JP2017012005A - Scaffolding material for liquid medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To use a liquid medium containing no agar, thereby avoiding the influence of growth inhibition by agar, eliminating the complexity of handling found in the liquid medium, and further, a scaffold for a liquid medium capable of supporting automated processing. Provide members. SOLUTION: A permeation base 11 of a non-woven fabric that comes into contact with a liquid medium 3 and absorbs and holds the liquid medium, and a semipermeable membrane that is placed on the permeation base and permeates the liquid medium absorbed by the permeation base. The compartmentalized culture unit 20 which is a plate body of a resin member having a thickness of at least 0.4 mm and which is in close contact with the permeable membrane portion 15 of the membrane and has a through hole portion 23 formed in the plate body. To be equipped with. [Selection diagram] Fig. 1

Description

  The present invention relates to a liquid medium scaffold member, and more particularly to a liquid medium scaffold member that is suitable for culturing biological species that are susceptible to growth inhibition by agar and has excellent partitioning properties.

  When isolating microorganisms that grow in nature, after collecting environmental samples such as soil and diluting them appropriately, they are applied to a plate-like agar medium in which nutrients are dissolved or inoculated on a slant-like agar medium. Was confirmed to grow. Then, by collecting the colonies of microorganisms and further applying and inoculating them on another agar medium, it is possible to isolate the microorganisms with increased purity and uniformity.

  In the case of a general agar medium, the medium contains about 1.5% agar. An agar medium can be prepared inexpensively and easily. Moreover, since agarose, which is the main component of agar, is unlikely to be a nutrient for microorganisms, it is easy to adjust the nutrients contained in the medium, and microorganisms can be selected through the medium. For this reason, the agar medium is widely used in the isolation and culture of microorganisms.

  According to the knowledge so far, it was thought that most microorganisms can be cultured on the agar medium described above. However, as the agar medium is improved, it has been considered that there are very few microorganisms that can be isolated and cultured on the agar medium among the microorganisms that grow in nature. According to recent researches on various microorganisms due to the development of DNA analysis, it is said that only about 1% of all species can be isolated and cultured on an agar medium. At present, it has not yet been clarified how components such as agarose contained in agar affect the growth of microorganisms.

  Thus, microorganisms that are difficult to isolate and culture on an agar medium are referred to as “difficult microorganisms”. The above-mentioned difficult-to-cultivate microorganisms are also attracting attention as unused resources because they are expected to improve the environment such as degradation of pollutants, pharmacological actions such as novel antibiotics, and other useful actions. Despite the fact that difficult-to-cultivate microorganisms exist, they are not effectively utilized because they cannot be isolated on an agar medium.

  Therefore, instruments and methods that do not depend on an agar medium have been proposed (see Patent Document 1). Patent Document 1 discloses the following. After collecting microorganisms on the surface of the microporous membrane, the collected surface is turned downward. A microporous membrane is fixed to a microporous membrane support composed of a holding ring and a base. The liquid medium is brought into contact with the surface opposite to the collection surface. The liquid medium can reach the microorganisms by permeating the microporous membrane. Microorganisms that have obtained medium nutrients will form colonies on the lower surface of the microporous membrane.

  However, in the case of the instrument disclosed in Patent Document 1, it is necessary to turn over the surface of the microporous membrane inoculated with microorganisms during the culture. Moreover, since a liquid culture medium is dripped on the upper side of a microporous membrane, the handling at the time of a subculture is inconvenient. In particular, when screening for new useful microorganisms, it is generally necessary to handle multiple samples. However, it is complicated and difficult to handle a large number of specimens with the instrument disclosed in Patent Document 1. For this reason, Patent Document 1 is a configuration that is notable as an example of application to a medium other than an agar medium, but is desired to be improved in ease of handling such as culture.

  Next, a culture apparatus specialized for microbial culture in a liquid medium has also been proposed (see Patent Document 2). The apparatus of Patent Document 2 is a form in which a porous hollow fiber membrane made of polyvinylidene fluoride or the like is suspended in a liquid medium culture tank, and microorganisms are injected into the hollow fiber membrane for culturing. By using the culture apparatus, it has become possible to increase the efficiency of microorganism isolation and culture. When culturing a target microorganism using the apparatus of Patent Document 2, it is necessary to accurately control the concentration of the microorganism when injected into the porous hollow fiber membrane. Moreover, the number of parts constituting the apparatus is large and complicated. For this reason, the operation is complicated, and the cost burden required for introducing the apparatus increases. Therefore, it is unsuitable when finishing a low cost and handling a large number of samples.

  From a series of circumstances, the inventors newly developed a medium member suitable for microbial culture in a liquid medium, and solved the conventional problem (see Patent Document 3). The culture medium member includes a permeation base part that absorbs a liquid culture medium and a permeable membrane part. Therefore, it is inexpensive because it has a simple structure. In addition, microorganisms collected on the permeable membrane part can be seeded, and the culture efficiency with the liquid medium was greatly improved.

  Thereafter, the efficiency of screening work for collected microorganisms and the like has been studied using a liquid medium. In this case, the use of industrial robots is an urgent task because of the need to efficiently and surely perform dispensing, etc., in order to collect samples containing microorganisms and grow microorganisms, and to add drugs necessary for growth. . Therefore, in order to be able to cope with the automation process, the culture member of Patent Document 3 is further improved and developed to complete a new culture member.

Japanese Patent No. 4439837 International Publication WO2007 / 023711 Japanese Patent Application Laid-Open No. 2012-200352

  The present invention has been made in view of the above points, and by using a liquid medium that does not contain agar, it is possible to avoid the influence of growth inhibition due to agar, and also eliminate the complexity of handling seen in the liquid medium, Provides a scaffold member for a liquid medium that can be used for automated processing.

  That is, the invention of claim 1 includes a permeation base part that contacts a liquid medium and absorbs and holds the liquid medium, and the liquid medium placed on the permeation base part and absorbed by the permeation base part. A liquid culture medium comprising: a permeable membrane portion of a semipermeable membrane to be permeated; and a partition culture portion that is in close contact with the permeable membrane portion and has a through-hole portion formed in the plate body. Related to scaffolding members.

  Invention of Claim 2 concerns on the scaffold member for liquid culture media of Claim 1 whose said osmosis | permeation stand part is a nonwoven fabric.

  The invention according to claim 3 relates to the scaffold member for liquid medium according to claim 1, wherein the permeation base is a nonwoven fabric made of regenerated cellulose fibers.

  The invention according to claim 4 relates to the scaffold member for liquid medium according to any one of claims 1 to 3, wherein the permeable membrane part is a semipermeable membrane of a regenerated cellulose film.

  The invention according to claim 5 relates to the scaffold member for liquid medium according to any one of claims 1 to 4, wherein a plurality of the through-hole portions in the compartment culture portion are formed.

  Invention of Claim 6 concerns on the scaffold member for liquid culture media of any one of Claim 1 thru | or 5 whose plate | board thickness of the said division culture part is at least 0.4 mm or more.

  The invention according to claim 7 relates to the scaffold member for liquid medium according to any one of claims 1 to 6, wherein the compartment culture part is a resin member.

  The invention according to claim 8 is the scaffold member for liquid medium according to any one of claims 1 to 7, wherein an adhesive is applied to the back side of the compartment culture part and is in close contact with the permeable membrane part. Related.

  According to the scaffold for liquid medium according to the first aspect of the present invention, a permeation base part that contacts the liquid medium and absorbs and holds the liquid medium, and is placed on the permeation base part and absorbs liquid to the permeation base part. To provide a permeable membrane part of a semipermeable membrane that allows the liquid medium to permeate, and a partition culture part that is in close contact with the permeable membrane part and has a through hole formed in the plate body By using a liquid medium that does not contain agar, it is possible to avoid the influence of growth inhibition due to agar, and it is possible to eliminate the complexity of handling that is observed in the liquid medium.

  According to the scaffold member for liquid medium according to the invention of claim 2, in the invention of claim 1, since the permeation base is a non-woven fabric, the liquid medium can be efficiently absorbed using capillary action.

  According to the scaffold member for liquid medium according to the invention of claim 3, in the invention of claim 1, since the permeation base is a nonwoven fabric made of regenerated cellulose fiber, it is well known in that it has little influence on biological species. Moreover, it is widely distributed and can be procured at low cost.

  According to the scaffold member for a liquid medium according to the invention of claim 4, in the invention of any one of claims 1 to 3, since the permeable membrane part is a semipermeable membrane of a regenerated cellulose film, It is possible to allow various nutrient components and the like to permeate through the entire membrane without any deviation. Furthermore, it is widely distributed and has the advantage that it can be obtained at low cost with little impact on biological species.

  According to the scaffold member for liquid medium according to the invention of claim 5, in the invention of any one of claims 1 to 4, a plurality of the through-hole parts in the compartment culture part are formed. It becomes a form that mimics a well plate and can be used for automated processing.

  According to the scaffold member for liquid medium according to the invention of claim 6, in the invention of any one of claims 1 to 5, since the plate thickness of the compartment culture part is at least 0.4 mm, The rigidity of the member itself can be satisfied.

  According to the scaffold member for liquid medium according to the invention of claim 7, in the invention of any one of claims 1 to 6, the partition culture part is a resin member. Can be formed.

  According to the scaffold member for a liquid medium according to the invention of claim 8, in the invention of any one of claims 1 to 7, an adhesive is applied to the back side of the compartment culture part and is in close contact with the permeable membrane part. Therefore, each through hole can be completely independent.

It is a disassembled perspective view of the scaffold member for liquid culture media of 1st Embodiment of this invention. It is a perspective view at the time of bonding a permeable membrane part. It is a fragmentary sectional view of the scaffold member for liquid culture media of a 1st embodiment. It is a schematic diagram at the time of use of the scaffold member for liquid culture media of 1st Embodiment. It is a perspective view at the time of packaging of the scaffold member for liquid culture media of a 1st embodiment. It is a perspective view of the scaffold member for liquid culture media of other embodiments. It is a photograph of the scaffold member for liquid culture media of Example 7.

  As described in the background art, a semi-solid agar medium is generally used by gelling a culture solution for the convenience of seeding (inoculation) and passage of cultured organism species, ease of storage, etc. It is. In particular, in the case of biological species such as microorganisms, most species are fixed by colony formation. In this case, although it is possible to adhere to the bottom of the culture vessel, it is difficult to completely grow in the liquid medium due to respiration and metabolism. Therefore, the liquid medium scaffold member of the present invention is a member that serves as a scaffold that enables the cultivation of a species to be cultured even in a highly fluid medium represented by a liquid medium.

  The liquid medium scaffold member 10A of the present invention and its usage will be described in order with reference to the exploded perspective view of FIG. The liquid medium scaffold member 10A of the first embodiment is a usage example that is installed in a petri dish and used for culture. An appropriate amount of the liquid medium 3 necessary for culture is poured into the dish portion 1. A permeation base 11 is provided in the dish 1, and the permeable membrane part 15 is placed immediately above the permeation base 11. A compartment culture unit 20 that is in close contact with the permeable membrane unit 15 is provided immediately above the permeable membrane unit 15. The compartment culture unit 20 is a plate, and a through hole 23 is formed in the plate. Eventually, a lid 2 covering the dish 1 is put on. Accordingly, the liquid medium scaffold member 10 </ b> A in the illustrated example includes the permeation platform 11, the permeable membrane unit 15, and the compartment culture unit 20.

  The permeation platform 11 is in contact with the liquid medium 3. The liquid medium 3 is absorbed in the permeation base 11 by capillary action and spreads and is held throughout the permeation base. Therefore, the material is not particularly limited as long as the material is permeable. Considering heat sterilization using an autoclave or the like, a heat resistant material is more preferable. In the liquid medium scaffold member, the upper surface of the permeable membrane portion 15 is placed at a position higher than the liquid surface of the liquid medium 3 (see FIG. 3). In addition to suppressing the outflow of biological species and the like cultured on the permeable membrane part 15, excess water due to the liquid medium is also suppressed. Therefore, in order to avoid that the liquid medium scaffold member 10 itself is buried in the liquid medium 3, the permeation base 11 needs to have an appropriate thickness. For example, absorbent cotton having a predetermined thickness, a plate body on which filter papers are stacked, and a polyurethane sponge-like material. In addition, woven fabrics such as cotton, hemp, and wool, and superposition of these woven fabrics can also be used.

  Further, the permeation base 11 is formed from a nonwoven fabric. Since the nonwoven fabric does not have a fiber texture and is non-uniform, it is likely to cause capillary action after being absorbed once, and the absorption efficiency of the liquid medium is high. As long as the material of the nonwoven fabric is a fibrous material, it can be used widely. For example, non-woven fabrics such as polyamide fiber, polyester fiber, acrylic resin, polyethylene fiber and polypropylene synthetic resin fiber, and felt fabric can be used. Synthetic resin fibers are excellent in chemical resistance, inexpensive and resistant to heat. Felt fabric is also inexpensive. A known method is appropriately used for forming the nonwoven fabric.

  Among these, the penetration table part 11 is formed from the nonwoven fabric which consists of a regenerated cellulose fiber. Nonwoven fabrics made from regenerated cellulose fibers, for example, in the case of nonwoven fabric production by the viscose method, are mainly sulfidized by adding carbon disulfide after immersing pulp derived from wood in an alkali solution such as sodium hydroxide, Viscose is prepared by dissolution, and after aging, it is discharged into an acid solution such as sulfuric acid in a fibrous form and superposed. Therefore, a solidification reaction occurs, and a cloth-like product made of only cellulose fibers can be obtained. Since all the regenerated cellulose fibers are formed from cellulose, it is expected that the regenerated cellulose fibers have less influence on the species to be cultured than agarose agar. Nonwoven fabric made of regenerated cellulose fibers is a widely distributed material and is well known in that it has little effect on biological species. Moreover, it can be procured at a low cost.

  For the permeable membrane portion 15 placed on the osmosis table 11, a material is selected that takes into account the heat resistance during sterilization by an autoclave or the like and the permeability of the liquid medium absorbed by the capillary phenomenon through the osmosis table 11. . Specifically, dissolved sugars (carbon source), nitrogen sources such as various peptones and yeast extracts, salts (various metal components), other low molecular compounds such as amino acids and vitamins, growth promoting factors and growth inhibition The property of permeating factors and antibiotics is required. For example, a membrane filter, an ultrafiltration membrane, etc. are mentioned. In these, nutrient components of the medium are supplied through the opening of the membrane. For this reason, the components in the supplied medium may be biased. In addition, if the liquid medium is excessively supplied, the species being cultured may flow out.

  From this point, a semipermeable membrane is used for the permeable membrane portion 15. This is because it is necessary to permeate the liquid medium sucked by the capillary action through the permeation base 11. As described above, the semipermeable membrane can permeate various nutrient components and the like dissolved in the liquid medium through the entire membrane. In addition, it is possible to prevent the species (microorganisms) inoculated and cultured on the permeable membrane portion 15 from passing through the permeable membrane portion and entering the permeation platform portion 11. Specific examples of the semipermeable membrane include an acetylcellulose membrane, a polyacrylonitrile membrane, a fluororesin membrane, a polyester polymer alloy membrane, and a collagen membrane.

  The semipermeable membranes such as the above-mentioned collagen membranes are expensive and fragile. Therefore, for the permeable membrane portion 15 described above, a semipermeable membrane of a regenerated cellulose film, that is, a cellophane film is used. The semipermeable membrane (cellophane film) of the regenerated cellulose film is the same component as the non-woven fabric made of the regenerated cellulose fiber described above. The only difference is that it is formed into a film by being prepared into viscose and ripened and then discharged into an acid solution such as sulfuric acid in the form of a film. Since all the semipermeable membranes (cellophane films) of the regenerated cellulose film are also formed from cellulose, it is expected to have less influence on the species to be cultured than agarose of agar. In particular, the cellophane film has a great advantage that it can be obtained at low cost.

  As is clear from the drawing, the compartment culture part 20 that is in close contact with the permeable membrane part 15 is plate-shaped (plate body), and a plurality of through-hole parts 23 are formed. That is, the liquid medium scaffold member 10A of the present invention has a form imitating a well-known multi-well plate or the like. Therefore, the microorganisms and cells to be cultured are seeded (inoculated) on the permeable membrane portion 15 in each through-hole portion 23. For this reason, the through hole 23 needs to have a certain depth. The plate thickness of the compartment culture unit 20 is defined in consideration of the moldability of the compartment culture unit 20, the ease of sticking work with the permeable membrane unit 15, the rigidity (strength) of the member itself, and the like. The plate thickness of the compartment culture unit 20 is at least 0.4 mm or more, preferably 1 mm or more, and more preferably around 2 mm. The upper limit of the thickness of the compartment culture unit 20 is not particularly limited. However, if the plate thickness is excessively increased, the molding process described later becomes difficult. In addition, since the thickness of the permeation platform 11 is also added, the liquid medium scaffolding member cannot fit in the petri dish or the like. Therefore, the upper limit of the plate thickness of the compartment culture unit 20 is approximately 15 mm.

  The compartment culture part 20 is preferably formed of a resin member. Convenience at the time of processing the resin member is high, and the through hole can be freely formed. There are no particular restrictions on the type of material resin of the compartment culture unit 20. Generally selected from resin types that are easy to mold. The compartment culture unit 20 is selected from a heat-resistant resin that does not easily undergo thermal deformation in consideration of sterilization using an autoclave or the like. In the case of gamma ray sterilization or the like, special heat resistance is not required.

  The compartment culture unit 20 includes a thermoplastic resin such as polyethylene resin (high density polyethylene resin, high molecular weight polyethylene), polypropylene resin, polyethylene terephthalate (PET) resin, polyamide resin, polycarbonate resin, polyvinyl chloride resin, acrylic resin, A polyimide resin or a polyurethane resin is exemplified. Examples of the thermosetting resin include phenol resin, diallyl phthalate resin, urea (urea) resin, melamine resin, epoxy resin, silicone resin, polyimide resin, and polyurethane resin.

  Here, the joining between each other will be described. Even if the permeable membrane part 15 is merely placed on the permeation base part 11, moisture and nutrients of the liquid medium 3 permeate. Therefore, it is not particularly required to join each other. The compartment culture part 20 is not only placed on the permeable membrane part 15 but also in close contact with each other. The compartment culture unit 20 includes a plurality of through-hole portions 23. For this reason, an accurate experiment cannot be performed if microorganisms seeded in one through hole enter the surrounding through hole. This is because it is necessary to completely separate the individual through hole portions. Specifically, as shown in the perspective view of FIG. 2, the adhesive 30 is evenly applied to the bottom surface 22 of the compartment culture unit 20. And the permeable membrane part 15 is affixed on the bottom face 22 of the division culture part 20. FIG. Reference numeral 21 denotes an upper surface of the compartment culture unit 20.

  As the adhesive 30, a resin adhesive used for resin bonding or processing is used. The adhesive 30 is selected from various resin components such as an epoxy resin system, an acrylate resin system, and an ethylene vinyl acetate resin. Of course, other than these, there is no particular limitation as long as it is a kind that exhibits sufficient adhesive ability and is heat resistant during sterilization. However, it is necessary to use a resin component that does not affect the growth of the species to be cultured.

  The schematic diagram of the partial vertical cross-section of FIG. 3 shows the positional relationship between the dish part 1, the permeation base part 11, the permeable membrane part 15, and the compartment culture part 20 in the liquid medium scaffold member 10A. In the drawing, the petri dish lid 2 (see FIG. 1) is omitted. The base 12 of the permeation base 11 contacts the dish 1 of the petri dish. The liquid culture medium 3 in the dish part 1 penetrates into the permeation base part 11 and reaches the top part 13 by capillary action. Since the table upper part 13 of the osmosis table part 11 and the film part lower surface 16 of the permeable film part 15 are in contact with each other, the liquid medium also permeates the permeable film part 15 and oozes out to the film part upper surface 17. Then, the medium nutrient reaches the through hole 23 of the compartment culture unit 20, and the biological species to be cultured can obtain nutrients, salts, and the like via the permeable membrane unit 15.

  Since the permeable membrane part 15 is stable together with the osmotic platform 11, and the permeable membrane part 15 is adhered to the compartment culture part 20, it can serve as a scaffold for proliferation. Note that the permeable membrane portion 15 may be attached to the permeation base portion 11 using a binder (adhesive) of a component that does not affect the growth. Alternatively, the permeable membrane portion 15 may be attached to the permeation base portion 11 with water absorption. The semi-permeable membrane (cellophane film) of the regenerated cellulose film absorbs moisture from the liquid medium and wets it, and is moderately viscous. From this point, the cellophane film is excellent.

  The organism species to be cultured is a microorganism. A plurality of microorganism colonies C are formed on the upper surface 17 of the membrane portion on the permeable membrane portion 15 in the through-hole portion 23 of the compartment culture unit 20. Microorganisms are not limited to eukaryotes such as yeast and filamentous fungi, but prokaryotes, archaea, or uncultured microorganisms are also envisaged. Also, E. coli infected with bacteriophage. coli. And Bacillus sp. It is also envisaged that fungi that have been infected with viruses, such as fungi that have undergone genetic recombination, gene transfer, or other transformations. This is particularly effective when it is desired to reduce the influence of agarose that has not been elucidated so far. It is also effective for culturing under conditions in which the hardness of the agar medium cannot be maintained, such as high temperature culture conditions such as thermophilic bacteria and thermophilic bacteria. Of course, the species to be cultured are not limited to microorganisms, but plankton of animals and plants, protozoa, algae, or tissue cells of animals and plants are also assumed.

  Usually, in culture using an agar medium as commonly used, it is impossible to replace the medium itself over time. However, in the case of a liquid medium, medium exchange during the culture is easy. Therefore, it is convenient for analyzing consumption components, product products, etc., and tracking changes over time such as metabolism. Moreover, as already stated, although the liquid medium which does not use agar is used, the solid surface of the agar medium can be substituted by the permeable membrane part. For this reason, there is little influence on the work efficiency of those engaged in the culture work without greatly changing the ease of handling when using the conventional agar medium.

  The schematic diagram of FIG. 4 is a usage example of the liquid medium scaffold member 10A. In FIG. 4A, a biological species such as a microorganism is previously seeded (inoculated) into the through hole 23 of the compartment culture unit 20 and cultured at the same site. A predetermined amount of solution is injected (injected) into each through hole 23 by the dispensing device 40. The solution for dispensing is arbitrary, and examples include nutrient solutions, antibiotics, staining solutions, antibody reagents, fluorescent reagents, and radioisotope labeling reagents. The through-hole portions 23 of the partition culture unit 20 to be dispensed are arranged on grid points in rows and columns, and each arrangement is extremely standardized. In the embodiment described later, a total of 100 through-hole portions 23 of 10 rows and 10 columns are formed (drilled).

  The dispensing device 40 in the illustrated example has a plurality of branched ends, and a replacement tip 41 is attached to each. A solution can be held in each exchange chip 41 (multi-channel compatible type or the like). By using the dispensing device 40 shown in the figure, the solution is injected into the plurality of through-hole portions 23 in a single process. The dispensing process can be performed manually, or by a work robot installed in a clean bench or a safety cabinet. Therefore, the liquid medium scaffold member 10A has a structure that can effectively cope with large-scale processing such as high throughput. Thereby, the efficiency of culture and isolation can be improved more than ever.

  The schematic diagram of FIG. 4B shows the upper surface 21 of the compartment culture unit 20. The liquid medium scaffold member 10 </ b> A is used to inoculate and inoculate a biological species such as a microorganism into the through hole 23 of the compartment culture unit 20. Therefore, for example, as shown in FIG. 4A, the colony 24 is discolored in some through holes 23 as a result of dispensing a labeling reagent or a staining solution into the through holes 23. As can be easily understood from the drawing, the liquid medium scaffold member 10A is easy to use because it has a form similar to an existing multi-well plate. For example, the liquid medium scaffold member can be inserted into a measuring device or stored as it is, and the work efficiency can be improved.

  For example, microorganisms that have prevailed and proliferated in a certain through hole 23 are less likely to cross with the adjacent through hole 23. Therefore, by repeating the sorting and culturing while paying attention to the inside of each through hole 23, it becomes possible to isolate the target microorganism relatively easily, and the efficiency of screening can be increased. Furthermore, different microorganisms can be cultured in the through hole 23 of the compartment culture unit 20 while sharing the same liquid medium.

  In addition, as shown in the schematic diagram of FIG. 4B, the display unit 27 can be provided on the upper surface 21 of the compartment culture unit 20. The display unit 27 includes numbers, alphabets, and various symbols. In the illustrated example, numbers 1 to 10 and alphabets A to J are used. The display unit 27 is used when specifying the positions of the plurality of through-hole portions 23.

  The perspective view of FIG. 5 shows a state in which one liquid medium scaffold member 10A is accommodated in a resin packaging bag 50 and the opening is sealed. This is an example of a form that assumes so-called packaging and sales. Thus, when accommodated in a packaging bag and sealed, biological contamination of the liquid medium scaffold member 10A is suppressed. Further, it can be assumed that the liquid medium scaffold member 10A is accommodated in the packaging bag 50 and is subjected to sterilization treatment such as gamma ray sterilization or heat sterilization. Therefore, the cleanliness at the time of storage etc. is maintained high. In addition, there is no restriction | limiting in the accommodation number of the scaffold members 10A for liquid culture media in the packaging bag 50, According to a use scale, it is appropriate.

  FIG. 6A is a perspective view of the liquid medium scaffold member 10B of the second embodiment. The liquid medium scaffold member 10B includes the permeation platform 11, the permeable membrane 15, and the compartment culture unit 20B. These are the same as the aforementioned liquid medium scaffold member 10A. However, as a feature of the compartment culture part 20B, there is a form and size provided with only one through-hole part 23. As an application of the liquid medium scaffold member 10B, for example, use in a petri dish having a diameter of about 3 cm or a 6-well culture plate (well plate) is assumed.

  FIG. 6B is a perspective view of the liquid medium scaffold member 10C of the third embodiment. The liquid medium scaffold member 10 </ b> C includes the permeation platform 11, the permeable membrane unit 15, and the compartment culture unit 20 </ b> C. These are also the same as the liquid medium scaffold member 10A. However, the compartment culture unit 20C in this example has a different shape from the compartment culture units 20A and 20B described above. In the drawing, the through hole portions 23 are arranged in a single row. The liquid medium scaffold member 10C of the third embodiment can be adapted to culture containers of various sizes by increasing or decreasing the number of installation. For example, a mouth portion such as a T flask can be taken in and out of a narrow container.

[Preparation of scaffold for liquid medium]
In producing the liquid medium scaffold members of Examples 1 to 7, the permeation platform and the permeable membrane were common. A non-woven fabric made of regenerated cellulose fibers (Futamura Chemical Co., Ltd., TCF # 408, total thickness 10 mm) is used as the permeation base, and a semi-permeable membrane of regenerated cellulose film, so-called cellophane film (Futamura Chemical) is used as the permeable membrane. Manufactured by Co., Ltd., PL # 300, film thickness 18 μm) was used.

<Example 1>
A dedicated acrylic resin suitable for the three-dimensional modeling apparatus (3D printer) and the same apparatus was used to form through holes in a plate-like object having 10 × 10 lattice points. The thickness after curing was 0.4 mm, a square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. An epoxy resin (manufactured by Scotch, Premier Gold Super Versatile) was applied as an adhesive to the back side of the compartment culture section thus prepared, and a cellophane film was adhered thereto. And it stood still at normal temperature and the adhesive agent was hardened. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain a liquid medium scaffold member of Example 1.

<Example 2>
Using the three-dimensional modeling apparatus (3D printer) of Example 1 and a dedicated polyamide resin (nylon resin) suitable for the same apparatus, through holes are formed in a plate-like object having 10 × 10 lattice points. did. The thickness after curing was 0.5 mm, a square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. The same adhesive as in Example 1 was applied to the back side of the compartment culture part thus prepared, and a cellophane film was adhered thereto. And it stood still at normal temperature and the adhesive agent was hardened. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain a liquid medium scaffold member of Example 2.

<Example 3>
Using the same acrylic resin as in Example 1, a three-dimensional modeling apparatus (3D printer) was used to form through-holes on a plate-like object having 10 × 10 lattice points. The thickness after curing was 0.4 mm, the square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. An epoxy ultraviolet curable resin (820SEL, manufactured by Kyoritsu Chemical Industry Co., Ltd.) was applied as an adhesive to the back side of the compartment culture section thus prepared, and a cellophane film was adhered thereto. And the ultraviolet-ray was irradiated on condition of 3000 mJ / cm < ² > under normal temperature, and the adhesive agent was hardened. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain a liquid medium scaffold member of Example 3.

<Example 4>
The same polyamide resin as in Example 2 was used, and a three-dimensional modeling apparatus (3D printer) was used to form a through-hole portion in a plate-like object having 10 × 10 lattice points. The thickness after curing was 0.5 mm, a square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. An ultraviolet curable resin common to Example 3 was applied as an adhesive on the back side of the compartment culture part thus prepared, and a cellophane film was adhered thereto. Then, the adhesive was cured by irradiating ultraviolet rays at room temperature under the same conditions as in Example 3. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain the liquid medium scaffold member of Example 4.

<Example 5>
Using the same acrylic resin as in Example 1, a three-dimensional modeling apparatus (3D printer) was used to form through-holes on a plate-like object having 10 × 10 lattice points. The thickness after curing was 2.0 mm, the square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. An ultraviolet curable resin common to Example 3 was applied as an adhesive on the back side of the compartment culture part thus prepared, and a cellophane film was adhered thereto. Then, the adhesive was cured by irradiating ultraviolet rays at room temperature under the same conditions as in Example 3. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain the liquid medium scaffold member of Example 5.

<Example 6>
The same polyamide resin as in Example 2 was used, and a three-dimensional modeling apparatus (3D printer) was used to form a through-hole portion in a plate-like object having 10 × 10 lattice points. The thickness after curing was 2.0 mm, the square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. An ultraviolet curable resin common to Example 3 was applied as an adhesive on the back side of the compartment culture part thus prepared, and a cellophane film was adhered thereto. Then, the adhesive was cured by irradiating ultraviolet rays at room temperature under the same conditions as in Example 3. Finally, the cell culture film-attached compartment culture part was overlapped on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain a liquid medium scaffold member of Example 6.

<Example 7>
The same polyamide resin as in Example 2 was used, and a three-dimensional modeling apparatus (3D printer) was used to form a through-hole portion in a plate-like object having 10 × 10 lattice points. The thickness after curing was 2.0 mm, the square with a side of 5 cm, and the diameter of the through hole was 3.8 mm. A polyolefin resin-based hot melt resin (manufactured by Sanyo Trading Co., Ltd., 356P) was applied as an adhesive to the back side of the compartment culture section thus prepared, and a cellophane film was adhered thereto. And it stood still at normal temperature and the adhesive agent was hardened. Finally, the cell culture film-attached compartment culture part was stacked on the permeation base part (regenerated cellulose fiber nonwoven fabric) to obtain a liquid medium scaffold member of Example 7.

[Production results]
The scaffold members for liquid culture media of Examples 1 and 2 have a hole-like function because the through-hole portion has a depth despite being thin. In addition, when the thickness is less than 0.4 mm, it becomes difficult to perform resin molding and sticking processing of the compartment culture part itself, and the durability of the member may be reduced. Therefore, it was assumed that a plate thickness of at least 0.4 mm was necessary.

  Examples 3 and 4 are examples in which the type of adhesive was changed. In particular, by using an ultraviolet curable resin, bending deformation generated in the compartment culture part or the like was further reduced. In the liquid medium scaffolds of Examples 1 and 2, deformation of cellophane was partially confirmed from the influence of moisture.

  Examples 5, 6, and 7 are examples in which the thickness of the compartment culture part is increased, and the shape is most stable with almost no deformation due to the rigidity of the compartment culture part itself. Moreover, since these through-hole parts have sufficient depth and the injection | pouring operation | work by a pipette etc. is easy, favorable workability | operativity is ensured.

  For reference, the liquid medium scaffold member of Example 7 was photographed. The photograph in FIG. 7 shows the scaffold member for liquid medium of Example 7 in which the cell culture film-attached compartment culture part is superimposed on the permeation base part (regenerated cellulose fiber nonwoven fabric), and the whole dish part (inner diameter) 8.5 cm). Although it is difficult to see on the photograph, numbers 1 to 10 in the row direction and letters A to J in the column direction were formed in a convex shape. In the photograph, the upper lid and liquid medium were omitted.

[Collection and culture]
The inventors tried to culture using a culture species as a microorganism in order to verify the effectiveness of the liquid medium scaffold member. The verified microorganisms were collected from the pond water on the university campus of the University of Tsukuba (Tsukuba City, Ibaraki Prefecture).

  Example 7 was used for the liquid medium scaffold member. The permeation base (regenerated cellulose fiber nonwoven fabric) was sterilized by heating with an autoclave. Similarly, the cell culture section with cellophane film attached was also sterilized by heating in an autoclave. In this way, all members of the liquid medium scaffold member of Example 7 were sterilized by dividing each member.

  As the liquid medium, an R2A powder medium (manufactured by Nippon Pharmaceutical Co., Ltd.) was prepared to a specified concentration and used. The liquid medium was dispensed into a sterilized plastic petri dish (inner diameter 8.5 cm) by a predetermined amount. However, the liquid surface of the liquid medium in the petri dish was set to an appropriate position lower than the upper part of the permeation base part of the liquid medium scaffold member.

  The pond water collected at the above university campus was appropriately diluted with sterilized water. Using a dispensing device (Gilson, Pipetteman), 2 μL of this diluted water was applied to the semipermeable membrane (cellophane film) of the regenerated cellulose film at the inner bottom of all through-hole portions of the liquid medium scaffold member of Example 7. Dispensing and inoculating. Then, the petri dish was covered with an upper lid and cultured in an incubator at 30 ° C. for 48 hours in a humidified state.

  After the above culture, the liquid medium scaffold member was visually observed. The discolored through hole was a colony of grown microorganisms. From this, it was confirmed that it is effective for culturing microorganisms. In the liquid medium scaffold member, existing dispensing devices can be used for the inoculation (inoculation) of microorganisms, and the through-holes are arranged in a standard lattice point, so continuous dispensing etc. Suitable for operation. Therefore, expectation is high for the high-throughput processing of the liquid medium scaffold member. Furthermore, it is not necessary to prepare a gel-like medium such as agar, and the medium can be cultured in a liquid medium. Therefore, it is possible to contribute to the improvement of the overall working efficiency as well as the simplification of the medium preparation.

  It has been shown that by employing the liquid medium scaffold member of the present invention, it is possible to cultivate relatively easily at a relatively low cost using only the liquid medium. Therefore, it has great significance as an alternative to an agar medium, and industrial applicability due to scale expansion can be greatly expected. In addition, it is possible to apply to automated processing by standardizing parts, and it is possible to further activate research on unused biological resources such as difficult-to-cultivate microorganisms that could not grow on conventional agar media .

DESCRIPTION OF SYMBOLS 1 Petri dish part 2 Petri dish cover part 3 Liquid culture medium 10A, 10B, 10C Liquid medium scaffold 11 Penetration base part 12 Base bottom part 13 Base top part 15 Permeable membrane part 16 Membrane part lower surface 17 Membrane part upper surface 20, 20B, 20C Compartment culture part 21 Upper surface of compartment culture part 22 Lower surface of compartment culture part 23 Through-hole part 27 Display part 30 Adhesive 40 Dispensing instrument 50 Packaging bag C Microorganism colony

Claims (8)

A permeation stand that contacts the liquid medium and absorbs and holds the liquid medium;
A semipermeable membrane permeable membrane portion that is placed on the osmosis table portion and allows the liquid medium that has been absorbed into the osmosis table portion to pass therethrough;
A scaffold member for a liquid medium, comprising: a plate body that is in close contact with the permeable membrane portion, wherein the plate body is formed with a through-hole portion.   The scaffold member for a liquid medium according to claim 1, wherein the permeation base is a nonwoven fabric.   The scaffold member for a liquid medium according to claim 1, wherein the permeation base is a nonwoven fabric made of regenerated cellulose fibers.   The scaffold member for a liquid medium according to any one of claims 1 to 3, wherein the permeable membrane part is a semipermeable membrane of a regenerated cellulose film.   The scaffold member for a liquid medium according to any one of claims 1 to 4, wherein a plurality of the through-hole portions in the compartment culture portion are formed.   The scaffold member for a liquid medium according to any one of claims 1 to 5, wherein a thickness of the compartment culture part is at least 0.4 mm or more.   The scaffold member for a liquid medium according to any one of claims 1 to 6, wherein the compartment culture part is a resin member.   The scaffold member for a liquid medium according to any one of claims 1 to 7, wherein an adhesive is applied to the back side of the compartment culture part and is in close contact with the permeable membrane part.

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