WO2009121726A1 - Detection and enumeration of microorganisms - Google Patents
Detection and enumeration of microorganisms Download PDFInfo
- Publication number
- WO2009121726A1 WO2009121726A1 PCT/EP2009/053295 EP2009053295W WO2009121726A1 WO 2009121726 A1 WO2009121726 A1 WO 2009121726A1 EP 2009053295 W EP2009053295 W EP 2009053295W WO 2009121726 A1 WO2009121726 A1 WO 2009121726A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- repair
- microorganisms
- medium
- growth medium
- salt
- Prior art date
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Classifications
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- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention concerns a method for detecting and enumerating viable microorganisms of the species Legionella pneumophila in a sample.
- the invention also includes a kit suitable for use in such a method. This method and kit enable viable microorganisms to be quantified more rapidly.
- Legionella bacteria are ubiquitous in wet or moist environments such as soil and non-marine aquatic habitats. They can also be found in warm and cold water installations, cooling towers of air conditioning systems and water humidifiers.
- Legionella especially Legionella pneumophila, are pathogens that can cause an acute bacterial pneumonia, generally known as "legionnaires disease", which is often lethal for infected individuals.
- PCR Polymerase Chain Reaction
- DNA polymerase to amplify a piece of DNA by in vitro enzymatic replication. During the progression of the technique the DNA generated is used as a template for replication which brings about a chain reaction in which the DNA template is exponentially amplified. PCR enables a single or few copies of a piece of DNA to be amplified by generating millions or more copies of the DNA piece. Typically such a method is described by Diederen et al., J Med Microbiol. 2007 Jan; 56 (Pt 1 ):94-101.
- PCR a drawback of PCR is that the samples tend to contain polymerisation reaction inhibitors and therefore do not consistently provide quantitative results. Furthermore, the technique relies upon a prior DNA purification step which can result in loss of DNA with the consequential underestimation of the Legionella present. To some extent these disadvantages are overcome by real-time PCR which is quantitative. However, the technique cannot distinguish between viable cells and non-viable cells.
- FISH fluorescent in situ hybridisation
- an oligonucleotide probe labelled by a fluorescent substance penetrates into the bacteria cells.
- the probe will attach itself to its target and will not be removed by any subsequent washing step.
- the bacteria in which the probe is fixed will then emit a fluorescent signal.
- This fluorescent signal may then be quantified by techniques such as flow cytometry, solid phase cytometry, or epifluorescent microscopy.
- a typical FISH technique is described by Dutil S et al J Appl Microbiol. 2006 May;100(5):955-63. However, using the FISH technique alone the total number of viable Legionella pneumophila could be detected but unfortunately the method could not exclusively identify only those Legionella pneumophila bacteria able to divide and by consequence make a colony.
- a further method for enumerating viable Legionella pneumophila involves ChemChrome V6 and is described by Delgado-Viscogliosi et al Appl Environ Microbiol. 2005 Jul;71 (7):4086-96.
- This method allows the quantification of Legionella pneumophila as well as discrimination between viable and non-viable bacteria. It combines specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6) and employing epifluorescent microscopy for the enumeration.
- ChemChrome V6 a bacterial viability marker
- this technique distinguishes between viable and non-viable cells it is not able to separately identify those colony-forming bacteria.
- US 20070218522 describes methods and compositions for detecting and quantifying viable Legionella and other heterotrophic aerobic bacteria the method includes the use of dipslides that include an absorbent medium, growth promoting and growth selective substances for rapid detection and quantification of micro-colonies of Legionella. This technique would not enumerates injured bacteria.
- EP 1329515 relates to a method of testing for the presence of microorganisms in a gaseous environment comprising hydrogen peroxide by bringing the gaseous environment into contact with an agar growth medium comprising a salt of pyruvic acid and allowing the development of colonies of the microorganisms.
- a growth medium such as a nutrient agar plate
- the plate count method remains the preferred choice of method for obtaining the total viable count.
- This generally means applying a sample suspected of containing the microorganism onto a plate containing a solid nutrient source or growth medium.
- plating Such a technique is generally referred to as plating.
- total viable count we mean the total number of bacteria capable of yielding a population discernible by the observer. Typically this will mean a visible colony on the surface of a growth medium such as nutrient agar plate.
- microorganisms such as Legionella pneumophila in the environment may be subject to one or more stresses which prevent the microorganism from growing and multiplying in its environmental situation.
- Such stressed microorganisms would not divide at all or form a visible colony under normal culturing conditions.
- a proportion of microorganisms cells will generally be stressed due to environmental conditions, such as starvation, presence of biocide, heat shock and desiccation.
- these cells may be in a vulnerable physiological state in which the technique of plating the microorganisms may exacerbate stressing of those already stressed microorganisms cells due to the presence of atmospheric oxygen. Furthermore this could lead to artifactual death of the stressed bacteria leading to an underestimation of the total viable count.
- ROS reactive oxygen species
- an objective of the present invention is to find a method for accurately enumerating Legionella pneumophila. This is especially so in regard to its standard method using the plating technique.
- a method for detecting and enumerating viable microorganisms in a sample suspected of containing said microorganisms we provide a method for detecting and enumerating viable microorganisms in a sample suspected of containing said microorganisms
- oxidative stress we mean an imbalance between the concentration of ROS (endogene production or exogene adduction) and the ability of the microorganisms to readily detoxify the reactive intermediates or efficiently repair the resulting damage.
- ROS endogene production or exogene adduction
- Such disruption of the normal metabolic processes of the microorganism can cause toxic effects due to the formation of free radicals and oxidising agents, such as peroxides, which may lead to damage to the components of the microorganisms cells, for instance DNA, proteins or lipids.
- Causing an effect on the metabolism of the microorganism means bringing about changes to natural internal chemical processes within the microorganism cell.
- references to endogenously means changes are brought about within the microorganism cell to reduce oxidative stress. This could for instance be changes to the metabolic processes within the microorganism. It may also include removal of ROS within the microorganism cell.
- the repair compound may be or include at least one compound that inhibits the formation of and/or degrades ROS. In general this would be achieved by modification of the metabolism.
- the repair compound may be or include at least one compound that indirectly inhibits the formation of and/or degrades the ROS. Such a compound that exerts an indirect effect on the ROS may do this by interfering with the metabolism of the microorganism. Such a compound may be regarded as indirectly reducing ROS endogenously for instance during aerobic respiration.
- the present method induces the repair of stressed Legionella pneumophila cells and thus more accurately provides a total viable count.
- the method reduces the amount of incubation time required. In general we find that the method can reduce the incubation time by several hours and in some cases at least one day. In some cases method of the present invention may reduce the incubation time by up to several days, e.g. up to five days, by comparison to the conventional method.
- inventive method can bring about a reduction of interfering microorganisms i.e. those microorganisms other than the Legionella pneumophila.
- the method of the present invention desirably involves contacting stressed Legionella pneumophila microorganism cells with at least one compound that inhibits the formation of and/or reduces and/or removes ROS and this tends to induce repair of the stressed cells.
- the Legionella pneumophila microorganism may be brought directly in contact with the repair compound upon collection of the sample.
- the container into which the sample of water, believed to contain the microorganism, is collected may already contain the repair compound.
- a sample of water containing the Legionella pneumophila may be diluted with dilution water containing repair compound for analysis purpose.
- the sample, optionally having been diluted may be brought into contact with the growth medium containing the repair compound or the repair compound may be applied after contacting the microorganism with the growth medium.
- One form of this invention desirably involves contacting said sample with a repair medium, preferably a non-selective repair medium, containing said repair compound and then bringing this into contact with a growth medium, preferably a selective growth medium.
- a repair medium is a liquid and more preferably a broth.
- the repair medium is a liquid this is suitably referred to as a liquid repair method.
- the sample is first introduced into a liquid medium containing the repair compound.
- the liquid repair method allows stressed bacteria to repair in a non-selective liquid medium.
- the liquid repair method will employ a broth as the liquid medium.
- the liquid medium containing the microorganisms will then be transferred to a growth medium.
- the stressed microorganisms would either have been repaired prior to transference to the growth medium or would repair upon contact with the growth medium. More preferably the growth medium is a selective growth medium. Typically the liquid medium containing the microorganisms will be plated onto a selective growth medium plate such as a selective agar growth medium plate.
- step (1 ) comprises contacting said sample with a growth medium, preferably a non-selective growth medium containing said repair compound, and then bringing this into contact with a repair medium also containing said repair compound.
- the repair medium is a nonselective repair medium, more preferably a solid, and particularly preferably a selective agar growth medium.
- the repair medium is a solid this is would be termed a solid repair method.
- the solid repair method will involve contacting the sample with a non-selective growth medium containing of the repair compound. Subsequently this can be brought into contact with a selective growth medium containing the repair compound.
- the non-selective growth medium can be a non-selective agar growth medium.
- the sample can be plated onto any non-selective agar and then a selective agar growth medium containing the compound or compounds that prevent the formation, reduces or removes the ROS is overlaid onto the non-selective agar growth medium.
- the sample may be applied to a selective growth medium which already contains the repair compound.
- a selective growth medium may be a selective agar growth medium. Plating of the sample may be carried out as described previously.
- the sample may be collected from water in the form of an aerosol.
- the aerosol may be located in a cooling tower or air conditionner.
- the water condensed from the aerosol before testing according to the method of the present invention comprises contacting said sample from aerosol with a dilution water containing a repair medium, preferably a non-selective repair medium containing said repair compound, and then bringing this into contact with a growth medium also containing said repair compound.
- the growth medium should be suitable for growth of Legionella pneumophila. Suitable growth medium types are documented in the literature and are well known to the skilled person. Normally the growth medium should contain activated carbon and cysteine.
- the selective growth medium is a selective agar growth medium and more preferably is a buffered charcoal yeast extract (BCYE) agar growth medium.
- BCYE growth medium would become selective by the addition of antibiotic supplement.
- a highly desirable BCYE growth medium with antibiotic is known as GVPC (Glycine, Vancomycine, Polymyxine B, Cycloheximide).
- the plating method is documented in the literature and is well known that the skilled person. Typically the method will involve applying a quantity of these samples of water onto agar gel that has been placed in a Petri dish. This may be termed a Petri dish method or an agar plating method.
- the aim of the agar plating is to spread an aliquot, typically 100 ⁇ l of water suspected of containing the microorganism, termed a bacterial suspension, onto a solid medium in a Petri dish. Glass beads or a cell scraper can be used to spread the bacterial suspension on the agar plate. After spreading, most of the liquid is absorbed by the agar and a thin layer with bacteria remains on the agar surface.
- incubation By incubation, bacterial growth in the form of colonies developed on the agar surface.
- the incubation will occur at a temperature best suited for the microorganism, which is well documented in the literature and known to the skilled person. Typically the temperature will be between 30°C and 50°C, for instance around 37°C.
- the repair compound should be added in an amount effective to reduce oxidative stress of the microorganism. Preferably this will be an amount effective to reduce or substantially remove ROS in the microorganism cell.
- the repair compound includes at least thioglycolic acid or its salts thereof.
- the thioglycolic acid is in the form of thioglycolate and usually in the form of the sodium salt.
- the thioglycolic acid or salts scavenge exogenously ROS.
- the repair compound includes at least one compound selected from the group consisting of catalase, ascorbic acid (or salt thereof), metabisulphurous acid (or salt thereof), dimethyl sulphoxide (DMSO), 3,3'-thiodipropionic acid (TDPA) (or salt thereof) and pyruvic acid (or salt thereof). These compounds have all been found to reduce or remove ROS. When ascorbic acid and pyruvic acid are used they are preferably present in a medium at a concentration of between 0.01 and 1 % by weight calculated as the sodium salt.
- DMSO is preferably used at a concentration between 0.01 and 0.1 % by weight and catalase is desirably present at a concentration in the range of 0.001 to 0.1 % by weight.
- the repair medium or growth medium will comprise both thioglycolic acid (or salt) and at least one of the group selected from catalase, ascorbic acid (or salt thereof), metabisulphurous acid (or salt thereof), dimethyl sulphoxide (DMSO), 3,3'-thiodipropionic acid (TDPA) (or salt thereof) and pyruvic acid (or salt thereof).
- thioglycolate and sodium pyruvate is especially preferred.
- the repair compound may be or include at least one compound that indirectly inhibits the formation of and/or degrades the ROS
- said compound may bring about reduced levels of ROS by interfering with the metabolism of the microorganism.
- Such compounds will include amino acids or their salts.
- a particularly preferred compound is glutamic acid or glutamate salt.
- the repair compound would include glutamic acid or glutamate salt, especially the sodium salt.
- glutamic acid or glutamate salt especially the sodium salt.
- the amount of glutamic acid or glutamate will be between 0.01 and 5% by weight calculated as the sodium salt.
- the repair compound will include both pyruvic acid or pyruvate (especially the sodium salt) together with glutamic acid or glutamate (especially as the sodium salt).
- This combination of pyruvic acid or pyruvate with glutamic acid or glutamate seems to induce a synergistic effect in that it allows a higher estimation (and therefore more accurate estimation) of culturable Legionella than either compound respectively used alone.
- this combination brings about a further reduction of lag phase during development of the Legionella pneumophila, in particular in a liquid medium.
- Such a reduction of lag phase in liquid medium results in a reduction of the time required to obtain a visible colony on agar plate.
- the amount of pyruvate and glutamate will be as stated previously. It is particularly preferred that the ratio of glutamate to pyruvate will be in the range between 1 :1 and 50:1 , especially between 5:1 and 20:1 and more especially between 7:1 and 15:1.
- Glutamate is not known to be an antioxidant. However, it s appear that indirectly glutamate could reduce the endogenous production of ROS naturally formed during growth or their consequences on macromolecules (oxidation).
- a keto acid and/or a reduced oxygen scavenging enzyme may also be desirable to include a keto acid and/or a reduced oxygen scavenging enzyme with the repair medium and/or growth medium.
- a keto acid and/or a reduced oxygen scavenging enzyme are not considered a repair compound according to the present invention. Nevertheless, it may be beneficial to include one or both of these compounds with any of the aforementioned repair compounds or combinations thereof. Detecting and quantifying the viable microorganisms can be carried out by any of the known technique is documented in the literature. Typically this will mean counting the visible colonies of the surface of the growth medium, such as nutrient agar plate.
- the method according to present invention facilitates the accurate quantitative determination for the existence of Legionella pneumophila. Furthermore the incubation time may be significantly reduced.
- the method is suitable for detecting Legionella pneumophila in samples derived from any of the group selected from industrial cooling waters, drinking waters, and natural waters.
- the present invention also incorporates a kit for more accurately detecting and enumerating viable microorganisms of the species Legionella pneumophila in a sample suspected of containing said microorganisms comprising: (1 ) at least one repair compound,
- the microorganisms are of the species Legionella pneumophila
- the repair compound directly or indirectly causes an effect on the metabolism to reduce the oxidative stress of the microorganism
- the kit may also contain any of the embodiments described in regard to the first aspect of the invention.
- the kit is suitable for use with the method of the present invention and enables more accurate enumeration of Legionella pneumophila.
- the following examples illustrate the invention.
- a suspension of Legionella pneumophila was added to 5 flasks containing 50 ml of sterile phosphate buffer (PBS) at final concentration of 10 8 bacteria/ml.
- PBS sterile phosphate buffer
- a biocide solution was added to obtain final concentrations in the range 10 to 30 mg/L.
- One flask was performed in parallel and served as control without biocide.
- the biocide used is a THPS (tetrakis(hydroxymethyl)phosphonium sulfate).
- Figure 1 shows the enumeration of culturable Legionella Pneumophila after biocide treatment on BCYE medium (squares) and BCYE medium plus 0.1 % pyruvate (diamonds).
- the presence of the biocide is to introduce stressing of the microorganism.
- the results show that in the presence of pyruvate a much higher microorganism count is achieved where the microorganisms are stressed. In the absence of biocide the microorganisms are unstressed. In this case it can be seen that the presence and absence of pyruvate give the same result. This demonstrates that the presence of the pyruvate stressed microorganisms of Legionella pneumophila are repaired and and thus a more accurate reading is provided.
- a suspension of Legionella pneumophila was added to 1 flask containing 50 ml of sterile phosphate buffer (PBS) at final concentration of 10 8 bacteria/ml.
- a biocide solution was added to obtain final concentration of 15 mg/L.
- the biocide used is a THPS (tetrakis(hydroxymethyl)phosphonium sulfate). After homogenization, suspension was incubated at 37 ⁇ 1 °C, in the dark and with agitation for 60 min. Biocide is eliminated by 2 washes in PBS (5,000 x g, 10 min) before bacterial counting.
- a suspension of Legionella pneumophila was added to 1 flask containing 1 L of sterile phosphate buffer (PBS) at final concentration of 3 x 10 2 bacteria/ L. After concentration by filtration, 2 aliquot of 100 ⁇ l from the same suspension is plated on GVPC agar plate (GVPC), GVPC supplemented with 0.1 % of pyruvate and 1 % of glutamic acid (GVPC+X). Numbers of colony were counted at 0, 3, 5 and 10 days after incubation at 37°C. The results are shown in Figure 3. At 3 days of incubation, no colony was visible on GVPC medium when 300 colonies could be already enumerated on GVPC supplemented medium.
- PBS sterile phosphate buffer
- Example 5 A suspension of Legionella pneumophila was added to 1 flask containing 50 ml of sterile phosphate buffer (PBS) at final concentration of 10 8 bacteria/ml. A biocide solution was added to obtain final concentration of 15 mg/l.
- the biocide used is a THPS (tetrakis(hydroxymethyl)phosphonium sulfate).
- Figure 5 shows the number of culturable Legionella pneumophila obtained on standard medium (BCYE) and number of culturable Legionella pneumophila obtained on standard medium supplemented with pyruvate after dilution in PBS (scratched bar) or PBS + Pyruvate (dark bar).
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Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2718477A CA2718477C (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of legionella pneumophila |
JP2011502329A JP5393771B2 (en) | 2008-04-04 | 2009-03-20 | Microbe detection and counting |
DK09728732.0T DK2262907T3 (en) | 2008-04-04 | 2009-03-20 | DETECTION AND NUMBER OF MICRO-ORGANISMS |
EP09728732.0A EP2262907B1 (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of microorganisms |
RU2010144840/10A RU2490327C2 (en) | 2008-04-04 | 2009-03-20 | METHOD OF DETECTING AND COUNTING VIABLE Legionella pneumophila MICROORGANISMS |
AU2009231361A AU2009231361B2 (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of microorganisms |
US12/934,834 US9181575B2 (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of microorganisms |
UAA201013074A UA101657C2 (en) | 2008-04-04 | 2009-03-20 | Detecting and enumerating microorganisms |
CN200980112246.5A CN101998999B (en) | 2008-04-04 | 2009-03-20 | Microorganism detection and counting |
MX2010010456A MX2010010456A (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of microorganisms. |
IL208159A IL208159A (en) | 2008-04-04 | 2010-09-15 | Detection and enumeration of microorganisms |
HRP20180438TT HRP20180438T1 (en) | 2008-04-04 | 2018-03-14 | Detection and enumeration of microorganisms |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0806135.0A GB0806135D0 (en) | 2008-04-04 | 2008-04-04 | Detection and enumeration of microorganisms |
GB0806135.0 | 2008-04-04 | ||
GB0900850.9 | 2009-01-20 | ||
GB0900850A GB0900850D0 (en) | 2009-01-20 | 2009-01-20 | Detection and enumeration of microorganisms |
Publications (1)
Publication Number | Publication Date |
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WO2009121726A1 true WO2009121726A1 (en) | 2009-10-08 |
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ID=40652736
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Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2009/053295 WO2009121726A1 (en) | 2008-04-04 | 2009-03-20 | Detection and enumeration of microorganisms |
Country Status (17)
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US (1) | US9181575B2 (en) |
EP (1) | EP2262907B1 (en) |
JP (1) | JP5393771B2 (en) |
KR (1) | KR20100131515A (en) |
CN (1) | CN101998999B (en) |
AU (1) | AU2009231361B2 (en) |
CA (1) | CA2718477C (en) |
DK (1) | DK2262907T3 (en) |
HR (1) | HRP20180438T1 (en) |
HU (1) | HUE035702T2 (en) |
IL (1) | IL208159A (en) |
MX (1) | MX2010010456A (en) |
MY (1) | MY159867A (en) |
NO (1) | NO2262907T3 (en) |
PT (1) | PT2262907T (en) |
RU (1) | RU2490327C2 (en) |
WO (1) | WO2009121726A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011151793A1 (en) * | 2010-06-03 | 2011-12-08 | Basf Se | Detection and enumeration of microorganisms |
EP3587554A1 (en) * | 2018-06-26 | 2020-01-01 | Inwatec GmbH & Co. KG | Method for accelerated determination of the concentration of living thermophilic bacteria in water-bearing installations |
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CN103748211B (en) | 2011-07-13 | 2016-05-18 | 食品安全测试***公司 | Be used for listerial culture medium and cultural method and detect listerial method |
EP2617833A1 (en) * | 2012-01-18 | 2013-07-24 | Centre National de la Recherche Scientifique (CNRS) | A method for specifically detecting living bacteria |
EP2868750A1 (en) * | 2013-10-30 | 2015-05-06 | Centre National de la Recherche Scientifique (CNRS) | A method for labeling specifically living bacteria comprising the use of modified monosaccharide compounds |
RU2542969C1 (en) * | 2014-01-09 | 2015-02-27 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Ставропольский государственный аграрный университет" | Method for air microbioassay |
EP3091081A1 (en) * | 2015-05-04 | 2016-11-09 | Centre National de la Recherche Scientifique (CNRS) | A method for labeling specifically living microorganisms comprising the use of modified monosaccharide compounds |
EP3091082A1 (en) * | 2015-05-04 | 2016-11-09 | Centre National de la Recherche Scientifique (CNRS) | A method for labeling specifically living bacteria comprising the use of modified non endogenous monosaccharide compounds |
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FR2834998B1 (en) | 2002-01-18 | 2004-04-02 | Millipore Sas | METHOD FOR MONITORING THE PRESENCE OF MICROORGANISMS IN A GASEOUS MEDIUM COMPRISING HYDROGEN PEROXIDE |
FR2845097B1 (en) * | 2002-10-01 | 2006-06-16 | Metis Biotechnologies | METHOD FOR DETECTING AND COUNTING MICROORGANISMS IN A SAMPLE |
US7901932B2 (en) * | 2005-03-17 | 2011-03-08 | Phigenics, Llc | Methods and compositions for rapidly detecting and quantifying viable Legionella |
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2009
- 2009-03-20 HU HUE09728732A patent/HUE035702T2/en unknown
- 2009-03-20 DK DK09728732.0T patent/DK2262907T3/en active
- 2009-03-20 AU AU2009231361A patent/AU2009231361B2/en not_active Ceased
- 2009-03-20 JP JP2011502329A patent/JP5393771B2/en not_active Expired - Fee Related
- 2009-03-20 CA CA2718477A patent/CA2718477C/en active Active
- 2009-03-20 EP EP09728732.0A patent/EP2262907B1/en active Active
- 2009-03-20 NO NO09728732A patent/NO2262907T3/no unknown
- 2009-03-20 US US12/934,834 patent/US9181575B2/en not_active Expired - Fee Related
- 2009-03-20 MX MX2010010456A patent/MX2010010456A/en active IP Right Grant
- 2009-03-20 PT PT97287320T patent/PT2262907T/en unknown
- 2009-03-20 RU RU2010144840/10A patent/RU2490327C2/en active
- 2009-03-20 CN CN200980112246.5A patent/CN101998999B/en not_active Expired - Fee Related
- 2009-03-20 MY MYPI2010004365A patent/MY159867A/en unknown
- 2009-03-20 WO PCT/EP2009/053295 patent/WO2009121726A1/en active Application Filing
- 2009-03-20 KR KR1020107024742A patent/KR20100131515A/en active Search and Examination
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2010
- 2010-09-15 IL IL208159A patent/IL208159A/en active IP Right Grant
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2018
- 2018-03-14 HR HRP20180438TT patent/HRP20180438T1/en unknown
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IL208159A (en) | 2017-10-31 |
DK2262907T3 (en) | 2018-04-09 |
AU2009231361A1 (en) | 2009-10-08 |
CA2718477A1 (en) | 2009-10-08 |
CN101998999B (en) | 2015-08-05 |
EP2262907B1 (en) | 2017-12-20 |
PT2262907T (en) | 2018-03-01 |
HRP20180438T1 (en) | 2018-04-20 |
HUE035702T2 (en) | 2018-05-28 |
RU2490327C2 (en) | 2013-08-20 |
KR20100131515A (en) | 2010-12-15 |
CN101998999A (en) | 2011-03-30 |
IL208159A0 (en) | 2010-12-30 |
NO2262907T3 (en) | 2018-05-19 |
RU2010144840A (en) | 2012-05-20 |
EP2262907A1 (en) | 2010-12-22 |
MX2010010456A (en) | 2010-12-20 |
JP5393771B2 (en) | 2014-01-22 |
MY159867A (en) | 2017-02-15 |
AU2009231361B2 (en) | 2012-08-02 |
CA2718477C (en) | 2014-05-13 |
US20110065145A1 (en) | 2011-03-17 |
US9181575B2 (en) | 2015-11-10 |
JP2011516051A (en) | 2011-05-26 |
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