Title of Invention

"A METHOD OF PREPARING A SAMPLE FOR THE MICROBIOLOGICAL ANALYSIS AND A UNIT THEREOF"

Abstract A method of preparing a sample for the microbiological analysis of a liquid which may contain microorganisms of several different types each having predetermined morphological characteristics, which method comprises the step of making a selection of the microorganisms from said different types of microorganisms, said step of making a selection comprising: the step of procuring a unit (201) for preparing said sample comprising a generally tubular body (202) within which are fixed a first (3) and a second (4) membrane 1 said body (202) having an opening (207) for introduction of said liquid, a first compartment (226) between said introduction opening (207) and said first membrane (3), a second compartment (227) between said first (3) and second (4) membranes and a third compartment (228) on the opposite side of said second membrane (4) from said first membrane (3), said first membrane (3) having a predetermined first pore diameter and said second membrane (4) a predetermined second pore diameter less than said predetermined first pore diameter of said first membrane (3); the step of passing a predetermined volume of said liquid from the first (226) to the second (227) compartment through said first membrane (3); the step of passing all the filtrate, having so reached the second compartment (227), from the second (227) to the third (228) compartment through said second membrane (4) in order to collect said sample on said second membrane (4) with said microorganisms selected from said different types of microorganisms which are those of which the size is less than the predetermined pore diameter of said first membrane (3) and greater than the predetermined pore diameter of said second membrane (4); and the step of retrieving said sample so collected on said second membrane (4); characterized in that the step of procuring a preparation unit comprises the step of selecting a preparation unit (201) in the body (202) of which is formed an auxiliary opening (232) capable of being unstopped communicating with said second compartment (227).
Full Text The present invention relates to a method of preparing a sample for the microbiological analysis,
The present invention concerns the preparation of a sample for the microbiological analysis of a liquid.
It is known that there are liquids which may contain different types of microorganisms, among which the microbiological analysis must only concern one or several types.
These liquids exist for example at the end of a manufacturing chain for monoclonal antibodies. More particularly, such liquids, bearing eukaryotes which have enabled the antibodies to be produced, may possibly be contaminated with bacteria and/or viruses.
The presence of the eukaryote cells may hinder the microbiological
analysis of the bacteria and viruses either by liberating toxins which may adversely
affect the growth of the microorganisms in the case of conventional detection on a
gel growth medium (detection of false negatives) or by perturbing the reading of
the results in the case of fast detection (detection of false positives) by
luminescence, fluorescence, by amplification or hybridization or by any other
method of analysis of the nucleic acids of those microorganisms.
In order to carry out a reliable analysis of this type of liquid, it is thus appropriate to prepare a sample in which selection has been made of only the
microorganisms (the bacteria and/or viruses in the example given above)
belonging to the types of microorganisms which it is desired to detect.
Methods are already known for preparing a sample for the analysis of a liquid in which the microorganisms to detect are selected by exploiting the differences in morphological characteristics (physical and/or chemical) between the different types of microorganisms.
More particularly, a method of preparation is already known comprising a step of selection consisting of adding into the liquid to analyze a reagent adapted to carry out a specific lysis of the microorganisms belonging to the types of microorganisms which it is not desired to keep in the sample, then of filtering the liquid on a membrane so as to collect on that membrane a sample containing solely the microorganisms to detect and which have not reacted specifically with the lysing agent (those having undergone the lysis being destroyed and having passed through the membrane). The sample so collected is then retrieved to be analyzed with conventional or fast microbiological analysis techniques.

Another method for forming the sample consists of separating the microorganisms to keep from the others by virtue of their differences in mass by centrifuging at low speed.
Another method also, comprising the step of procuring a unit with two membranes, each having a different diameter of pore (one for removing the contaminants, and the second for collecting the microorganisms), is already known from document US 2004/0219628.
The invention concerns preparing a sample according to a similar method while at the same time having improved performance and being simpler and more convenient to implement.
To that end it provides a method of preparing a sample for the microbiological analysis of a liquid which may contain microorganisms of several different types each having predetermined morphological characteristics, which method comprises the step of making a selection of the microorganisms from said different types of microorganisms, said step of making a selection comprising:
- the step of procuring a unit for preparing said sample comprising a generally tubular body within which are fixed a first and a second membrane, said body having an opening for introduction of said liquid, a first compartment between said introduction opening and said first membrane, a second compartment between said first and second membranes and a third compartment on the opposite side of said second membrane from said first membrane, said first membrane having a predetermined first pore diameter and said second membrane a predetermined second pore diameter less than said predetermined first pore diameter of said first membrane;
- the step of passing a predetermined volume of said liquid from the first to the second compartment through said first membrane;
- the step of passing all the filtrate, having so reached the second compartment, from the second to the third compartment through said second membrane in order to collect said sample on said second membrane with said microorganisms selected from said different types of microorganisms which are those of which the size is less than the predetermined pore diameter of said first membrane and greater than the predetermined pore diameter of said second membrane; and
- the step of retrieving said sample so collected on said second
membrane;
characterized in that the step of procuring a preparation unit comprises the step of selecting a preparation unit in the body of which is formed an auxiliary opening capable of being unstopped communicating with said second compartment.
In the method according to the invention, the sample formed of the microorganisms to analyze (bacteria and/or viruses for example) is collected on the second membrane after having been separated from the undesired microorganisms (for example eukaryote cells) of too great a size to pass through the first membrane.
Such a selection of the microorganisms on the basis of their differences in size is particularly effective, often more so than in the aforementioned methods of the prior art.
An explanation of this appears to be that, in the case of centrifuging, a portion of the microorganisms to collect is taken by centrifugal force with the microorganisms to eliminate; and, in the case of chemical lysis, that the lysing agent is never perfectly selective such that a small but non-zero proportion of the microorganisms to collect undergoes lysis and is thus not collected on the membrane, whereas in the method according to the invention (selection by size), such marginal effects are negligible, or even non-existent.
According to preferred features, for reasons of simplicity and convenience with regard both to manufacture and to use:
- the step of retrieving said sample comprises the step of unstopping said opening, the step of depositing an agent for lysing said microorganisms selected on said second membrane by said auxiliary opening then the step of passing the lysate so formed from the second to the third compartment through said second membrane; and/or
- the step of procuring a preparation unit comprises the step of selecting a preparation unit of which the body comprises a first portion to which is fixed said first membrane and a second portion to which is fixed said second membrane, said portions being detachable from each other.
It is thus possible, either by virtue of a body formed in two detachable portions, or by virtue of an aperture provided in that body, to have access to the second membrane and more particularly to the face of the second membrane on which the sample has been collected.
According to other preferred features, for the same reasons as those stated above:
- the steps of passing said liquid through said membranes are performed by placing said third compartment under reduced pressure;
- the steps of passing said liquid through said membranes are performed by placing said first compartment under increased pressure;
- the steps of passing said liquid through said membranes are performed by centrifuging said preparation unit;
- the step of procuring a preparation unit comprises the step of selecting as said first membrane a membrane of pore diameter greater than 1 µm;
- the step of procuring a preparation unit comprises the step of selecting as said second membrane a membrane of pore diameter less than 0.65 µm ; and/or
- the step of procuring a preparation unit comprises the step of selecting as said second membrane a membrane of pore diameter less than 0.1 µm;
According to a second aspect, the invention also concerns a unit for preparing a sample suitable for the implementation of the method as set forth above, comprising a generally tubular body within which are fixed a first and a second membrane, said body having an opening for introduction of said liquid, a first compartment between said introduction opening and said first membrane, a second compartment between said first and second membranes and a third compartment on the opposite side of said second membrane from said first membrane, said first membrane having a predetermined first pore diameter and said second membrane a predetermined second pore diameter less than said predetermined first pore diameter of said first membrane, said unit being adapted to pass a predetermined volume of said liquid from the first to the second compartment by said first membrane then to pass all the filtrate, having so reached the second compartment, from the second to the third compartment
through said second membrane in order to collect said sample on said second membrane; said body also comprising means for retrieving said sample so collected on said second membrane; characterized in that said retrieval means comprise an opening capable of being unstopped formed in said body and communicating with said second compartment.
The preparation unit is thus designed to make it possible to pass the entirety of the predetermined volume through the two membranes (with the exception of course of the parts retained by the membranes), and furthermore, to have access to the sample by virtue of the means for retrieving the sample.
It will be noted that a simple filtering unit with several stages, for example a solution clarification unit, would not be able to serve as a preparation unit according to the invention, since such a filtering unit is not made with membranes, would not be adapted to pass all the filtrate arriving in an intermediate compartment through the second stage (a residual filtrate would always remain) and/or would lack means for retrieving the sample.
According to preferred features, for reasons of simplicity and convenience with regard both to manufacture and to use, said first membrane is permeable to the air when wet.
In this manner, when the liquid has been entirely emptied from the first compartment and the first membrane comes into contact with the air on the side which is turned towards that compartment, that membrane, by virtue of the sufficient size of its pores, remains permeable to the air (no bubble point phenomenon) such that the air may penetrate by that membrane into the second compartment to enable the liquid which it contains to be evacuated.
According to other preferred features, for the same reasons as those stated above:
- the pores of said first membrane are of diameter greater than 1 µm;
- the pores of said second membrane are of diameter less than 0.65 µm;
- the pores of said second membrane are of diameter less than 0.1 µm;
- said body comprises a first portion to which is fixed said first membrane and a second portion to which is fixed said second membrane ;
- said body comprises a cup delimiting said third compartment; and/or
- said cup is detachable.
The features and advantages of the invention will appear from the following description, given by way of preferred but non-limiting example, with reference to the accompanying drawings in which:
- Figure 1 is a cross-section view of a preparation unit useful to the understanding of the invention;
- Figure 2 is a view of the unit similar to Figure 1 but in which the two portions of a body of that unit which were nested together have been separated;
- Figure 3 is a view similar to Figure 1 of a second embodiment of the preparation unit; and
- Figure 4 is a view similar to Figure 1 of a third embodiment of the preparation unit.
The preparation unit 1 illustrated in Figure 1 comprises a generally tubular body 2 within which are fixed a membrane 3, a membrane 4 and two porous supports 5 and 6.
The membranes 3 and 4 are of polyvinylidene fluoride (PVDF), the membrane 3 having pores of diameter equal to 5 µm and the membrane 4 having pores of diameter equal to 0.4 µm.
The body 2 is formed by a portion 9 and a portion 10 with one nested in the other. Portion 9 has an opening 7 for introduction of the liquid while portion 10 has an opening 8 for outlet of that liquid.
Portion 9 has a first cylindrical part 15 delimiting the opening 7 on one side and on the opposite side joining a second part 16 which is also cylindrical and of diameter less than that of part 15, parts 15 and 16 being connected to each other by a transverse annular wall 17.
Adjacent the transverse wall 17 is fixed the porous support 5 and is sealed the membrane 3, which bears by its face 12 against the porous support 5 from the same side as the opening 7 (Figure 1).
Portion 10 comprises a first cylindrical part 20 of diameter substantially equal to the diameter of the cylindrical part 15, a second cylindrical part 21 of diameter substantially equal to the diameter of the cylindrical part 16, and a third cylindrical part 22 of diameter less than the diameter of part 21 and delimiting the opening 8.
Portions 20 and 21 are connected to each other by a transverse annular wall 23 whereas portions 21 and 22 are connected to each other by a transverse annular wall 24.
Adjacent the annular wall 23 is fixed the porous support 6 and is sealed the membrane 4, which bears by its face 14 against the porous support 6, the face 13 of that membrane facing the porous support 5 (Figure 1).
Portion 9 with membrane 3 delimits a compartment 26 for admission of the liquid to filter, portions 9 and 10 in the nested state delimiting with membranes 3 and 4 an intermediate compartment 27 whereas portion 10 with membrane 4 delimits a compartment 28 for evacuation of the liquid.
In the nested state of portion 9 in portion 10 of the body 2, the outwardly turned surface of the cylindrical part 16 faces the inner surface of the cylindrical part 20 while they bear against each other by elastic deformation so as to maintain portions 9 and 10 fastened together.
A description will now be made of the different steps of preparing a sample from a liquid to analyze which may contain eukaryote cells and bacteria whose presence it is desired to detect among those cells.
At a first step, the operator connects the cylindrical part 22 to a vacuum source (a vacuum pump for example) to place compartment 28 under reduced pressure and delivers a predetermined volume (for example 10 ml) of liquid into compartment 26,
Placing compartment 28 under reduced pressure gives rise to the placing under reduced pressure of compartment 27 such that the predetermined volume of liquid is sucked and passes through membrane 3 and support 5 to occupy compartment 27.
The pores of membrane 3 are dimensioned such that only the microorganisms of greatest size, here the eukaryote cells, are retained by that

membrane, whereas the other microorganisms, here the bacteria, pass through it and porous support 5 to arrive in the filtrate occupying compartment 27.
Furthermore, the large size of the pores of membrane 3 mean that even when that membrane is wet, it remains permeable to the air (absence of bubble point phenomenon) such that the air can penetrate into compartment 27 by membrane 3.
As this membrane allows the air to pass, the filtrate contained in compartment 27 then flows through membrane 4 and support 6 to occupy compartment 28 and then leave body 2 through the outlet opening 8.
The pores of membrane 4 are dimensioned so as to retain the bacteria which it is desired to detect.
Once these operations have been carried out, the operator disconnects the vacuum source from the cylindrical part 22.
Next he separates portions 9 and 10 by sliding portion 9 with respect to portion 10 such that they disengage from each other as illustrated in Figure 2.
Membrane 4 and in particular face 13 of that membrane on which the sample has been collected is thus rendered accessible.
It is then possible to extract that membrane to place it in contact with a gel growth medium and then to place that assembly in an incubator to incubate the bacteria.
Another solution consists of treating that membrane (after having or not having extracted it from portion 10) by depositing thereon from the face 13 side a reagent revealing the presence of the ATP of the bacteria by luminescence or another possibility is a reagent making it possible to identify the bacteria by fluorescence
Still another solution consists of analyzing the DMA of the bacteria collected on membrane 4 by a conventional method (of PCR type, for example).
It is also possible, if it is desired to retrieve the sample independently of the membrane, to deposit on that membrane an agent for lysing bacteria.
By again connecting the unit to a source of vacuum, the lysate containing the biological material of the bacteria then passes through membrane 4 then compartment 28 to be collected in a container disposed beyond opening 8.

The liquid retrieved in this container may then be analyzed by a fast microbiological detection method (by luminescence for example).
The combined use of the unit according to the invention with the fast detection methods for the microorganisms makes it possible to verify in short periods of time and on a large scale all kinds of liquid samples that may contain several types of microorganisms.
Another embodiment of the preparation unit is represented in Figure 3.
Generally, for similar members the same references have been used, to which the number 100 has been added.
The preparation unit 101 is adapted to be placed in a centrifuge so as to make the liquid pass through the two membranes without having to place the preparation unit under reduced pressure.
In this embodiment, the annular 24 and cylindrical 22 parts have been eliminated whereas cylindrical part 121 is extended with respect to part 21 of the preceding embodiment such that it is possible to fit onto it a cup 130 belonging to body 102 to extract the second filtrate when the liquid comes out from the opening 108 or else to retrieve a lysate in that cup, once the second filtrate has been emptied, when it is desired to retrieve the sample independently from membrane 4 (by depositing a lysing agent on that membrane).
The cup is thus adapted to be separated from portion 110 (by sliding along cylindrical part 121) either to empty the second filtrate or to obtain access to the lysate in order to perform a fast microbiological detection on that lysate, for example by adding into the cup a reagent revealing the presence of ATP by luminescence.
Another embodiment is represented in Figure 4.
In this embodiment, the preparation unit 201 has a similar structure to preparation unit 101 apart from an opening 232, obturated in fluid-tight manner by a removable stopper 233, which is provided laterally in cylindrical part 220 of portion 210 of body 202.
Once the sample has been prepared and the stopper 233 removed, this opening makes it possible if needed to obtain access to compartment 227 and in particular to face 13 of membrane 3, for example to deposit a lysing agent there,

without having to separate portions 209 and 210 of body 202, the biological material of the microorganisms then being retrieved in cup 230.
In a variant, the pores of membrane 4 are of diameter less than 0.1 ^im so as to collect bacteria and/or viruses on the membrane.
In still another variant, the liquid is passed through membranes 3 and 4 by placing the admission chamber 26 under increased pressure for example using a piston engaged in cylindrical part 15 by leaving between the piston and the liquid a volume of air. When it is actuated, the piston gives rise to the displacement of the volume of liquid as well as the volume of air compressed between the piston and liquid, such that compartment 26 is placed under increased pressure. The liquid therefore penetrates into compartment 27 and is then flushed by the air pushed by the piston, which air in turn penetrates into compartment 27 through membrane 3 which remains permeable to air.
In a still further variant, the microorganisms to detect are not bacteria or viruses but yeasts (of sufficiently small size not to be retained by the first membrane) or molds for example.
In yet another variant, the liquid to analyze does not contain eukaryotes but other types of microorganisms (to separate from the microorganisms to detect) such as yeasts or else filamentous fungi.
In again another variant, the two portions of the body of the preparation unit snap-fit together and the separation of the portions is carried out for example by breaking the snap-fitting feet.
In still another variant, the two portions form a single piece and are connected by a frangible zone to be broken in order to separate them from each other.
In yet another variant the membrane collecting the sample is not sealed to the body of the preparation unit but is pressed against an 'O' ring seal and/or the membranes are formed of Polyethersulfone (PES), polycarbonate, polyester or another possibility is cellulose ester.
The present invention is not limited to the embodiment described and represented but covers any variant form.








We Claim:
1. A method of preparing a sample for the microbiological analysis of a liquid which may contain microorganisms of several different types each having predetermined morphological characteristics, which method comprises the step of making a selection of the microorganisms from said different types of microorganisms, said step of making a selection comprising:
the step of procuring a unit (201) for preparing said sample comprising a generally tubular body (202) within which are fixed a first (3) and a second (4) membrane 1 said body (202) having an opening (207) for introduction of said liquid, a first compartment (226) between said introduction opening (207) and said first membrane (3), a second compartment (227) between said first (3) and second (4) membranes and a third compartment (228) on the opposite side of said second membrane (4) from said first membrane (3), said first membrane (3) having a predetermined first pore diameter and said second membrane (4) a predetermined second pore diameter less than said predetermined first pore diameter of said first membrane (3);
the step of passing a predetermined volume of said liquid from the first (226) to the second (227) compartment through said first membrane (3);
the step of passing all the filtrate, having so reached the second compartment (227), from the second (227) to the third (228) compartment through said second membrane (4) in order to collect said sample on said second membrane (4) with said microorganisms selected from said different types of microorganisms which are those of which the size is less than the predetermined pore diameter of said first membrane (3) and greater than the predetermined pore diameter of said second membrane (4); and
the step of retrieving said sample so collected on said second membrane (4); characterized in that the step of procuring a preparation unit comprises the step of selecting a preparation unit (201) in the body (202) of which is formed an auxiliary opening (232) capable of being unstopped communicating with said second compartment (227).
2. A method as claimed in claim 1, wherein the step of retrieving said sample comprises the step of unstopping said opening (232), the step of depositing an agent for lysing said microorganisms selected on said second membrane (4) by said auxiliary opening (232)

then the step of passing the lysate so formed from the second (227) to the third (228) compartment through said second membrane (4).
3. A method as claimed in anyone of claims 1 or 2, wherein the step of procuring a preparation unit comprises the step of selecting a preparation unit (201) of which the body (202) comprises a first portion (209) to which is fixed said first membrane (3) and a second portion (210) to which is fixed said second membrane (4), said portions being detachable from each other.
4. A method as claimed in any one of claims 1 to 3, wherein the steps of passing said liquid through said membranes (3, 4) are performed by placing said third compartment (28) under reduced pressure.
5. A method as claimed in any one of claims 1 to 3, wherein the steps of passing said liquid through said membranes (3, 4) are performed by placing said first compartment (26) under increased pressure.
6. A method as claimed in any one of claims 1 to 3, wherein the steps of passing said liquid through said membranes (3, 4) are performed by centrifuging said preparation unit (201).
7. A method as claimed in any one of claims 1 to 6, wherein the step of procuring a preparation unit (201) comprises the step of selecting as said first membrane (3) a membrane of pore diameter greater than 1 µm.
8. A method as claimed in any one of claims 1 to 7, wherein the step of procuring a preparation unit comprises the step of selecting as said second membrane (4) a membrane of pore diameter less than 0.65 µm.
9. A method as claimed in claim 8, wherein the step of procuring a preparation unit comprises the step of selecting as said second membrane a membrane of pore diameter less than 0.1 µm.
10. A unit for preparing a sample suitable for the implementation of the method as claimed in any one of claims 1 to 9, comprising a generally tubular body (202) within which are fixed a first (3) and a second (4) membrane, said body (202) having an opening (207) for introduction of said liquid, a first compartment (226) between said introduction opening (207) and said first membrane (3), a second compartment (227) between said first (3) and second (4) membranes and a third compartment (228) on the opposite side of said second

membrane (4) from said first membrane (3), said first membrane (3) having a predetermined first pore diameter and said second membrane (4) a predetermined second pore diameter less than said predetermined first pore diameter of said first membrane (3); said unit being adapted to pass a predetermined volume of said liquid from the first to the second compartment by said first membrane (3) then to pass all the filtrate, having so reached the second compartment (227), from the second to the third compartment through said second membrane (4) in order to collect said sample on said second membrane (4); said body also comprising means (232) for retrieving said sample so collected on said second membrane (4); characterized in that said retrieval means comprise an opening (232) capable of being unstopped formed in said body (202) and communicating with said second compartment (227).
11. A unit as claimed in claim 10, wherein said first membrane (3) is permeable to the air when wet.
12. A unit as claimed in claim 11, wherein the pores of said first membrane (3) are of diameter greater than 1 µm.
13. A unit as claimed in any one of claims 10 to 12, wherein the pores of said second membrane (4) are of diameter less than 0.65 µm.
14. A unit as claimed in claim 13, wherein the pores of said second membrane (4) are of diameter less than 0.1 µm.
15. A unit as claimed in any one of claims 10 to 14, wherein said body (202) comprises a first portion (209) to which is fixed said first membrane (3) and a second portion (210) to which is fixed said second membrane (4).
16. A unit as claimed in any one of claims 10 to 15, wherein said body (202) comprises a cup (230) delimiting said third compartment (228).
17. A unit as claimed in claim 16, wherein said cup (230) is detachable.

Documents:

1235-DEL-2007-Abstract-(24-09-2010).pdf

1235-DEL-2007-Abstract-(29-04-2011).pdf

1235-del-2007-abstract.pdf

1235-DEL-2007-Claims-(24-09-2010).pdf

1235-del-2007-claims.pdf

1235-del-2007-Correspondence Others-(20-03-2012).pdf

1235-DEL-2007-Correspondence Others-(29-04-2011).pdf

1235-DEL-2007-Correspondence-Others-(24-09-2010).pdf

1235-del-2007-correspondence-others-1.pdf

1235-del-2007-correspondence-others.pdf

1235-DEL-2007-Description (Complete)-(24-09-2010).pdf

1235-del-2007-description (complete).pdf

1235-del-2007-drawings.pdf

1235-DEL-2007-Form-1-(24-09-2010).pdf

1235-DEL-2007-Form-1-(29-04-2011).pdf

1235-del-2007-form-1.pdf

1235-del-2007-form-18.pdf

1235-DEL-2007-Form-2-(24-09-2010).pdf

1235-DEL-2007-Form-2-(29-04-2011).pdf

1235-del-2007-form-2.pdf

1235-DEL-2007-Form-3-(29-04-2011).pdf

1235-del-2007-form-3.pdf

1235-del-2007-form-5.pdf

1235-del-2007-GPA-(20-03-2012).pdf

1235-DEL-2007-GPA-(24-09-2010).pdf

1235-DEL-2007-Petition-137-(29-04-2011).pdf

abstract.jpg


Patent Number 248578
Indian Patent Application Number 1235/DEL/2007
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 26-Jul-2011
Date of Filing 08-Jun-2007
Name of Patentee MILLIPORE CORPORATION
Applicant Address 290 CONCORD ROAD, BILLERICA, MASSACHUSETTS 01821, USA
Inventors:
# Inventor's Name Inventor's Address
1 SEBASTIEN RIBAULT 10, RUE DU CANAL, F-67380 LINGOLSHEIM, FRANCE
2 GAEL WAICHE 7, RUE ELISABETH JUNECK, F-67120 MOLSHEIM, FRANCE
PCT International Classification Number G01N33/48; G01N21/01
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0605796 2006-06-27 France