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WO1999002649A1 - Support de recuperation et de culture de micro-organismes - Google Patents

Support de recuperation et de culture de micro-organismes Download PDF

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Publication number
WO1999002649A1
WO1999002649A1 PCT/GB1998/002015 GB9802015W WO9902649A1 WO 1999002649 A1 WO1999002649 A1 WO 1999002649A1 GB 9802015 W GB9802015 W GB 9802015W WO 9902649 A1 WO9902649 A1 WO 9902649A1
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WO
WIPO (PCT)
Prior art keywords
recovery
growth
cells
reagents
growth medium
Prior art date
Application number
PCT/GB1998/002015
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English (en)
Inventor
Peter Jeremy Stephens
Patrick Druggan
Original Assignee
Oxoid Limited
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
Priority claimed from GBGB9714594.0A external-priority patent/GB9714594D0/en
Application filed by Oxoid Limited filed Critical Oxoid Limited
Priority to EP98932405A priority Critical patent/EP1002053A1/fr
Publication of WO1999002649A1 publication Critical patent/WO1999002649A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66

Definitions

  • This invention relates to growth of microorganisms, and concerns reagents for use in the recovery and growth of rmcroorganisms, for instance in techniques for detecting microorganisms, for example in food samples.
  • pathogenic microorganisms such as Listeria and Salmonella are capable of proliferating very rapidly under e right conditions, the presence of even a single viable cell of such organisms in a foodstuff may give rise to serious infection after the foodstuff has been ingested by a human. It is therefore necessary for such organisms to be detectable even when present at extremely low levels.
  • a typical conventional technique for detecting a target microorganism of interest involves incubating a sample in a pre-enrichment broth, commonly buffered peptone water (BPW) for 18 to 24 hours under conditions which encourage microorganism recovery and growth, so that any organisms present can proliferate in the sample and attain population levels which are more readily detectable.
  • This step is known as a pre- enrichment, recovery or resuscitation step.
  • Portions of the pre-enrichment culture are then subcultured into selective enrichment broths and incubated for a further 20 to 24 hours.
  • the selective enrichment broths are designed to inhibit growth of comparatively innocuous non-target microorganisms and so favour the growth of the target microorganisms. This step is known as a selective enrichment step.
  • the target organisms are then identified.
  • Conventional processing involves subculturing enrichment broths onto selective differential agar plates and incubating for 20 to 24 hours. Suspected colonies of target organism are identified by visual examination, and selected suspected colonies are removed from the plates, purified and identified, eg using triple sugar iron (TSI) agar and lysine iron (LSI) agar slopes and serological tests. Purification, identification and confirmation can take up to 48 hours.
  • TSI triple sugar iron
  • LSI lysine iron
  • the culture stage of pre-enrichment still remains essential. Direct inoculation of a naturally contaminated sample into selective enrichment media often results in a failure to recover the organisms of interest. This may occur because some healthy cells are often killed when added to selective culture media and thus if they are present in low numbers this may lead to the death of the entire population of the organism of interest.
  • Pre-enrichment allows the number of organisms to increase, thus reducing the detrimental effects of the initial killing.
  • sublethally damaged cells are more sensitive to selective agents than undamaged cells, and the concentration of selective agents used is unlikely to facilitate any recovery of injured organisms.
  • pre-enrichment allows the cells of interest to repair any lesions and thus regain their resistance to selective agents, prior to enrichment. In many cases it may be necessary to recover small numbers of damaged cells and thus both events occur during pre-enrichment, ie. damaged cells are able to repair and grow to much larger numbers before they are exposed to selective agents.
  • Cells entering a food as an environmental contaminant may be starved, desiccated or previously exposed to disinfectants or antibiotics.
  • cells that have been exposed to relatively mild stresses such as chilling and changes in oxygen level may also show evidence of sublethal damage/stress.
  • the present invention concerns reagents for use in the recovery and growth of microorganisms that have particular, but not exclusive, application as a recovery medium for use in a pre-enrichment step.
  • the present invention provides reagents for use in the recovery and growth of microorganisms, comprising
  • Toxic oxidising species include hydrogen peroxide, hydroxyl radicals, superoxide anions and singlet oxygen. Toxic oxidising species may be generated internally by cells by various different mechanisms, or may be generated in the growth medium.
  • the expression "generating low levels of toxic oxidising species” means that levels of toxic oxidising species generated in use of the medium are below those to which healthy cells of the microorganism(s) of interest are sensitive. It is difficult to quantify the expression "generating low levels of toxic oxidising species” by reference to the specific amounts of hydrogen peroxide, hydroxyl radicals, superoxide anions, singlet oxygen etc generated. However, attempts can instead be made to quantify compounds that are a source of toxic oxidising species and to measure the total amount of oxidising species.
  • Riboflavin is an important source of toxic oxidising species, generating such species on exposure to light.
  • An optimised growth medium has been produced which contains 0.045mg/l riboflavin, and experiments involving adding riboflavin to the optimised medium have shown that performance of the medium is not significantly affected until more than 0.2mg/l riboflavin is added.
  • the growth medium desirably contains not more than 0.245 mg riboflavin per litre of suspension.
  • the main sources of riboflavin in conventional dehydrated culture media are yeast and liver peptone, so it is appropriate for the growth medium to include such ingredients in small amounts and/or in forms low in riboflavin.
  • Riboflavin only produces toxic oxidising species on exposure to light, so an alternative approach is to maintain the growth medium in light- free conditions during storage and use.
  • Riboflavin levels are conveniently determined by high performance liquid chromatography (HPLC).
  • the level of hydrogen peroxide equivalent of the growth medium is preferably less than about 0.03mM, more preferably less than about 0.02mM.
  • vitamins particularly B vitamins, may also act as sources of toxic oxidising species, so these should be present in the growth medium at suitably low levels.
  • OXYRASE enzyme is a known recovery agent that aids recovery of stressed or damaged cells.
  • OXYRASE is an enzyme composition which is made from sterilised bacterial membrane fragments, and which is know to be an effective oxygen-reducing enzyme used to produce anaerobic conditions.
  • the OXYRASE enzyme is described in technical bulletins distributed by Oxyrase, Inc. of Ohio and is further described by Adler et al in J. Bacteriology, August 1981, 326-332, and in a paper by H I Adler in Critical Reviews of Biotechnology 10: 118 (1990) entitled "The Use of Microbial Membranes to Achieve Anaerobiosis". See also US Patents Nos. 4476224, 4996073 and 5204853 and WO88/04319.
  • the word OXYRASE is a trade mark of Oxyrase, Inc, from whom OXYRASE enzyme is available as a frozen suspension.
  • OXYRASE enzyme is used in freeze-dried form, for ease of handling and storage and to increase shelf life.
  • OXYRASE enzyme may be freeze-dried, e.g. using conventional freeze-drying techniques. For freeze-drying it is necessary to use the OXYRASE enzyme in a form without lactate and succinate, as freeze-drying is otherwise not possible. If necessary, lactate and succinate must be removed from the OXYRASE enzyme, eg by centrifuging, prior to freeze-drying.
  • the hydrogen donor should be present in sufficient amount for functioning of the enzymes contained in OXYRASE. Suitable donors include lactic acid, succinic acid, formic acid, alpha glycerol phosphate and salts of these materials. A mixture of hydrogen donors may be used.
  • the currently preferred hydrogen donor is succinate, e.g. sodium succinate.
  • a millimolar concentration of the hydrogen donor, in use, is generally ample to remove all dissolved oxygen. Good results have been obtained with hydrogen donor at a concentration of 15mM.
  • the hydrogen donor may be included as an ingredient of the growth medium, providing a 2 component recovery system, or may be presented as a separate reagent, eg in the form of a vial of liquid reagent, providing a 3 component recovery system. In the latter case, the growth medium may be more versatile and multi-purpose.
  • the reagents may be presented in solid form, eg impregnated into agar, typically by adding agar to reagent broth in an amount 15g of agar per litre of broth. In this case it may be appropriate to use OXYRASE at increased concentration, eg at up to 10 times the concentration that would otherwise be appropriate.
  • OXYRASE enzyme causes the cells to grow anaerobically thus bypassing any internal pathways that produce toxic oxidising species such as hydrogen peroxide.
  • OXYRASE enzyme also contains catalase which removes external hydrogen peroxide which is why when it is added to poor peptones at high concentrations it is still effective at improving recovery. Additionally, free nucleic acid and lipid material in die OXYRASE preparation may absorb toxic oxidising species. Additional catalase may optionally be included in the reagents.
  • optimised microorganism recovery and growth it is necessary to optimise the nature and amount of growth medium and the amount of OXYRASE enzyme. Poorer performing growth medium combined wiui high concentrations of OXYRASE enzyme, and optimised growth medium combined with lower concentrations of OXYRASE enzyme, will achieve similar results in terms of speed and extent of microorganism recovery and growth.
  • OXYRASE enzyme is expensive so it is therefore beneficial to use it at the lowest concentration possible. For this reason, use of an optimised growth medium is highly desirable.
  • the combination of use of a growth medium generating low levels of toxic oxidising species together with OXYRASE enzyme enables significantly improved recovery, i.e. better and faster recovery, of stressed microorganism cells than is possible with conventional growth media. Not only are more stressed cells recoverable, but also recovery of all cells takes place in less time than under traditional conditions. Both the recovery medium and OXYRASE act to reduce oxidative stress, which is believed to be involved in cell recovery in a number of ways:
  • the growth medium contributes only low levels of toxic oxidising species, e.g. hydrogen peroxide, hydroxyl radicals, superoxide anions and singlet oxygen, to the medium, possibly generated by exposure to oxygen, light and/or high temperatures, eg on autoclaving.
  • toxic oxidising species e.g. hydrogen peroxide, hydroxyl radicals, superoxide anions and singlet oxygen
  • OXYRASE causes the cells to grow anaerobically thus bypassing any internal pathways that provide toxic oxidising species such as hydrogen peroxide.
  • OXYRASE also contains catalase which removes external hydrogen peroxide which is why when it is added to poor growth media at high concentration it is still effective at improving recovery. Additionally, free nucleic acid and lipid material in the OXYRASE preparation may absorb toxic oxidising species.
  • the growth medium may be otherwise generally conventional in formulation.
  • Typical ingredients include peptone, sodium chloride, buffer such as orthophosphate.
  • the reagents of the invention will work for a very wide range of microorganisms, bodi facultative and obligate anaerobes. It is also expected that the reagents of the invention will be effective with cells exposed to may different types of stress: so far they have been shown to be effective with cells damaged by heat, acid/salt combinations, hypochlorite and irradiation.
  • the reagents may be used for enriching the population of a target microorganism in a sample, which is the conventional first step in detecting a target microorganism of interest, e.g. Salmonella, in a sample such as a sample of a foodstuff, beverage or an environmental sample.
  • a target microorganism of interest e.g. Salmonella
  • the reagents of the invention may, for example, be used as the pre-enrichment medium in methods of enriching the population of a target microorganism in a sample as disclosed in the specification of our copending British Patent Application No. 9714594.0 filed 11th July 1997 (pursued in a PCT application), which conveniently also involves the use of timed-release capsules containing selective agents.
  • the reagents of the invention also find use in other techniques requiring recovery and growth of microorganisms, particularly microorganisms in stressed, damaged or debilitated condition.
  • the reagents of the invention could also be used as an alternative to the traditional Buffered Peptone Water (BPW) in a pre-enrichment step either in a conventional 20-24h incubation period or in a shortened incubation period followed by an earlier transfer to selective enrichment media. They could be used with die most sensitive rapid detection techniques such as PCR whereby low numbers of target organism (where low numbers still means greater than 10 3 cells/ml for successful PCR detection) can be detected in a very dense background flora of non-target organisms, thus possibly removing the need for any form of selective enrichment. Generation of such levels of target organisms is not possible in all situations with current pre-enrichment because of the Jameson Effect preventing increase in the target population before recovery is complete. With faster recovery of all stressed cells, using the reagents of the invention, this may enable high enough numbers of target cells to be generated before the Jameson Effect takes place.
  • PCR Peptone Water
  • the reagents of the invention find application in many areas in addition to the area of foodborne pathogen isolation.
  • the reagents will be of interest for research purposes as well as for use in commercial testing in the recovery of so called "viable but non- culturable" microorganisms, particularly those present in soil and water samples.
  • viable but non- culturable microorganisms particularly those present in soil and water samples.
  • the conditions created by use of reagents of the invention are highly realistic when compared to conditions micToorganisms might be exposed to in natural environments. Apart from when encountering a host defence system that uses oxidative stress as a means to kill invading cells it is unlikely for a microorganism to be expected to recover and grow in the presence of riboflavin and hydrogen peroxide.
  • the recovery conditions created by use of the reagents of the invention very closely resemble those of an animal intestine.
  • Figures 1 and 2 are graphs of absorbance (600nm) versus time (h) illustrating the recovery of heat- injured Salmonella typhimurium using a conventional medium ( Figure 1) and reagents in accordance with the invention ( Figure 2);
  • Figure 3 is a bar chart illustrating the effect of recovery conditions on the recovery of low levels of heat injured Salmonella typhimurium, showing the number of cells recovered relative to those obtained using a standard conventional medium (Buffered Peptone Water (BPW) from Oxoid Limited) expressed as difference in log I0 cells recovered/ml; and
  • BPW Peptone Water
  • Figure 4 is a further bar chart illustrating the effect of recovery conditions on the recovery of low levels of heat injured Salmonella typhimurium, showing the number of cells recovered relative to those obtained using a standard conventional medium (Buffered Peptone Water (BPW) from Oxoid Limited) expressed as difference in log 10 cells recovered/ml.
  • BPW Bitered Peptone Water
  • Optimised reagents for use in the recovery and growth of microorganisms comprise
  • the growth medium (a) is intended to be dissolved in distilled water in the amount of 4.275g of medium in 225ml of water to produce a broth having the following composition:
  • the peptone component of the pre-enrichment broth formulation is a meat based product comprising: % dry weight meat meal 82.3 tryptone 13.9 yeast extract 2.5 disodium hydrogen orthophosphate 1.3
  • the riboflavin content of the growth medium was measured by the technique described in Example 3, and was determined to be 0.045 mg/1.
  • the freeze-dried OXYRASE enzyme (b) is produced by freeze-drying in known manner OXYRASE enzyme obtained from Oxyrase, Inc. , after removal of succinate and lactate if present.
  • OXYRASE enzyme obtained from Oxyrase, Inc.
  • This amount of freeze-dried OXYRASE enzyme should be rehydrated with 2mls of sterile distilled water and mixed gently to avoid frothing.
  • the sodium succinate in the growth medium acts as a hydrogen donor for the enzymes contained in the OXYRASE, and is present in an amount that equates to 15mM.
  • Example 1 the sodium succinate is omitted from the growth medium and is present as a separate reagent in the form of a vial of liquid sodium succinate.
  • the reagents thus constitute a 3 component recovery system comprising:
  • the reagents are otherwise the same as in Example 1, and are used generally the same way, but with me sodium succinate being added to me growth medium after rehydration.
  • the sodium succinate solution may be used to rehydrate the freeze-dried OXYRASE enzyme.
  • Hydrogen peroxide levels in pre-enrichment media were analysed using a YSI Model 2700 Select Biochemistry Analyser (Yellow Springs Instrument Company, Ohio, U.S.A.).
  • the sensor probe is covered by a membrane which in instruments for detecting glucose and otiier similar metabolites would contain immobilised oxidase enzyme.
  • Sample material containing the appropriate metabolite would diffuse into die membrane where the oxidase enzyme would rapidly oxidise the text metabolite and produce hydrogen peroxide which in turn would be detected at the electrode.
  • the oxidase membrane was replaced with a non-enzymatic protein membrane. This allowed hydrogen peroxide in the sample to diffuse directly through to the electrode.
  • the electrode consists of a platinum anode and a silver cathode. Hydrogen peroxide is oxidised at the platinum electrode producing a flow of detectable electrons:
  • the platinum anode is capable of oxidising many substances other than hydrogen peroxide.
  • the membrane contains an inner layer consisting of a very thin film of cellulose acetate.
  • the film readily passes hydrogen peroxide but excludes chemical compounds with molecular weights above approximately 200 Daltons. The film, however, can still be penetrated by compounds such as hydrogen sulphide, low molecular weight mercaptens, hydroxylamines, hydrazines, phenols and anilines.
  • a vial of freeze-dried Salmonella typhimurium (Colworth House Microbiology Culture Collection No. 3073) was opened aseptically and its contents rehydrated in 1 ml of Heart Infusion Broth (HUB, Difco, 0038-17-7).
  • a quantity (0.5ml) of the reconstituted culture was inoculated into 90 ml of fresh HIB and incubated shaking (80 rev min "1 ) in a 250 ml conical flask in a waterbath at 37° C. After 20 h a sterile loop was used to remove a small volume of this broth culture and inoculate a slope of Heart Infusion Agar (HIA, Difco, 0044-17). This was incubated for 20 h at 37°C to give a dense lawn of growth and then stored at 4°C until required. Fresh slopes were made every 14 days.
  • a mid-exponential phase culture of Salmonella typhimurium was set up by transferring, by loop, a small portion of the slope culture into 9 ml of HIB. This was then incubated at 37 °C for 20 h without shaking.
  • 1 ml of this stationary phase culture was used to inoculate 90 ml of pre-warmed HIB in a 250 ml conical flask held in a shaking waterbath (80 rev min 1 ) at 37°C. This culture was incubated for 3-4 h until its optical density (OD) at 600 nm against a HIB blank was 0.300.
  • a submerged-coil heating apparatus This comprised a narrow bore stainless steel coil submerged in a thermostatically controlled waterbath, generally as described in Cole and Jones 1990, Letters in Applied Microbiology 11, 233-235. Before every experiment the coil was sterilized, flushed through several times with sterile distilled water and pre-heated to the appropriate temperature. The temperature was verified using a calibrated electronic probe held in the waterbath.
  • the kinetics of recovery and growth of heat-injured Salmonella were measured in a number of different pre-enrichment media; including Buffered Peptone Water, optimised peptone medium, yeast extract peptone medium and optimised peptone medium plus OXYRASE (0.2 units/ml). All media were prepared and handled according to the manufacturer's guidelines.
  • the second portion of the 1 in 25 diluted culture of heat-injured cells was serially diluted, up to 10 9 fold, through the pre-enrichment media under investigation.
  • the same first dilution was used before separately diluting the culture in each of the different broths.
  • Each dilution was then used to inoculate up to 4 Bioscreen honeycomb plates. Up to 100 wells were filled (400 ⁇ l) for each dilution.
  • the inoculated honeycomb plates were placed in the reading chamber of a Labsystems Bioscreen (Life Sciences International) where they were incubated at 37 °C and shaken at medium intensity for 5 sec prior to every reading.
  • Measurements of the OD of all wells were made at a wavelength of 600 nm at 15 min intervals for 48 h. The data generated were then converted into Microsoft Excel format and where appropriate, processed into growth curves. The inoculum level was estimated from a most probable number (MPN) calculation on the number of positive and negative wells in the Bioscreen plates. Purity of Salmonella cultures, in Bioscreen plates and microtitre plates used for MPNs, were checked by streaking random well contents onto HIA and XLD agar (Oxoid CM469).
  • the second portion of the 1 in 25 diluted culture was also used for an MPN analysis of cell numbers surviving and growing in the different pre-enrichment media.
  • the dilutions containing the fewest cells were placed into 96 well microtitre plates (200 ⁇ l well "1 ). At least two plates were filled for each dilution. The plates were sealed with a plastics film (ICN How, UK) before being incubated at 37°C. After 48 h incubation the number of wells in which growth had occurred, ie. the number of turbid wells, was assessed visually, was recorded and an MPN estimate calculated using a standard MPN equation.
  • a heating time of 15 min was chosen as this generated a large population of injured cells but not too many dead cells.
  • Figures 1 and 2 are Bioscreen printouts (showing absorbance at 600nm versus time in hours) illustrating lag times of heat injured Salmonella typhimurium cells (injured by treatment at 53.5 °C for 15 minutes) incubated at 37°C with different recovery media at inoculum levels obtained by 10 "5 dilution of a 2 x 10 8 cells/ml population of injured cells.
  • Figure 1 shows results using a conventional unmodified Buffered Peptone Water (BPW) from Oxoid Limited
  • Figure 2 shows results using the reagents generally as described in Example 1, in the form of optimised peptone medium as described in Example 1 but not including succinate, with fresh OXYRASE (including succinate at the same level used in Example 1) added to give a working concentration of OXYRASE of 0.2 units/ml.
  • BPW Peptone Water
  • FIG. 1 A comparison of Figures 1 and 2 shows much more rapid and uniform recovery of heat injured cells using reagents in accordance with the invention as compared with conventional BPW.
  • Example 4 To compare the effect of riboflavin levels in growth media on the recovery times of heat- injured Salmonella cells, experiments were carried out using the technique described in Example 4 using the optimised growth medium of Example 1 (which contains riboflavin in an amount of 0.045 mg/1) ("optimised peptone", used as a control) and the optimised peptone with various amounts of riboflavin added prior to autoclaving of the media.
  • the results are shown graphically in Figure 4. The results show that adding up to 0.2 mg/1 riboflavin has no significant effect on recovery, but at higher levels of added riboflavin recovery deteriorates significantly.
  • the growth medium of the invention should contain riboflavin at a concentration not exceeding 0.245 mg/1.

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Abstract

L'invention porte sur des réactifs destinés à être utilisés dans la récupération et la culture de micro-organismes. Cette invention comprend (a) un support de culture générant de faibles taux d'espèces oxydantes toxiques; (b) un donneur d'hydrogène; et (c) une enzyme OXYRASE lyophilisée. La combinaison de l'utilisation d'un support de culture générant de faibles espèces oxydantes toxiques et de l'enzyme OXYRASE permet d'améliorer considérablement la récupération, c'est-à-dire de récupérer de manière plus fiable et plus rapide des cellules de micro-organismes soumises à un stress qu'il n'est possible de le faire avec les supports de culture traditionnels. Non seulement un plus grand nombre de cellules soumises à un stress sont récupérables, mais la récupération de toutes les cellules s'effectue en moins de temps que dans les conditions traditionnelles.
PCT/GB1998/002015 1997-07-11 1998-07-10 Support de recuperation et de culture de micro-organismes WO1999002649A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP98932405A EP1002053A1 (fr) 1997-07-11 1998-07-10 Support de recuperation et de culture de micro-organismes

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB9714594.0 1997-07-11
GBGB9714594.0A GB9714594D0 (en) 1997-07-11 1997-07-11 Detection of microorganisms
EP97307332.3 1997-09-19
EP97307332 1997-09-19
GB9721396.1 1997-10-09
GBGB9721396.1A GB9721396D0 (en) 1997-07-11 1997-10-09 Improvements in or relating to growth of microorganisms

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WO1999002649A1 true WO1999002649A1 (fr) 1999-01-21

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EP2314669A1 (fr) 2009-10-26 2011-04-27 Sarl, Polyor Substrats matriciels carbones pour l'obtention de bactéries biofertilisantes
JP2013517769A (ja) * 2010-01-22 2013-05-20 日立化成株式会社 複数病原菌検出の方法
EP2684588A1 (fr) 2012-07-13 2014-01-15 Polyor SARL Filtres macro/microporeux pour l'incubation et le diagnostique de l'activité microbiologique d'echantillons environnementaux

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Publication number Priority date Publication date Assignee Title
GB0022556D0 (en) 2000-09-14 2000-11-01 Oxoid Ltd Improvements in or relating to selective agents for biological cultures
US9274101B2 (en) 2001-04-20 2016-03-01 Biolog, Inc. Methods and kits for obtaining a metabolic profile of living animal cells
FR2834998B1 (fr) 2002-01-18 2004-04-02 Millipore Sas Procede de controle de la presence de micro-organismes dans un milieu gazeux comprenant du peroxyde d'hydrogene
FR2845097B1 (fr) 2002-10-01 2006-06-16 Metis Biotechnologies Procede de detection et de comptage de microorganismes dans un echantillon
IT1393642B1 (it) 2009-04-08 2012-05-08 Gruppo Meccaniche Luciani Srl Calzatura con sistema di aerazione ottenuto con processo ad iniezione diretta su tomaia
EP2302029A1 (fr) 2009-09-29 2011-03-30 Fundacion Gaiker Dispositif portable d'enrichissement, aliquotage et test pour micro-organismes et toxines
CN108026562A (zh) * 2015-09-03 2018-05-11 3M创新有限公司 富集和检测靶微生物的方法

Citations (5)

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