EP1119638A1 - Detection of cells in a liquid sample - Google Patents

Detection of cells in a liquid sample

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Publication number
EP1119638A1
EP1119638A1 EP99969120A EP99969120A EP1119638A1 EP 1119638 A1 EP1119638 A1 EP 1119638A1 EP 99969120 A EP99969120 A EP 99969120A EP 99969120 A EP99969120 A EP 99969120A EP 1119638 A1 EP1119638 A1 EP 1119638A1
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EP
European Patent Office
Prior art keywords
cells
sample
added
filtered
detection
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Application number
EP99969120A
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German (de)
French (fr)
Inventor
Frank Nitzsche
Guido Eggers
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Individual
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Individual
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Publication of EP1119638A1 publication Critical patent/EP1119638A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the invention relates to a method for the detection of cells in a sample in liquid form, in which a substance is added to the sample which leads to the formation of a specific enzyme in active cells, and the cells are filtered off with a membrane filter.
  • Such a method is known from DE 38 55 762 Tl.
  • the coliform bacteria present in a liquid are concentrated using a filter, the pores of which are sufficiently small to retain the bacteria.
  • This filter is then placed on a nutrient medium to increase the number of cells.
  • This proliferation is promoted in such a way that a microcolony visible to each individual bacterium is formed under fluorescence conditions.
  • These microcolonies are then irradiated with light of a certain wavelength so that the number of fluorescent microcolonies can be counted.
  • the invention is therefore based on the object of providing a method in which a much faster detection of cells capable of reproduction in a sample is possible.
  • This object is achieved with a generic method in which, immediately after filtering off the cells with a membrane filter, the filtered cells are examined fluorescence-optically with the aid of a microscope.
  • the advantage of the method is that this examination can be carried out on a single cell.
  • the sample drawn does not therefore have to be enriched or multiplied, as in classic microbiology, in order to subsequently examine a cell population.
  • the sample is examined directly after the membrane filtration without an intermediate growth step, and the method allows information about the enzyme formation capacity of a cell in the sample after only one hour.
  • the fluorescent dye can be added at any stage of the process before the microscopic examination. In various applications, however, it is advantageous if the fluorescent dye follows the filtration is added to the cells filtered off since this can reduce the background radiation.
  • a polycarbonate filter is advantageously used to separate the cells from the sample. These filters are particularly well suited to filtering off the microorganism sought by choosing an appropriate pore size.
  • An advantageous embodiment provides that a substance is added to the cells, which opens the cell walls at least in places. This makes it easier for the inducer of the enzyme sought and the fluorescent dye to enter the cell.
  • the method according to the invention is particularly suitable for food samples. It is advantageous if the sample is a liquid or liquefied food.
  • the method is particularly suitable for the analysis of beverages, such as preferably beer or non-alcoholic beverages.
  • the first process step is to convert the sample material to be examined into a liquid, aqueous form. If foods such as cheese or jam are to be examined, these substances must be liquefied in a suitable manner. In the figure the substance to be examined is designated with the reference number 1.
  • the aqueous form of the sample material to be examined in the Erlenmeier flask has the reference number 2. If the sample is a drink, the addition of the sample material is unnecessary since the sample 2 is already in liquid form.
  • the inducer 3 of the enzyme sought is added to this sample.
  • an inducer for galactosidase e.g. Galactose added.
  • a substance 5 can also be added subsequently or simultaneously, which opens the cell walls of the sought-after microorganisms at different locations.
  • Mutanolysin for example, can be used as the cell wall-opening substance.
  • cell wall-accessible substances that can pass through the cell wall without digestion.
  • the sample After an exposure time and exposure temperature corresponding to the substrate, the sample is filtered through a polycarbonate membrane filter 6 with a pore size corresponding to the microorganism sought. The sample thereby arrives in the collecting container 7 and the microorganisms sought remain on the membrane filter 8.
  • a fluorescent dye 4 is added to the filtered cells remaining on the filter.
  • a reagent specific for galactosidase such as fluorescindigalactoside, is added.
  • the membrane filter 8 is then examined fluorescence optically. This is carried out with the aid of a microscope 9. In microscopic image 10, the cells which have the enzyme activity sought are illuminated according to the fluorescent dye.
  • the number of luminous dots or the intensity of the luminosity can be used to easily deduce the contamination of the sample with lactobacilli capable of reproduction.
  • the result takes into account both the number of microorganisms per sample volume and the enzyme activity and thus the ability of the cells found to proliferate.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (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)

Abstract

The invention relates to a method for detecting cells in a sample, according to which a substance is added which in the presence of active cells results in the formation of a certain enzyme. The cells are then examined by fluorescence microscopy. In the microscopic image the cells presenting the enzyme activity for which they are being screened become luminous in accordance with the fluorescent dye used. This method permits the very rapid detection of cells capable of multiplication.

Description

Verfahren zum Nachweis von Zellen in einer in flüssiger Form vorliegenden Probe Method for the detection of cells in a sample in liquid form
Die Erfindung betrifft ein Verfahren zum Nachweis von Zellen in einer in flüssiger Form vorliegenden Probe, bei dem der Probe ein Stoff zugesetzt wird, der bei aktiven Zellen zur Bildung eines bestimmten Enzyms führt, und die Zellen mit einem Membranfilter abfiltriert werden.The invention relates to a method for the detection of cells in a sample in liquid form, in which a substance is added to the sample which leads to the formation of a specific enzyme in active cells, and the cells are filtered off with a membrane filter.
Ein derartiges Verfahren ist aus der DE 38 55 762 Tl bekannt. Bei diesem Verfahren werden die in einer Flüssigkeit vorliegenden coliformen Bakterien mittels eines Filters, dessen Poren genügend klein sind, um die Bakterien zurückzuhalten, konzentriert. Dieser Filter wird anschließend auf ein Nährmedium aufgelegt, um die Zellzahl zu vermehren. Diese Vermehrung wird derart gefördert, daß eine unter Fluoreszensbedingungen sichtbare Mikrokolonie zu jedem einzelnen Bakterium gebildet wird. Diese Mikrokolonien werden anschließend mit Licht einer bestimmten Wellenlänge bestrahlt, so daß die Anzahl der fluoreszierenden Mikrokolonien gezählt werden kann.Such a method is known from DE 38 55 762 Tl. In this method, the coliform bacteria present in a liquid are concentrated using a filter, the pores of which are sufficiently small to retain the bacteria. This filter is then placed on a nutrient medium to increase the number of cells. This proliferation is promoted in such a way that a microcolony visible to each individual bacterium is formed under fluorescence conditions. These microcolonies are then irradiated with light of a certain wavelength so that the number of fluorescent microcolonies can be counted.
Dieses Verfahren hat jedoch den Nachteil, daß die Durchführung des gesamten Verfahrens vom Konzentrieren der Bakterien bis zum Auszählen der fluoreszierenden Mikrokolonien mehrere Stunden in Anspruch nimmt und das Verfahren daher zeit- und arbeitsintensiv ist. Ähnliche Verfahren sind aus der DE 38 52 825 T2, der WO 96/14431 AI und der US 4,242,447 A bekannt. Alle diese Verfahren haben jedoch den Nachteil, daß zum Nachweis der Keime mehrere Stunden benötigt werden.However, this method has the disadvantage that it takes several hours to carry out the entire method from concentrating the bacteria to counting the fluorescent microcolonies, and the method is therefore time-consuming and labor-intensive. Similar methods are known from DE 38 52 825 T2, WO 96/14431 AI and US 4,242,447 A. However, all of these methods have the disadvantage that several hours are required to detect the germs.
Der Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren bereitzu- stellen, bei dem ein weit schnellerer Nachweis von vermehrungsfähigen Zellen in einer Probe möglich ist.The invention is therefore based on the object of providing a method in which a much faster detection of cells capable of reproduction in a sample is possible.
Diese Aufgabe wird mit einem gattungsgemäßen Verfahren gelöst, bei dem direkt anschließend an die Abfiltrierung der Zellen mit einem Membranfilter die abfiltrierten Zellen mit Hilfe eines Mikroskopes fluoreszenz- optisch untersucht werden.This object is achieved with a generic method in which, immediately after filtering off the cells with a membrane filter, the filtered cells are examined fluorescence-optically with the aid of a microscope.
Der Vorteil des Verfahrens liegt darin, daß diese Untersuchung an einer einzelnen Zelle durchgeführt werden kann. Die gezogene Probe muß daher nicht wie bei der klassischen Mikrobiologie zunächst angereichert oder vermehrt werden, um anschließend eine Zellpopulation zu untersuchen. Bei dem erfindungsgemäßen Verfahren wird die Probe direkt anschließend an die Membranfiltration ohne zwischengeschalteten Vermehrungsschrittunter- sucht und das Verfahren erlaubt schon nach einer Stunde eine Aussage über die Enzymbildungsfähigkeit einer Zelle in der Probe.The advantage of the method is that this examination can be carried out on a single cell. The sample drawn does not therefore have to be enriched or multiplied, as in classic microbiology, in order to subsequently examine a cell population. In the method according to the invention, the sample is examined directly after the membrane filtration without an intermediate growth step, and the method allows information about the enzyme formation capacity of a cell in the sample after only one hour.
Der Fluoreszenzfarbstoff kann in jedem Verfahrensstadium vor der mikros- kopischen Untersuchung zugegeben werden. Vorteilhaft ist es bei verschiedenen Anwendungsfällen jedoch, wenn der Fluoreszenzfarbstoff nach der Filtration den ab filtrierten Zellen zugegeben wird, da dadurch die Hintergrundstrahlung verringert werden kann.The fluorescent dye can be added at any stage of the process before the microscopic examination. In various applications, however, it is advantageous if the fluorescent dye follows the filtration is added to the cells filtered off since this can reduce the background radiation.
Zur Trennung der Zellen von der Probe wird vorteilhafterweise ein Poly- karbonatfilter verwendet. Diese Filter sind besonders gut geeignet, über die Wahl einer entsprechenden Porengröße den gesuchten Mikroorganismus ab- zufiltrieren.A polycarbonate filter is advantageously used to separate the cells from the sample. These filters are particularly well suited to filtering off the microorganism sought by choosing an appropriate pore size.
Eine vorteilhafte Ausgestaltung sieht vor, daß den Zellen eine Substanz zugegeben wird, die die Zeil wände zumindest stellenweise öffnet. Dadurch wird der Zutritt des Inducers des gesuchten Enzyms und des Fluoreszenz- farbstoffes in die Zelle erleichtert.An advantageous embodiment provides that a substance is added to the cells, which opens the cell walls at least in places. This makes it easier for the inducer of the enzyme sought and the fluorescent dye to enter the cell.
Das erfindungsgemäße Verfahren eignet sich vor allem für Lebensmittelproben. Hierbei ist es günstig, wenn die Probe ein flüssiges oder verflüssigtes Lebensmittel ist. Insbesondere für die Untersuchung von Getränken, wie vorzugsweise Bier oder alkoholfreien Getränken ist das Verfahren besonders geeignet.The method according to the invention is particularly suitable for food samples. It is advantageous if the sample is a liquid or liquefied food. The method is particularly suitable for the analysis of beverages, such as preferably beer or non-alcoholic beverages.
Ein Ausfühungsbeispiel der Erfindung ist in der Figur dargestellt und wird im folgenden näher erläutert.An exemplary embodiment of the invention is shown in the figure and is explained in more detail below.
Der erste Verfahrensschritt liegt darin, das zu untersuchende Probenmaterial in eine flüssige, wässrige Form zu überführen. Wenn Lebens- mittel, wie beispielsweise Käse oder Marmelade untersucht werden sollen, müssen diese Stoffe auf geeignete Weise verflüssigt werden. In der Figur ist der zu untersuchende Stoff mit der Bezugsziffer 1 bezeichnet. Die im Erlenmeierkolben vorliegende wässrige Form des zu untersuchenden Probenmaterials hat die Bezugsziffer 2. Wenn die Probe ein Getränk ist, erübrigt sich die Zugabe des Probenmaterials, da die Probe 2 schon in flüssiger Form vorliegt.The first process step is to convert the sample material to be examined into a liquid, aqueous form. If foods such as cheese or jam are to be examined, these substances must be liquefied in a suitable manner. In the figure the substance to be examined is designated with the reference number 1. The aqueous form of the sample material to be examined in the Erlenmeier flask has the reference number 2. If the sample is a drink, the addition of the sample material is unnecessary since the sample 2 is already in liquid form.
Zu dieser Probe wird der Inducer 3 des gesuchten Enzyms zugesetzt. Um z.B. Lactobazillen nachzuweisen, wird ein Inducer für Galactosidase, z.B. Galactose, zugesetzt.The inducer 3 of the enzyme sought is added to this sample. To e.g. Detecting lactobacilli is an inducer for galactosidase, e.g. Galactose added.
Letztlich kann auch anschließend oder gleichzeitig eine Substanz 5 zuge- setzt werden, die Zellwände der gesuchten Mikroorganismen an verschiedenen Stellen öffnet. Als zellwandöffnende Substanz kann beispielsweise Mutanolysin verwendet werden. Es besteht jedoch auch die Möglichkeit, zellwandgängige Substanzen zu verwenden, die ohne Aufschluß die Zeil wand passieren können.Ultimately, a substance 5 can also be added subsequently or simultaneously, which opens the cell walls of the sought-after microorganisms at different locations. Mutanolysin, for example, can be used as the cell wall-opening substance. However, there is also the possibility of using cell wall-accessible substances that can pass through the cell wall without digestion.
Nach einer dem Substrat entsprechenden Einwirkzeit und Einwirktemperatur wird die Probe über einen Polykarbonatmembranfilter 6 mit einer dem gesuchten Mikroorganismus entsprechenden Porengröße filtriert. Die Probe gelangt dabei in den Auffangbehälter 7 und die gesuchten Mikroorganismen verbleiben auf dem Membranfilter 8.After an exposure time and exposure temperature corresponding to the substrate, the sample is filtered through a polycarbonate membrane filter 6 with a pore size corresponding to the microorganism sought. The sample thereby arrives in the collecting container 7 and the microorganisms sought remain on the membrane filter 8.
Den auf dem Filter verbleibenden abfiltrierten Zellen wird ein Fluoreszenzfarbstoff 4 zugegeben. Zum Nachweis von Lactobazillen wird ein für die Galactosidase spezifisches Reagenz wie Fluorescindigalactosid zugegeben. Der Membranfilter 8 wird anschließend fluoreszenzoptisch untersucht. Dies wird mit Hilfe eines Mikroskops 9 durchgeführt. Im mikroskopischen Bild 10 leuchten die Zellen, die über die gesuchte Enzymaktivität verfügen, dem Fluoreszenzfarbstoff entsprechend auf.A fluorescent dye 4 is added to the filtered cells remaining on the filter. To detect lactobacilli, a reagent specific for galactosidase, such as fluorescindigalactoside, is added. The membrane filter 8 is then examined fluorescence optically. This is carried out with the aid of a microscope 9. In microscopic image 10, the cells which have the enzyme activity sought are illuminated according to the fluorescent dye.
Aus der Anzahl der Leuchtpunkte oder der Intensität der Leuchtkraft kann auf einfache Weise auf die Kontamination der Probe mit vermehrungsfähigen Lactobazillen zurückgeschlossen werden. Das Ergebnis berücksichtigt sowohl die Anzahl der Mikroorganismen pro Probevolumen als auch die Enzymaktivität und somit Vermehrungsfähigkeit der gefundenen Zellen. The number of luminous dots or the intensity of the luminosity can be used to easily deduce the contamination of the sample with lactobacilli capable of reproduction. The result takes into account both the number of microorganisms per sample volume and the enzyme activity and thus the ability of the cells found to proliferate.

Claims

Patentansprüche: Claims:
1. Verfahren zum Nachweis von Zellen in einer in flüssiger Form vorliegenden Probe (2), bei dem der Probe (2) ein Stoff (3) zugesetzt wird, der bei aktiven Zellen zur Bildung eines bestimmten Enzyms führt, und die Zellen mit einem Membranfilter (8) abfiltriert werden, dadurch gekennzeichnet, daß direkt anschließend die abfiltrierten Zellen mit Hilfe eines Mikroskopes (9) fluoreszenzoptisch untersucht werden.1. A method for the detection of cells in a sample (2) in liquid form, in which a sample (3) is added to the sample (2), which leads to the formation of a certain enzyme in active cells, and the cells with a membrane filter (8) are filtered off, characterized in that the cells filtered off are subsequently examined fluorescence-optically with the aid of a microscope (9).
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die Zellen mit einem Polykarbonatfilter abfiltriert werden.2. The method according to claim 1, characterized in that the cells are filtered off with a polycarbonate filter.
3. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß der Fluoreszenzfarbstoff (4) nach der Filtration den ab filtrierten Zellen zugegeben wird.3. The method according to any one of the preceding claims, characterized in that the fluorescent dye (4) is added to the filtered cells after filtration.
4. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß den Zellen eine Substanz (5) zugegeben wird, die die Zellwände zumindest stellenweise öffnet.4. The method according to any one of the preceding claims, characterized in that a substance (5) is added to the cells, which opens the cell walls at least in places.
5. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß die Probe (1 , 2) ein Lebensmittel ist. 5. The method according to any one of the preceding claims, characterized in that the sample (1, 2) is a food.
6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß die Probe (2) ein flüssiges oder verflüssigtes Lebensmittel ist.6. The method according to any one of the preceding claims, characterized in that the sample (2) is a liquid or liquefied food.
7. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß die Probe (2) ein Getränk, vorzugsweise Bier oder ein alkoholfreies Getränk wie etwa Milch, Limonade oder Fruchtsaft ist. 7. The method according to any one of the preceding claims, characterized in that the sample (2) is a drink, preferably beer or a non-alcoholic drink such as milk, lemonade or fruit juice.
EP99969120A 1998-09-11 1999-09-09 Detection of cells in a liquid sample Withdrawn EP1119638A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE1998141588 DE19841588A1 (en) 1998-09-11 1998-09-11 Detecting cells especially in food or drink by adding a material giving rise to an enzyme followed by optical fluorescence examination
DE19841588 1998-09-11
PCT/DE1999/002867 WO2000015832A1 (en) 1998-09-11 1999-09-09 Detection of cells in a liquid sample

Publications (1)

Publication Number Publication Date
EP1119638A1 true EP1119638A1 (en) 2001-08-01

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP99969120A Withdrawn EP1119638A1 (en) 1998-09-11 1999-09-09 Detection of cells in a liquid sample

Country Status (4)

Country Link
EP (1) EP1119638A1 (en)
AU (1) AU1149300A (en)
DE (2) DE19841588A1 (en)
WO (1) WO2000015832A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004011822A1 (en) * 2004-03-11 2005-09-29 Cognis Deutschland Gmbh & Co. Kg Quality assurance system for the detection of microorganisms

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4242447A (en) * 1978-11-29 1980-12-30 Bioresearch Rapid detection of bacteria
EP0101398B1 (en) * 1982-07-21 1986-06-18 Packard Instrument Company, Inc. Method of concentrating and measuring unicellular organisms
DE3852825T2 (en) * 1987-11-05 1995-05-18 James D Berg QUICK PROCEDURE FOR DETECTING COLIFORM MICROORGANISMS.
US5510243A (en) * 1994-06-21 1996-04-23 Gelman Sciences, Inc. Multiple chromogen enzyme targeting (MCET) for use in bacterial contamination monitoring
JPH11505405A (en) * 1994-11-07 1999-05-21 ステュディーエン・サメンベルキングスフェアバンド・ブラームス・ウォーター Enzymatic detection method of Escherichia coli type bacteria or Escherichia coli (E. coli)
DE19608320A1 (en) * 1996-02-22 1997-08-28 Biosquant Gmbh Rapid, high- sensitivity determination of microbial quality of water
IT1284178B1 (en) * 1996-06-27 1998-05-08 Isrim S C A R L PROCEDURE FOR THE RAPID DETECTION OF THE PRESENCE OF BACTERIAL CELLS IN A SAMPLE EVEN IF IN A VERY LIMITED NUMBER.

Non-Patent Citations (1)

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Title
See references of WO0015832A1 *

Also Published As

Publication number Publication date
WO2000015832A1 (en) 2000-03-23
DE19981818D2 (en) 2001-09-27
DE19841588A1 (en) 2000-03-23
AU1149300A (en) 2000-04-03

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