Abstract:
Device for spraying a reagent onto a support adapted to retain microorganisms on a predetermined surface. The device includes a spraying bell as well as a nozzle for emitting a jet of droplets of the reagent into a spraying chamber comprised by the bell, the device also including an absorbent pad mounted against the bell transversely to the jet and closing the chamber from the opposite side to the nozzle with the exception of a circular central opening provided in the pad, the diameter of the central opening being adapted to enable a portion of the jet, when the device faces the support and is at a predetermined distance therefrom, to pass through the central opening over its entire area and be deposited on the whole of the predetermined surface of the support.

Description:
BACKGROUND 
     The present invention concerns a spraying device for fast microbiological analysis. 
     At present, checking the microbiological quality of liquids, gases or surfaces in the context of industrial and medical activities has to conform to strict standards. 
     On account of this, the industrial players and health authorities must have tools at their disposal making it possible to detect microbiological contaminations as soon as possible, in order to be able to correct these in good time and at reduced cost. 
     In practice, microbiological monitoring is carried out on a gel growth medium where microorganisms, after having been collected on a microporous membrane, are cultured until they have been rendered visible to the naked eye. 
     The incubation periods vary from one microorganism to another but are in general at least 24 hours and sometimes more for slower growth microorganisms (such as mycobacteria) or because the microorganisms have been stressed by environmental conditions. 
     To render the detection more rapid, another approach consists of reducing the minimum duration of culture (or even, for some microorganisms, of eliminating it completely) by basing the detection of the microorganisms on their metabolic activity. 
     A universal metabolic marker, most commonly adenosine triphosphate (ATP) contained in living microorganisms, is measured by bringing it into contact with a reagent revealing the presence of ATP by luminescence (termed a “bio luminescence reagent”) which enables the presence of microorganisms to be noticed without having to wait for colonies to form on a gel growth medium and to become visible to the naked eye. 
     The quantity of light emitted is a function of the mass of ATP and thus the number of microorganisms. 
     A device for detection by fast microbiology is already known which is commercialized by the applicant under the name Milliflex Rapid®, and which comprises:
         a station for filtering a volume of liquid onto a membrane so as to capture the microorganisms that may be contained in the liquid on the membrane;   a station for spraying a reagent revealing the presence of ATP by luminescence facing which the operator places the membrane, after the filtering step and after having rendered the ATP of the microorganisms accessible (by a step of lysis of the microorganisms for example), for the reagent to be deposited; and   a station for measuring the quantity of light emitted in response to the depositing of the reagent revealing the presence of ATP by luminescence, facing which the operator places the membrane, after the spraying step, the light emitted by the membrane being collected by a CCD camera and processed and then analyzed to detect the presence of microorganisms on that membrane.       

     The spraying station is provided with a spraying device comprising a sprayer spraying droplets emitted in the form of a jet onto the membrane in the ambient air situated above the membrane. 
     SUMMARY 
     The invention concerns the provision of a device of the same type that both has better performance and is more practical while remaining reliable in terms of the risks of contaminations of the support to analyze. 
     To that end it provides a device for spraying a reagent onto a support for the fast microbiological analysis of said support, said support being adapted to retain microorganisms on a predetermined surface, characterized in that said device comprises a spraying bell as well as a nozzle for emitting a jet of droplets of said reagent into a spraying chamber comprised by said bell, said device also comprising an absorbent pad mounted against said bell transversely to said jet and closing said chamber from the opposite side to said nozzle with the exception of a circular central opening provided in said pad, the diameter of said central opening being adapted to enable a portion of said jet, when said device faces said support and is at a predetermined distance therefrom, to pass through said central opening over its entire area and be deposited on the whole of said predetermined surface of said support. 
     The spraying device according to the invention, provided with a bell and an absorbent pad used in combination, makes it possible to keep the (relatively volatile) droplets of the sprayed jet confined such that it is thus possible to group together the measuring station and the spraying station into the same analysis chamber. 
     More particularly, in the absence of effective confinement of the jet of droplets, such grouping together would be rendered impossible since those (relatively volatile) droplets would risk being deposited on the entire surface of the analysis chamber including the measuring station. As this chamber may be contaminated by extraneous ATP (ATP which is naturally present on the surfaces), putting the reagent in contact with that extraneous ATP would generate light which would risk perturbing the analysis of that emitted by the support. 
     Furthermore, the pad of this device is shaped so as to collect the droplets situated at the periphery of the jet and liable to enter into contact with the bell. This is because it is particularly important to trap those droplets since, having entered into contact with the bell (which may also be contaminated by extraneous ATP), they constitute potential sources of contamination of the support by extraneous ATP if the paths of those droplets were to end their travel on that support. 
     The pad thus makes it possible to select only the portion of the jet of droplets adapted to uniformly cover the whole of the surface of the support to analyze, without risk of contaminating it. 
     Furthermore, the diameter of the opening of the pad, which is a function of the distance to the support from the pad, means that the beam of sprayed droplets leaving by the opening of the pad over the entire area of that opening uniformly covers the entire surface to treat of the support and solely that surface in order to avoid any risk of contamination by the droplets coming into contact with the environment close to the support. 
     According to features that are preferred for reasons of simplicity and convenience of use, said spraying chamber is delimited by a wall substantially transverse to said jet, facing said pad and at the center of which is disposed said nozzle, by a side wall of which one edge joins to the periphery of said substantially transverse wall, as well as by said pad disposed against said side wall; 
     According to still other features that are preferred for the same reasons as above, said side wall has at least one frusto-conical portion against which is disposed said pad and of which the taper is adapted to orientate the droplets which are at the periphery of said jet towards said pad. 
     That frusto-conical portion enables the droplets which do not have sufficient energy to rebound to flow along that portion and then to be absorbed by the pad and, for those which have sufficient speed to rebound, to orientate the rebounds towards the pad itself but not in the direction of its central opening such that those droplets are also trapped by the pad. 
     According to still other preferred features:
         said side wall comprises, between said transverse wall and said frusto-conical portion against which is disposed said pad, an intermediate portion also adapted to orientate the droplets which are at the periphery of said jet towards said pad and of lesser taper than that of said frusto-conical portion against which is disposed said pad;   said transverse wall comprises a frusto-conical portion of taper between that of said intermediate portion and that of said frusto-conical portion against which is disposed said pad; and/or   said device also comprises a ring for retaining said pad against said bell.       

     According to still other preferred features, at the center of said ring, there is provided a circular opening of greater diameter than the diameter of the opening provided in said pad. 
     The difference in diameter means that a portion of the pad is visible from the support such that in case the droplets strongly rebound on the support they land and are trapped on the pad and not on the ring (since in that case they would risk falling back onto the support, carrying extraneous ATP). 
     According to still other preferred features:
         said ring has snap-fitting means adapted to cooperate with complementary snap-fitting means comprised by said bell;   said device also comprises a reservoir and a pump disposed between said nozzle and said reservoir;   said reservoir is slidingly mounted relative to said bell, said pump being adapted to make a said jet of droplets come out of said nozzle on sliding of said reservoir in the direction of said bell;   said reservoir comprises a body provided with an annular bearing collar; and/or       

     said reservoir has snap-fitting means adapted to cooperate with complementary snap-fitting means comprised by said bell. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       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: 
         FIG. 1  is a perspective view of a spraying device according to the invention; 
         FIG. 2  is a view similar to  FIG. 1  but with part of the device cut away; 
         FIG. 3  is an elevation view in section taken along a median plane of symmetry of the device and of a filter cassette provided with a microporous membrane to analyze disposed under the device using a robot arm; 
         FIG. 4  is a similar view to  FIG. 3  but in which a liquid reservoir of the device is represented in the position it occupies after sliding towards a spraying bell of the device to actuate the ejection of a jet of droplets; 
         FIG. 5  is an enlarged view of the filter cassette and of the end of the spraying device which faces the cassette, in which arrows show different possible paths for the droplets of sprayed reagent; and 
         FIG. 6  is a perspective view of the device into which a protective plug has been fitted, this assembly being placed in a packaging tray. 
     
    
    
     DETAILED DESCRIPTION 
     The spraying device  1  illustrated in  FIGS. 1 and 2  comprises a reservoir  2 , a confinement bell  3 , a spraying unit  4 , a porous pad  5  and a fixing ring  6  ( FIG. 2 ). 
     The reservoir  2  is slidingly mounted relative to the bell  3  and comprises a body  10  of plastics material as well as a cover  11 . 
     The body  10  has a first cylindrical portion  12 , a second cylindrical portion  13 , an annular collar  14  and two legs  15 . 
     The cylindrical portion  12  delimits a housing  17  for receiving the reagent which is obturated by the cover  11 . 
     The cylindrical portion  13  is situated in line with the portion  12  on the opposite side to the cover  11  and has an outer diameter less than that of the portion  12 . 
     A screw thread  16  is formed in the neighborhood of the opposite end of portion  13  to that connected to portion  12 . 
     The collar  14  is transversely attached to the rest of the body at the junction of portions  12  and  13 . 
     The two legs  15  which are diametrically opposite each other project from the opposite side of the collar to the cover  11  Each leg has a tooth  18  projecting from the opposite side to the other leg  15 . 
     The cover  11  has two ducts  20  and  21 . 
     A filter unit  22  (and respectively  23 ) is screwed into duct  20  (and respectively duct  21 ). 
     The filter unit  22  is an air filter whereas unit  23  is a liquid filter. 
     Means  24  for identification of the reagent and/or of the device may be stuck or engraved around the reservoir. 
     The confinement bell  3  is of plastics material formed as a single piece. 
     The bell comprises a first portion  31  ( FIG. 3 ) delimiting a housing  32  for receiving the spraying unit  4  and a second portion  33  delimiting together with the pad  5  a spraying and confinement chamber  34 . 
     Portion  31  of the bell is formed from a first cylindrical portion  35 , from a second cylindrical portion  36  and from an annular collar  37 . 
     Portion  35 , which is of greater diameter than portion  36 , is joined by one end to that portion (portions  35  and  36  being in line with each other) and by the other end to the collar  37 , the latter being connected transversely to portion  35 . 
     Collar  37  has two diametrically opposite oblong apertures  38  ( FIG. 1 ) into which the legs  15  of the reservoir are snap fitted. 
     In the snap-fitted state of the reservoir in the bell, the collars  14  and  37  face each other with the teeth  18  of the snap-fitting feet  15  being situated on the opposite side of the collar  37  to the collar  14 . 
     Portion  33  of the bell has a significantly flared first frusto-conical portion  40 , a second very slightly flared portion  41  (here this portion is practically cylindrical, its taper is practically nil) and a third portion  42  also frusto-conical and of moderate degree of flare, against which the pad  5  is disposed. 
     The degree of taper of portion  40  is thus greater than that of portion  41  and less than that of portion  42 . 
     Cylindrical portion  35  is joined to portion  40  via portion  36 . 
     Portion  40  forms a wall substantially transverse to the direction of spraying of the reagent and faces the pad  5  whereas portions  41  and  42  form a lateral wall extending from the periphery of portion  40  to the pad  5 . 
     This portion also comprises an annular collar  43  transversely connected to portion  41  as well as an annular lip  44  connected to the opposite peripheral end of portion  42  to that joined to portion  41 . 
     The bell is also provided with reinforcing fins  45  at portion  36  and reinforcing fins  46  at portion  42  that are attached to collar  43  ( FIG. 1 ). 
     The absorbent pad  5 , visible in  FIG. 2 , is of cellulose wadding and takes the form of a disc  50  in which a circular central opening  51  is formed. The pad is disposed against the lip  44  of portion  42  and closes the spraying chamber  34  except for the central opening  51  which enables said chamber to communicate with the outside of the device. 
     The ring  6  is of plastics material and takes the form of a disc  60  in which a circular opening  61  is formed at its center and is provided with a snap-fitting collar  62  at its periphery. 
     The ring also has a series of cut-outs  63  at its periphery. 
     Pad  5  is disposed between the lip  44  and the ring  6  with the ring snap fitted against the lip. 
     The diameter of the opening  51  here is 40 mm whereas the diameter of the opening  61  is 65 mm such that the pad has a portion  52  ( FIG. 2 ) hidden by portion  60  and a portion  53  which projects inwardly of the bell and which is not covered by portion  60  of ring  6 . 
     The spraying unit  4  comprises a pump  70  and a spraying nozzle  71  as well as a screw-fitting plug  72  which are illustrated in  FIG. 2 . 
     Via a duct  73  the pump communicates on one side with the housing  17  of the reservoir and on the other side with the nozzle  71 , the duct  73  continuing through the nozzle so as to issue in the housing  34  of the bell ( FIG. 2 ). 
     The plug  72  as well as the pump  70  are disposed in the housing  32  ( FIG. 3 ) within portion  35 , with the plug  72  screwed into the screw thread  16  of the cylindrical portion  13 . 
     Nozzle  71  has a frusto-conical portion  77  housed in portion  36  of which one end issues in the confinement housing  34  as well as an annular collar  74  transversely connected to the opposite end of the frusto-conical portion to that emerging in housing  34 , which abuts against portion  36  ( FIG. 3 ). 
     Nozzle  71  is provided to emit a jet of microscopic droplets by mechanical actuation of the pump  70  engaged by a sliding movement of the reservoir  2  towards the bell  3  while bearing on the collar  14  (for example with the assistance of a robot arm). 
     Device  1  is delivered in the packaging illustrated in  FIG. 6  and takes the form of a rigid tray  75  provided with a cover (not shown), a cylindrical plug  76  being engaged around the bell, the plug abutting against the collar  43  in order to protect the confinement chamber. 
     A description will now be given with the help of  FIGS. 3 to 5  of how the reagent contained in the reservoir of the device is deposited on a support in the form of microscopic droplets. 
     The support illustrated here is a cassette  80  comprising a filter membrane  81  having useful surface  82  corresponding to a diameter equal to 49 mm and a body  83  surrounding the membrane  81 . 
     Once the filtration of the microorganisms has been made onto the membrane  81  and after having made the ATP of the microorganisms retained on the surface  82  accessible (for example by a step of lysis of the microorganisms), the cassette  80  is placed, with the assistance of a robot arm  85  ( FIG. 3 ) under the spraying device  1  at a predetermined distance (here 19 mm) from the absorbent pad  5 . This device, from which the plug  76  has been removed beforehand, is fixedly held to a frame (not shown) of the analysis device. 
     A motorized unit (not shown) is then actuated to bear against the collar  14  of the reservoir so as to make the reservoir  2  slide towards the bell  3 , the latter remaining immobile ( FIG. 4 ), and so causing the actuation of the pump  70  to eject a jet of microscopic droplets into the chamber  34  through the nozzle  71 . 
     The motorized unit is then actuated to release the pressure it exerts on the collar  14  so as to allow the reservoir  2  to resume its initial position relative to the bell  3  ( FIG. 3 ) so as to be ready to perform a new spraying operation. 
     The majority of the micro-droplets cross the bell through the openings  51  and  61  without entering into contact with either the bell or the absorbent pad, those droplets being deposited onto the support evenly and homogenously over the entire useful surface  82  to be analyzed. 
     The diameter of the opening  51  and the distance from the pad  5  relative to the surface  82  are thus determined on the basis of the spatial dimensions of the jet of droplets in order for a high proportion (approximately 90%) of those droplets to pass through the opening  51  of the pad  5  over the entire area of that opening before being deposited on the entire surface  82 . 
     A small proportion of the micro-droplets (the remaining 10%), situated at the periphery of the jet, are stopped by the absorbent pad  5  by entering directly into contact with it or else after having encountered the internal surfaces of the portions  40  to  42  of the bell and after having flowed along them to the pad  5  or after having rebounded on that surface so as to land on the pad  5 . 
     More particularly and as illustrated in  FIG. 5 , in which different possible paths of the droplets are represented by the arrows A to H, the taper of the portions  40  to  42  is chosen such that, whatever the location at which the droplet rebounds, it is directed after its rebound towards portions  52  and  53  of the absorbent pad  5  and not in the direction of the central opening  51 . 
     This is because the coming into contact of the droplets with the bell leads to a risk of potential contamination by the extraneous ATP that may be present on the bell such that it is sought to trap in the absorbent pad  5  all those droplets (paths A to C) in order to avoid them falling back onto the membrane  81  carrying extraneous ATP which they may have captured on the bell. 
     It will be noted that the frusto-conical portion  40  is as flared as possible in order to attempt to minimize the risks of contact with the droplets. 
     Moreover portion  41  is practically cylindrical so as to reduce the dimensions of the bell. 
     The degree of taper of portion  42  and of portion  41  are chosen to orientate the rebounds of the droplets as desired without the bell assuming dimensions that are too large. 
     The major proportion of the droplets which pass through the opening  51  is directly absorbed by the support (paths D and E) or rebounds slightly without coming back into contact with the device (path F). 
     However, where the rebound leads to a contact with the device (paths G and H), it will be noted that the annular portion  53  of the absorbent pad projecting inwardly of the bell makes it possible in that case to trap those droplets which then come back into contact with the absorbent pad and not into contact with the ring  6  of which the opening  61  is of greater diameter than that of the opening  51  for that reason (since in that case they then risk falling back onto the support, carrying extraneous ATP). 
     It can be noted that surface  82  and solely that surface receives the droplets of the jet, path D being the most extreme path possible. Beyond that, the droplets are captured by the pad such that no droplet risks coming into contact with the body  83  of the cassette  80  and then falling back onto the membrane  81 , risking contaminating it with the extraneous ATP. 
     The support thus treated is then available for the microbiological analysis for example by moving the cassette  80  to the measuring station (not shown) within the same chamber in order to measure the quantity of light emitted in response to putting the reagent in contact with the ATP of the microorganisms that may be present on the membrane using a photomultiplier for example. 
     The device according to the invention may be used to treat several membranes, on each spraying operation the volume of liquid which was sprayed is replaced in the housing  17  by the air entering that housing through the pump  70 . 
     It is also possible to regularly fill that housing by injecting a volume of reagent through the filter unit  23  using a syringe, the unit  22  forming a vent in that case. 
     Where the filter cassette is provided with a membrane of different diameter and thus of different useful surface, it is possible to adjust the distance separating that membrane from the pad  5  by moving the device  1  vertically in order for the jet of droplets to cover the whole of the surface of that membrane and solely that surface. 
     The device may be a single-use device but may also be used as many times as necessary provided that the pad is not saturated with liquid. 
     As a variant the spraying device may also be used in any microbiological analysis method requiring a pre-treatment step aimed at eliminating the extraneous ATP that the membrane contains by spraying that reagent on the membrane before having rendered the ATP of the microorganisms accessible (for example by lysis) in order for it to react and solely eliminate the extraneous ATP, it being possible to carry out the lysis step after neutralization of the sprayed reagent to eliminate the extraneous ATP. 
     In still another variant the spraying device may be used for any other type of reagent intended for microbiological analysis which it is sought to deposit uniformly over a surface while ensuring that the sprayed reagent is well confined. 
     The present invention is not limited to the embodiments described and represented but encompasses any variant form thereof.