WO2010086565A1 - Procede d'isolement ou de denombrement de microorganismes sur un milieu de culture gelose - Google Patents
Procede d'isolement ou de denombrement de microorganismes sur un milieu de culture gelose Download PDFInfo
- Publication number
- WO2010086565A1 WO2010086565A1 PCT/FR2010/050146 FR2010050146W WO2010086565A1 WO 2010086565 A1 WO2010086565 A1 WO 2010086565A1 FR 2010050146 W FR2010050146 W FR 2010050146W WO 2010086565 A1 WO2010086565 A1 WO 2010086565A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- culture medium
- agar culture
- suspension
- sample
- microorganisms
- Prior art date
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 74
- 229920001817 Agar Polymers 0.000 title claims abstract description 59
- 239000008272 agar Substances 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 55
- 244000005700 microbiome Species 0.000 title claims abstract description 38
- 238000010899 nucleation Methods 0.000 claims abstract description 56
- 239000000725 suspension Substances 0.000 claims abstract description 52
- 238000003892 spreading Methods 0.000 claims abstract description 40
- 238000000151 deposition Methods 0.000 claims abstract description 5
- 238000006073 displacement reaction Methods 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 description 24
- 238000002955 isolation Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 230000000813 microbial effect Effects 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 9
- 238000009826 distribution Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 238000010835 comparative analysis Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 238000011534 incubation Methods 0.000 description 3
- 230000009191 jumping Effects 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000013459 approach Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
Definitions
- the field of the invention is that of the analysis of target microorganisms in a complex sample. More particularly, the present invention relates to a method for isolating or even counting microorganisms on an agar culture medium from a liquid sample to be analyzed or a suspension of microorganisms.
- Isolation of microorganisms on agar culture medium, from a liquid sample to be analyzed or a suspension of microorganisms, is a step often indispensable to many microbiological analysis processes. This step is used in particular to carry out identifications, to verify the microbial purity of a sample or to carry out a bacterial count by counting the isolated colonies thus obtained.
- the sample to be analyzed contains an undetermined amount of germs that can range from zero to more than 10 8 bacteria per milliliter (ml).
- a determined volume of each dilution thus produced is then spread on agar medium.
- the Petri dishes corresponding to each of the dilutions are then incubated in a thermostated chamber. After microbial growth, it is possible to select the petri dish of agar medium whose microbial load on the box is sufficiently low to distinguish and possibly transplant isolated colonies.
- EP-O 242 114 which describes an apparatus and a method for seeding a culture medium with a sample.
- the method consists in making several segments of spreading from an inoculum. These segments are in the form of an arc and are made by means of four different spreading heads. A dilution effect of the sample is obtained by partial overlap of the subsequent segments.
- the method described in the document is in fact very close to the manual reference isolation method which consists of producing several segments of spreading from a single inoculum by performing overlapping segments in order to load the seeding means. in bacteria and to obtain a depletion of bacteria during the subsequent spreading segment.
- FR-A-2 694 570 describes a method and a system for depositing bacterial solutions on a culture medium using a stylet in fluid communication via a hose to a bacterial solution dispenser and fed by means of a cylinder.
- the bacterial solution is deposited in the form of spirals or spots, by rotational displacement of the culture medium on an ad hoc platform at the same time that the bacterial solution is poured onto the medium.
- Such a method can not be considered as an isolation method insofar as the bacterial solution is discharged throughout the movement of the medium by rotation. Indeed, there is no depletion in bacterial solution and therefore in bacteria.
- a first objective of the present invention is therefore to provide a process for isolating microorganisms that is more efficient than the methods of the state of the art.
- a second object of the present invention is to provide a method of enumeration more efficient than the methods of the state of the art.
- a third objective of the present invention is to provide a method for isolating, or even counting, microorganisms making it possible to obtain isolated colonies on a very wide range of microorganism charges.
- a fourth objective of the present invention is to provide a method for isolating, or even counting, microorganisms to obtain a reliable evaluation of the microorganism load of the sample or initial suspension (e).
- a fifth object of the present invention is to provide a method of isolating, or even counting, microorganisms exploitable on a reduced surface of agar culture medium.
- a second object of the present invention relates to a method of enumeration of microorganisms on an agar culture medium comprising the steps of:
- the rupture and the restoration of the contact between the seeding means and the agar culture medium during the displacement of said means can be likened to a jump of said means.
- This jump allows the seeding means to be depleted in sample or suspension. Indeed, as long as the seeding means is in contact with the agar culture medium, it entrains the liquid by capillary drainage.
- said seeding means is removed from the surface of the culture medium, until the liquid recoil. There is no longer any entrainment of the liquid vein. During this recess, the seeding means carries with it a fraction of sample or suspension remained attached to said seeding means.
- the displacement of the seeding means, while the latter is out of contact with the culture medium, is continued in a direction substantially parallel to the surface of said culture medium.
- This displacement must be sufficient so that, when the contact between the seeding means and the agar culture medium is restored, this new contact point is sufficiently far from the last point of contact before rupture, to avoid any contact between the seeding medium and the previous spreading. Indeed, such contact may lead to sample transfer or suspension of the first spread, and associated bacteria, on the seeding means thus limiting the depletion phenomenon.
- the seeding means resumes its horizontal displacement on the agar culture medium, causing the fraction of sample or suspension remained attached, by capillary drainage and allowing a new spread of this fraction.
- the factors that influence the amount of sample or suspension retained on the seeding means during the contact break are essentially: the wettability of the agar culture medium;
- the seeding means has a multitude of contact surfaces with said culture medium.
- Such seeding means may be for example an applicator used with the PREVI TM Isola system, as protected in the patent application WO-A-2005071055.
- the seeding means has a single contact surface with said culture medium.
- Such means may be for example a oese, a platinum loop or a swab.
- the displacement of the seeding means may advantageously be a rectilinear movement.
- Straight movement means one or more rectilinear segments, possibly of different directions. Such a movement is conventionally used in the traditional process of isolation with a platinum handle or loop.
- the displacement of the seeding means is a curvilinear displacement.
- Such a movement is the one used in the PREVI TM Isola system.
- the displacement of the seeding means follows the edges of the petri dish when it is a round box.
- this curvilinear movement makes it possible to increase the length of the spreading.
- the method according to the invention can be implemented by means of an automated system.
- a particularly suitable system is the PREVI TM Isola system marketed by the applicant.
- the number of times that the contact between said seeding means and the surface of the agar culture medium is broken and restored is between 2 and 6 times.
- the volume of sample or suspension deposited on the agar culture medium is between 10 and 1000 ⁇ l.
- the spreading segments are of variable lengths. Indeed, for the same isolation, it may be advantageous to produce successive spreading segments of different lengths. This is particularly the case for samples suspected of being excessively loaded with microorganisms.
- Several spreading segments of limited length will make it possible to deplete the seeding means very rapidly, on a very small surface of culture medium.
- the subsequent plating segments are longer in length to allow isolated colonies to be obtained.
- FIG. 1 represents the views of a comparative analysis between an isolation carried out according to the traditional method and isolations carried out according to various procedures of the method of the invention, with a bacterial suspension at approximately 10 8 CFU / ml.
- FIG. 2 represents the views of a comparative analysis between an isolation carried out according to the traditional method and isolations carried out according to various procedures of the method of the invention, with a bacterial suspension at approximately 10 7 CFU / ml.
- FIG. 3 represents the snapshots of a comparative analysis between an isolation carried out according to the traditional method and an isolation carried out according to the method of the invention, followed by a count of the bacterial colonies, with suspensions having variable bacterial charges.
- FIG. 4 represents the snapshots of a comparative analysis between a count of bacterial colonies, made according to the traditional method and a count of bacterial colonies, made according to the method of the invention, as well as the evaluation of the volume distribution factor. bacterial suspension after each contact resumption.
- Example 1 Obtain isolated colonies from a highly contaminated solution on a reduced agar surface:
- Figure IA no jump (suspension at approximately 10 8 CFU / ml)
- Figure IB 5 jumps (suspension at approximately 10 8 CFU / ml)
- Figure IC 6 jumps (suspension at approximately 10 8 CFU / ml) - Figure ID: 7 jumps (suspension at approximately 10 8 CFU / ml)
- Figure 2A no jump (suspension at approximately 10 7 CFU / ml)
- Figure 2B 3 jumps (suspension at approximately 10 7 CFU / ml)
- Figure 2C 6 jumps (suspension at approximately 10 7 CFU / ml)
- the width (10 cm) of the Petri dish is not sufficient to allow isolated colonies to be obtained with spreading by the traditional method, namely by displacing the means of spreading without making jump ( Figure IA).
- Example 2 Interests of a jumping spread for the production of a model allowing a reliable count of the microbial load of a sample on an agar medium:
- Procedure 100 ⁇ l of solutions loaded with Staphylococcus aureus at different concentrations obtained by successive 10-fold dilutions are deposited on the edge of an agar culture medium. This volume is then spread manually and rectilinearly using the applicator used on the PREVI TM Isola system. A control spread is achieved without jumping with the applicator. From a deposit identical to the control condition, a spread comprising 4 successive jumps is carried out in parallel, these 4 jumps defining 5 distinct zones, designated zone 1 to zone 5.
- line A corresponds to the results obtained with a bacterial load of between 80,000 and 130,000 CFU.
- Line B corresponds to the results obtained with a bacterial load of between 8,000 and 13,000 CFU.
- Line C corresponds to the results obtained with a bacterial load of between 800 and 1300 CFU.
- Line D corresponds to the results obtained with a bacterial load of between 80 and 130 CFU.
- line A, right column, zones 4 and 5 have respectively 67 and 34 isolated colonies.
- Line B, zones 3, 4 and 5 have respectively 116, 24 and 6 isolated colonies.
- Zone 4 has no colony.
- zones 1, 2 and 3 have respectively 115, 14 and 1 isolated colonies. The charge is quite low, zones 4 and 5 are virgin of all bacteria.
- zone 1 which is the deposition zone of the 100 ⁇ l of suspension
- zone 2 the volume deposited is of the order of 15% of the initial volume.
- zone 3 it is between 1 and 2% of the volume that is deposited.
- the number of colonies is approximately divided by ten.
- zone-level solution volume distribution data By combining zone-level solution volume distribution data with the corresponding enumeration values, it is possible by a simple mathematical approach to design a computational model that allows an evaluation of the initial microbial load present in the base sample. In view of the improvement that besides the automation of the seeding, this method would allow by a single seeding to accurately count the microbial load of a solution over a wide range of microbial load.
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (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)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2010800061289A CN102300999A (zh) | 2009-01-30 | 2010-01-29 | 在琼脂培养基上分离或计数微生物的方法 |
EP10707619A EP2391727A1 (fr) | 2009-01-30 | 2010-01-29 | Procede d'isolement ou de denombrement de microorganismes sur un milieu de culture gelose |
US13/143,879 US20110275110A1 (en) | 2009-01-30 | 2010-01-29 | Method for isolating or counting microorganisms on an agar culture medium |
JP2011546924A JP5628207B2 (ja) | 2009-01-30 | 2010-01-29 | 寒天培養培地上の微生物を単離又は計数する方法 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0950591A FR2941709B1 (fr) | 2009-01-30 | 2009-01-30 | Procede d'isolement ou de denombrement de microorganismes sur un milieu de culture gelose |
FR0950591 | 2009-01-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2010086565A1 true WO2010086565A1 (fr) | 2010-08-05 |
Family
ID=40810707
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2010/050146 WO2010086565A1 (fr) | 2009-01-30 | 2010-01-29 | Procede d'isolement ou de denombrement de microorganismes sur un milieu de culture gelose |
Country Status (6)
Country | Link |
---|---|
US (1) | US20110275110A1 (fr) |
EP (1) | EP2391727A1 (fr) |
JP (1) | JP5628207B2 (fr) |
CN (1) | CN102300999A (fr) |
FR (1) | FR2941709B1 (fr) |
WO (1) | WO2010086565A1 (fr) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103535183B (zh) * | 2013-08-14 | 2016-04-06 | 上海雪榕生物科技股份有限公司 | 提高液体菌种发菌点的简易接种方法 |
CA2985854C (fr) * | 2015-04-23 | 2023-10-17 | Bd Kiestra B.V. | Une methode et un systeme de comptage automatise de colonie microbienne a partir d'un echantillon preleve d'un support plaque |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0242114A2 (fr) | 1986-04-18 | 1987-10-21 | Vista Laboratories Ltd. | Méthode et dispositif d'ensemencement d'un milieu de culture |
FR2694570A1 (fr) | 1992-08-06 | 1994-02-11 | Jalenques Francois | Procédé et appareil d'ensemencement de solutions sur un milieu de culture possédant des possibilités de programmation du type de dépôt de la solution. |
WO1998041610A2 (fr) * | 1997-03-17 | 1998-09-24 | Canadian Space Agency | Procede et appareil destines a inoculer automatiquement des specimens de bacteries a des milieux de culture a partir de recipients contenant des specimens cliniques |
WO2005071055A1 (fr) | 2004-01-22 | 2005-08-04 | Medvet Science Pty Ltd | Dispositif de peignage microbien |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61219378A (ja) * | 1985-03-22 | 1986-09-29 | ラモツト・ユニヴア−シテイ・オ−ソリテイ−・フオ−・アプライド・リサ−チ・エンド・インダストリアル・デイヴエロプメント・リミテツド | 微生物培地移送装置 |
US6251624B1 (en) * | 1999-03-12 | 2001-06-26 | Akzo Nobel N.V. | Apparatus and method for detecting, quantifying and characterizing microorganisms |
CN1970727A (zh) * | 2006-12-06 | 2007-05-30 | 刘放 | 一种微生物平板培养基连续接种装置 |
-
2009
- 2009-01-30 FR FR0950591A patent/FR2941709B1/fr not_active Expired - Fee Related
-
2010
- 2010-01-29 US US13/143,879 patent/US20110275110A1/en not_active Abandoned
- 2010-01-29 WO PCT/FR2010/050146 patent/WO2010086565A1/fr active Application Filing
- 2010-01-29 CN CN2010800061289A patent/CN102300999A/zh active Pending
- 2010-01-29 EP EP10707619A patent/EP2391727A1/fr not_active Ceased
- 2010-01-29 JP JP2011546924A patent/JP5628207B2/ja not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0242114A2 (fr) | 1986-04-18 | 1987-10-21 | Vista Laboratories Ltd. | Méthode et dispositif d'ensemencement d'un milieu de culture |
FR2694570A1 (fr) | 1992-08-06 | 1994-02-11 | Jalenques Francois | Procédé et appareil d'ensemencement de solutions sur un milieu de culture possédant des possibilités de programmation du type de dépôt de la solution. |
WO1998041610A2 (fr) * | 1997-03-17 | 1998-09-24 | Canadian Space Agency | Procede et appareil destines a inoculer automatiquement des specimens de bacteries a des milieux de culture a partir de recipients contenant des specimens cliniques |
WO2005071055A1 (fr) | 2004-01-22 | 2005-08-04 | Medvet Science Pty Ltd | Dispositif de peignage microbien |
Non-Patent Citations (1)
Title |
---|
See also references of EP2391727A1 |
Also Published As
Publication number | Publication date |
---|---|
FR2941709B1 (fr) | 2011-03-18 |
JP5628207B2 (ja) | 2014-11-19 |
CN102300999A (zh) | 2011-12-28 |
JP2012516144A (ja) | 2012-07-19 |
EP2391727A1 (fr) | 2011-12-07 |
US20110275110A1 (en) | 2011-11-10 |
FR2941709A1 (fr) | 2010-08-06 |
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