JP2005080506A - Sampler and sampling system for microorganism in low-temperature liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sampler designed to perform sampling of a microorganism in a low-temperature liquid in an on-line system without causing leakage of the low-temperature liquid. <P>SOLUTION: The sampler for the microorganism in the low-temperature liquid is designed to detect the microorganism existing in the low-temperature liquid. The sampler for the microorganism is equipped with a membrane filter made of a fluororesin, a pair of annular gaskets made of the fluororesin and sandwiching the peripheral part of the membrane filter from both sides, a pair of holders sandwiching the membrane filter and the pair of annular gaskets from both sides and sealing therein and an elastic member provided between at least one surface of the holder and at least one surface of the gasket and pressing the gasket. The membrane filter is taken out after passing the low-temperature liquid therethrough and brought into contact with a culture medium or dipped in the liquid culture medium to thereby carry out culturing. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a sampler for sampling microorganisms in a cryogenic liquid, and a sampling system equipped with such a sampler.

  In the pharmaceutical industry and the food industry, a low-temperature liquid such as liquid nitrogen is used to rapidly cool a chemical or food. It is obvious that it is not preferable that the low-temperature liquid used for such a rapid cooling treatment is contaminated with microorganisms because the microorganisms adhere to food and medicine and the microorganisms may grow in the distribution process.

  For this reason, an apparatus for sterilizing a liquefied gas such as liquid nitrogen through a filter is known.

  However, there is a fundamental desire to evaluate such a quenching system itself and to sample microorganisms in the cryogenic liquid in order to detect microbial contamination of the cryogenic liquid.

  Conventionally, in order to sample microorganisms in a cryogenic liquid, after collecting a certain amount of cryogenic liquid in a container and evaporating the cryogenic liquid, the microorganisms adhering to the container are examined (see, for example, Patent Document 1), The inner surface of the container was rinsed to examine microorganisms in the rinse solution. However, with such a method, the cryogenic liquid can be microbially contaminated during sampling. Further, since the cryogenic liquid evaporates while sampling the cryogenic liquid in the container, it is difficult to measure the exact volume of the taken-out cryogenic liquid sample. Thus, the conventional sampling method has a problem that it is inferior in reliability.

  On the other hand, in order to evaluate low-temperature microbial contamination online, a method of evaluating the number of microorganisms (hereinafter referred to as the membrane method) by flowing a low-temperature liquid through the membrane filter and culturing the microorganisms collected by the membrane filter is considered. . The membrane method is widely used when a liquid at room temperature is used (bacterial inhibition performance test method of JIS K3823 ultrafiltration module).

However, it has been found that when a cryogenic liquid is handled by a membrane filter, there is a problem that the cryogenic liquid leaks unless the seal structure is devised.
JP 2000-185710 A

  An object of the present invention is to provide a sampler that can sample microorganisms in a cryogenic liquid online without causing leakage of the cryogenic liquid, and a sampling system equipped with such a sampler.

  A sampler of microorganisms in a cryogenic liquid according to one aspect of the present invention includes a membrane filter made of fluororesin, a pair of fluororesin annular gaskets sandwiching a peripheral portion of the membrane filter from both sides, the membrane filter and 1 A pair of holders that enclose and sandwich a pair of annular gaskets, and an elastic member that is provided between at least one side of the holder and the gasket and that presses the gasket; The filter is taken out and brought into contact with a medium or immersed in a liquid medium and cultured to enable detection of microorganisms present in the cryogenic liquid.

  A sampling system for microorganisms in a cryogenic liquid according to another aspect of the present invention includes the above-described sampler, a first pipe connecting the cryogenic liquid source to be sampled and the sampler, and the first pipe if necessary. And a means for cooling the sampler so that the cryogenic liquid passing through the pipe and the sampler is kept in a liquid state, and the membrane filter is taken out after allowing the cryogenic liquid to pass through. The microorganisms present in the cryogenic liquid can be detected by bringing them into contact with the medium or immersing them in a liquid medium and culturing. In addition, when a low temperature liquid can be hold | maintained in a liquid state, it is not necessary to necessarily provide a cooling means.

  The sampling system of the present invention has a second pipe connected to a first pipe connecting the cryogenic liquid source and the sampler and supplying a sterilizing gas of the same type as the cryogenic liquid. The sterilized cryogenic liquid liquefied by the cooling means in one pipe is passed through the sampler, and after passing the sterilized cryogenic liquid, the membrane filter is taken out and brought into contact with the medium or immersed in the liquid medium and cultured. May enable blank sampling of microorganisms present in the sterile cryogenic liquid.

  In the sampling system of the present invention, a plurality of samplers may be connected in parallel.

  According to the present invention, microorganisms in a cryogenic liquid can be sampled online without causing leakage of the cryogenic liquid.

  The sampler of microorganisms in a cryogenic liquid according to the present invention is based on a membrane method, and includes a membrane filter, a pair of annular gaskets sandwiching the peripheral edge of the membrane filter from both sides, and a membrane filter and a pair of annular gaskets on both sides. And a pair of holders that are sandwiched and sealed.

  As a material for the membrane filter and the gasket, a fluororesin such as polytetrafluoroethylene (PTFE) is used. PTFE has a wide use temperature range of 200 ° C. to −200 ° C., and exhibits durability even at a low temperature liquid such as liquid nitrogen. In the following, the fluororesin will be described as being representative of PTFE.

  A gasket made of PTFE produces sealability by elastic deformation at room temperature. However, when the temperature is lowered from room temperature to liquid nitrogen temperature, the elastic deformation generated at room temperature is not elastic deformation and loses sealability. In PTFE, it is indispensable to generate a new elastic strain by applying a new force according to the temperature in order to maintain the sealing property. That is, plastic deformation that does not tend to recover the elastic deformation of the PTFE gasket at room temperature when it is instantaneously cooled to a low temperature by simply tightening the PTFE membrane filter and the pair of PTFE gaskets from both sides with a pair of holders at room temperature. Therefore, the sealing performance is lost, and the possibility that the cryogenic liquid leaks from the connecting portion of the pair of holders increases. Further, even when PTFE is returned from a low temperature to a normal temperature, it takes several hours to several days for the elastic strain to recover to a level before being exposed to the low temperature. Therefore, in order to prevent microbial contamination other than the sample, it is important to apply a new stress in real time to PTFE in response to a change in temperature to generate a new elastic strain in PTFE and always maintain a sealing property.

  Therefore, in the sampler of the present invention, an elastic member (for example, a coil spring) is provided between at least one side of the holder and the PTFE gasket to press the PTFE gasket. An elastic member may be provided between the holders on both sides and the PTFE gasket. In such a sampler, when a PTFE membrane filter and a pair of PTFE gaskets are tightened with a pair of holders from both sides at room temperature and then cooled to a low temperature, the elastic deformation of the PTFE gasket tends to become plastic deformation. Since a new elastic strain at a low temperature can be given to the PTFE gasket by the pressing force, it is possible to prevent the low temperature liquid from leaking from the connecting portion of the pair of holders. Therefore, in the sampler of the present invention, the microorganisms in the cryogenic liquid can be sampled online without causing leakage of the cryogenic liquid. In addition, even when the temperature returns to room temperature, the sealing performance is always maintained despite the change in temperature, so that new microbial contamination can be prevented.

  FIG. 1 shows an exploded sampler of microorganisms in a cryogenic liquid according to an embodiment of the present invention. As shown in FIG. 1, for example, a PTFE membrane filter 1 having a pore diameter of 0.1 μm is supported by a support filter 2. A pair of PTFE annular gaskets 3 and 3 are provided so as to sandwich the peripheral edge of the PTFE membrane filter 1 (and the support filter 2) from both sides. All of these members are sandwiched from both surfaces by stainless steel upstream and downstream holders 4, 4 and sealed between them. Specifically, the PTFE gasket 3, the support filter 2, the PTFE membrane filter 1, and the PTFE gasket 3 are accommodated in a recess provided in the downstream holder 4, and the upstream holder 4 is screwed to tighten the holders together. In FIG. 1, a coil spring 5 is provided as an elastic member between the upstream holder 4 and the upstream PTFE gasket 3 to press the PTFE gasket 3. For this reason, when the low-temperature liquid is passed, stress can be applied by the coil spring 5 to cause a new strain in the PTFE gasket 3, and leakage of the low-temperature liquid can be prevented. After passing the cryogenic liquid, the sampler is disassembled and the PTFE membrane filter 1 is taken out and brought into contact with the medium and cultured, or immersed in the liquid medium and sonicated and then cultured in the liquid medium. The presence and number of microorganisms present in the cryogenic liquid can be examined.

  FIG. 2 schematically shows a sampling system for microorganisms in a cryogenic liquid according to an embodiment of the present invention. A valve (V4) 102 that allows the liquid nitrogen 101 to flow out is attached to the tank 100 that stores the liquid nitrogen 101 that is a sampling target. The sampling system of the present invention is connected downstream of the valve (V4) 102 (the connecting portion is shown downstream of V4). This sampling system has a container 10 containing liquid nitrogen 11 as a cooling means. A coil upstream pipe 12, a coil 13, a coil downstream pipe 14, a valve (V2) 15, a sampler upstream pipe 16, a sampler 17 shown in FIG. 1, and a sampler downstream pipe 18 are sequentially connected downstream of the valve (V4) 102. (The first pipe in FIG. 2 is the coil upstream pipe 12, coil 13, coil downstream pipe 14 and sampler upstream pipe 16 between the valve (V4) 102 and the sampler 17). A measurement cylinder 19 is provided at the outlet of the sampler downstream pipe 18. The coil 13 and the sampler 17 are immersed in the liquid nitrogen 11 in the container 10. A vent pipe 20 having a valve (V1) 21 is connected to the coil downstream pipe 14. Further, a nitrogen gas pipe 22 having a valve (V3) 23 is connected to the coil upstream pipe 12. This nitrogen gas pipe 22 is provided with a sterilization filter 24. In FIG. 2, one sampler is immersed in the liquid nitrogen 11 in the container 10, but a plurality of samplers may be switched and provided in parallel.

  Note that the sampler 17 is not necessarily immersed in the liquid nitrogen 11 in the container 10, and the sampler may be disposed outside the liquid nitrogen 11 as long as the condition that the liquid nitrogen does not vaporize is satisfied. For example, a sampler may be disposed at a position indicated by a broken line 17 ′ in FIG. 2 (in this case, a connection portion is provided upstream of the sampler).

  Further, when there is no need for blank sampling, it is not necessary to provide devices such as the nitrogen gas pipe 22, the valve (V3) 23, and the sterilization filter 24. Therefore, most simply, as shown by a broken line, downstream of the valve (V4) 102, the valve (V2 ′) 15 ′, the sampler upstream pipe 16 ′, the sampler 17 ′, the vent pipe 20 ′, and the valve (V1 ′) A configuration in which 21 'is provided may be used.

  Blank sampling and sampling of microorganisms in liquid nitrogen 101 were performed using the sampling system of FIG. Hereinafter, these operation procedures will be described.

(A) Blank sampling First, after sterilizing devices including V1, V2, V3, V4 and the sampler, each device is set. If necessary, the sterilized nitrogen gas may be supplied to prevent microbial contamination without immersing the coil 13 in the liquid nitrogen 11 while a device such as the sampler 17 is attached. With V4 closed, open V1 and V3. After sterilizing the nitrogen gas with the sterilizing filter 24, the nitrogen gas is supplied to the coil 13 immersed in the liquid nitrogen 11 accommodated in the container 10 via the V 3 and the nitrogen gas pipe 22, and passes through the coil 13. Allow to liquefy in between. This produces sterile liquid nitrogen. After confirming liquefaction of nitrogen at the outlet of the vent pipe 20, V1 is closed.

  Next, V2 is opened, blank liquid nitrogen liquefied in the coil 13 is passed through the sampler 17 (blank sampling), and the amount thereof is examined by the measuring cylinder 19. Thereafter, V3 and V2 are closed.

  Next, the connection upstream of V1 and V2 is disconnected, the equipment containing V1 and V2 is removed, the sampler 17 is placed in the clean bench, and the temperature of the sampler 17 is returned to room temperature. Thereafter, the sampler 17 is disassembled to remove the PTFE membrane filter, which is transferred onto a desired medium to culture the microorganism.

(B) Sampling of microorganisms in liquid nitrogen 101 As in (A), after sterilizing the devices including V1, V2, V3, V4 and the sampler, each device is set. From the state where V4 is closed, V1 and V4 are opened. It is confirmed that the liquid nitrogen 101 in the tank 100 flows out from the outlet of the vent pipe 20.

  Next, V2 is opened, V1 is closed, the liquid nitrogen 101 in the tank 100 is passed through the sampler 17 (sampling), and the amount thereof is examined by the measuring cylinder 19. Thereafter, V4 and V2 are closed.

  Next, the connection upstream of V1 and V2 is disconnected, the equipment containing V1 and V2 is removed, the sampler 17 is placed in the clean bench, and the temperature of the sampler 17 is returned to room temperature. Thereafter, the sampler 17 is disassembled to remove the PTFE membrane filter, which is transferred onto a desired medium to culture the microorganism.

  In addition, after blank sampling, before removing the blank sampler, the microorganisms in liquid nitrogen may be sampled using another sampler in parallel, and then both samplers may be removed and cultured.

  Separately, a test was conducted to examine the integrity of the membrane filter before and after sampling. In this test, a sampler is placed in ethanol, and a gas is supplied to the sampler while changing the pressure to examine the pressure at which bubbling occurs. This test was performed immediately after assembling the sampler at room temperature and after passing liquid nitrogen through the sampler. As a result, in all cases, the pressure at which bubbling occurred was 0.2 MPa, and it was confirmed that the membrane filter was not damaged (maintained integrity) even when liquid nitrogen was passed.

  Actually, after sampling microorganisms in liquid nitrogen contaminated with microorganisms using the sampling system shown in FIG. 2, the membrane filter is placed on a desired medium or immersed in a liquid medium and cultured for 10 days. The results of examining the number of microorganisms are shown in Table 1.

As can be seen from Table 1, the sampling system shown in FIG. 2 enables on-line sampling of microorganisms in liquid nitrogen.

The figure which decomposes | disassembles and shows the sampler of the microorganisms in the cryogenic liquid which concerns on one Embodiment of this invention. The figure which shows the structure of the sampling system of the microorganisms in the cryogenic liquid which concerns on one Embodiment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... PTFE membrane filter, 2 ... Support filter, 3 ... PTFE annular gasket, 4 ... Holder, 5 ... Coil spring, 10 ... Container, 11 ... Liquid nitrogen, 12 ... Coil upstream piping, 13 ... Coil, 14 ... Coil downstream piping 15 ... Valve (V2), 16 ... Sampler upstream piping, 17 ... Sampler, 18 ... Sampler downstream piping, 19 ... Measuring cylinder, 20 ... Vent piping, 21 ... Valve (V1), 22 ... Nitrogen gas piping, 23 ... Valve (V3), 24 ... sterile filter, 100 ... tank, 101 ... liquid nitrogen, 102 ... valve (V4).

Claims (3)

A fluororesin membrane filter,
A pair of fluororesin annular gaskets sandwiching the peripheral edge of the membrane filter from both sides;
A pair of holders for enclosing the membrane filter and a pair of annular gaskets sandwiched from both sides;
An elastic member provided between at least one side of the holder and the gasket and pressing the gasket;
In a cryogenic liquid characterized by enabling detection of microorganisms present in the cryogenic liquid by removing the membrane filter after passing through the cryogenic liquid and bringing it into contact with the culture medium or immersing in a liquid culture medium and culturing. Microbial sampler. A sampler according to claim 1;
A first pipe connecting a cryogenic liquid source to be sampled and the sampler;
Means for cooling at least a portion of the first pipe and the sampler so that the cryogenic liquid passing through the first pipe and the sampler is maintained in a liquid state;
In a cryogenic liquid characterized by enabling detection of microorganisms present in the cryogenic liquid by removing the membrane filter after passing through the cryogenic liquid and bringing it into contact with the culture medium or immersing in a liquid culture medium and culturing. Microbial sampling system. A second pipe connected to a first pipe connecting the cryogenic liquid source and the sampler to supply a sterilizing gas of the same type as the cryogenic liquid, and the sterilizing gas is cooled in the first pipe; Pass the sterilized cryogenic liquid liquefied by means through the sampler, pass the sterilized cryogenic liquid, and then remove the membrane filter and contact with the medium or immerse in the liquid medium and culture in the sterilized cryogenic liquid The sampling system according to claim 2, wherein blank sampling of microorganisms to be performed is enabled.

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