FR2485732A1 - Automatic sample isolation for analysing cultures of microorganism - eliminates need for chemical additives so sample is accurately representative - Google Patents

Abstract

Appts. is described for automatically injecting liq. samples in sterile conditions into sterile test tubes. Appts. comprises a horizontal carousel table with a series of test tube pockets on a pitch circle centred on a vertical axis of rotation of the plate. The table is rotated intermittently in steps such that a pocket advances circumferentially by a distance equal to the pitch between pockets. When a pocket reaches the injection station it allows its test tube to fall coaxially on to an upper horizontal stop. The upper horizontal stop is swung away to allow the tube to descend further on to a lower horizontal stop which pivots in opposition to the upper stop. At this stage an injection needle is withdrawn upwards from a sterilisation chamber to a height which allows the test tube to be moved beneath the needle by transverse movement of a carriage. The injection needle is flexibly piped to a sterilisable vessel from which sample liq. is injected into the test tube where it is passed through a fine filter. The fine filter separates the tube into two compartments. Pref. the filter comprises a glass fibre membrane of 0.2 micron porosity on a porous backing disc of sintered stainless steel. The sampling is used partic. for making repeated analyses in monitoring and controlling the culture of microorganisms. Stable samples can be isolated without resorting to chemical additives. Such additives can dilute the sample and/or impair the accuracy of subsequent tests. The sample is now strictly representative of the culture at the moment of sampling.

Description

The present invention relates to a method and apparatus for sterile and automatic collection of liquid samples in sterile test tubes.

 Most microorganism culture methods require taking regular samples over time in order to carry out analyzes, dosages and measurements to monitor the evolution of this culture.

 The liquid sample collectors that currently exist do not allow satisfactory stabilization of the sample taken, the composition of the latter changing over time and therefore requiring rapid analysis.

In the prior art, in an attempt to stabilize the sample taken, it has been proposed to introduce chemical additives into the sample. However, such an attempt has not proved satisfactory since it results in three major drawbacks, namely
- the dilution provided by the additive;
- possible interference of the additive with a subsequent dosing method;
- the inertia of the inactivation of the microorganism by this additive.

 To remedy these problems, the Applicant has developed a method and an apparatus which make it possible to obtain samples that are rigorously representative of the state of the culture at the actual time of sampling.

 The essential means of the present invention consists in implementing a filtration operation avoiding the addition of chemical additives.

 Thanks to such filtration, it is possible to rapidly separate the cultivated microorganism from its culture medium.

 This filtration ensures the biological and biochemical stability of the sample, which allows either its immediate analysis or its conservation for later analysis.

 The apparatus for sterile and automatic collection of liquid samples in sterile test tubes according to the present invention comprises a mobile barrel-type support on which rests a plurality of test tubes intended to receive individually according to a determined cycle samples from a sterilizable chamber external to said device, said mobile support being actuated by means of a rotation system, a set of flaps supported by an axis acting in an open-close mode in opposition, a device for conveying the test pieces in two parts, of which the one is placed on a sliding carriage which, when it moves, brings it to test against an injection needle with an upward-downward movement and connected to the sterile chamber readable by a flexible hose by means of which the sample contained in said chamber is introduced into the test piece to undergo filtration, said needle, before m Ise in place of the test tube for the injection of the sample, located in a sterilization chamber, and a store for collecting the test pieces.

The apparatus according to the present invention is also remarkable for the characteristics below taken individually or in combination
the rotation system of the mobile support consists of a toothed wheel, a rotating pin and a rotation arm provided with a pawl,
the device for conveying the test pieces has the form of a chimney consisting of a fixed part and a mobile part,
- it further includes a cam programmer actuated by an electric motor as well as a sequential clock,
each test piece is in two parts separated from each other by a disc or pellet carrying a filtering membrane, the test piece being closed at each of its ends by a plasma plug
- The membrane is a glass fiber membrane with a porosity of approximately 0.2 e and the pellet is a sintered stainless steel pellet.

 The method for sterile and automatic collection of liquid samples in sterile test tubes by means of the apparatus according to the present invention consists in performing a filtration of the sample during which sterile neutral gas causes said sample to pass through a filtering membrane arranged in the upper part of 11 specimen.

According to a characteristic of the process of the invention, the sterile neutral gas is chosen from Neon, Argon, Krypton,
Xenon, Radon, and any gas which does not affect the sample taken.

According to another characteristic of the iS vention process, the filtered volume is a function of the volume of the sampling chamber and of the pressure exerted in the sample.
and te, / the pressure applied varies as a function of the composition of the sample to be filtered.

 Various advantages and features of the present invention will emerge from the detailed description below.

The invention is illustrated, by way of nonlimiting example, in the accompanying drawings in which
Fig. 1 is a perspective view of the apparatus of the invention.

 Fig. 2 is a block diagram for explaining the present invention.

 Fig. 3 is a section showing the internal structure of a test piece.

 Fig. 4 is a diagram of the sterilizable sample chamber external to the device from which the sample is conveyed in the injection needle.

 The apparatus illustrated in FIGS. 1 and 2 comprises at its upper part a mobile support of the barrel type 1.

This barrel 1 can receive several test pieces E; for example, its capacity can be 16 test tubes. The barrel 1 can rotate by means of a rotation system constituted by a toothed wheel 2 made integral with the barrel 1 by means of a rotating pin 3 and locked by a pawl locking the toothed wheel 2 on an arm of rotation 4.

 Thanks to the means described above, a test piece E is brought above an upper flap 6- forming part of a set with an internal flap 7 which is supported on an axis 5, said set of flaps 6,7 operating according to an open-close mode in opposition. During the rotation of the axis 5, the test tube E disposed above the upper flap 6 passes through a conveying device consisting of two parts, a fixed chimney 8 and a movable chimney 9.

A sliding carriage 10 supports the movable chimney 9 as well as a sterilization chamber 11 for an injection needle 12. At the bottom of the apparatus is a magazine 13 for collecting the test pieces E. The apparatus according to the present invention also comprises a cam programmer 15 actuated by an electric motor as well as a sequential clock, not shown.

 In FIG. 3, the internal structure of a test piece E has been illustrated. Such a test piece E consists of two parts designated respectively by 16 and 17 which are separated by a disc or pad 18 on which a filter membrane 19 rests. A gasket 20 seals between the two parts 16, 17 of test piece E while each end is closed by a plug 21.

By way of example, the disc or pellet 18 is made of sintered stainless steel and the membrane 19 is a “whatman” glass fiber membrane with a porosity of 0.2 F (biological sterility standard). The stopper 21 is a usual plasma type stopper.

Before explaining in detail the operation of the apparatus according to the present invention, it is important to specify how the sample is conveyed in the injection needle. For this purpose, reference is made to FIG. 4.

 FIG. 4 illustrates a sterilizable sampling chamber 14 connected to the bottom valve V1 of a closing tank 22. The chamber 14which is independent of the apparatus of the invention, is connected to the injection needle 12 by means of a flexible hose F. The operation of the chamber 14 will be specified below and a description will now be given of that of the apparatus according to the present invention.

To use this device, it is necessary to have a source of electricity, compressed air, steam, gas sterile from the sterilizable sampling chamber 14.

The barrel IX loaded with the test tubes E sterilized beforehand, performs a partial rotation by means of the aforementioned rotation system constituted by the toothed wheel 2, the rotating axis 3 and the ratchet arm 4. I1 as a result that a test tube E is brought above the upper flap 6 closing the fixed chimney 8 in its upper part. At this moment, the axis 5 of the flaps pivots, which overlaps the opening of the flap 6 and the closing of the flap 7 which closes the lower part of the movable chimney 9. The test tube
E thus falls on the flap 7. During this phase, the injection needle 12 is located in the sterilization chamber 11 in vapor circulation.

 The synchronization of the movements rapidly allows the needle 12 to exit the sterilization chamber 11 by moving the carriage 10, the placing of the test piece E under the needle 12 and the introduction of the latter. in the test piece E. The sample contained in the chamber 14 is then conveyed in the test piece E via the injection needle 12.

 This operation finished, the needle 12 leaves the test tube E, the carriage 10 reposition the test tube E on the flap 7 and the needle-12 returns to the sterilization chamber 11. The axis 5 of the flaps 6 , 7 pivots, which causes the opening of the shutter 7 and the closing of the shutter 6; the test piece E then falls into the magazine 13. The apparatus is thus in position for a new cycle.

The delivery of the sample contained in the tank 22 to the injection needle 12 via the chamber 14 comprises different phases: firstly, a sterilization phase of the chamber 14 during which the valves V1 , V2 and V5 (see Figure 4) are closed while the valves V3 and V4 are open. During this phase, VA vapor is introduced; this vapor sterilizes the chamber 14, the needle 12 and the chamber 11. Secondly, a purging and cooling phase takes place; the valve V3 is closed and the valve V2 is open which receives sterile neutral gas GN, for example nitrogen, the effect of which is to purge the condensates of vapor. Then the valves
V2 and V4 are closed and the valve V5 is open and supplied with FR refrigerant. Chamber 14 is cooled by circulation of the FR refrigerant. Simultaneously, the injection needle 12 leaves the sterilization chamber 11 to enter the test tube E. We then proceed to the filling phase of the chamber 14. For this purpose, the valve V5 is closed and the chamber 14 is at this time in a slight vacuum. The valve V1 is then open and the withdrawal liquid P fills the chamber 14. In the next phase, the valve V1 is closed and the valves V4 and V2 are open.

The sterile neutral gas GN pushes the sample contained in the chamber 14 in the test tube E via the hose F and the needle 12. The fact of refrigerating the chamber 14 after sterilization makes it possible to obtain a temperature and a constant voids in it. The main purpose of this operation is to eliminate the influence of pressure and temperature in the chamber on the volume of the sample.

 As described above, the sample arrives in the upper part of the test tube E. The pressure of the sterile neutral gas GN applied pushes the liquid through the filtering membrane 19 until the pressure is equalized between the lower and upper parts 17, 16 of the test piece E, the function of the membrane 19 being to separate the liquid substrate and the microorganism thus preventing any evolution of the filtered sample. The filtered volume depends on the volume of the sampling chamber 14 and on the pressure exerted in the test piece. LR pressure varies according to the composition of the sample to be filtered. According to the present invention, the cam programmer 15 controls the automation of the operation of the chamber 14 in conjunction with that of the apparatus. The operations indicated above are carried out using pneumatic cylinders actuated by mini-valves controlled by the cams of the programmer.

The apparatus of the invention always operates under sterile conditions and, during the very short time when the needle 12 is between the sterilization chamber 11 and the test tube E, the needle 12 is inside a bellows (not shown) which seals between the support of the needle 12 and the chamber li or the specimen
E. This bellows is sterilized at the same time as the sterilization chamber 11. It will however be noted that, in the event that the needle is temporarily in non-sterile conditions, this fact would be of little importance. The bottom line is that the inside of the fermenter is not in contact with the outside air, which is the case. The few external contaminants which could contaminate the needle 12 will be automatically retained by the filter membrane 19 in the upper part 16 of the test piece E and will not be able to develop due to the absence of the culture medium.

 The present invention therefore provides a method and apparatus for sterile and automatic collection of liquid samples in sterile test tubes. The invention allows samples to be taken in situ from industrial fermentation tanks.

 The invention is not limited to the embodiment described and includes its technical equivalents.

Claims (11)

 1. Apparatus for sterile and automatic collection of liquid samples in sterile test tubes, characterized in that it comprises a mobile support of the barrel type (1) on which rests a plurality of test tubes (E) intended to receive individually according to a determined cycle of samples coming from a sterilizable chamber (14) external to said device, said mobile support (1) being actuated by means of a rotation system (2,3,4), a set of flaps (6 , 7) supported on an axis (5) acting in an open-close mode in opposition, a conveying device (8, 9) of the test pieces (E) in two parts, one of which (9) is arranged on a carriage sliding (10) which, during its movement, brings the test piece (E) opposite an injection needle (12) driven in an up-down movement and connected to the sterilizable chamber (14) by a hose by means of which the sample contained in said chamber (14) is introduced into s the test piece (E) to undergo filtration, said needle (12), before the positioning of the test piece (E) for the injection of the sample located in a sterilization chamber (11), and a store (13) for collecting test pieces (E).  2. Apparatus according to claim 1, characterized in that the rotation system (2,3,4) of the movable support (I) consists of a toothed wheel (2), a rotating pin (3) and a rotation arm (4) provided with a ratchet.  3. Apparatus according to one of claims 1 or 2, characterized in that the conveying device (8, 9) of the test pieces (E) has the form of a chimney consisting of a fixed part (8) and d 'a movable part (9).  4. Apparatus according to any one of claims 1 to 3, characterized in that it further comprises a cam programmer (15) actuated by an electric motor, as well as a sequential clock. 5. Apparatus according to any one of claims 1 to 4, characterized in that each test piece (E) is in two parts (16,17) separated from each other by a disc or pad (18) carrying a filter membrane (19) , the specimen (E) being closed at each of its ends by a plasma plug (21).  6. Apparatus according to claim 5, characterized in that the membrane (19) is a glass fiber membrane with a porosity of substantially 0.2 and the pellet (18) is a pellet of sintered stainless steel. 7. Method for sterile and automatic collection of liquid samples in sterile test tubes by means of the apparatus according to any one of claims 1 to 6, characterized in that it consists in filtering the sample (P) during of which sterile neutral gas (GN) causes said sample (P) to pass through a filter membrane (19) disposed in the upper part (16) of the test piece (E).  8. Method according to claim 7, characterized in that the sterile neutral gas (GN) is chosen from: Neon, Ar gon, Yrypton, xenon, Radon, and any gas which does not alter the sample - i - sampled --9 A method according to any one of claims 7 or 8, characterized in that the filtered volume is a function of the volume of the sampling chamber (14) and the pressure exerted in the test piece (E). 10. Method according to any one of claims 7 to 9, characterized in that the pressure applied varies according to the composition of the sample to be filtered. 11. Method according to any one of claims 7 to 10, characterized in that it consists in carrying out a sterilization phase of the sampling chamber (14) during which the valves (V1 V2 and V5) are then closed that the valves (V3 and V4) are open, steam (V2) then being introduced via the valve (V3), to perform a purge and cooling phase by closing the valve (V3) and opening of the valve (V2) which receives sterile neutral gas (GN) in order to purge the steam condensates, to stop said purging phase by closing the valves (V2 and V4), to cool the chamber (14) by circulating refrigerant (FR) by opening the valve (V5), then proceed to the filling phase of the chamber (14) after closing the valve (V5), the chamber (14) being under a slight vacuum, by opening the valve V1, the withdrawal liquid (P) thus filling the chamber (14).