JP5066537B2 - Microorganism culture equipment - Google Patents

Description

  The present invention relates to an apparatus for generating a gradient of nutrient components or a gradient of growth conditions in a solid medium when microbial culture or dilution plate culture is performed on a solid medium. The present invention relates to a predictable culture apparatus.

  It is no exaggeration to say that applied microbiology has been developed by this pure separation method since Robert Koch developed the gelatin plate culture method in 1881. It has long been said that with this technology, only 1% or less of the microorganisms present in the environment can be separated.

  In recent years, DNA has been isolated directly from the environment without microbial separation and culture, and the microbiota can be analyzed from the genetic level, and this microbial population that cannot be separated has been named VBNC (ViableBut Nonculturable). Microorganisms that have not been known so far have been attracting attention.

  However, the whole picture of these 99% of non-separable microorganisms is still left in mystery. The major cause of this is that there are too many culture factors (dissolved oxygen concentration, carbon dioxide concentration, redox potential, culture temperature, medium component concentration, salt concentration, medium pH, etc.), and the remaining 99% of microorganisms can be separated. In order to do so, a huge amount of time and manpower is expected, and the current situation is that he is hesitant in front of the treasure mountain.

  In recent years, due to advances in genetic engineering, there are many researches that start with genes in the field of applied microbiology, and such research is in a trend that seems to be leading edge. Research that seeks and applies new microbial resources using traditional microbial screening methods is often thought of as time-consuming, labor-intensive, and old-fashioned research.

  However, the inventors believe that it is necessary to review the screening of microorganisms once again in order to dig up new genetic resources. By the way, Horikoshi et al. Showed us the existence of a new microbial world called the “alkali world” by moving only one pH to the alkali side, and gave us a glimpse of a new gene resource. But nevertheless, the whole picture of microorganisms inhabiting the soil remains enveloped in the veil.

  On the other hand, when looking at the separation of microorganisms from the natural world, the culture conditions from Koch to Horikoshi are still “points”, and the inventors have sought to expand from “lines” to “planes” and further to multi-dimensions. The idea to do is never accepted in the past.

  As mentioned above, the pure plate separation method using a solid medium described in the original paper titled `` Methods for Pathogenic Microorganisms '' by Robert Koch in 1881 was the same as the method to date until a small improvement of RJPetri in 1887. It continues to be used as it is.

Now that we have described the history of microbiological research methods, we must take up the “medium selection method, a diffusion method in gelatin useful for microbiological research” published by Robert Koch's disciple Martinus W. Beijerinck in 1889. This method is known as the oxanograph method and is still used today, particularly in antibiotic and vitamin research. The method published by Alexander Fleming in 1929 for the determination of penicillin is exactly the application of this oxanographic method. That is, the concentration gradient generated when the test substance diffuses in the solid medium is quantified by inhibiting the growth of the test bacteria. Several methods for investigating the growth characteristics of known microorganisms and the effects of food preservatives have been proposed using such a substance diffusion phenomenon in a solid medium.
JWTWimpenny et.al.:J.Gen.Microbiol.,130,2921-2926 (1984) (Non-patent Document 1);
JWTWimpennyet.al .; FEMSMicrobiol., 40,263-267 (1987) (non-patent document 2);
PJMcClure, et.al .: Lett. Appl. Microbiol., 9, 95-99 (1989) (non-patent document 3);
ACPeters, et.al.:Binary, 3,147-155 (1991) (non-patent document 4);
K, McGrath, et.al., Int. Biodeter. Biodegrad., 29, 45-52 (1992) (non-patent document 5);
FA Gentile et.al .: LifeSci., 50,287-293 (1992) (non-patent document 6);
LVThomaset.al.:Int.J.Food.Microbiol., 17,289-301 (1993) (Non-patent Document 7);
LVThomaset.al.:Appl.Environ.Microbiol.,62,2006-2012(1996) (non-patent document 8);
N.Rattanasomboonet.al.:Int.J.Food.Sci.Technol.,36,369-376 (2001) (Non-patent document 9);
EZPanagouet.al.:Appl.Environ.Microbiol., 71,392-399 (2005)) (Non-Patent Document 10).

  As an extension of these research methods, there is an attempt to reach a steady state of material diffusion (DE Caldwell et.al.:Can.J.Microbiol.,19,53-58 (1972) (non- Patent Document 11)).

Japanese Patent Laid-Open No. 7-170973 (Patent Document 1) discloses that a sample in which a plurality of types of microorganisms are mixed is processed by specific gravity gradient centrifugation, and the microorganisms are selected according to the specific gravity, or the sample is contained in an electrolytic solution. And a method for selecting microorganisms by installing a filter for selecting microorganisms of a predetermined size in the movement path of the microorganisms.
Furthermore, in Japanese Patent Application Laid-Open No. 2004-329122 (Patent Document 2), cell culture is performed under conditions in which gradients are given to various culture conditions for promoting cell tissue regeneration to form tissues. An apparatus is described.

  In reviewing the proposals for the analysis of microbial growth characteristics of the oxanograph method described so far, it is possible to point out a large fatal defect as a scientific methodology. Before presenting the problem, a specific example of known technology will be shown.

  That is, the operation of applying the test substance into the solid medium can be carried out in various forms, and the filter paper piece impregnated with the specimen substance solution / the solution itself is allowed to stand on the solid medium surface solidified in advance. Alternatively, after solidifying the solution containing the sample substance with agar, etc., overlay or contact the agar layer not containing the sample substance, or embed the test substance in the form of crystals or powder in the agar. Start spreading.

  Although there is a difference in these diffusion initiation forms, these known techniques do not have any chronological and quantitative grasp as to what concentration gradient the target substance diffuses in the solid medium. Moreover, there is no attempt to control this material diffusion. That is, in further detail, since the material diffusion is a physical phenomenon, the present inventors think that the calculation of the diffusion coefficient by theoretical numerical analysis and the prediction of the material diffusion are possible. The technical paper does not describe any logical attempts.

J.W.T.Wimpenny et.al.:J.Gen.Microbiol.,130,2921-2926 (1984) J.W.T.Wimpennyet.al .; FEMSMicrobiol., 40,263-267 (1987) P.J.McClure, et.al .: Lett. Appl. Microbiol., 9, 95-99 (1989) A.C.Peters, et.al.: Binary, 3,147-155 (1991) K, McGrath, et.al., Int. Biodeter. Biodegrad., 29, 45-52 (1992) F.A.Gentile et.al.:Life Sci., 50,287-293 (1992) L.V.Thomaset.al.:Int.J.Food.Microbiol., 17,289-301 (1993) L.V.Thomaset.al.:Appl.Environ.Microbiol.,62,2006-2012 (1996) N.Rattanasomboonet.al.:Int.J.Food.Sci.Tech-nol.,36,369-376 (2001) E.Z.Panagou et.al.:Appl.Environ.Microbiol.,71,392-399 (2005)) (D.E.Caldwell et.al.:Can.J.Microbiol.,19,53-58 (1972)

Japanese Unexamined Patent Publication No. 7-170973 JP 2004-329122 A

  An object of the present invention is to provide a microorganism culturing apparatus in which a plurality of different culture factors can be selected and a gradient of these culture factors can be formed in one incubator.

  Another object of the present invention is to provide a microorganism culture apparatus that can form and control gradients of different culture factors in an incubator such as the same petri dish.

  Still another object of the present invention is to provide a microorganism culturing apparatus capable of generating a gradient of nutrient components or growth conditions in a solid medium.

  In the present invention, a method in which a solution containing a microorganism sample (specimen substance) is solidified with agar or the like and then an agar layer not containing the specimen substance is brought into contact with the solution.

  Next, a plurality of model specimen substances were selected from the microorganism culture factors, and numerical analysis was performed using the diffusion equation as an error function by experimental verification to quantitatively grasp how these substances diffuse into the solid medium.

  Furthermore, from the diffusion coefficient of the obtained model compound, the measured value was compared with the theoretical value, and the aim was to estimate the concentration distribution of the medium component factor in the agar medium. Simultaneously with these numerical analyses, an culturing apparatus as an embodiment is designed, and a method for controlling the diffusion of medium component factors is devised to generate arbitrary culture conditions.

  That is, the culture apparatus of the present invention can fill a part of the side wall of the incubator having a filling chamber filled with a microorganism sample with the first agar containing the first component that affects the cultivation of the microorganism. A first storage part is formed, and the first storage part and the filling chamber of the incubator are partitioned by a removable first partition plate, and the other part of the side wall of the incubator has an influence on the culture of the microorganism. A second container that can be filled with the second agar containing the second component is formed, and the second container and the filling chamber of the incubator are partitioned by a removable second partition plate; This achieves the above object.

  In one embodiment, the 3rd accommodating part which can be filled with the 3rd agar containing the 3rd component which affects the culture | cultivation of this microorganism is formed in the bottom part of the said incubator.

  In one embodiment, the incubator is formed in a box shape, the first accommodating portion is formed on one side wall of the incubator, and the second accommodating portion is disposed on another side wall adjacent to the first accommodating portion. Is formed.

  In one embodiment, the incubator is formed in a substantially cylindrical shape, and the first accommodating portion and the second accommodating portion are formed on the peripheral wall of the incubator.

  In one embodiment, three or more accommodating parts are formed on the side wall of the incubator.

  In one embodiment, the first agar and the second agar are different in at least one of medium component concentration, salt concentration, and medium pH.

  The method for screening microorganisms of the present invention is a method for screening microorganisms contained in a microorganism sample using the culture apparatus, and cultivating the microorganisms in a first container formed on the side wall of the incubator. Filling the first agar containing the first component containing the first component to affect the first agar layer, removing the first partition plate and placing the first agar layer in the filling chamber of the incubator A step of exposing, and a step of filling the second storage portion formed on the side wall of the incubator with a second agar containing a second component affecting the cultivation of the microorganism to form a second agar layer. Removing the second partition plate and exposing the second agar layer to the filling chamber of the incubator, and filling the incubator filling chamber with a fluid solid medium containing a microorganism sample. Solidifying the process, whereby There is achieved.

  In one embodiment, the solid medium does not contain a first component and a second component.

  Incidentally, although the multidimensional culture method disclosed in the present invention continuously changes the culture factor, if the factor achieved in the culture medium is regarded as a non-continuous gradient and is assumed to be 100 stages, the culture vessel developed in three dimensions Will be 1 million steps. If the tolerance for growth factors of microorganisms is considered to be 10 levels corresponding to the factors, one culture container is estimated to be equivalent to 1000 traditional dishes, and an effective comprehensive culture method using this device It will be.

FIG. 1 is a schematic view of a culture apparatus. FIG. 2 is a schematic view showing a state in which the culture apparatus is filled with agar. FIG. 3 is a top view of the two-element gradient square culture apparatus. FIG. 4 is a top view of the three-element gradient round culture apparatus. FIG. 5 is a cross-sectional view of a four-element gradient round deep culture apparatus. FIG. 6 is a top view of the three-element gradient square culture apparatus. FIG. 7 is a top view of the three-element gradient square culture apparatus. FIG. 8 is a top view of the four-element gradient round culture apparatus. FIG. 9 is a top view of the five-element gradient round culture apparatus. FIG. 10 is a top view of the six-element gradient round culture apparatus. FIG. 11 is a top view of the seven-element gradient round culture apparatus. FIG. 12 shows a cross-sectional view of the diffusion measuring device. FIG. 13 shows a concentration distribution in which arginine diffuses in the agar (diffusion distance 3 inches). FIG. 14 shows a concentration distribution in which glycine diffuses in the agar (diffusion distance 3 inches). FIG. 15 shows a concentration distribution in which arginine diffuses in the agar (diffusion distance 5 inches). FIG. 16 shows a concentration distribution in which glycine diffuses in the agar (diffusion distance 5 inches). FIG. 17 shows a concentration distribution in which glycerin diffuses in the agar (diffusion distance 5 inches). FIG. 18 shows the concentration distribution in which glucose diffuses in the agar (diffusion distance 5 inches). FIG. 19 shows how to derive the diffusion equation (formula (1)) and the error function integral formula (formula (2)) used in the analysis of the present invention. FIG. 20 shows the diffusion coefficient D obtained from the concentration distribution of arginine shown in FIGS. 13 and 15 by the equation (2) in FIG. FIG. 21 shows the diffusion coefficient D obtained from equation (2) in FIG. 18 from the glycine concentration distribution shown in FIG. 14 and FIG. FIG. 22 shows the diffusion coefficient D obtained from the glycerin concentration distribution shown in FIG. FIG. 23 shows the diffusion coefficient D obtained from the glucose concentration distribution shown in FIG. 18 by equation (2) in FIG. FIG. 24 shows a comparison between the concentration distribution theoretical value calculated from the arginine diffusion coefficient D obtained from the equation (2) in FIG. 19 and the actual measurement value. FIG. 25 shows a comparison between the theoretical value of the concentration distribution calculated from the diffusion coefficient D of glycine obtained from the equation (2) in FIG. 19 and the actual measurement value. FIG. 26 shows a comparison between the theoretical value of the concentration distribution calculated from the diffusion coefficient D of glycerin obtained from the equation (2) in FIG. 19 and the actual measurement value. FIG. 27 shows a comparison between the theoretical value of the concentration distribution calculated from the glucose diffusion coefficient D obtained from the equation (2) in FIG. 19 and the actual measurement value. FIG. 28 shows the simulation results of the estimated concentration distribution on the 10th day of the diffusion time under various initial concentration C ₀ conditions calculated from the arginine diffusion coefficient D obtained from equation (2) in FIG. FIG. 29 shows a cross-sectional view of a diffusion measuring device with a dialysis function (diffusion distance 5 inches). FIG. 30 shows a concentration distribution pattern in which diffusion was examined with a gradient-formed agar containing 5% glycerin using a diffusion measurement device with a dialysis function (diffusion distance 5 inches). FIG. 31 shows a cross-sectional view of a diffusion measuring device (diffusion distance 5 inches) having a dialysis function and refluxing a gradient forming component solution having a constant concentration instead of gradient forming agar. FIG. 32 shows a diffusion measurement apparatus (diffusion distance: 5 inches) having a dialysis function and refluxing a gradient-forming component solution having a constant concentration instead of gradient-forming agar to perform diffusion with a gradient-forming component solution containing 5% glycerin. It shows the examined density distribution pattern. FIG. 33 is a perspective view of the culture apparatus 1 in which a reflux / dialysis function is added to the three-element gradient round culture apparatus. FIG. 34 compares the diffusion reach distances of the phosphate buffer when the reflector is installed and when it is not installed on the fifth day of diffusion. FIG. 35 is a front view of an incubator for measuring anaerobic bacteria such as lactic acid bacteria. 36 is a cross-sectional view of the incubator shown in FIG. FIG. 37 is a graph showing a pH gradient when Mcllvaine buffer is used. FIG. 38 is a graph showing a pH gradient when Britton-Robinson buffer is used. FIG. 39 is a schematic diagram for explaining the concentration gradient direction of the dissolved gas. FIG. 40 is a schematic diagram for explaining the concentration gradient direction of the dissolved gas. FIG. 41 is a calibration curve of dissolved carbon dioxide gas concentration. FIG. 42 is a perspective view of the culture apparatus. FIG. 43 is a cross-sectional view of the culture apparatus. FIG. 44 is a graph showing a state where a dissolved oxygen concentration gradient is formed. FIG. 45 is a graph showing the formation state of the dissolved oxygen concentration gradient. FIG. 46 is a graph showing a dissolved carbon dioxide concentration gradient in the case of 10% carbon dioxide aeration. FIG. 47 is a graph showing the dissolved carbon dioxide concentration gradient when 20% carbon dioxide gas is passed. FIG. 48 is a schematic diagram of ORP gradient formation using an electrochemical technique. FIG. 49 is a graph showing an ORP odor formed by a combination method of redox agents. FIG. 50 is an explanatory diagram of ORP gradient formation using an electrochemical method.

  The present invention will be described in more detail with reference to the accompanying drawings.

  1 to 3 are conceptual diagrams of a microorganism culture apparatus A according to the present invention.

  This culture apparatus A includes an incubator 1 having a filling chamber 10 filled with a microorganism sample in the center. The incubator 1 is formed in a box shape with an upper opening using a transparent plate such as plastic or glass.

  A first container 11 that can be filled with the first agar containing the first component is formed on one side wall of the incubator 1, and the first container 11 and the filling chamber 10 of the incubator 1 are removed. It is partitioned by a possible first partition plate 14.

  That is, frames 17 having a square cross section are fixed to the four corners of the incubator 1, and the first partition plate 14 is disposed between the pair of frames 17 so as to be slidable upward. Specifically, the frame 17 is provided with a groove for inserting the end portion of the first partition plate 14, or the frame 17 is provided with a locking piece (not shown). The part is attached to these grooves or locking pieces so as to be vertically movable.

  A second container 12 that can be filled with the second agar containing the second component is formed on the side wall of the incubator 1 adjacent to the first container 11, and the second container 12 and the incubator 1 The filling chamber 10 is partitioned by a removable second partition plate 15. When attaching the 2nd partition plate 15 to the incubator 1, it can be comprised similarly to the case of the 1st partition plate 14. FIG.

  The bottom surface of the incubator 1 is formed as a flat surface, and the third container 13 that can be filled with the third agar containing the third component at a high concentration is formed at the bottom of the incubator 1. A partition plate is not necessary for the third accommodating portion 13.

  The first component, the second component, and the third component each affect the culture of microorganisms, and examples thereof include known medium components and medium pH adjusters.

  In particular, a combination of (a) nitrogen source compounds as the first component, (b) carbon source compounds as the second component, and (c) trace salt compounds as the third component is preferable.

  The concentration of the first component in the first agar is preferably about 10 to 1%.

  The concentration of the second component in the second agar is preferably about 10 to 1%.

  The concentration of the third component in the third agar is preferably about 0.01 to 0.001%.

  The microorganism sample filled in the filling chamber 10 of the incubator 1 refers to all the samples containing microorganisms, and typically contains microorganisms, agar, and water (also referred to as a solid medium). This microbial sample usually does not contain the first component, the second component and the third component.

  This microorganism sample is filled in the filling chamber 10 of the incubator 1 in a fluid state.

  The shape, size, and the like of the incubator 1, the filling chamber 10, and the storage units 11 to 13 can be set as appropriate. Moreover, the number of accommodating parts can also be set arbitrarily according to the purpose. A lid is usually attached to the upper end opening of the incubator 1. Specific examples of these will be described later.

  Screening of microorganisms using the culture apparatus A having the above-described configuration can be performed as follows.

  In the state which attached the partition plates 14 and 15 to the 1st and 2nd accommodating parts 11 and 12 of the culture apparatus A of this invention, the fluid 1st agar containing the 1st component in the 1st accommodating part 11 To form a first agar layer 21 (FIG. 2). Similarly, the second agar layer 22 is formed by filling the second storage portion 12 with the fluid second agar containing the second component. Further, the third agar layer 23 is formed by filling the bottom of the filling chamber 10 of the incubator 1 with the fluid third agar containing the third component.

  Although the thickness of the 1st agar layer 21, the 2nd agar layer 22, and the 3rd agar layer 23 can be set suitably, for example, it can be set as the same thickness, respectively.

  After the first agar layer 21 and the second agar layer are solidified, the first and second partition plates 14 and 15 are removed, respectively. The first agar layer 21 and the second agar layer 22 are each exposed to the filling chamber 10 of the incubator 1.

  Next, the filling chamber 10 of the incubator 1 is filled with fluidized agar (solid medium) containing a microorganism sample and solidified.

  Thereafter, the culturing apparatus A is allowed to stand at an appropriate culturing temperature, and the appearance of microbial colonies is awaited in the agar layer 24 filled in the filling chamber 10.

  During this time, the first, second, and third agar layers 21-23 diffuse the first to third components into the agar layer 24 in the filling chamber 10, and the diffusion starts at the central agar layer 24. A concentration gradient of the first to third components depending on the elapsed time is formed. As a result, a colony of microorganisms appears in accordance with the concentration of the medium component diffused from the upstream by “some noodles” or “the method of grabbing the cocoon”.

  The shape of the incubator 1 is not limited to that shown as a conceptual diagram in FIG. 1, and various shapes such as a generally used round shape can be used. Representative shapes are illustrated in FIGS.

  The culture apparatus A shown in FIG. 4 has three accommodating portions 11 to 13 formed adjacent to the inner surface of a bottomed cylindrical incubator 1. That is, four spacers 26 are fixed to the inner surface side of the peripheral wall of the incubator 1, and the partition plate 16 is slidably attached to the inner end of the spacer 26. The partition plate 16 may be formed of a single sheet, or may be formed for each of the different accommodating portions.

  After the first to third agar layers are formed by filling the first to third agar containing the first to third components in the respective accommodating portions 11 to 13, the partition plate 16 is slid upward. As a result, each agar layer is exposed to the filling chamber 10 of the incubator 1. The filling chamber 10 is filled with agar containing a microorganism sample and solidified.

  In addition, the position of each accommodating part 11-13 does not need to adjoin, and you may install in any position in the container 1 depending on the direction of gradient formation. Although not shown in this drawing, a lid 9 is attached to cover the same (the same is true in the drawings of the following embodiments, and the description is omitted).

  The culture apparatus A shown in FIG. 5 is obtained by deepening the round incubator 1 shown in FIG. 4, and a fourth accommodating portion 28 is formed at the bottom, and this portion contains the fourth component. The fourth agar can be filled and solidified.

  In FIG. 5, the 1st-3rd agar can be filled and solidified in the 1st-3rd accommodating parts 11-13. After the agar containing these four kinds of medium components is solidified, the partition plate 16 is removed, and the agar containing other culture components or optional components not included in the agar layer formed in the first to fourth storage portions. Is filled into the filling chamber 10 of the incubator 1.

  In FIG. 5, the storage portions 11 to 13 for filling and solidifying the gradient-formed agar cannot be clearly identified in this drawing, but are not limited to the positions as shown in the plan view of FIG. You may install in any position in the container 1 depending on the direction of formation.

  Three or more accommodating portions may be formed on the side wall of the incubator 1. FIG. 6 is a top view of the culture apparatus A having three accommodating portions.

  In this culturing apparatus A, the incubator 1 is formed in a hexagonal cross section, and its three wall surfaces serve as partition plates 14 to 16, and has a substantially U-shaped cross section outside the incubator 1 corresponding to the partition plates 14 to 16. A wall 30 is provided.

  That is, unlike the structure shown in FIGS. 1-4, the 1st-3rd accommodating parts 11-13 are the examples installed in the position protruded outside from the center filling chamber 10. FIG.

  In addition, in FIG. 6, the accommodating parts 11-13 are not limited to the position shown in this drawing, You may install in any position of the wall surface of the incubator 1 depending on the direction of gradient formation.

  The storage portions 11 to 13 in the figure are filled and solidified with gradient-formed agar. After solidification, the partition plates 14 to 16 are removed, and the filling chamber 10 is filled with other culture components not included in the agar layer in the storage units 11 to 13 or agar containing optional components.

  FIG. 7 is a top view of the square culture apparatus A having three accommodating portions.

  In this culturing apparatus A, the incubator 1 is formed in a hexagonal cross section, and its three wall surfaces serve as partition plates 14 to 16, and has a substantially U-shaped cross section outside the incubator 1 corresponding to the partition plates 14 to 16. A wall 30 is provided.

  That is, unlike the structure shown in FIGS. 1-4, the accommodating parts 11-13 are installed in the position protruded outside from the filling chamber 10 of the incubator 1 like FIG.

  In addition, the 1st-3rd accommodating parts 11-13 are not limited to the position shown to this drawing in the figure, Depending on the direction of gradient formation, it installs in which position of the side surface of the incubator 1. Also good.

  In the figure, the first to third accommodating portions 11 to 13 are filled and solidified with gradient-formed agar. After solidification, the partition plates 14 to 16 are removed, and the filling chamber 10 is filled with other culture components that are not included in the agar layers of the first to third storage units 11 to 13 or any components.

  FIG. 8 is a top view of a substantially round culture apparatus A having four accommodating portions.

  In this culture apparatus A, a partition plate 19 having a substantially U-shaped cross section is slidably attached to the inner surface side of the incubator 1 having an octagonal cross section.

  That is, the first to fourth accommodating portions 11 to 13 and 28 in the figure are filled and solidified with gradient-formed agar. After solidification, the partition plate 19 is removed from the incubator 1, and agar containing other culture components or optional components not included in the agar layers formed in the first to fourth storage portions 11 to 13 and 28, respectively. Fill the filling chamber 10. In addition, the 1st-4th accommodating parts 11-13, 28 in a figure are not limited to the position shown to this drawing, Depending on the direction of desired gradient formation, it installs in which position in a container. May be.

  FIG. 9 is a top view of a substantially round culture apparatus A having five accommodating portions.

  In the figure, the first to fifth accommodating parts are filled with gradient-formed agar and solidified. After solidification, the partition plate 19 is removed and the filling chamber 10 is filled with agar containing other culture components or optional components not included in the agar layer formed in the first to fifth storage units.

  In addition, the 1st-5th accommodating part in a figure is not limited to the position shown in this drawing, You may install in any position in a container depending on the direction of gradient formation.

  FIG. 10 is a top view of the round incubator 1 having six accommodating portions.

  Gradient agar is filled and solidified in the first to sixth storage units. After solidification, the partition plate 19 is removed and the filling chamber 10 is filled with agar containing other culture components or optional components not included in the agar layer formed in the first to sixth storage units.

  In addition, the 1st-6th accommodating part which fills and solidifies gradient formation agar in the figure is not limited to the position shown in this drawing, It installs in which position in a container depending on the direction of gradient formation. Also good.

  FIG. 11 is a top view of the round incubator 1 having seven accommodating portions. In the figure, gradient forming agar is filled and solidified in the first to seventh accommodating portions. After solidification, the partition plate is removed and the filling chamber 10 is filled with agar containing other culture components or optional components not included in the agar layer formed in the first to seventh accommodation portions. In addition, the 1st-7th accommodating part in a figure is not limited to the position shown in this drawing, You may install in any position in a container depending on the direction of gradient formation.

  In the above-described microorganism culture apparatus A of the present invention, the measurement results obtained by examining what concentration gradient the components diffuse from the agar containing the medium components are presented and clearly shown in the prior art. To clarify the whole picture of the concentration distribution of substance diffusion in a solid medium.

  In the incubator 1 illustrated in FIG. 1 to FIG. 11, the substance diffusion is expected to diffuse in a fan shape not only in the linear direction but also at a certain angle from the accommodating portion. The apparatus shown in FIG. 12 was used.

  The measuring device 3 includes a pipe 32 that can be filled with agar, and an insertion rod 34 that is inserted into one end of the pipe 32. A thread groove 36 is formed at one end of the pipe 32, and a part of the insertion rod 34 is screwed into the thread groove 36, so that the insertion rod 34 rotates in the pipe 32 by rotating the insertion rod 34. It is configured to be movable.

  That is, in the pipe 32 having a constant inner diameter, the agar 39 to be diffused and the gradient-formed agar 40 containing a high concentration of medium components are brought into contact with each other to be solidified. The agar layer is fed out by the feeding mechanism 38, and the agar layer is cut out at intervals of 2 mm, and the component concentration of each part is measured. The surface of the agar 40 that was not in contact with the agar 39 was sealed with Saran Wrap (registered trademark) or the like in order to prevent drying during the experiment period.

  Specifically, the tip 32i of the pipe 32 was filled with 1.5% agar containing 1% arginine hydrochloride (neutralized and near pH 5). After a predetermined time at a diffusion temperature of 27.5 ° C., slice the agar layer to a thickness of 2 mm as described above, heat extract with 0.01 N hydrochloric acid, dilute as appropriate, and quantify arginine with an automatic amino acid analyzer. did. In this case, the diffusing agar layer 39 corresponds to the central portion 32j of the pipe 32, and its length is 3 inches.

  FIG. 13 shows the medium components when the gradient-formed agar 40 containing medium components is 0.5 inches, the diffusion agar 39 is 3 inches, the initial concentration is 1% arginine hydrochloride, and the diffusion temperature is 27.5 ° C. Is a graph in which the distance from the tip of the gradient-forming agar containing γ is plotted on the X-axis and the arginine concentration of that portion is plotted on the Y-axis.

  The portion with a distance of about 1.3 cm corresponds to the gradient-forming agar 40 containing medium components. As can be seen from the figure, the diffusion tip of arginine reaches the tip of the agar layer 39 on the tenth day of diffusion. In this measurement, an apparatus using acrylic resin was used. However, since the tip portion 35 is a partition to hold the agar layer, the portion acts as a steel body, and the diffusion direction of arginine is reflected and reversed at the tip portion 35. As seen from the concentration distribution data on the 40th day, the arginine concentration in the apparatus tends to be homogenized.

  In summary, for a diffusion distance of 3 inches, the desired concentration gradient can be maintained up to about 10 days after the start of diffusion.

  FIG. 14 shows the state of diffusion under the same conditions when used in gradient-formed agar containing 1% glycine as a high concentration component. The graph shown in FIG. 14 is the result obtained using the apparatus shown in FIG.

  A 1.5% agar layer containing 1% glycine is filled and solidified at the tip 32i of the pipe 32 in FIG. The agar layer was sliced to a thickness of 2 mm at a diffusion temperature of 27.5 ° C. at a predetermined time, extracted by heating with 0.01 N hydrochloric acid, diluted as appropriate, and glycine was quantified with an automatic amino acid analyzer. In this case, the diffusing agar layer 39 corresponds to 32j in FIG. 12, and its length is 3 inches.

  In the case of microbial culture that is completed in about 10 days, satisfactory results can be obtained with the incubator 1 of this standard. More specifically, immediately after the agar layers 21 to 22 in the first to third storage units 11 to 13 are solidified, a microbial sample is seeded in the filling chamber 10 to perform “some noodles” or “a method of grasping a candy”. In this method, a colony of microorganisms appears corresponding to the concentration of medium components diffused from upstream.

  Alternatively, a method may be employed in which the microorganism sample is waited without being seeded until a desired concentration gradient is formed, and the microorganism sample is seeded after the diffusion time so that a microorganism colony appears. Alternatively, after the seeding of the microorganism sample, the incubator 1 is stored at a low temperature where the microorganism cannot grow until the desired concentration gradient is formed, and then transferred to the optimum growth temperature after the desired concentration gradient is formed. Alternatively, a method of causing colonies of microorganisms to appear may be used.

  As shown in the previous section, the component concentration of the gradient-forming agar containing a high concentration of medium components decreases with the diffusion time, and the driving force for component diffusion significantly decreases with the passage of time. Further, maintaining the concentration gradient of the medium components over a long period of time can be achieved by increasing the diffusion distance.

  The distance of 3 inches used in Example 1 is a length set because a commercially available petri dish generally used for microbial culture has a diameter of 9 cm. Thus, the diffusion distance was extended to 5 inches for examination.

  For the diffusion measurement, the apparatus shown in FIG. 12 is used, and the tip 32i of the pipe 32 in FIG. 12 is filled and solidified with a 1.5% agar layer 40 containing 1% arginine hydrochloride (neutralized and near pH 5). The agar layer was sliced to a thickness of 2 mm at a diffusion time of 27.5 ° C., extracted with heat with 0.01 N hydrochloric acid, diluted as appropriate, and arginine was quantified with an automatic amino acid analyzer.

  In this case, the diffusing agar layer 39 corresponds to the intermediate portion 32j of the pipe 32 in FIG. 12, and its length is 5 inches.

  FIG. 15 shows a case where a gradient-forming agar containing a high-concentration medium component is 0.5 inches, a diffusion agar is 5 inches, an arginine having an initial concentration of 1%, and a diffusion temperature of 27.5 ° C. Is a graph in which the distance from the tip of the gradient-forming agar containing γ is plotted on the X-axis, and the arginine concentration of that portion is plotted on the Y-axis. The portion with a distance of about 1.3 cm corresponds to gradient-formed agar containing a high concentration component.

  From the figure, the diffusion tip reaches about the 15th day, and then the diffusion direction is reflected and reversed by the wall surface. As far as the concentration distribution data on the 40th day is seen, the arginine concentration in the apparatus is the diffusion distance described in Example 1. Similar to 3 inches, it tends to be homogenized.

  FIG. 16, FIG. 17, and FIG. 18 show the diffusion state under the same conditions when used in gradient-forming agar containing 1% glycine, 5% glycerin and 5% glucose as high concentration components, respectively.

  From these results, it can be seen that in the case of microbial culture that is completed in about 15 days, satisfactory results can be obtained with the incubator 1 of this standard.

  FIG. 16 is a graph obtained as follows.

  The apparatus shown in FIG. 12 is used for the diffusion measurement, and the tip 32i of the pipe 32 in FIG. 12 is filled and solidified with a 1.5% agar layer containing 1% glycine (neutralized to have a pH of about 5). The agar layer was sliced to a thickness of 2 mm at a diffusion temperature of 27.5 ° C. at a predetermined time, extracted by heating with 0.01 N hydrochloric acid, diluted as appropriate, and glycine was quantified with an automatic amino acid analyzer. In this case, the diffusing agar layer 39 corresponds to 32j in FIG. 12, and its length is 5 inches.

  FIG. 17 is a graph obtained as follows.

  For the diffusion measurement, the apparatus shown in FIG. 12 is used, and a 1.5% agar layer containing 5% glycerin is filled and solidified at the tip 32i of the pipe 32 in FIG. The agar layer was sliced to a thickness of 2 mm at a diffusion temperature of 27.5 ° C. at a predetermined time, heated and extracted with deionized water, diluted as appropriate, and glycerin was quantified by high performance liquid chromatography. In this case, the diffusing agar layer 39 corresponds to the central portion 32j of the pipe 32 in FIG. 12, and its length is 5 inches.

  For the diffusion measurement, the apparatus shown in FIG. 12 is used, and the tip 32i of the pipe 32 in FIG. 12 is filled and solidified with a 1.5% agar layer containing 5% glucose. The agar layer was sliced to a thickness of 2 mm at a diffusion temperature of 27.5 ° C. at a predetermined time, heated and extracted with deionized water, diluted as appropriate, and glucose was quantified by high performance liquid chromatography. In this case, the diffusing agar layer 39 corresponds to the central portion 32j of the pipe 32 in FIG. 12, and its length is 5 inches.

  From the substance concentration distribution in the solid medium of the four types of compounds shown in the previous section, this substance diffusion can be understood and analyzed as diffusion when initially distributed at a constant concentration over a wide area.

Formula 1 in FIG. 19 (Source: Barlow Physical Chemistry (bottom) (5th edition), p845), D: diffusion coefficient, C ₀ : initial substance concentration, x: diffusion distance (distance from the tip of the gradient-forming agar), y: distance of gradient agar, t: diffusion time. This integration can be performed by calculating the diffusion coefficient D by using the error function integral equation shown in equation (2).

The diffusion coefficient D of the measurement data of FIG. 15 (arginine) described in Example 2 was calculated by the same error function integration equation (2) using the commercially available numerical analysis software Maple. In the case of arginine shown in FIG. 20, the value of the diffusion coefficient D fluctuates as the distance from the gradient-forming agar decreases and as the diffusion time increases, but it has been found that the value converges to a certain value from a certain point. From the measured value of the convergent part, the diffusion coefficient D of arginine under the same conditions is 7.34 × 10 ⁻⁶ ± 2.36 × 10 ⁻⁷ m ^{2 / s (} n = 86, CV = 3. 2%). When used in gradient-formed agar containing 1% glycine, 5% glycerin and 5% glucose as high concentration substances, the diffusion coefficient D was determined by the same calculation method.

The results shown in FIG. 21, FIG. 22, and FIG. 23 are obtained, respectively, and from the convergent portion of the calculated values as with arginine, glycine: D = 1.07 × 10 ⁻⁵ ± 4.76 × 10 ⁻⁷ m ² / s (N = 91, C.V. = 4.4%); Glycerin; D = 9.16 × 10 ⁻⁶ ± 4.32 × 10 ⁻⁷ m ² / s (n = 200, C.V. = 4 .2%); glucose; D = 5.36 × 10 ⁻⁶ ± 1.72 × 10 ⁻⁷ m ² / s (n = 134, C.V. = 3.1%) .

  For the four compounds described in Example 3, using the calculated diffusion coefficients, the component concentrations (theoretical values) at the respective diffusion times and diffusion distances can be calculated by equation (2) in FIG. it can. The result of comparing the theoretical value with the actual measurement value in the case of arginine is shown in FIG.

  From the figure, it was verified that the theoretical value and the measured value were in good agreement until the 15th day of the diffusion time, and that the error function integral formula for calculating the diffusion coefficient was valid. As for the other three substances shown in Example 3, good agreement between the theoretical values and the actually measured values is recognized as shown in FIG. 25 (glycine), FIG. 26 (glycerin), and FIG. 27 (glucose).

  As described in Example 4, it was proved that the error function integral formula for calculating the diffusion coefficient is valid within the diffusion time until the tip of the diffusion material reaches the container wall. The examples described so far show the arginine concentration of gradient-formed agar at 1%. This concentration setting is generally set based on common sense of microbial culture, but it may be desirable to set a different concentration gradient pattern depending on the target experiment, such as targeting undernourished microorganisms. Therefore, as a result of simulating the initial concentration at the 5-fold stage with the error function integral formula for calculating the diffusion coefficient, the result shown in FIG. 28 was obtained.

  Although the diffusion coefficient is expected to change slightly depending on the initial concentration, such a simulation is a guideline necessary for carrying out the present invention.

  As described above, a good agreement between the theoretical value and the actually measured value is recognized within the diffusion time until the tip of the diffusion material reaches the container wall, but the actually measured value becomes higher than the theoretical value thereafter. As described in Example 2, this phenomenon is presumed that the diffusion direction is reflected and reversed by the container wall and turned back. It was estimated that the concentration gradient could be maintained for a longer time if a diffusion passage for the diffusion material was prepared on the container wall. For this purpose, a dialysis membrane was installed instead of a wall, and a system was set up to remove the diffusing substance with water circulating outside the dialysis membrane (called diffusion with dialysis function).

  FIG. 29 shows a conceptual diagram of the measuring apparatus.

  This apparatus has a pipe 42 filled with agar, and a dialysis membrane 44 fixed to the upper end of the pipe 42, and the upper end of the pipe 42 is connected to a cylindrical part 48 formed on the lower surface of the holder 46. It is connected. A dialysis chamber 50 is formed in the cylindrical portion 48. In the figure, 52 is a dialysis water inlet, 54 is a dialysis water outlet, 56 is diffusion agar, and 58 is a gradient agar containing medium components (diffusion substances). The surface of the agar filled in 58 that was not in contact with the diffusion agar 56 was sealed with Saran Wrap (registered trademark).

  As a result of continuously passing about 500 ml of dialysis water per day into the dialysis chamber 50 having a volume of about 1 ml using this apparatus, in the case of glycerin as a diffusion material, as shown in FIG. Diffuse reflection can be significantly suppressed.

  The continuous liquid flow rate is about 500 ml, but is not limited to this, and can be determined by experiments individually and appropriately depending on the type of each diffusing substance or the concentration used.

  In this embodiment, a cellulosic material membrane used for protein desalting used in the biochemical field was used as the dialysis membrane 44. However, the membrane is not limited to this, and any material can be used if the material is a semipermeable membrane. The use of a more efficient nonwoven fabric, hollow fiber membrane, nylon mesh sheet, or the like can be exemplified.

  The diffusion with a dialysis function described in the previous section can significantly suppress the folding of the substance at the tip of diffusion.

  Another problem of the material diffusion of the present invention is that the concentration in the agar near the gradient-formed agar contained as a high concentration component decreases as the diffusion time becomes longer (the diagram described in Example 1). 13-14, FIGS. 15-18 demonstrated in Example 2).

  In other words, the component concentration of the gradient-forming agar containing the high-concentration component decreases with the diffusion time, and the driving force for diffusion of the component decreases remarkably with the passage of time. Since the diffusion driving force depends on the component concentration of gradient-forming agar containing a high-concentration component, the driving force can be maintained by changing the gradient-forming agar to a solution having a constant concentration.

  The present inventors refer to this method as diffusion with a reflux function, but it is also possible to create a concentration gradient together with the dialysis function described in Example 6.

  FIG. 31 shows an apparatus diagram.

  This apparatus has a pipe 42 filled with diffusion agar 56 and a dialysis membrane 44 fixed to the upper end of the pipe 42, and the upper end of the pipe 42 is connected to a cylindrical part 48 formed on the lower surface of the holder 46. It is connected. A dialysis chamber 50 is formed in the cylindrical portion 48. A medium component inlet 75 and a medium component outlet 76 are formed at the lower end of the pipe 42, respectively. In the figure, 52 is a dialysis water inlet and 54 is a dialysis water outlet.

  The space of the gradient agar 56 occupies a volume of about 1 ml with a pipe 42 having an inner diameter of 1 cm. A 20-fold volume of the diffusing material solution is refluxed in this portion (liquid feeding speed of about 50 ml per day).

  An example of a diffusion experiment using glycerin is shown in FIG. The number of days that the diffusion tip reaches the tip of the container takes about 15 days when reflux is not performed (see FIG. 16), but it is shortened to 10 days and is effective for maintaining a high concentration gradient. It is. The liquid feeding speed is about 50 ml, but is not limited to this, and can be determined by experiments individually and appropriately depending on the type of each diffusing substance or the concentration employed.

  From the examples described so far, the diffusion coefficient was obtained from the quantitative grasp of the diffusion of the four model compounds in the agar, and the agreement with the actual measurement value was verified.

  From these results, the present inventors completed a device having a dialysis function and a reflux function. Here, the present inventors propose a third method for controlling material diffusion. The diffusion phenomenon in the agar was vaguely presumed that the substance moved in the agar so as to soak up, but I learned that it would be easier to understand if the wave would propagate. Invented a method to control diffusion.

  Using a diffusion measuring apparatus shown in FIG. 12, a disk (not shown) having a thickness of 2 mm is installed as a reflector between the agar 39 to be diffused and the gradient forming agar 40 containing a high concentration component, and a constant aperture is provided at the center of the disk. The state of diffusion was investigated by opening an opening. The diffusion material used at this time is 0.2 M phosphate buffer (pH 6.8), and data for a diffusion time of 5 days are shown in FIG.

  Compared with the case where there is no reflector, it can be seen that diffusion is suppressed depending on the opening diameter, and the diffusion tip does not reach the diffusion position for 12 to 48 hours.

  FIG. 33 is a perspective view of a culture apparatus A in which a round incubator having three accommodating portions is provided with a reflux / dialysis function.

  This culture apparatus A is arranged between a cylindrical body 62 having a bottom, a ring-shaped semipermeable membrane 64 arranged in the cylindrical body 62, and a peripheral wall of the cylindrical body 62 and the semipermeable membrane 64. A plurality of spacers 66.

  The cylindrical body 62, the semipermeable membrane 64, and the spacers 66 form three accommodating portions 67 to 69. Further, a dialysis chamber 70 for dialysis water is formed on the peripheral wall of the cylindrical body 62 at a position facing these accommodating portions 67 to 69. These accommodating portions 67 to 69 and 70 form a sealed space.

  The storage portions 67 to 69 are respectively supplied with gradient forming component solutions containing medium components, and the dialysis chamber 70 is supplied with dialysis water.

  That is, in each accommodating part 67-69, the inflow port and outflow port for recirculating | circulating a culture medium component solution are each formed. The dialysis chamber 70 is formed with an inlet and an outlet for dialysis water.

  The filling chamber 63 formed in the central part of the culture apparatus A is filled with agar of the microorganism sample and solidified, and each of the storage parts 67 to 69 contains the first to third medium components. Is supplied and refluxed, and dialysis water is supplied to the dialysis chamber 70 to reflux.

  The first to third components are diffused from the storage portions 67 to 69 to the agar layer filled in the filling chamber 63, and each component is discharged from the agar layer by the dialysis water in the dialysis chamber 70.

  According to this culturing apparatus A, since the substance diffusion can be brought into a steady state, even when the culturing time has passed, a certain culturing condition can be maintained at a certain point for a long time.

  The accommodating portions 67 to 69 and the dialysis chamber 70 through which the dialysis water is passed are not limited to the positions shown in this drawing, and may be installed at any position in the container depending on the direction of gradient formation. . Although not shown in this drawing, a lid covering the same is attached.

(PH gradient culture method)
Since lactic acid bacteria are facultative anaerobes, they are cultured under conditions close to anaerobic conditions in which other aerobic bacteria are difficult to grow. In this example, in order to set continuous pH conditions under conditions close to anaerobic, culture using a vinyl tube is performed under a pH 3.0 to 5.0 gradient.

  As shown in FIGS. 35 to 36, the incubator 74 includes a casing 71 obtained by cutting a commercially available vinyl tube into a predetermined size, and a lid made of silicone that plugs openings at both ends of the casing 71. 72. The incubator 74 was used after being sterilized by gas in a state where the opening of the tube 71 was plugged.

  BCP (Bromcresol Purple) -added plate agar medium (Nissui Pharmaceutical) was used as the basic medium.

  Medium is pH 3.0, 3.5, 4.0, 4.5, 5.0 double Mcllvaine buffer (based on citric acid and phosphate) or double Britton- Robinson buffer (based on phosphoric acid, acetic acid and boric acid) 5ml and 2x BCP medium 5ml separately sterilized by autoclave are mixed in a clean bench, and the total volume is 10ml as shown in Fig. 36. It was created by overlaying.

  For the pH measurement, the agar in the tube was pushed out, and the pH was measured in 0.5 cm increments from the lower part (acid side). The pH meter used was a Lacom tester pH meter (piercing electrode / waterproof type).

  As a result of measuring the pH change at 50 ° C. of the pH gradient medium prepared as described above, the pH was stable after 24 hours and 48 hours in both cases of Mcllvaine buffer and Britton-Robinson buffer (FIG. 37, 38). From this, it was considered that a pH gradient environment sufficient for separation of lactic acid bacteria was established in the main culture system.

  According to this method, it can be isolated from the natural world of strains that could not be isolated by ordinary screening. In particular, a screening technique for thermophilic lactic acid bacteria can be provided.

  In addition, continuous pH conditions can be set under conditions close to anaerobic in one culture.

(Two-dimensional gradient culture method consisting of oxygen and carbon dioxide)
In this example, attention was given to gas as a growth factor of difficult-to-cultivate microorganisms, and a culture apparatus A with dissolved oxygen and dissolved carbon dioxide concentration gradients was developed.

  For this purpose, a device having a dissolved gas gradient as shown in FIGS.

  The dissolved oxygen concentration was measured using a fiber optic oxygen sensor. In order to measure the dissolved carbon dioxide concentration, the pH of the LB medium containing various dissolved carbon dioxide was measured, and the dissolved carbon dioxide concentration was calculated from the calibration curve (FIG. 41).

  An outline of the prototype device is shown in FIGS.

  As shown in FIG. 39, if carbon dioxide gas is supplied to the central part of the cylindrical culture apparatus A, the carbon dioxide gas diffuses in the concentric direction from the center, so that a concentration gradient is given. As shown in FIG. 40, by supplying oxygen to the upper part of the culture apparatus A, oxygen diffuses downward from the upper part, so that an oxygen concentration gradient is created.

  Specific examples of the culture apparatus A are shown in FIGS.

  In this culture apparatus A, a carbon dioxide introduction pipe 81 for introducing carbon dioxide into the central part of a cylindrical main body 80 whose upper end and lower end are closed by a lid is disposed, and a space part 82 above the main body 80 is provided. An air introduction rod 83 for introducing air into the inside is provided. Further, an air exhaust rod 84 that exhausts the air in the space 82 is provided.

  The carbon dioxide introduction pipe 81 and the air introduction rod 83 are each formed using a porous film. Therefore, carbon dioxide gas and air leak to the outside through the pores of the carbon dioxide introduction pipe 81 and the air introduction rod 83, respectively.

  The main body 80 of the culture apparatus A is filled with the agar medium 85, and carbon dioxide gas and air are supplied from the carbon dioxide introduction pipe 81 and the air introduction bowl 83, respectively, and when a predetermined time has elapsed, as shown by the arrows in FIG. In the central part of the agar medium 85, the carbon dioxide gas concentration is higher than that in the peripheral part, and in the upper part of the agar medium 85, the oxygen concentration is higher than in the lower part.

  FIG. 41 shows a dissolved carbon dioxide concentration calibration curve.

Carbon dioxide gas was blown into the LB medium to prepare various solutions. The pH of these solutions was measured using a pH glass electrode and dissolved carbon dioxide using a carbon dioxide sensor.
(1) Gradient dissolved oxygen concentration gradient: A volume of 65 ml of the culture apparatus A space is aerated at a rate of 225 ml / min for one day, and then the aeration is stopped to maintain the steady state from the first day to the third day. It was. The dissolved oxygen concentration range was 0.4 mg / l to 85 mg / l (FIG. 44). FIG. 44 shows the formation state of the dissolved oxygen concentration gradient.

The two figures show the results of two independent experiments (Figures 44-45).
(2) Dissolved carbon dioxide concentration gradient: The dissolved carbon dioxide concentration gradient when carbon dioxide was continuously aerated was stable with respect to the depth of the medium on the first day. On the third day, a concentration difference occurred depending on the depth of the medium. The range of the concentration difference was 68 mg / l to 120 mg / l with 10% carbon dioxide aeration, and 92 mg / l to 185 mg / l with 20% carbon dioxide aeration (FIGS. 46-47).
FIG. 46 shows the dissolved carbon dioxide concentration gradient when 10% carbon dioxide gas is passed, and FIG. 47 shows the dissolved carbon dioxide concentration gradient when 20% carbon dioxide gas is passed.

A mixed gas of carbon dioxide and nitrogen was vented. 46, the continuous line (green line) indicates the case where 1% carbon dioxide gas was aerated for 1 day; red line: aeration for 1 day; blue line: aeration for 3 days.
(Conclusion) The culture apparatus A capable of creating a concentration gradient of dissolved carbon dioxide and oxygen in a three-dimensional space in the XY plane and the Z-axis direction is useful for screening microorganisms that respond to the gradient of dissolved gas.

(Method of forming redox potential (ORP) odor)
Since the establishment of a method for purely separating and culturing microorganisms, the center of microbiology has been based on a single microbial species that can be separated and cultured, and the separated and cultivated microorganisms have contributed to us in a wide range of industrial fields.

  However, it is widely recognized that “living but not cultivated” microorganisms are the original form of most microorganisms in the natural environment, including pathogenic microorganisms, food microorganisms, soil microorganisms, and environmental microorganisms. It has emerged as a major issue in general science. Pasteur, a traditional method since Koch, has made colonies formed on a plate agar medium containing nutrients, and the isolated and cultured microorganisms were targeted for research and application.

  However, the microbial species that can be handled by such a technique is only about 1%, for example, only 0.3% for soil microorganisms and 0.001% for marine microorganisms. The main reason for this is that there are too many culture factors (culture temperature, medium composition, pH, etc.).

In nature, the oxidation reduction potential (ORP) of the environment in which microorganisms exist generally indicates a value between O ₂ (+810 mV) and H ₂ (−420 mV), and the microorganisms that inhabit There are many cases in which the type of ORP varies depending on the value of the ORP. Generally, if the ORP is low, the anaerobic degree is high, and if it is high, the aerobic degree is high.

  In order to form ORP, it is common to use a method using a redox agent and air oxidation. It is suggested that the method of forming the ORP gradient using the electrochemical method as in the present invention can form and maintain the ORP gradient better and faster than the method using the oxidant and air oxidation. . By forming an ORP gradient, it is possible to make the separation conditions multidimensional from “points” to “lines”, and it is considered that several percent of microorganisms that cannot be cultured can be purely cultured. In addition, if a methodology for searching for microorganisms using this technology is established, the scale of industries using microorganisms is expected to be several times the current level.

  As a method for controlling the ORP of the microorganism culture, it is possible to certify any ORP by combining an oxidizing agent and a reducing agent, but it changes with the passage of time. Therefore, in general, a method is adopted in which a place where a target ORP is formed as a result in a natural environment is searched and microorganisms living in the place are collected. However, in this method, it is very difficult to search for a specific microorganism that can inhabit and grow under a special ORP that is considered not to be formed in the natural environment, and there is no simple and efficient method.

  Therefore, the purpose of this embodiment is to artificially form an ORP gradient using an electrochemical method and to isolate microorganisms that can grow in a special ORP environment. Furthermore, in order to confirm the ORP not only by the color change of the redox dye but also by numerical values, a needle-like platinum electrode and a reference electrode are prepared, and an experiment is actually performed using them.

  FIG. 48 is a schematic diagram of ORP gradient formation using an electrochemical method.

  In general, since microorganisms are often searched for in the presence of air, the reducing agent is oxidized by air and ORP gradually rises. Thus, in order to maintain an almost constant ORP at all times, it is necessary to continue to add a reducing agent and / or an oxidizing agent as appropriate. As a result, excessive addition of the drug suppresses the growth of microorganisms, The problem is that it is not possible to collect microorganisms that can live in a specific ORP environment to be searched.

  In order to solve the above-mentioned problems, in the present invention, an electrochemical method that can be continuously supplied at any time and can be automatically controlled is selected as a means for ORP control.

  As shown in FIG. 50, this system includes a closed container 92 for storing a microorganism search sample 91, a positive electrode 93 and a negative electrode 94 inserted into the sample 91 through the opening of the container 92, And a rubber plug 95 that closes the opening.

  The positive electrode 93 and the negative electrode 94 are connected to a voltage variable DC power source 97 through a conductive wire 96.

  A direct current voltage is applied between the insoluble anode 93 and the cathode 94, and an arbitrary voltage is set according to the purpose to energize. As a result, the electrochemical anode oxidizing power and cathode reducing power can be supplied to the microbial habitat at any time, and the monitor ORP electrode installed at a fixed point should not be accumulated excessively. If the set value is deviated, ORP control is possible in which the supplied power is turned on and off.

The features of this embodiment are as follows.
Item 1. A microorganism comprising a step of inserting an anode and a cathode into a culture environment of a microorganism and applying a voltage between both electrodes, and a step of searching for a microorganism that has formed a colony at the surface of each electrode or at any place between both electrodes. Screening method.
Item 2. In order to improve the efficiency of claim 1, the method further includes a step of appropriately adding a redox dye to the culture environment of the microorganism.

  If an arbitrary ORP environment is formed in the microbial habitat environment by using the system of the present invention, and microorganisms that can inhabit / proliferate can be separated even in a special ORP environment that is not formed in the conventional natural environment, unknown or / or high addition The search for useful microorganisms capable of producing valuable substances and enzymes can be realized easily and efficiently.

In order to optimize ORP control and / or ORP odor formation efficiency, a small amount (about 0.01 to 10 mM) of a redox dye (for example, methylene blue, methyl viologen, benzyl viologen which does not adversely affect the growth of microorganisms) , Neutral red, Janus green, thionine, o-phenanthroline, potassium ferricyanide, potassium ferrocyanide, etc.) may be used as an electron carrier (electron carrier) in the environment.
(Experimental example)
Agar powder was added to 100 ml of deionized water so that the amount was 1.5% and melted. After the agar melted, before the agar solidified, a potassium ferricyanide solution and a potassium ferrocyanide solution were added to 100 mM, respectively.

  Half of the open container was filled with potassium ferrocyanide agar.

  After the agar solidified, potassium ferricyanide agar was then poured. The electrode was inserted into the agar gel so that the distance between the anode and the cathode was 288 mm, and a DC voltage of 2 V was applied. An ORP electrode composed of a needle-shaped platinum electrode and a reference electrode was sequentially inserted at regular intervals, and the ORP after 1 hour was measured.

  A closed acrylic tube was also measured in the same procedure. As a result of the measurement, when energization is performed under these conditions, it takes 288 hours to form an ORP odor by the combination method of redox agents as shown in FIG. The formation of an ORP scent is now possible.

  It was confirmed that the open container showed a higher ORP value than the closed acrylic tube.

  This was suggested to be affected by air oxidation and container size. As a result of the measurement, it was confirmed that the open container was more diffused and the potential was higher than the closed acrylic tube. This was suggested to be affected by air oxidation and container size.

  Using this principle, microorganisms that can grow in special ORP environments where general microorganisms cannot live have special metabolic systems, enzymes, etc., and have the potential to produce special substances. It is thought that there is. In addition, in an ORP environment region where microorganisms do not inhabit (or are difficult to), it means that the growth of microorganisms is suppressed.

  Based on the above facts, 1) If special microorganisms are searched for and used, it may have the ability to produce unknown substances and / or high value-added substances. 2) Microorganism growth suppression By constantly forming the ORP region, it is possible to relatively easily realize the growth control of microorganisms in foods, drinking water and other various articles containing water and in the natural environment.

  In addition, the opposed multidimensional gradient microorganism culture apparatus of the present invention, for example, the search and separation of all kinds of microorganisms including useful microorganisms from the natural world and specimen samples, simple and comprehensive analysis of the growth characteristics of microorganisms, It can be used for all kinds of experiments that require comprehensive setting of complex culture conditions such as exhaustive analysis of useful substance production conditions, and can be used for a wide range of applications in various bio-related industries. In particular, it can be applied to animal cell culture and plant tissue culture, and can also be used for applications such as examination under a plurality of conditions such as the effect of specific substances on microorganisms, animal cells, and plant cells.

DESCRIPTION OF SYMBOLS 1 Incubator 10 Filling chamber 11 1st accommodating part 12 2nd accommodating part 13 3rd accommodating part 14 1st partition plate 15 2nd partition plate 16 3rd partition plate 21 1st agar layer 22 2nd agar Layer 23 Third agar layer 24 Central agar layer

Claims (8)

A part of a side wall of an incubator having a filling chamber filled with a microbial sample is formed with a first container that can be filled with a first agar containing a first component that affects the cultivation of the microorganism, The first storage part and the filling chamber of the incubator are partitioned by a removable first partition plate, and the second part containing the second component that affects the cultivation of the microorganisms is formed in the other part of the side wall of the incubator. Apparatus in which a second storage part capable of filling the agar is formed, and the second storage part and the filling chamber of the incubator are partitioned by a removable second partition plate. The culture apparatus according to claim 1, wherein a third housing portion that can be filled with a third agar containing a third component that affects the culture of the microorganism is formed at the bottom of the incubator. The incubator is formed in a box shape, the first accommodating portion is formed on one side wall of the incubator, and the second accommodating portion is formed on another side wall adjacent to the first accommodating portion. The culture apparatus according to 1. The culture apparatus according to claim 1, wherein the incubator is formed in a substantially cylindrical shape, and the first accommodation portion and the second accommodation portion are formed on a peripheral wall of the incubator. The culture apparatus according to claim 1, wherein three or more accommodating portions are formed on a side wall of the incubator. The culture apparatus according to claim 1, wherein the first agar and the second agar are different in at least one of a medium component concentration, a salt concentration, and a medium pH. A method for screening microorganisms contained in a microorganism sample using the culture apparatus according to claim 1, wherein a first container that is formed on a side wall of the incubator has a first effect on culture of the microorganisms. Filling a first agar containing one component to form a first agar layer, removing the first partition plate and exposing the first agar layer to a filling chamber of an incubator, Filling the second agar containing the second component affecting the culture of the microorganism into the second container formed on the side wall of the incubator to form a second agar layer, the second Removing the partition plate and exposing the second agar layer to the filling chamber of the incubator; and filling and solidifying the filling chamber of the incubator with a fluid solid medium containing a microorganism sample. A method for screening microorganisms to be included. The method according to claim 7 , wherein the solid medium does not contain a first component and a second component.