JP5167898B2 - Sampling apparatus and sampling method using the same - Google Patents

Description

  The present invention relates to a sampling device for collecting minute objects contained in a fluid (liquid) and a sampling method using the same, and in particular, microorganisms such as cells contained in a fluid (liquid) in a sterile state. The present invention relates to a sampling device for collecting and a sampling method using the same.

In bioreactors installed in pharmaceuticals, food and beverage production lines, it is necessary to inspect for microbial contamination in order to guarantee product quality.
Conventionally, in order to collect (sample) microorganisms, the microorganisms are sampled aseptically after steam sterilization of the sampling line. However, when steam sterilization was used in this way, the possibility of poor sterilization could not be denied. A sampling vessel for sampling microorganisms is connected to a conduit connecting the bioreactor. And after sampling the microorganisms, the conduit is open from the connection part (connection port) with the bioreactor to the sampling container, so it cannot be said that there is no invasion of microorganisms from the outside. .
Therefore, a fluid sampling device using a sample container that can sample a fluid in an aseptic state has been proposed (see, for example, Patent Document 1). This device performs aseptic sampling into a sterilized sample container through a connector that is aseptically removable.
JP 2005-181336 A

 In the above fluid sampling device, when analyzing the collected sample, it has been necessary to collect the sample from the sample container by hand and process it. For this reason, there is a risk that microorganisms may enter the sample container from the outside. From these facts, even if a trace amount of microorganism was detected from the sample, there was no confirmation that the microorganism was derived from the sample.

 The present invention has been made in view of the above circumstances, and provides a sampling device capable of collecting minute objects contained in a fluid (liquid) in a sterile state and a sampling method using the same. With the goal.

  The sampling device according to the present invention includes a membrane filter, and a sample collection unit formed of a transparent film that is covered on one surface side of the membrane filter and forms a space between the membrane filter, A supporting member that supports the membrane filter from the other surface, and the other side of the membrane filter are provided to hold the sample collection unit and the supporting member, and the filtrate that has passed through the membrane filter flows in. A filtrate inflow part, a sample introduction pipe provided in the sample collection part so as to communicate with the space, and a filtrate discharge pipe connected to the filtrate inflow part.

  It is preferable that a chemical solution injection tube is provided so that the tip of the membrane filter faces the other surface.

  The sampling method according to the present invention is a sampling method using the sampling device according to the present invention, in which a liquid sample is introduced into the space of the sample collection unit, and a micro object contained in the liquid sample is removed. After being collected on one surface of the membrane filter, the collected bacterial cells are stained by spraying a chemical solution from the other surface side of the membrane filter, and then on one surface side of the membrane filter. A transparent film is closely attached.

  According to the sampling device of the present invention, a membrane filter, and a sample collecting section formed of a transparent film that is covered on one surface side of the membrane filter and forms a space between the membrane filter, A support member that supports the membrane filter from the other surface, and a filtrate that is provided on the other surface side of the membrane filter, holds the sample collection unit and the support member, and passes through the membrane filter. Since it has an inflowing filtrate inflow part, a sample introduction pipe provided to communicate with the space in the sample collection part, and a filtrate discharge pipe connected to the filtrate inflow part, after completion of sampling, Since the sample collection part is completely sealed and the device itself is used for microscopic observation, the sample is manually removed from the sample container as before. Not only there is no need to collect, can be prevented microorganisms from entering the sample of interest from an external device. Therefore, it can be confirmed that all the detected microorganisms such as the bacterial cells are derived from the sample, and strict microorganism detection can be performed.

  According to the sampling method of the present invention, by using the sampling device according to the present invention, a liquid sample is introduced into the space of the sample collection unit, and a micro object contained in the liquid sample is removed from the membrane filter. After collecting on one surface, spray a chemical solution from the other surface side of the membrane filter to stain the collected cells, and then adhere the transparent film to one surface side of the membrane filter Therefore, after sampling is completed, the sample collection part is completely sealed, and the apparatus itself is used for microscopic observation, so that it is not necessary to collect a sample from a sample container through a manual operation, Microorganisms can be prevented from entering the target sample from the outside of the apparatus. Therefore, it can be confirmed that all the detected microorganisms such as the bacterial cells are derived from the sample, and strict microorganism detection can be performed.

The best mode of the sampling apparatus of the present invention and the sampling method using the same will be described.
This embodiment is specifically described for better understanding of the gist of the invention, and does not limit the present invention unless otherwise specified.

1A and 1B are schematic configuration diagrams showing an embodiment of a sampling apparatus according to the present invention, in which FIG. 1A is a plan view and FIG. 1B is a cross-sectional view taken along line AA in FIG.
The sampling device 10 of this embodiment includes a sample collection unit 13 composed of a membrane filter 11 and a transparent film 12, a support member 14, a filtrate inflow unit 15, a sample introduction pipe 16, a filtrate discharge pipe 17, A chemical solution injection pipe 18 and a holding member 19 are schematically configured.

The sample collection unit 13 is provided so as to cover the membrane filter 11 and the one surface 11 a side of the membrane filter 11, that is, provided so as to cover the one surface 11 a of the membrane filter 11. And a transparent film 12 that forms a space 20. Moreover, the membrane filter 11 and the transparent film 12 are joined without a gap, and the space 20 is formed by these.
Moreover, as for the sample collection part 13, the part enclosed with the dashed-two dotted line shown in FIG.1 (b) may be detachable with the holding member 19. FIG.

The support member 14 is provided on the other surface 11 b side of the membrane filter 11 and supports the membrane filter 11 from the other surface 11. The support member 14 prevents the membrane filter 11 from being deformed or damaged by the pressure of the liquid flowing into the space 20 of the sample collection unit 13.
The filtrate inflow portion 15 is shaped like a mortar and is provided on the other surface 11 b side of the membrane filter 11 to hold the sample collection portion 13 and the support member 14 and permeate the membrane filter 11. This is the part where the filtrate flows.

  The sample introduction tube 16 is provided in the sample collection unit 13 so as to communicate with the space 20. Further, the end of the sample introduction tube 16 opposite to the side connected to the sample collection unit 13 is detachably connected to a bioreactor or the like via a known aseptic connector. A liquid containing micro objects such as microorganisms is introduced from the bioreactor or the like into the sample collection unit 13 through the sample introduction tube 16. Therefore, in order to send the liquid from the bioreactor or the like to the sample collection unit 13 in the middle of the sample introduction tube 16 and between the sample collection unit 13 and the bioreactor or the like, Abbreviation) is provided.

  The filtrate discharge tube 17 is connected to the bottom of the filtrate inflow portion 15 and discharges the filtrate that has passed through the membrane filter 11 to the outside of the apparatus. Further, the end of the filtrate discharge pipe 17 opposite to the side connected to the sample collection unit 13 is detachably connected to a filtrate collection tank (container) through a known aseptic connector. It has become. Further, a liquid feed pump (not shown) such as a peristaltic pump is provided in the middle of the filtrate discharge pipe 17 in order to discharge the filtrate in the filtrate inflow portion 15.

  The chemical solution injection tube 18 is provided from the other surface 11b side of the membrane filter 11 so that the tip 18a faces the other surface 11b. The chemical solution injection tube 18 is used to prevent microorganisms such as cells collected on the one surface 11a of the membrane filter 11 from treating the cell wall of the cells or DAPI (4 ′, 6-diamino-2-phenyllinole). For spraying various dye solutions.

  The holding member 19 holds the sample collection unit 13, the support member 14, the filtrate inflow unit 15, the sample introduction pipe 16, the filtrate discharge pipe 17, and the chemical solution injection pipe 18. In the sample collection unit 13, a bioreactor or the like It is for performing stably operation which collects micro objects, such as microorganisms contained in the liquid introduced from.

The membrane filter 11 is a porous film-like filter in which a large number of uniform and fine pores (micropores) are formed.
Examples of the material for the porous film forming the membrane filter 11 include one or more selected from polycarbonate, polytetrafluoroethylene, polyester, cellulose acetate, and the like.
In addition, the size of the micropores of the membrane filter 11 is appropriately adjusted according to the size of the micro object to be collected. However, since the size of many cells is 0.5 μm or more, the size is 0.1 μm. It is preferable that it is -0.45 micrometer or less, and it is more preferable that it is about 0.20 micrometer from the condition of clogging.

  As the transparent film 12, a transparent and excellent resin film is used. For example, a film made of a polyester resin such as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene naphthalate (PEN); polyethylene ( Film made of polyolefin resin such as PE) and polypropylene (PP); Film made of polyfluorinated ethylene resin such as polyvinyl fluoride, polyvinylidene fluoride and polytetrafluoroethylene; Polyamide resin such as nylon 6, nylon 6,6 A film made of a vinyl polymer such as polyvinyl chloride (PVC), ethylene-vinyl acetate copolymer, polyvinyl alcohol, vinylon; polymethyl methacrylate, polyethyl methacrylate, polya Ethyl acrylic acid, acrylic made of a resin film such as polybutyl acrylate; of polystyrene films; films made of polycarbonate (PC); consisting of polyarylate film; and a film made of polyimide and the like. Among these films, a non-fluorescent film subjected to a non-fluorescent treatment is used when observation is performed with an epifluorescence microscope. Moreover, the shrink film which shrink | contracts by applying heat can also be used.

The support member 14 is a mesh-like member made of metal, synthetic resin, or the like, and the mesh size is set larger than the fine pores of the membrane filter 11.
As the metal or the synthetic resin that forms the support member 14, a material that does not deteriorate due to corrosion or the like by a liquid introduced into the sample collection unit 13 from a bioreactor or the like is used. Examples of the metal include stainless steel. As a synthetic resin, the thing similar to resin which makes said transparent film 12 is mentioned, for example.

  As a material forming the filtrate inflow portion 15, a metal or a synthetic resin that is not deteriorated due to corrosion or the like by a liquid introduced into the sample collection portion 13 from a bioreactor or the like is used. Examples of the metal include stainless steel. As a synthetic resin, the thing similar to resin which makes said transparent film 12 is mentioned, for example.

  The sample introduction tube 16 and the filtrate discharge tube 17 are flexible, and a metal or a synthetic resin that does not deteriorate due to corrosion or the like by a liquid introduced into the sample collection unit 13 from a bioreactor or the like is used. Examples of the metal include stainless steel. As a synthetic resin, the thing similar to resin which makes said transparent film 12 is mentioned, for example.

  As a material forming the chemical solution injection pipe 18, a metal or a synthetic resin that does not deteriorate due to corrosion or the like by a liquid introduced into the sample collection unit 13 from a bioreactor or the like is used. Examples of the metal include stainless steel. As a synthetic resin, the thing similar to resin which makes said transparent film 12 is mentioned, for example.

  The material of the holding member 19 is not particularly limited, but a metal or a synthetic resin that does not deteriorate due to corrosion or the like by the liquid introduced into the sample collection unit 13 from a bioreactor or the like is preferable.

  Next, the operation of the sampling device 10 will be described with reference to an example of a method for sampling cells using the sampling device 10.

A liquid sample (liquid sample) is introduced into the space 20 of the sample collection unit 13 of the sampling device 10 via the sample introduction tube 16 connected to the bioreactor by an aseptic connector. At this time, a liquid sample is introduced from the bioreactor into the space 20 of the sample collection unit 13 using a liquid feed pump (A) such as a peristaltic pump provided in the sample introduction pipe 16.
In addition, a liquid feed pump (B) such as a peristaltic pump provided in the filtrate discharge pipe 17 is provided at almost the same time as the introduction of the liquid sample into the sample collecting unit 13 is started or after a while after the introduction is started. In use, the discharge of the filtrate that permeates the membrane filter 11 and flows into the filtrate inflow portion 15 is started.

  At this time, the liquid sample (A) provided in the sample introduction pipe 16 and the liquid delivery pump (B) provided in the filtrate discharge pipe 17 are adjusted, and the liquid sample introduced into the sample collection unit 13. Is equal to the flow rate (flow velocity) of the filtrate discharged from the filtrate inflow portion 15, and pressure is applied to the space above the membrane filter 11 (the space on the one surface 11a side of the membrane filter 11). Do not. Thereby, damage to the membrane filter 11 and the transparent film 12 can be prevented.

  The liquid sample that has flowed into the space 20 of the sample collection unit 13 passes through the membrane filter 11 and is discharged from the filtrate inflow unit 15 to the outside of the sampling device 10 through the filtrate discharge pipe 17. In addition, the cells contained in the liquid sample are collected on one surface 11 a of the membrane filter 11.

  After the filtration of the liquid sample is completed, the cell surface treatment chemical solution of the bacterial cells and DAPI (4 ′, 6-diamino-2-phenyllinole) are stained from the chemical solution injection tube 18 to the other surface 11b of the membrane filter 11. A chemical solution is sprayed to stain the cells collected on the one surface 11 a of the membrane filter 11. Thus, if a chemical | medical solution is spread | dispersed from the other surface 11b side of the membrane filter 11, a chemical | medical solution permeate | transmits the membrane filter 11, and it can dye | stain a microbial cell. On the other hand, even if a microorganism enters from the outside of the sampling device 10 to the other surface 11b side of the membrane filter 11, the microorganism cannot pass through the membrane filter 11 at a bacterial level. There will be no mixed state with the bacteria collected on the surface 11a. Therefore, in this invention, only the microbial cells derived from a liquid sample can be collected.

  After the staining of the bacterial cells is completed, the transparent film 12 is brought into close contact with the one surface 11a side of the membrane filter 11. As a result, the sample introduction tube 16 is also sealed, and the sample collection unit 13 is completely sealed. In order to bring the transparent film 12 into close contact with the membrane filter 11, the air or gas in the space 20 of the sample collection unit 13 is removed by a vacuum pump or the like, and the space 20 is decompressed, or a shrink film is used. In this case, the film is instantaneously heated to about 100 ° C. to shrink the film.

After the adhesion of the transparent film 12 is completed, the sample introduction tube 16 is removed from the bioreactor, the filtrate discharge tube 17 is removed from the filtrate collection tank (container), and the sampling device 10 is placed on the stage of the fluorescence microscope.
Then, the cells collected on the one surface 11a of the membrane filter 11 are observed with an epifluorescence microscope.
Note that if an oil-resistant film is used as the transparent film 12, oil can be applied to the surface thereof, and observation by oil immersion is also possible.

  According to the sampling apparatus 10 of this embodiment and the sampling method using the sampling apparatus 10, the sample collection unit 13 is completely sealed after the sampling is completed, and the apparatus itself is used for microscope observation. In addition, it is not necessary to manually collect a sample from the sample container, and microorganisms can be prevented from entering the target sample from the outside of the apparatus. Therefore, it can be confirmed that all the detected microorganisms such as the bacterial cells are derived from the sample, and strict microorganism detection can be performed.

In this embodiment, the sampling apparatus 10 is used for microscopic observation after the end of sampling. However, the present invention is not limited to this, and the sample collection unit 13 is attached to and detached from the holding member 19 as described above. Only the sample collection unit 13 may be used for microscopic observation.
In this embodiment, the case where the microorganisms contained in the liquid sample are sampled has been exemplified. However, the present invention is not limited to this, and other minute objects contained in the fluid (liquid) are sampled and the minute objects are sampled. The present invention can also be applied when observing a microscope.

It is a schematic block diagram which shows one Embodiment of the sampling device which concerns on this invention, (a) is a top view, (b) is sectional drawing which follows the AA line of (a).

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Sampling apparatus, 11 ... Membrane filter, 12 ... Transparent film, 13 ... Sample collection part, 14 ... Support member, 15 ... Filtrate inflow part, 16 ... Sample Introducing pipe, 17 ... filtrate discharging pipe, 18 ... chemical liquid injection pipe, 19 ... holding member, 20 ... space.

Claims (3)

A sample collection unit comprising a membrane filter and a transparent film that is covered on one surface side of the membrane filter, forms a space between the membrane filter, and can be in close contact with the membrane filter ; ,
A support member for supporting the membrane filter from the other surface;
Provided on the other surface side of the membrane filter, holding the sample collection part and the support member, and a filtrate inflow part into which a filtrate that has passed through the membrane filter flows,
A sample introduction tube provided in the sample collection part so as to communicate with the space;
A filtrate discharge pipe connected to the filtrate inflow portion;
A sampling apparatus comprising:   The sampling apparatus according to claim 1, wherein a chemical solution injection tube is provided so that the tip of the membrane filter faces the other surface. A sampling method using the sampling device according to claim 1 or 2,
A liquid sample is introduced into the space of the sample collection unit, and a minute object contained in the liquid sample is collected on one surface of the membrane filter, and then from the other surface side of the membrane filter. A sampling method comprising spraying a chemical solution to stain the collected bacterial cells, and then bringing the transparent film into close contact with one side of the membrane filter.

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