer Platte gewachsenen Kolonie in einem Gefäß mit einem Fassungsvermögen von 250 ml hergestellt. Das Gefäß wurde über Nacht bei 37 °C ± 2 °C unter Schütteln in einem Wasserbad bebrütet. Die Suspension wurde anhand von Kultivierungstechniken untersucht, in sterile Flaschen umgefüllt und bei 4 °C ±2 °C bis zur weiteren Verwendung aufbewahrt.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.
Sporen von Bacillus subtilis var niger (NCTC 10073) wurden als mikrobiologische Tracer eingesetzt. Diese Sporen wurden 3 Mal mit Hilfe von Zentrifugation gewaschen und mit sterilem, destilliertem Wasser wieder in Suspension gebracht. Die Konzentration der Stammlösung von B. subtilis betrug ca. 2 x 10u KBE/ml. Eine 1 -Liter-Suspension aus Bacillus subtilis wurde für den Einsatz in diesen Tests hergestellt, indem die Stammlösung auf ca. 2,5 x 109 KBE/ml mit sterilem, destiUiertemWasser verdünnt und 40 Minuten lang auf 60 °C zur Abtötung eventuell vorhandener vegetativer Organismen erhitzt wurde. Die Bestimmung der für die Tests verwendeten Sporensuspension ergab eine Endkonzentration an B. subtilis Sporen von 2,26 x 109 KBE/ml.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.
Vorbereitung der Verneblungssuspension:Preparation of the nebulization suspension:
Die Suspension von E. coli ATCC 8739 wurde zwei Wochen bei 4 °C + 2°C vor dem Einsatz aufbewahrt. Eine Nachbestimmung anhand von Kultivierungstechniken zeigte, daß die E. coli Konzentration während dieser Zeit konstant blieb. Einleitende Tests wurden mit verschiedenen Konzentrationen der Test- und Tracerkeime durchgeführt, um die geeigneten Konzentrationen für die Endprüfungen zu ermitteln.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.
Die Konzentration der Stammsuspension von E. coli ATCC 8739 betrug 2,1 x 1010 KBE/ml. Die Herstellung einer Verneblungssuspension von 10 ml erfolgte durch Mischen von 5-ml-Suspensionen von E. coli (verdünnt mit destilliertem Wasser, ergibt 7,6 x 103 bis 2,8 x 104 KBE/ml) und einer 5-ml-Suspension von B. subtilis (verdünnt mit destilliertem Wasser, ergibt 4,0 x 103 KBE/ml). Die Endsuspension wurde innerhalb von 30 Minuten vernebelt (aerosolisiert).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.
Aero sol-Prüfstand :Aero sol test bench:
Der Prüfstand besteht aus einem Verneblungsapparat des Typs Collison 3-jet Nebulizer zur Erzeugung von Bakterienaerosolen in einer geschlossenen und abgedichtetenThe test bench consists of a nebulizer of the Collison 3-jet nebulizer type for the generation of bacterial aerosols in a closed and sealed
Kammer. Die Aerosolkammer ist mit einem 1 Meter langen Rohrsystem aus Edelstahl mit einem Innendurchmesser von 45 mm verbunden. Der Filterkopf des Sartorius Luftkeimsammlers ist an diese Rohrleitung mittels eines entsprechenden Adapters angeschlossen. Der Eingang zur Kammer ist durch ein leistungstarkes Luftfilter geschützt.Chamber. 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.
Der Sartorius MD 8 airscan Luftkeimsammler (Serien-Nr. 9607001) mit Kalibrierzertifikat, Schlauch und Filterkopf wurde gemäß der Bedienungsanleitung betrieben.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.
Gelatinemembranfilter:Gelatin membrane filter:
Es wurden sterile Gelatinemembranfilter mit einer Porengröße von etwa 3,0 μm, die jeweils als Probe A, B, C, D, E und F gekennzeichnet waren, eingesetzt. Die modifizierten Gelatinemembranfilter A, B, C, D und E enthielten verschiedene, oben in der Tabelle genannte Mengen Trimethylammonioacetat als osmosprotektive Substanz.Sterile gelatin membrane filters with a pore size of approximately 3.0 μm, each identified as sample A, B, C, D, E and F, were used. The modified gelatin membrane filters A, B, C, D and E contained various amounts of trimethylammonioacetate mentioned in the table above as an osmosprotective substance.
Testverfahren:Test procedure:
9 ml der zu vernebelnden Suspension wurden in den Collison Vernebungsapparat eingegeben (1 ml wurde zum mikrobiologischen Nachweis zurückbehalten). Der jeweilige Gelatinemembranfilter wurde in den Filterkopf gelegt und an den Prüfstand (wie oben beschrieben) angeschraubt.9 ml of the suspension to be nebulized were introduced into the Collison nebulizer (1 ml was retained for microbiological detection). The respective gelatin membrane filter was placed in the filter head and screwed onto the test stand (as described above).
Das MD8 airscan wurde auf einen Luftdurchsatz von 7,5 m3/h eingestellt. Das MD8 airscan wurde eingeschaltet, und nach 15 Sekunden wurde der Collison Verneblungsapparat durch Anschluß an eine Druckgasleitung mit 180 kPa aktiviert. Der Verneblungsapparat wurde 1 Minute lang aktiviert, und das MD8 wurde danach ausgeschaltet.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.
Die Gelatinemembranfilter wurden entweder nach dieser 1 -minütigen Beaufschlagung mit keimhaltigem Aerosol (angesaugt mit Hilfe des Luftkeimsammlers MD8) direkt oder nach einer weiteren 8 minütigen Beaufschlagung mit filtrierter Luft auf die TSBA Platten aufgelegt und wie weiter oben beschrieben bebrütet und vermessen.The 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.
Die in der Erfindungsbeschreibung verwendeten %-Angaben sind Masseprozente. The percentages used in the description of the invention are percentages by mass.

Claims

Patentansprüche claims
1. Gelatinemembranfilter zur Sammlung von Mikroorganismen aus Gasen, dadurch gekennzeichnet, daß die Gelatinemembranfilter osmoprotektive Substanzen enthalten.1. Gelatin membrane filter for the collection of microorganisms from gases, characterized in that the gelatin membrane filter contains osmoprotective substances.
2. Gelatinemembranfilter nach Anspruch 1 , dadurch gekennzeichnet, daß die Gelatinemembranfilter die osmoprotektiven Substanzen in einer Menge enthalten, die wenigstens zu einer Verdopplung der Anzahl lebensfähiger Mikroorganismen führt im Vergleich zu Gelatinemembranfiltern ohne die osmoprotektiven Substanzen, wenn die Gelatinemembranfilter zur Sammlung der Mikroorganismen eine Minute und weitere 8 Minuten mit einem Luftstrom von 7,5 m3/h durchströmt werden.2. Gelatin membrane filter according to claim 1, characterized in that the gelatin membrane filters contain the osmoprotective substances in an amount which leads at least to a doubling of the number of viable microorganisms in comparison to gelatin membrane filters without the osmoprotective substances if the gelatin membrane filters for collecting the microorganisms one minute and air flow of 7.5 m 3 / h for a further 8 minutes.
3. Gelatinemembranfilter nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Gelatinemembranfilter Trimethylammonioacetat als osmoprotektive Substanz enthalten.3. Gelatin membrane filter according to claim 1 and 2, characterized in that the gelatin membrane filter contain trimethylammonioacetate as an osmoprotective substance.
4. Gelatinemembranfilter nach Anspruch 1 bis 3, dadurch gekennzeichnet, daß die Gelatinemembranfilter ein Bindemittel enthalten.4. gelatin membrane filter according to claim 1 to 3, characterized in that the gelatin membrane filter contain a binder.
5. Gelatinemembranfilter nach den Ansprüchen 1 bis 4, die durch Phaseninversion aus einer wässrigen homogenen Membranziehlösung bestehend aus folgenden Komponenten erhältlich sind: a) Gelatine mit einem Anteil von 4,6 bis 5,6 % an der gesamten Membranziehlösung, b) Ethanol mit einem Anteil von 38 bis 46 % an der gesamten Membranziehlösung, c) wenigstens einer osmoprotektiven Substanz mit einem Anteil an der gesamten5. Gelatin membrane filter according to claims 1 to 4, which are obtainable by phase inversion from an aqueous homogeneous membrane drawing solution consisting of the following components: a) gelatin with a share of 4.6 to 5.6% of the total membrane drawing solution, b) ethanol with a 38 to 46% of the total membrane drawing solution, c) at least one osmoprotective substance with a share of the total
Membranziehlösung, der wenigstens zu einer Verdopplung der Anzahl lebensfähiger Mikroorganismen führt im Vergleich zu Gelatinemembranfiltern ohne diese osmoprotektiven Substanzenen, wenn die Gelatinemembranfilter zur Sammlung der Mikroorganismen eine Minute und weitere 8 Minuten mit einem Luftstrom von 7,5 m3/h durchströmt werden und d) Bindemittel mit einem Anteil von 0,02 bis 0, 1 % an der gesamten Membranziehlösung.Membrane drawing solution which at least doubles the number of viable microorganisms compared to gelatin membrane filters without these osmoprotective substances if the gelatin membrane filters for collecting the microorganisms are flowed through for one minute and a further 8 minutes with an air flow of 7.5 m 3 / h and d) binders with a share of 0.02 to 0.1% of the total Membrane drawing solution.
6. Gelatinemembranfilter nach Anspruch 5, dadurch gekennzeichnet, daß als osmoprotektive Substanz Trimethylammonioacetat mit einem Anteil von 0,005 bis 0,75 % bezogen auf den Gehalt an der Gelatine in der Membranziehlösung vorhanden ist.6. Gelatin membrane filter according to claim 5, characterized in that trimethylammonioacetate is present as an osmoprotective substance with a proportion of 0.005 to 0.75% based on the content of the gelatin in the membrane drawing solution.
7. Verfahren zur Herstellung von Gelatinemembranfiltern nach den Ansprüchen 1 bis 6, indem a) eine wässrige homogene Membranziehlösung, die mindestens Gelatine und Ethanol enthält, hergestellt, b) ein dünner Film aus der Membranziehlösung auf einer Unterlage ausgebreitet, c) der dünne Film zur Ausbildung einer gelierten Phase Luft ausgesetzt, d) die gelierte Phase zur Nachbehandlung in ein Fällbad eingebracht und e) der Gelatinemembranfilter getrocknet wird, dadurch gekennzeichnet, daß f) der Membranziehlösung wenigstens eine osmoprotektive Substanz hinzugefügt wird.7. A method for producing gelatin membrane filters according to claims 1 to 6, 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 Formation of a gelled 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, characterized in that f) at least one osmoprotective substance is added to the membrane drawing solution.
8. Verfahren zur Herstellung von Gelatinemembranfiltern nach Anspruch 7, dadurch gekennzeichnet, daß die osmoprotektive Substanz der Membranziehlösung in einer Menge hinzugefügt wird, die wenigstens zu einer Verdopplung der Anzahl lebensfähiger Mikroorganismen führt im Vergleich zu Gelatinemembranfiltern ohne diese osmoprotektiven Substanzenen, wenn die Gelatinemembranfilter zur Sammlung der Mikroorganismen eine Minute und weitere 8 Minuten mit einem Luftstrom von 7,5 m3/h durchströmt werden. 8. A process for the preparation of gelatin membrane filters according to claim 7, characterized in that the osmoprotective substance is added to the membrane drawing solution in an amount which at least doubles the number of viable microorganisms in comparison to gelatin membrane filters without these osmoprotective substances when the gelatin membrane filters are collected the air flow through the microorganisms for one minute and a further 8 minutes with 7.5 m 3 / h.
9. Verfahren zur Herstellung von Gelatinemembranfiltern nach Anspruch 7 und 8, dadurch gekennzeichnet, daß als osmoprotektive Substanz Trimethylammonioacetat mit einem Anteil von 0,005 bis 0,75 % bezogen auf den Gehalt an der Gelatine zu der Membranziehlösung hinzugefügt wird.9. A process for the preparation of gelatin membrane filters according to claim 7 and 8, characterized in that trimethylammonioacetate is added as an osmoprotective substance with a proportion of 0.005 to 0.75% based on the content of the gelatin to the membrane drawing solution.
10. Verfahren zur Herstellung von Gelatinemembranfiltern nach den Ansprüchen 7 bis 9, dadurch gekennzeichnet, daß ein Bindemittel mit einem Anteil von 0,02 bis 0,1 % an der gesamten Membranziehlösung hinzugefügt wird.10. A process for the preparation of gelatin membrane filters according to claims 7 to 9, characterized in that a binder is added in a proportion of 0.02 to 0.1% of the total membrane drawing solution.
11. Verfahren nach den Ansprüchen 7 bis 10, dadurch gekennzeichnet, daß gemäß Schritt d) die gelierte Phase zur Nachbehandlung in ein erstes Verfestigungsbad aus Methylacetat, das einen Anteil von 10 bis 20% eines Alkohols, vorzugsweise Methanol, enthält, eingebracht wird.11. The method according to claims 7 to 10, characterized in that according to step d) the gelled phase for aftertreatment is introduced into a first solidifying bath made of methyl acetate, which contains a proportion of 10 to 20% of an alcohol, preferably methanol.
12. Verfahren zur Herstellung von Gelatinemembranfiltern nach den Ansprüchen 1 bis 6, indem a) eine wässrige homogene Membranziehlösung, die mindestens Gelatine und Ethanol enthält, hergestellt, b) ein dünner Film aus der Membranziehlösung auf einer Unterlage ausgebreitet, c) der dünne Film zur Ausbildung einer gelierten Phase Luft ausgesetzt, d) die gelierte Phase zur Nachbehandlung in ein Fällbad eingebracht und e) der Gelatinemembranfilter getrocknet wird, dadurch gekennzeichnet, daß gemäß Schritt d) die gelierte Phase in einem ersten Verfestigungsbad aus Methylacetat, einem Alkohol, vorzugsweise Methanol mit einem Anteil von 10 bis 20% und Trimethylammonioacetat als osmoprotektiver Substanz mit einem Anteil von weniger als 2 % an der gesamten Lösung des ersten Verfestigungsbades nachbehandelt wird. 12. A method for producing gelatin membrane filters according to claims 1 to 6, 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 Formation of a gelled 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, characterized in that, according to step d), the gelled phase in a first solidification bath made of methyl acetate, an alcohol, preferably methanol a share of 10 to 20% and trimethylammonioacetate as an osmoprotective substance with a share of less than 2% of the total solution of the first solidification bath.
13. Verfahren nach Anspruch 12, dadurch gekennzeichnet, daß der Membranziehlösung ein Bindemittel mit einem Anteil von 0,02 bis 0, 1 % hinzugefügt wird. 13. The method according to claim 12, characterized in that a binder with a proportion of 0.02 to 0.1% is added to the membrane drawing solution.
EP98955551A 1997-11-13 1998-11-07 Gelatine membrane filters and method for producing the same Withdrawn EP1019179A1 (en)

Applications Claiming Priority (3)

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DE19750215A DE19750215C1 (en) 1997-11-13 1997-11-13 Gelatin membrane filter increasing number of viable micro-organisms collected from gas
DE19750215 1997-11-13
PCT/EP1998/007118 WO1999025465A1 (en) 1997-11-13 1998-11-07 Gelatine membrane filters and method for producing the same

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DE19814715A1 (en) * 1998-04-02 1999-10-07 Sartorius Gmbh Gelatin membrane filters, process for their preparation and their use
DE60220989T2 (en) * 2001-02-05 2008-03-13 Millipore Corp., Billerica DETECTION OF MICROORGANISMS
GB2387130A (en) 2002-04-04 2003-10-08 Fluid Technologies Plc Hollow fibre filter membrane unit with microorganism detector, and associated usage
FR2917095B1 (en) * 2007-06-07 2009-07-17 Biomerieux Sa DEVICE FOR LYING MICROORGANISMS PRESENT IN AN ENVIRONMENTAL OR CLINICAL SAMPLE AND EXTRACTING NUCLEIC ACIDS FROM SAID MICROORGANISMS FOR ANALYSIS.
JP5245379B2 (en) * 2007-12-04 2013-07-24 株式会社日立プラントテクノロジー Collection device and analysis system using the same
EP2305789A4 (en) * 2008-06-27 2013-12-25 Hitachi Plant Technologies Ltd Cartridge of microbial cell-capturing carrier, carrier treating device and method for counting microbial cells
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EP3336515B1 (en) 2016-12-14 2022-05-11 Airbus Defence and Space GmbH Hand-held air sampler, including filter magazine for storing and positioning filter elements

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