WO2022249400A1 - Soil microorganism observation device - Google Patents
Soil microorganism observation device Download PDFInfo
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- WO2022249400A1 WO2022249400A1 PCT/JP2021/020233 JP2021020233W WO2022249400A1 WO 2022249400 A1 WO2022249400 A1 WO 2022249400A1 JP 2021020233 W JP2021020233 W JP 2021020233W WO 2022249400 A1 WO2022249400 A1 WO 2022249400A1
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- nutrient solution
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- 239000002689 soil Substances 0.000 title claims abstract description 53
- 244000005700 microbiome Species 0.000 title claims abstract description 49
- 235000015097 nutrients Nutrition 0.000 claims abstract description 91
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 238000002955 isolation Methods 0.000 claims description 52
- 239000011521 glass Substances 0.000 claims description 12
- 238000007599 discharging Methods 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract description 6
- 230000003993 interaction Effects 0.000 description 9
- 238000013459 approach Methods 0.000 description 5
- 239000000470 constituent Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005459 micromachining Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Abstract
This soil microorganism observation device has: a chamber part for culturing soil microorganisms; a nutrient liquid flow channel part through which a nutrient liquid flows; a separation part that communicates the chamber part and the nutrient liquid flow channel part; an optically transparent culturing plate having an air flow channel that allows air to flow in and out of the separation part; and an air pressure control unit that forms air bubbles by feeding air through the air flow channel into a fluid inside of the separation part, and that, by controlling the air pressure, changes the size of the air bubbles to change the effective flow channel area of the separation part.
Description
本発明は、土壌微生物のコロニー（細胞集団）を観察するための土壌微生物観察デバイスに関する。
The present invention relates to a soil microorganism observation device for observing colonies (cell populations) of soil microorganisms.
土壌は不透明であるため、直接的な観察が難しく、その生態系についての研究は未解明の部分が多い。
Because the soil is opaque, it is difficult to directly observe it, and many aspects of its ecosystem remain unexplained.
土壌研究のアプローチのひとつとして、マイクロ流体デバイス的アプローチが知られている。マイクロ流体デバイス的アプローチは、土壌の物理的性質などを再現したマイクロデバイスを構築し、その中における微生物を観察することで、土壌エコシステムを解明するアプローチである。
A microfluidic device approach is known as one of the approaches to soil research. The microfluidic device approach is an approach to elucidate the soil ecosystem by constructing a microdevice that reproduces the physical properties of soil and observing microorganisms in it.
非特許文献１と非特許文献２は、マイクロ流体デバイス的アプローチに使用される観察デバイスを開示している。非特許文献１は、脂質膜を利用した流路を有する培養プレートを用いた観察デバイスを開示している。非特許文献２は、網目状流路を利用した流路を有する培養プレートを用いた観察デバイスを開示している。
Non-Patent Document 1 and Non-Patent Document 2 disclose an observation device used in a microfluidic device approach. Non-Patent Document 1 discloses an observation device using a culture plate having a channel using a lipid membrane. Non-Patent Document 2 discloses an observation device using a culture plate having channels using mesh-like channels.
本発明の目的は、土壌微生物を培養するチャンバ部と栄養液を流す栄養液流路部との間の隔離度を制御しながら、土壌微生物のコロニーを観察可能にする土壌微生物観察デバイスを提供することである。
SUMMARY OF THE INVENTION An object of the present invention is to provide a soil microorganism observation device that enables observation of colonies of soil microorganisms while controlling the degree of isolation between a chamber portion for culturing soil microorganisms and a nutrient solution channel portion for flowing nutrient solution. That is.
実施形態に係る土壌微生物観察デバイスは、土壌微生物を培養するチャンバ部と、栄養液を流す栄養液流路部と、前記チャンバ部と前記栄養液流路部を連通する隔離部と、前記隔離部に空気を出入りさせる空気流路とを有する光学的に透明な培養プレートを備える。土壌微生物観察デバイスはさらに、前記空気流路を介して前記隔離部内の液体中に空気を送り込むことで気泡を形成するとともに、前記空気の圧力を制御することにより、前記気泡の大きさを変化させて前記隔離部の実効流路面積を変化させる空気圧制御部を備える。
A soil microorganism observation device according to an embodiment includes a chamber portion for culturing soil microorganisms, a nutrient fluid flow path portion for flowing a nutrient fluid, an isolation portion for communicating between the chamber portion and the nutrient fluid flow path portion, and the isolation portion. and an optically transparent culture plate with air channels for letting air in and out of the chamber. The device for observing soil microorganisms further forms air bubbles by sending air into the liquid in the isolation section through the air flow path, and changes the size of the air bubbles by controlling the pressure of the air. and an air pressure control section for changing the effective flow area of the isolation section.
土壌微生物を培養するチャンバ部と栄養液を流す栄養液流路部との間の隔離度を制御しながら、土壌微生物のコロニーを観察可能にする土壌微生物観察デバイスが提供される。
A soil microorganism observation device is provided that enables observation of colonies of soil microorganisms while controlling the degree of isolation between a chamber portion for culturing soil microorganisms and a nutrient solution channel portion for flowing nutrient solution.
以下、図１ないし図５を参照して、実施形態に係る土壌微生物観察デバイス４０について説明する。
A soil microorganism observation device 40 according to an embodiment will be described below with reference to FIGS.
図１は、土壌微生物観察デバイス４０を模式的に示す上面図である。図１に示すように、土壌微生物観察デバイス４０は、光学的に透明な培養プレート１０を有する。
FIG. 1 is a top view schematically showing the soil microorganism observation device 40. FIG. As shown in FIG. 1, the soil microorganism observation device 40 has an optically transparent culture plate 10. As shown in FIG.
培養プレート１０は、土壌微生物を培養する４つのチャンバ部２１と、栄養液を流す栄養液流路部２２と、チャンバ部２１のそれぞれと栄養液流路部２２を連通する４つの隔離部２３と、隔離部２３のそれぞれに空気を出入りさせる４つの空気流路２４とを有する。
The culture plate 10 includes four chamber portions 21 for culturing soil microorganisms, a nutrient fluid channel portion 22 for flowing a nutrient fluid, and four isolation portions 23 for communicating the chamber portions 21 with the nutrient fluid channel portion 22 respectively. , and four air passages 24 for allowing air to flow in and out of each of the isolation portions 23 .
培養プレート１０はまた、栄養液流路部２２に栄養液を供給する栄養液供給口２５と、栄養液流路部２２から栄養液を排出する栄養液排出口２６と、土壌微生物を含む培養液６１をチャンバ部２１のそれぞれに流し込むための４つの培養液設置部２７とを有する。
The culture plate 10 also includes a nutrient supply port 25 for supplying the nutrient solution to the nutrient solution channel portion 22, a nutrient solution discharge port 26 for discharging the nutrient solution from the nutrient solution channel portion 22, and a culture solution containing soil microorganisms. 61 into each of the chamber sections 21.
図２は、図１の土壌微生物観察デバイス４０のＡ－Ａ断面図である。図２は、便宜上、チャンバ部２１と培養液設置部２７は培養液６１で満たされ、栄養液流路部２２は栄養液６２で満たされ、隔離部２３は培養液６１と栄養液６２の混合液で満たされものとして描かれている。
FIG. 2 is an AA cross-sectional view of the soil microorganism observation device 40 of FIG. 2, for the sake of convenience, the chamber part 21 and the culture solution setting part 27 are filled with the culture solution 61, the nutrient solution channel part 22 is filled with the nutrient solution 62, and the isolation part 23 is the mixture of the culture solution 61 and the nutrient solution 62. It is depicted as being filled with liquid.
図１と図２から分かるように、チャンバ部２１は円柱形状の空間であり、栄養液流路部２２は、上側から視て、直方体の両端面が円弧状の曲面に丸められた略直方体状の空間である。栄養液供給口２５は、栄養液流路部２２の一方の端部から上方に延びており、培養プレート１０の上面に開口している。栄養液排出口２６は、栄養液流路部２２の他方の端部から上方に延びており、培養プレート１０の上面に開口している。
As can be seen from FIGS. 1 and 2, the chamber part 21 is a cylindrical space, and the nutrient solution channel part 22 has a substantially rectangular parallelepiped shape in which both end faces of the rectangular parallelepiped are rounded into arcuate curved surfaces when viewed from above. is the space of The nutrient solution supply port 25 extends upward from one end of the nutrient solution channel portion 22 and opens to the upper surface of the culture plate 10 . The nutrient solution outlet 26 extends upward from the other end of the nutrient solution channel portion 22 and opens to the upper surface of the culture plate 10 .
隔離部２３は、チャンバ部２１に比べて、幅寸法も高さ寸法も小さい空間である。空気流路２４は、チャンバ部２１に対する連絡部と栄養液流路部２２に対する連絡部と間における隔離部２３のほぼ中央から上方に延びており、培養プレート１０の上面に開口している。培養液設置部２７は、隔離部２３の反対方向にチャンバ部２１から延びており、そのチャンバ部２１とは反対側の端部において、上方に延びており、培養プレート１０の上面に開口している。培養液設置部２７は、高さ寸法はチャンバ部２１と同じであるが、幅寸法は隔離部２３よりもさらに小さい。
The isolation section 23 is a space that is smaller in width and height than the chamber section 21 . The air channel 24 extends upward from approximately the center of the isolation part 23 between the communicating part for the chamber part 21 and the communicating part for the nutrient solution channel part 22 and opens to the upper surface of the culture plate 10 . The culture solution setting portion 27 extends from the chamber portion 21 in the direction opposite to the isolation portion 23, extends upward at the end portion opposite to the chamber portion 21, and opens to the upper surface of the culture plate 10. there is The culture medium setting portion 27 has the same height dimension as the chamber portion 21 , but is smaller in width dimension than the isolation portion 23 .
図２に示すように、培養プレート１０は、加工された上部ガラス板１１の下面に、平板状の下部ガラス板１２を貼り合わせて作製される。上部ガラス板１１には、チャンバ部２１と栄養液流路部２２と隔離部２３と培養液設置部２７に対応する溝が下面側に形成される。さらに、上部ガラス板１１には、空気流路２４と栄養液供給口２５と栄養液排出口２６に対応する孔と、培養液設置部２７のチャンバ部２１とは反対側の端部に連通する孔が形成される。
As shown in FIG. 2, the culture plate 10 is produced by bonding a flat lower glass plate 12 to the lower surface of the processed upper glass plate 11 . The upper glass plate 11 has grooves corresponding to the chamber portion 21 , the nutrient solution flow path portion 22 , the separation portion 23 and the culture solution setting portion 27 formed on the lower surface side. Furthermore, in the upper glass plate 11, holes corresponding to the air flow path 24, the nutrient solution supply port 25, and the nutrient solution outlet 26 are communicated with the end portion of the culture solution setting portion 27 opposite to the chamber portion 21. A hole is formed.
上部ガラス板１１の溝や孔は、マイクロ加工技術を利用して形成される。溝や孔は、マイクロ加工により形成されるが、それらのサイズは問わない。たとえば、栄養液流路部２２は、流路幅が２０μｍ程度である。
The grooves and holes of the upper glass plate 11 are formed using microfabrication technology. The grooves and holes are formed by micromachining, but their size does not matter. For example, the nutrient solution channel portion 22 has a channel width of about 20 μm.
必要な溝や孔を形成した上部ガラス板１１の下面に、平板状の下部ガラス板１２が貼り合わせることによって、培養プレート１０が完成する。
The culture plate 10 is completed by attaching the flat lower glass plate 12 to the lower surface of the upper glass plate 11 in which necessary grooves and holes are formed.
図１と図２に示すように、土壌微生物観察デバイス４０はまた、隔離部２３に空気を供給するとともに、空気の圧力を制御する空気圧制御部４１を備える。空気圧制御部４１は、たとえば、ポンプやコンプレッサーで構成される。空気圧制御部４１は、空気チューブ４２によって、空気流路２４に流体的に接続されている。
As shown in FIGS. 1 and 2, the soil microorganism observation device 40 also includes an air pressure control section 41 that supplies air to the isolation section 23 and controls the pressure of the air. The air pressure control unit 41 is composed of, for example, a pump and a compressor. The air pressure controller 41 is fluidly connected to the air flow path 24 by an air tube 42 .
空気圧制御部４１は、空気チューブ４２と空気流路２４を介して隔離部２３内の液体（培養液６１と栄養液６２の混合液）中に空気を送り込むことで気泡（空気層）を形成する。空気圧制御部４１はまた、空気の圧力を制御することにより、気泡の大きさを変化させて隔離部２３の実効流路面積を変化させることができる。ここで、隔離部２３の実効流路面積とは、隔離部２３の壁面と気泡の間の面積を意味する。これにより、空気圧制御部４１は、チャンバ部２１と栄養液流路部２２の間の隔離度を調整することができる。
The air pressure control unit 41 forms air bubbles (air layer) by feeding air into the liquid (mixture of the culture solution 61 and the nutrient solution 62) in the isolation unit 23 through the air tube 42 and the air flow path 24. . The air pressure control unit 41 can also change the effective flow area of the isolation unit 23 by changing the size of the air bubbles by controlling the air pressure. Here, the effective channel area of the isolation portion 23 means the area between the wall surface of the isolation portion 23 and the air bubbles. Thereby, the air pressure control section 41 can adjust the degree of isolation between the chamber section 21 and the nutrient solution channel section 22 .
図３は、図２の培養プレートのＢ－Ｂ断面図であり、隔離部２３内の液体（培養液６１と栄養液６２の混合液）中に気泡が形成された様子を示している。図３の最も上側の隔離部２３には気泡が形成されていないが、図３の上から２番目の隔離部２３内の液体中には気泡７１が形成され、図３の上から３番目の隔離部２３内の液体中には気泡７２が形成され、図３の最も下側の隔離部２３内の液体中には気泡７３が形成されている。
FIG. 3 is a BB cross-sectional view of the culture plate in FIG. 2, showing how bubbles are formed in the liquid (mixed liquid of the culture solution 61 and the nutrient solution 62) in the isolation part 23. FIG. Although no bubbles are formed in the uppermost isolation portion 23 in FIG. 3, bubbles 71 are formed in the liquid in the second isolation portion 23 from the top in FIG. Air bubbles 72 are formed in the liquid in the isolation part 23, and air bubbles 73 are formed in the liquid in the lowermost isolation part 23 in FIG.
気泡が形成されていない図３の最も上側の隔離部２３の実効流路面積は最大である。この場合、チャンバ部２１と栄養液流路部２２の間の隔離度は０％である。
The uppermost isolation part 23 in FIG. 3, in which bubbles are not formed, has the largest effective channel area. In this case, the degree of isolation between the chamber portion 21 and the nutrient solution channel portion 22 is 0%.
気泡７２の大きさは気泡７１の大きさよりも大きい。したがって、図３の上から３番目の隔離部２３の実効流路面積は、図３の上から２番目の隔離部２３の実効流路面積よりも小さい。すなわち、図３の上から３番目の隔離部２３の隔離度は、図３の上から２番目の隔離部２３の隔離度よりも高い。これにより、図３の上から２番目のチャンバ部２１内の培養液と比較して、図３の上から３番目のチャンバ部２１内の培養液の方が、栄養液流路部２２内の栄養液との間のインタラクションが小さく制御される。
The size of the bubble 72 is larger than the size of the bubble 71. Therefore, the effective channel area of the third separating portion 23 from the top in FIG. 3 is smaller than the effective channel area of the second separating portion 23 from the top in FIG. That is, the isolation degree of the third isolation portion 23 from the top in FIG. 3 is higher than the isolation degree of the second isolation portion 23 from the top in FIG. As a result, the culture solution in the third chamber portion 21 from the top in FIG. The interaction with the nutrient solution is small and controlled.
気泡７３の大きさは気泡７２の大きさよりも大きく、さらに、気泡７３は隔離部２３の全体の空間を占有している。このため、図３の最も下側の隔離部２３の実効流路面積はゼロである。この場合、チャンバ部２１と栄養液流路部２２の間の隔離度は１００％である。これにより、図３の最も下側のチャンバ部２１内の培養液と栄養液流路部２２内の栄養液との流通はなく、両者間のインタラクションがゼロに制御される。
The size of the bubble 73 is larger than the size of the bubble 72, and the bubble 73 occupies the entire space of the isolation part 23. Therefore, the effective flow area of the lowermost isolation portion 23 in FIG. 3 is zero. In this case, the degree of isolation between the chamber portion 21 and the nutrient solution channel portion 22 is 100%. As a result, there is no circulation between the culture solution in the lowermost chamber portion 21 and the nutrient solution in the nutrient solution channel portion 22 in FIG. 3, and the interaction between them is controlled to be zero.
図１と図４に示すように、土壌微生物観察デバイス４０はさらに、栄養液供給口２５を介して栄養液流路部２２に栄養液を供給する栄養液供給部４５を備える。栄養液供給部４５は、供給チューブ４６によって、栄養液供給口２５に流体的に接続されている。たとえば、栄養液供給部４５は、シリンジで構成される。また、たとえば、栄養液供給部４５は、栄養液を常に送り出す。
As shown in FIGS. 1 and 4 , the soil microorganism observation device 40 further includes a nutrient solution supply section 45 that supplies nutrient solution to the nutrient solution channel portion 22 via the nutrient solution supply port 25 . The nutrient solution supply part 45 is fluidly connected to the nutrient solution supply port 25 by a supply tube 46 . For example, the nutrient solution supply part 45 is configured by a syringe. Also, for example, the nutrient solution supply unit 45 always delivers the nutrient solution.
栄養液供給部４５から供給される栄養液は栄養液流路部２２内を栄養液排出口２６に向かって流れる。図５に示すように、栄養液排出口２６には排出チューブ４７が流体的に接続されており、余分な液体は排出チューブ４７から排出される。
The nutrient solution supplied from the nutrient solution supply portion 45 flows through the nutrient solution flow path portion 22 toward the nutrient solution discharge port 26 . As shown in FIG. 5, a drain tube 47 is fluidly connected to the nutrient solution outlet 26 and excess liquid is drained from the drain tube 47 .
以下、土壌微生物観察デバイス４０を用いた観察処理について説明する。
Observation processing using the soil microorganism observation device 40 will be described below.
まず、準備段階として、培養液設置部２７から土壌微生物を含む培養液を流し込む。ここでは、流し込む培養液の量は１０μＬである。培養液は、一例では、１０μＬの培養液に含まれる土壌微生物が１個体程度になるように、土壌を水で希釈した溶液である。別の例では、培養液は、１０μＬの培養液に含まれる土壌微生物が１個体程度になるように、土壌から分離した微生物を水に混ぜた溶液である。
First, as a preparatory stage, a culture solution containing soil microorganisms is poured from the culture solution installing portion 27 . Here, the amount of the culture medium to be poured is 10 μL. The culture solution is, for example, a solution obtained by diluting soil with water so that 10 μL of the culture solution contains about one individual soil microorganism. In another example, the culture solution is a solution in which microorganisms separated from soil are mixed with water so that 10 μL of the culture solution contains about 1 individual soil microorganism.
この準備段階では、１つのチャンバ部２１に連通する培養液設置部２７以外の培養液設置部２７をセロハンテープなどで塞ぎ、かつ栄養液流路部２２の圧力を低めに設定し、培養液設置部２７に培養液を流し込みながらチャンバ部２１に存在している空気を押し出す。これにより、チャンバ部２１と培養液設置部２７が培養液で満たされる。隔離部２３は細く形成されているため、多くの培養液が栄養液流路部２２に流れ出ることが防がれる。培養液の流し込みの終了後は、図２に示すように、培養液設置部２７の開口は蓋２９で塞がれる。
In this preparatory stage, the culture solution setting portions 27 other than the culture solution setting portion 27 communicating with one chamber portion 21 are closed with cellophane tape or the like, and the pressure of the nutrient solution flow path portion 22 is set to be low, and the culture solution is set. Air existing in the chamber part 21 is pushed out while the culture solution is poured into the part 27 . As a result, the chamber part 21 and the culture solution installing part 27 are filled with the culture solution. Since the separating portion 23 is formed thin, a large amount of the culture solution is prevented from flowing out to the nutrient solution channel portion 22 . After the pouring of the culture solution is completed, the opening of the culture solution installing portion 27 is closed with the lid 29 as shown in FIG.
次に、空気圧制御部４１により、隔離部２３内の培養液中に空気を送り込むことで気泡（空気層）を形成する。さらに、空気圧制御部４１により、送り込む空気の圧力を制御することにより、気泡の大きさを制御する。これにより、隔離部２３の実効流路面積を変化させ、栄養液流路部２２に対するチャンバ部２１の隔離度を調整する。栄養液流路部２２に対するチャンバ部２１の隔離度は、一定に保ってよいし、時間の経過とともに変更してもよい。
Next, the air pressure control unit 41 sends air into the culture solution in the isolation unit 23 to form air bubbles (air layers). Further, the size of the air bubbles is controlled by controlling the pressure of the air to be sent by the air pressure control section 41 . Thereby, the effective channel area of the isolation part 23 is changed, and the degree of isolation of the chamber part 21 from the nutrient solution channel part 22 is adjusted. The degree of isolation of the chamber section 21 from the nutrient solution flow path section 22 may be kept constant or may be changed over time.
続いて、栄養液供給部４５により、栄養液流路部２２に栄養液を供給する。栄養液は、土壌微生物を育成するための栄養素を含む液体栄養剤であり、たとえば、ＬＢ培地である。
Subsequently, the nutrient solution supply unit 45 supplies the nutrient solution to the nutrient solution channel unit 22 . A nutrient solution is a liquid nutrient containing nutrients for growing soil microorganisms, and is, for example, an LB medium.
観察対象のチャンバ部２１は、たとえば図２に示すように、カメラ５０によって光学的に観察される。
The chamber part 21 to be observed is optically observed by a camera 50, for example, as shown in FIG.
ここで、準備段階における培養液の流し込みは、観察目的に応じて、任意の個数のチャンバ部２１に対しておこなってよい。たとえば、１つのチャンバ部２１内の土壌微生物のコロニーと栄養液との間のインタラクションの観察が目的であれば、ただ１つのチャンバ部２１だけに培養液を流し込めばよい。あるいは、２つのチャンバ部２１内の土壌微生物のコロニーの間のインタラクションの観察が目的であれば、２つのチャンバ部２１に培養液を流し込めばよい。同様の考えから、３つまたは４つのチャンバ部２１内の土壌微生物のコロニーの間のインタラクションの観察が目的であれば、それぞれ、３つまたは４つのチャンバ部２１に培養液を流し込めばよい。
Here, the pouring of the culture medium in the preparation stage may be performed into any number of chamber parts 21 according to the purpose of observation. For example, if the purpose is to observe the interaction between the colony of soil microorganisms and the nutrient solution in one chamber portion 21 , the culture solution may be poured into only one chamber portion 21 . Alternatively, if the purpose is to observe the interaction between the colonies of soil microorganisms in the two chambers 21 , the two chambers 21 may be filled with the culture solution. Based on the same idea, if the purpose is to observe the interaction between the colonies of soil microorganisms in the three or four chambers 21, the three or four chambers 21 may be filled with the culture solution, respectively.
そのほか、４つのチャンバ部２１に培養液を流し込んだ状態においても、上記の観察をおこうことも可能である。たとえば、観察対象の１つのチャンバ部２１に対応する隔離部２３を除く他の隔離部２３中の気泡を大きくして、観察対象以外のチャンバ部２１と栄養液流路部２２の間の隔離度を１００％にすることにより、１つのチャンバ部２１内の土壌微生物のコロニーと栄養液との間のインタラクションを観察することが可能である。あるいは、観察対象の２つまたは３つのチャンバ部２１に対応する隔離部２３を除く他の隔離部２３中の気泡を大きくして、観察対象以外のチャンバ部２１と栄養液流路部２２の間の隔離度を１００％にすることにより、２つまたは３つのチャンバ部２１内の土壌微生物のコロニーの間のインタラクションを観察することが可能である。また、４つのチャンバ部２１のすべてが観察対象であれば、４つのチャンバ部２１と栄養液流路部２２の間の隔離度を制御すればよい。
In addition, the above observation can be made even when the culture solution is poured into the four chambers 21 . For example, by increasing the size of air bubbles in the isolation portions 23 other than the isolation portion 23 corresponding to one chamber portion 21 to be observed, the degree of isolation between the chamber portions 21 other than the observation object and the nutrient solution flow path portion 22 is 100%, it is possible to observe the interaction between the colonies of soil microorganisms and the nutrient solution in one chamber portion 21 . Alternatively, by enlarging the air bubbles in the isolation portions 23 other than the isolation portions 23 corresponding to the two or three chamber portions 21 to be observed, the air bubbles are 100%, it is possible to observe the interaction between the colonies of soil microorganisms in two or three chambers 21 . Moreover, if all the four chambers 21 are to be observed, the degree of isolation between the four chambers 21 and the nutrient fluid channel 22 may be controlled.
このように、実施形態の土壌微生物観察デバイス４０によれば、土壌微生物を培養するチャンバ部２１と栄養液を流す栄養液流路部２２との間の隔離度を制御しながら、土壌微生物のコロニーを観察することが可能となる。
As described above, according to the soil microorganism observation device 40 of the embodiment, while controlling the degree of isolation between the chamber portion 21 for cultivating soil microorganisms and the nutrient fluid flow channel portion 22 for flowing the nutrient fluid, colonies of soil microorganisms can be detected. can be observed.
また、複数のチャンバ部２１のそれぞれと栄養液流路部２２との間の隔離度を制御しながら、複数のチャンバ部２１内の土壌微生物のコロニー間のインタラクションを観察することが可能となる。
In addition, while controlling the degree of isolation between each of the plurality of chambers 21 and the nutrient solution flow path 22, it is possible to observe the interaction between colonies of soil microorganisms in the plurality of chambers 21.
実施形態では、培養プレート１０のチャンバ部２１と隔離部２３と空気流路２４と培養液設置部２７の個数が４つの例を説明したが、これら要素の個数は、これに限定されるものでなく、任意に変更されてよい。土壌微生物のコロニーと栄養液の間のインタラクションの観察する目的に対しては、培養プレート１０のチャンバ部２１と隔離部２３と空気流路２４と培養液設置部２７の個数は１つであってもよい。
In the embodiment, an example in which the number of chamber portions 21, isolation portions 23, air channels 24, and culture medium setting portions 27 of the culture plate 10 is four has been described, but the number of these elements is limited to four. can be changed arbitrarily. For the purpose of observing the interaction between the colony of soil microorganisms and the nutrient solution, the number of the chamber portion 21, isolation portion 23, air flow path 24, and culture solution setting portion 27 of the culture plate 10 is one. good too.
なお、本発明は、上記実施形態に限定されるものではなく、実施段階ではその要旨を逸脱しない範囲で種々に変形することが可能である。また、各実施形態は適宜組み合わせて実施してもよく、その場合組み合わせた効果が得られる。更に、上記実施形態には種々の発明が含まれており、開示される複数の構成要件から選択された組み合わせにより種々の発明が抽出され得る。例えば、実施形態に示される全構成要件からいくつかの構成要件が削除されても、課題が解決でき、効果が得られる場合には、この構成要件が削除された構成が発明として抽出され得る。
It should be noted that the present invention is not limited to the above-described embodiments, and can be variously modified in the implementation stage without departing from the gist of the present invention. Further, each embodiment may be implemented in combination as appropriate, in which case the combined effect can be obtained. Furthermore, various inventions are included in the above embodiments, and various inventions can be extracted by combinations selected from a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiments, if the problem can be solved and effects can be obtained, the configuration with the constituent elements deleted can be extracted as an invention.
１０…培養プレート
１１…上部ガラス板
１２…下部ガラス板
２１…チャンバ部
２２…栄養液流路部
２３…隔離部
２４…空気流路
２５…栄養液供給口
２６…栄養液排出口
２７…培養液設置部
２９…蓋
４０…土壌微生物観察デバイス
４１…空気圧制御部
４２…空気チューブ
４５…栄養液供給部
４６…供給チューブ
４７…排出チューブ
５０…カメラ
６１…培養液
６２…栄養液
７１，７２，７３…気泡
DESCRIPTION OFSYMBOLS 10... Culture plate 11... Upper glass plate 12... Lower glass plate 21... Chamber part 22... Nutrient solution channel part 23... Separation part 24... Air channel 25... Nutrient solution supply port 26... Nutrient solution outlet 27... Culture solution Installation part 29 Lid 40 Soil microorganism observation device 41 Air pressure control part 42 Air tube 45 Nutrient solution supply part 46 Supply tube 47 Discharge tube 50 Camera 61 Culture solution 62 Nutrient solution 71, 72, 73 … bubbles
１１…上部ガラス板
１２…下部ガラス板
２１…チャンバ部
２２…栄養液流路部
２３…隔離部
２４…空気流路
２５…栄養液供給口
２６…栄養液排出口
２７…培養液設置部
２９…蓋
４０…土壌微生物観察デバイス
４１…空気圧制御部
４２…空気チューブ
４５…栄養液供給部
４６…供給チューブ
４７…排出チューブ
５０…カメラ
６１…培養液
６２…栄養液
７１，７２，７３…気泡
DESCRIPTION OF
Claims (4)
- 土壌微生物を培養するチャンバ部と、
栄養液を流す栄養液流路部と、
前記チャンバ部と前記栄養液流路部を連通する隔離部と、
前記隔離部に空気を出入りさせる空気流路と、
を有する光学的に透明な培養プレートと、
前記空気流路を介して前記隔離部内の液体中に空気を送り込むことで気泡を形成するとともに、前記空気の圧力を制御することにより、前記気泡の大きさを変化させて前記隔離部の実効流路面積を変化させる空気圧制御部と、
を備える土壌微生物観察デバイス。 a chamber for cultivating soil microorganisms;
a nutrient solution channel portion for flowing the nutrient solution;
an isolation part that communicates the chamber part and the nutrient solution channel part;
an air flow path that allows air to flow in and out of the isolation section;
an optically transparent culture plate having
Air is sent into the liquid in the isolation section through the air flow path to form air bubbles, and by controlling the pressure of the air, the size of the air bubbles is changed to change the effective flow in the isolation section. a pneumatic control unit that changes the road area;
A device for observing soil microorganisms. - 前記培養プレートは、前記チャンバ部を複数備えるとともに、前記隔離部を１つの前記チャンバ部ごとに１つずつ備え、
複数の前記隔離部のそれぞれは、複数の前記チャンバ部の対応する１つを前記栄養液流路部に連通する、
請求項１に記載の土壌微生物観察デバイス。 The culture plate includes a plurality of the chambers, and includes one separation unit for each chamber,
each of the plurality of isolation portions communicates a corresponding one of the plurality of chamber portions with the nutrient fluid channel portion;
The soil microorganism observation device according to claim 1. - 前記培養プレートは、
マイクロ加工された第１のガラス板と、
前記第１のガラス板に貼り付けられた平板状の第２のガラス板と、
を有する、
請求項１または２に記載の土壌微生物観察デバイス。 The culture plate is
a micromachined first glass plate;
A flat second glass plate attached to the first glass plate;
having
The soil microorganism observation device according to claim 1 or 2. - 前記培養プレートは、
前記栄養液流路部に栄養液を供給する栄養液供給口と、
前記栄養液流路部から栄養液を排出する栄養液排出口と、
を有し、
前記栄養液供給口を介して前記栄養液流路部に栄養液を供給する栄養液供給部をさらに備える、
請求項１から３までのいずれかひとつに記載の土壌微生物観察デバイス。
The culture plate is
a nutrient solution supply port for supplying the nutrient solution to the nutrient solution channel;
a nutrient solution outlet for discharging the nutrient solution from the nutrient solution channel;
has
further comprising a nutrient solution supply unit that supplies nutrient solution to the nutrient solution flow channel through the nutrient solution supply port;
A device for observing soil microorganisms according to any one of claims 1 to 3.
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