Abstract:
A supplying system for an automatic inoculating machine for a sample on a substrate, characterized in that it includes a turret mobile in rotation, preferably around an axis substantially vertical, and an arm mobile in rotation, preferably around an axis substantially horizontal, carried by the turret, at a distal end of the aforesaid arm including means to fix a stylus, and means for raising or lowering the arm.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the field of automatic devices for inoculating a culture substrate with a sample to be analyzed, usually a substantially liquid sample. It relates more particularly to a system to take and inoculate the sample, for example on a substrate in a Petri dish. 
       BACKGROUND OF THE INVENTION 
       [0002]    In an automatic inoculating device, each sample is taken in a sample tank by the automatic device using a stylus, and then distributed on the substrate surface using the same stylus. Between each inoculation, it is proper to clean the stylus to avoid contamination of the next sample . . . . 
         [0003]    In the automatic devices of the prior art, stylus is dipped in several tanks, the first one containing a disinfectant, the following two containing sterile water for rinsing the disinfectant. Such a method is described in the document U.S. Pat. No. 5,547,872 o Schalkowsky et al. 
         [0004]    The stylus must be moved from one area of sample collection in the corresponding tank to an inoculating area in which the sample must be distributed, generally in a spiral pattern, then to each cleaning station and then into each cleaning tank. In all cases, the moving of the stylus is linearly, for example through a drive rack. 
         [0005]    At each position, the stylus must be lowered or raised, for example, to dip into the sample tank or in a tank of cleaning fluid. 
         [0006]    The maintenance of the devices of the prior art is delicate. In addition, they do not have a satisfactory reliability for the realization of the pattern of the inoculation. 
         [0007]    The invention aims to provide a system that can answer to the previously mentioned drawbacks, particularly a simple and inexpensive system, allowing for easy, reliable and efficient handling of the sample and possibly of the cleaning products. 
       SUMMARY OF THE INVENTION 
       [0008]    According to the invention, such a supplying system of an automatic inoculating device for a sample on a substrate is characterized in that it comprises a mobile turret in rotation, preferably around a substantially vertical axis, and an arm mobile in rotation, preferably around a substantially horizontal axis, carried by the aforesaid turret, a distal end of the aforesaid arm including means to fix a stylus, and means for raising or lowering the aforesaid arm. The means for raising or lowering the aforesaid arm includes a movable piston, from bottom to top and vice versa, the aforesaid piston being arranged, preferably near the axis, so that the arm is resting on an upper end of the aforesaid piston. 
         [0009]    The means for fixing the stylus are preferably fixing means without tools. They may advantageously comprise:
       a ring intended to be fixed, preferably glued, on the stylus;   means of axial and transverse positioning of the ring relative to the distal arm, and   means of keeping the ring on the distal arm.       
 
         [0013]    The axial and transverse positioning means may include a conical surface on the ring, and a conical surface of housing formed in the distal end, the aforesaid conical surfaces having substantially the same angle, and preferably expected to trap one in the other. The means of holding in place the ring may include a peripheral rib on the ring, and a nut provided for screwing on the cap and cooperating with the rib to keep the ring gripping with the extremity. 
         [0014]    The stylus is advantageously formed in one extremity of a flexible pipe. Preferably, this pipe is fixed so that it is fully accessible and mountable and/or removable without tools. To ensure that only the pipe may be contaminated by the sample, the pipe has preferably a length so that it provides an internal volume sufficient to store a sample volume sufficient for the inoculation. In order that this length is properly disposed in the controller, it may include a roll, to wind around a portion of the pipe. 
         [0015]    In addition, the system of the invention advantageously comprises motorized pumping means, preferably with a syringe, placed upstream of the pipe, provided for at least sucking up and distributing the sample by the stylus. It may also include dispensing means settled so that the pumping means can also suck up at least one product in a relevant tank, and then reject that aforesaid product by the stylus. 
         [0016]    Preferably, the means of distribution are settled so that the pumping means can draw two products, independently of one another, each product having a specific reserve, and discharge independently each product by the stylus. Advantageously, one product is a disinfectant, preferably an alcohol; the other is rinse aid, preferably distilled water. 
         [0017]    The system advantageously comprises means to adapt a Petri dish expected to contain the substrate. 
         [0018]    The invention also relates to a method of inoculating a Petri dish using a system according to the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]    Several embodiments of the invention will be described below, as no limiting examples, with reference to the accompanying drawings in which: 
           [0020]      FIG. 1  illustrates schematically the operation of an inoculating automatic device according to the invention; 
           [0021]      FIG. 2  is a section which illustrates schematically a system according to the invention for the supply of the automatic device of  FIG. 1 , the system comprising an arm mounted on a rotating turret; 
           [0022]      FIG. 3  is a half-section illustrating means for fastening a stylus to the extremity of the arm of  FIG. 2 ; 
           [0023]      FIGS. 4 and 5  illustrate different modes of implementation of a process of inoculating according to the invention; 
           [0024]      FIG. 6  is a sectional view of storage and sampling area for a product to be inoculated in the automatic device of  FIG. 1 ; 
           [0025]      FIG. 7  is a sectional view of means for cleaning the stylus of  FIG. 3 ; 
           [0026]      FIG. 8  is a view similar to  FIGS. 4 and 5 , in which the Petri dish is square and the inoculating is made in the form of straight lines parallel to each other; and, 
           [0027]      FIG. 9  is a view similar to  FIG. 8 , where inoculation takes the form of squares substantially homothetic. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0028]      FIG. 1  illustrates a system of automatic system to inoculate a substrate  2  with a sample to be analyzed  3 . In the illustrated example, the substrate is contained in a Petri dish  4 , under the shape of a gel, and the product  3  is substantially liquid. 
         [0029]    The illustrated system includes a supply area  11  for the sample to be tested and an inoculating area  12 . It includes means  10  to collect the sample  3  in the supply area and deposit it, at least partially, on the surface of the substrate  2 . The automatic device includes a tray  6 , turning around a vertical axis X 6 . The tray  6  constitutes a support for the Petri dish  4 . It is at least indirectly rotated around its axis X 6  by a motor  7 . 
         [0030]    A method of inoculating according to the invention is more particularly described with reference to  FIGS. 4 and 5 . 
         [0031]    The taking means include a turret  13 , mobile in rotation around a vertical axis X 13 , under the action of a motor  15 . The turret is equipped with an arm  14 . The arm  14  is movable in a vertical plane, relative to the turret  13 , rotating around a substantially horizontal axis X 14  carried by the turret  13 . A distal extremity  16  of the arm  14  carries a flexible pipe  17  with one extremity  18  of the aforesaid pipe, extending downwards from the arm  16  forms a stylus  18 . The turret will be described in more details with reference to  FIGS. 2 and 3 . Preferably, the pipe is made of a material not adhering, for example POLY (TETRAFLUOROETHYLENE). This arrangement is particularly advantageous in that it limits the adhesions, especially when a sample has a relatively thick and sticky consistency. 
         [0032]    The automatic device of  FIG. 1  includes further cleaning means  20  for the stylus. In the illustrated example, the cleaning means  20  include a pour tank  21 , draining means  22  for discharging effluent from the cleaning tank  21  and retention vessel  23  for the aforesaid discharged effluents by pumping. In the example shown, the draining means  22  include a diaphragm pump. The retention vessel is closed by a plug  24 . The plug is pierced by a vent  25 , having the shape of a pipe. This pipe  25  is equipped with a filter  26  of 0.2 μm, so the atmosphere is protected from any possible microbial contamination. The pour tank  21  and its use will be more particularly described with reference to  FIG. 7 . 
         [0033]    The automatic device of  FIG. 1  also includes reserves  31  and  32  for containing liquid for cleaning the stylus  18 . Preferably each of these reserves is removable and can be replaced by a full one, whenever necessary. Each reserve  31 . 32  is shaped like a bottle closed by a plug  33  with a vent  34  for maintaining the interior of the cylinder at atmospheric pressure as to the transfer of liquid it contains. 
         [0034]    Each vent  34  is fitted with a filter  35 , e.g. a filter 0.2 μm, to ensure the sterility of the liquid contained in the corresponding reserve. An initial reserve  31  of the two reserves contains a disinfectant  36 , alcohol  36  in the example shown. The second reserve  32  contains rinse aid  37 , distilled water  37  in the example shown. 
         [0035]    The automatic device includes further pumping means  40  and supplying means  41  for different fluids  3 ,  36 ,  37  manipulated by the automatic device  1 . In the example illustrated, the pumping means includes a syringe  40  engineered by a cylinder  42  in which slides a piston  43 . The piston is driven by a motor  44 , preferably a stepper motor. 
         [0036]    The supplying means are here schematized by three valves  50 ,  51 . A first valve  50  among these three valves includes two positions; the first position, shown in the figure, can suck up or reject a fluid through the pipe  17 . The second position of the first valve  50  allows connecting the syringe  40  with a supplying pipe  52  for cleaning fluid  36 ,  37 . The pipe  52  comprises two parts  521  and  522 , each engaging with a respective reserve  31 . 32  of cleaning fluid. 
         [0037]    Each second and third valve  51  is assigned to a respective reserve of  31 . 32  cleaning fluid  36 ,  37 . In a first opened position  51 A each valve  51  allows the fluid to flow in the respective pipe  52 . In a second closed position  51 B, shown in  FIG. 1 , each valve  51  prevents the respective liquid to flow out in the pipe  52 . 
         [0038]    A sensor  55  is arranged on each side piece  521 . 522 . The sensor  55  is provided to detect the absence or presence of liquid in the side piece. The absence of liquid in one of the side pieces commands the stopping of the automatic device  1  and the replacement or filling the corresponding reserve. 
         [0039]    It is preferable that the sample taken from the pipe is maintained sufficiently downstream of the syringe  40 , so that it cannot be contaminated by the sample. 
         [0040]    Thus, the pipe is provided with a sufficient length so that its internal volume may contain a sufficient sample for inoculating. To enable the arrangement of this length of the pipe inside of the automatic device  1 , it includes a roll  57  round which is wound a portion  17 A of pipe  17 . Preferably, the roll  57  includes a shaped screw along which the pipe portion  17 A is arranged, and preferably fixed by snap inside the screw. 
         [0041]    Thus prepared, the pipe ( 17 ) is visible and accessible throughout its length. It is assembled by interlocking and snap, so it is removable without tools. 
         [0042]    In the example shown, the automatic device includes a stainless steel body, on which are arranged the various elements that compose it. The body is not shown in  FIG. 1 . 
         [0043]    The body includes a substantially horizontal platform  62 , particularly visible in  FIGS. 2 ,  6 ,  7  and  8 . 
         [0044]    We will now describe generally a cycle of inoculating. 
         [0045]    A sample is first stored in the supply area, for example in a container  60 . 
         [0046]    The arm  14  is brought in a sampling position  14 A so that the stylus  18  is above the container  60 . The arm  14  is then lowered so that the stylus dips to a PA depth into the sample. The valve  50  being in the position  50 A, a sufficient portion of the sample is sucked up into the pipe using the syringe  40 . 
         [0047]    The arm is then raised, and then brought into a position  14 B by rotating the turret  13  around its axis X 13 , particularly illustrated in  FIG. 1 , for the inoculation of the substrate  2 . The arm  14  is lowered again so that the stylus is close enough to the substrate to deposit the sample with the desired precision. By a combination of rotating movements of the turret  13  around its axis X 13  and the platform  6  around its axis X 6 , the sample is automatically deposited according to a previously defined pattern. The pattern may be a spiral or a combination of points and/or circles or arcs of concentric circles, as illustrated with reference to  FIGS. 4 and 5 . 
         [0048]    Once inoculating is completed, the arm is raised, and then taken to a cleaning position  14 C by rotation of the turret  13  around its axis X 13 , to clean the stylus. The remaining sample still in the pipe is expelled into the discharge tank  21 , using the syringe  40 ; the first valve  50  is always in the position  50 A. Then the arm  14  is lowered so that the stylus  18  dips in the discharge tank. 
         [0049]    The valve  50  is placed in the position  50  B, the third valve  51  is kept closed, in position  51 B, the second valve  51  is placed in the position  51 A, and the syringe is operated so that it fills with alcohol  36 . Then, the positions of the first valve  50  and the second valve  51  are reversed, and the piston  43  is pushed inside the cylinder  42 , so that the alcohol  36  is expelled into the tank  21 , by browsing the entire length of the pipe  17 . The inside of the pipe  17  is thus fully disinfected. The exterior of the pipe at the location of the stylus is disinfected since the tank  21  is filled with alcohol. 
         [0050]    The valve  50  is returned to the position  50  B, the second valve  51  is kept closed, in position  51 B, the third valve  51  is placed in the position  51 A, and the syringe is operated so that it fills with distilled water  37 . Then, the positions of the first valve  50  and the third valve  51  are reversed, and the piston  43  is pushed inside the cylinder  42 , so that distilled water is expelled into the tank  21 , by browsing the entire length of the pipe  17 . The inside of the pipe  17  is thoroughly rinsed. The exterior of the pipe at the location of the stylus is flushed since the tank  21  is filled with distilled water  37 . 
         [0051]    A new cycle can then be started. 
         [0052]    The sample is maintained downstream of the syringe; this syringe and the upstream portion of the pipe contain alternatively only alcohol  36  or water  37 . This is that residual water that serves as a liquid piston between the piston  43  of syringe  40  and the sample, when the sample is handled, first to take and then to inoculate it. 
         [0053]    We will now describe the turret  13  in reference to  FIG. 2 . The turret base  61  includes a substantially disc-shaped and equipped with a peripheral skirt  610 . The platform  62  includes a circular orifice  63 . A raised edge  64  is formed in the platform  62  in the periphery of the orifice  63 . The skirt  610  is provided for covering the raised edge  64 , so that they contribute together to prevent the penetration of liquid and/or of solid inside the body  65  of the automatic device  1 . 
         [0054]    Drive means  66  extends below the base to inside the body. They are fastened, at least indirectly, to the motor  15  of the turret  13 . The base also carries a clevis  67  that defines the fail over horizontal axis X 14  of the arm  14  and bearing the aforesaid arm. Opposite to its distal extremity  16 , relative to the axis X 14 , the arm includes a proximal extremity  68  on which is fixed a counterweight  69 , so that the fail over of the arm is substantially without effort. The proximal extremity  68  and counterweight  69  are directly above the base  61 . 
         [0055]    A cylinder  71  extends vertically upward from the base  61 . The cylinder is placed close to the clevis  67 , between the clevis and the distal extremity  16 . The arm rests by its own weight on the upper extremity  72 , moving vertically, of the cylinder  71 . Thus, the arm  14  is movable in a vertical plane carried by the turret  13 . The distal extremity  16  of arm  14  rises or falls with the extremity  72  of the cylinder  71 . A hemispherical cap  73  covers and protects the inside of the turret  13 . 
         [0056]      FIG. 3  describes a particular arrangement for fastening the pipe to the extremity  16  of the arm  14 . This assembly includes the following elements, each substantially for revolution, and mounted coaxially with each other:
       a housing  75  running through the extremity  16  from top to bottom;   a ring  76  intended to be glued on the pipe, the part of the pipe exceeding downstream the ring is forming the stylus  18 ;   a nut  77  for maintaining the ring in the housing         
         [0060]    The housing  75  includes, from bottom to top, a cylindrical portion  91  of small diameter, sufficient for the stylus  18  to be insert through, then a conical widening portion  82  and a cylindrical portion of large diameter  84  forming with the conical portion an escarpment  85 . The interior portion  85  is threaded. 
         [0061]    The ring  76  comprises, upstream to downstream, a conical portion  91  gradually widening at an angle identical to the portion  82  of the housing  75 . The conical portions are provided to cooperate each other to position transversely and longitudinally the ring in the housing  75 , likewise the stylus relatively to the extremity  16  of arm  14 . The largest diameter of the portion  91  is greater than the largest diameter of the portion  82 , so that the portion  91  extends beyond the portion  82 , inside the cylindrical portion  94 . Beyond the conical portion  91 , the ring includes an annular rib  93  extending radially beyond the conical portion, and a cylindrical portion  94 , radially away from the rib  93 . 
         [0062]    The nut  77  includes an axial cylindrical drilling  96 , provided for the passage of the cylindrical portion  94  of the ring  76  and an anterior face  97  scheduled to come to bear on the rib  93  of the ring. Thus, when the assembly is achieved, the nut being in tune with the tapping of the housing  75 , the anterior surface of the nut comes to bear against the rib and keeps the ring in position in the housing. Thus, the stylus is then maintained in a fixed and defined position relative to the extremity  16  of the arm. 
         [0063]    We will now describe the reasons for improved inoculating a Petri dish  4 . 
         [0064]    In the automatic device of the prior art, the sample is inoculated in a pattern forming a spiral on the substrate. The pattern is achieved by moving radially the stylus at a constant linear speed, while the Petri dish turns on itself to constant angular velocity. This method is particularly advantageous in that it allows to gradually reducing the surface density in sample as it goes away from the center of the Petri dish. However, interpretation of results is complicated and requires special charts specific to the used inoculating device. The risks of misinterpretation are important. 
         [0065]    It is proposed according to the invention to produce patterns as concentric circles  99 . The density varies with the distance from the center of the Petri dish, as the spirals of the prior art, but it remains constant on a same circle, the interpretation is simplified because it depends no longer on the subtended angle under which the result of the culture is analyzed. In the example shown in  FIG. 4 , the pattern includes three groups of three close circles together. The circles of each group are very close; they have a density very close. Thus, each group corresponds noticeably to a determined concentration. To improve the accuracy of results, the automatic device advantageously comprises means for varying the speed of rotation of the Petri dish so that the density is substantially identical to the circles of the same group. 
         [0066]    In the operating mode of  FIG. 5 , shown on a reduced scale, circles have been confined to arcs of circle  99 . This pattern produced similar results. However, it avoids depositing the sample on previously inoculated area when achieving the circle. 
         [0067]    We will now describe the sampling area  11 , with reference to the section of  FIG. 6 . Area  11  includes a circular orifice  101  in platform  62 ; the peripheral edge of the hole is shaped like a raised edge  102 . A cylindrical tank  103  is disposed in the orifice  101 . A skirt  104  extends from the upper edge  105  of the container and comes to rest on the platform  62 , around the aforesaid raised edge  102 . The skirt  104  covered the aforesaid raised edge  102 , so that they contribute together to prevent the penetration of liquid and/or of solid inside the body  65  of the automatic device  1 . 
         [0068]    The product to be inoculated, that is to say the sample  3  is contained in a cup  106  whose upper edge  107  rests on the upper edge  105  of the container  103 . Thus, a sample  3  may be supplied or removed from the automatic device without the risk of spilling in the body of the device that remains protected by the container  103 . In addition, if the product  3  is spilled in the container, it is removable and can be removed for cleaning. 
         [0069]    During the taking of the sample, the stylus  18  is expected to dip of a depth PA, measured at the edge  107  of the cup  106 . 
         [0070]    We will now describe the operation of the pouring tank and the cleaning process, referring to  FIG. 7 . 
         [0071]    The pour tank  21  has substantially a shape of revolution around a vertical axis. It includes two coaxial bowls  111 ,  112 , having a common base  113 . The interior bowl  111  is designed specifically to receive the stylus  18  and cleaning fluids  36 ,  37 . Its shape is narrow, so it offers radially sufficient, but without excess, space to provide the stylus and allow a flow of liquid  36 . 37  around the stylus. The outer bowl  112  is designed to recover the liquid flowing inside the bowl  111  when it overflows. Both bowls  111 , 112  include drain pipes respectively  114 . 115 , formed in the base  113 , and which join here to form only one  116 , connected to the emptying pump  22 . 
         [0072]    As the turret  13  and the container  103 , the discharge tank  21  is inserted into an orifice  117  of the platform  62 . The tank  21  includes a skirt  119  which extends from an upper edge  121  of the outer bowl  112  and covers a raised edge  118  of the orifice  117 . The device, as explained above, for the container  103  protects the interior  65  and can make the tank  21  easily removable, especially for cleaning. 
         [0073]    When inoculating the substrate  2  is completed, as previously explained, the extremity of the arm  14  is rotated to a position  14 C in which the stylus  18  is above the tank  21 , preferably above the outer bowl  112 , so that the pipe  17  is purged from the sample remaining not used for inoculating. 
         [0074]    Then the arm is moved at position  14 C 1 , in which the stylus  18  is dipped at a depth of PB into the interior bowl  111 . The stylus is maintained at this position during the disinfection operation. As previously described, during this operation, alcohol  36  flows into the pipe so it drains out of the stylus  18 . Alcohol then fills the internal bowl above its upper rim  120  since it overflows inside the outer bowl. Thus, the level of alcohol in the interior bowl  111  is always the same, substantially equal to the upper rim  120  of the bowl  111 . The depth PB is chosen higher than the PA taking depth of the sample in the bowl  106 . This step ensures the external disinfection of the stylus  18 , on any height that may have been contaminated during the taking of the sample. 
         [0075]    Then the arm is moved into position  14 C 2 , in which the stylus  18  is dip at a depth PC into the interior bowl  111 . The stylus is maintained in this position during the flushing operation. As previously described, during this operation, water  37  flows through the pipe to drain out off the stylus  18 . The water then fills the internal bowl since it overflows the outer bowl, above its upper rim  120 . Thus, the water level inside the bowl  111  is always the same, substantially equal to the upper rim  120  of the bowl  111 . The depth PC is chosen higher than depth PB previously used for disinfection. This process ensures that alcohol previously used for cleaning out the stylus  18  is thoroughly rinsed and will not accidentally sterilized future inoculating. 
         [0076]      FIGS. 8 and 9  illustrate two modes of implementation for an inoculating method according to the invention. In these examples, the Petri dishes  4  are square. 
         [0077]    In the example shown in  FIG. 8 , the inoculation is done in a form of straight lines  131  parallel to each other, having substantially the same length. Lines  131  are grouped into three groups of three lines. The lines of the same group have a density approximately the same between themselves. The group represented on the left includes three lines of high density, the group represented on the right has three lines of low density, and the middle group includes three lines of intermediate density. 
         [0078]    In the example shown in  FIG. 9 , the inoculation is done in a form of straight lines grouped in square  132 . The squares are homothetic themselves around the same center. The squares  132  are grouped into three groups of two squares. The lines of all the squares of the same group have a density approximately the same between themselves. The most inside represented group include lines with high density, the most outside group includes lines with low density, and the intermediate group includes intermediate-density lines. 
         [0079]    Of course, the invention is not limited to the previous described examples. 
         [0080]    Thus, rather than a turret, it may be provided means of linear displacement for the stylus. 
         [0081]    Also, instead of being provided removable, the pouring tank or the container of the sample zone may be provided fixed and shaped by stamping directly into the platform of the automatic device. 
         [0082]    The pouring tank, as schematically illustrated in  FIG. 1 , may have a rectangular shape, and consist of two compartments separated by a wall for the pouring from one compartment to another. 
         [0083]    Instead of only circular patterns, it can besides be planned particular patterns with in particular points more or less thick.