WO2022249400A1 - Dispositif d'observation de micro-organismes du sol - Google Patents

Dispositif d'observation de micro-organismes du sol Download PDF

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Publication number
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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WO
WIPO (PCT)
Prior art keywords
nutrient solution
air
isolation
chamber
channel
Prior art date
Application number
PCT/JP2021/020233
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English (en)
Japanese (ja)
Inventor
真奈美 伊藤
和宏 高谷
Original Assignee
日本電信電話株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日本電信電話株式会社 filed Critical 日本電信電話株式会社
Priority to PCT/JP2021/020233 priority Critical patent/WO2022249400A1/fr
Publication of WO2022249400A1 publication Critical patent/WO2022249400A1/fr

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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
    • 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

Definitions

  • the present invention relates to a soil microorganism observation device for observing colonies (cell populations) of soil microorganisms.
  • 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.
  • 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.
  • 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.
  • a soil microorganism observation device 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.
  • 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.
  • FIG. 1 is a top view schematically showing a soil microorganism observation device according to an embodiment.
  • FIG. 2 is a schematic diagram showing the AA cross section of the device for observing soil microorganisms in FIG.
  • FIG. 3 is a schematic diagram showing a BB section of the culture plate of FIG.
  • FIG. 4 is a schematic diagram showing a CC cross section of the device for observing soil microorganisms in FIG.
  • FIG. 5 is a schematic diagram showing a DD section of the device for observing soil microorganisms in FIG.
  • a soil microorganism observation device 40 according to an embodiment will be described below with reference to FIGS.
  • FIG. 1 is a top view schematically showing the soil microorganism observation device 40.
  • the soil microorganism observation device 40 has an optically transparent culture plate 10. As shown in FIG. 1, the soil microorganism observation device 40 has an optically transparent culture plate 10.
  • 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 .
  • 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.
  • 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.
  • the chamber part 21 is a cylindrical space
  • 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 .
  • 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 .
  • 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.
  • 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.
  • the nutrient solution channel portion 22 has a channel width of about 20 ⁇ m.
  • 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.
  • 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 .
  • 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.
  • 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 .
  • 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.
  • the uppermost isolation part 23 in FIG. 3, in which bubbles are not formed, has the largest effective channel area.
  • the degree of isolation between the chamber portion 21 and the nutrient solution channel portion 22 is 0%.
  • 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.
  • 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.
  • 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 .
  • the nutrient solution supply part 45 is configured by a syringe.
  • the nutrient solution supply unit 45 always delivers the nutrient solution.
  • 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 .
  • a drain tube 47 is fluidly connected to the nutrient solution outlet 26 and excess liquid is drained from the drain tube 47 .
  • a culture solution containing soil microorganisms is poured from the culture solution installing portion 27 .
  • the amount of the culture medium to be poured is 10 ⁇ L.
  • the culture solution is, for example, a solution obtained by diluting soil with water so that 10 ⁇ L of the culture solution contains about one individual soil microorganism.
  • the culture solution is a solution in which microorganisms separated from soil are mixed with water so that 10 ⁇ L of the culture solution contains about 1 individual soil microorganism.
  • 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.
  • 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.
  • a nutrient solution is a liquid nutrient containing nutrients for growing soil microorganisms, and is, for example, an LB medium.
  • the chamber part 21 to be observed is optically observed by a camera 50, for example, as shown in FIG.
  • 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.
  • the culture solution may be poured into only one chamber portion 21 .
  • the two chambers 21 may be filled with the culture solution.
  • the three or four chambers 21 may be filled with the culture solution, respectively.
  • the above observation can be made even when the culture solution is poured into the four chambers 21 .
  • 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 .
  • the air bubbles are 100%, it is possible to observe the interaction between the colonies of soil microorganisms in two or three chambers 21 .
  • the degree of isolation between the four chambers 21 and the nutrient fluid channel 22 may be controlled.
  • 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.
  • 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.
  • 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.
  • 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.

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  • General Engineering & Computer Science (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Dispositif d'observation des micro-organismes du sol comprenant une partie de chambre pour cultiver des micro-organismes du sol ; une partie de canal d'écoulement de liquide nutritif à travers laquelle un liquide nutritif s'écoule ; une partie de séparation reliant la partie de chambre et la partie de canal d'écoulement de liquide nutritif ; un plateau de culture optiquement transparent ayant un canal d'écoulement d'air permettant à l'air d'entrer et de sortir de la partie de séparation ; et une unité de régulation de la pression d'air formant des bulles d'air en introduisant de l'air à travers le canal d'écoulement d'air dans un fluide à l'intérieur de la partie de séparation, et qui, en régulant la pression d'air, modifie la taille des bulles d'air pour modifier la surface effective du canal d'écoulement de la partie de séparation.
PCT/JP2021/020233 2021-05-27 2021-05-27 Dispositif d'observation de micro-organismes du sol WO2022249400A1 (fr)

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PCT/JP2021/020233 WO2022249400A1 (fr) 2021-05-27 2021-05-27 Dispositif d'observation de micro-organismes du sol

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PCT/JP2021/020233 WO2022249400A1 (fr) 2021-05-27 2021-05-27 Dispositif d'observation de micro-organismes du sol

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481077A (zh) * 2020-12-01 2021-03-12 北京理工大学 一种微流控灌流培养装置及其灌流方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481077A (zh) * 2020-12-01 2021-03-12 北京理工大学 一种微流控灌流培养装置及其灌流方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BORER BENEDICT, TECON ROBIN, OR DANI: "Spatial organization of bacterial populations in response to oxygen and carbon counter-gradients in pore networks", NATURE COMMUNICATIONS, vol. 9, no. 1, 1 December 2018 (2018-12-01), XP093008780, DOI: 10.1038/s41467-018-03187-y *
KHOSHMANESH KHASHAYAR, ALMANSOURI ABDULLAH, ALBLOUSHI HAMAD, YI PYSHAR, SOFFE REBECCA, KALANTAR-ZADEH KOUROSH: "A multi-functional bubble-based microfluidic system", SCIENTIFIC REPORTS, vol. 5, no. 1, 22 September 2015 (2015-09-22), XP093008777, DOI: 10.1038/srep09942 *
TÄUBER SARAH, LIERES ERIC, GRÜNBERGER ALEXANDER: "Dynamic Environmental Control in Microfluidic Single‐Cell Cultivations: From Concepts to Applications", SMALL, WILEY, vol. 16, no. 16, 1 April 2020 (2020-04-01), pages 1906670, XP093008776, ISSN: 1613-6810, DOI: 10.1002/smll.201906670 *
VAN STEIJN VOLKERT, KLEIJN CHRIS R., KREUTZER MICHIEL T.: "Predictive model for the size of bubbles and droplets created in microfluidic T-junctions", LAB ON A CHIP, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 10, no. 19, 1 January 2010 (2010-01-01), UK , pages 2513, XP093008779, ISSN: 1473-0197, DOI: 10.1039/c002625e *

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