JP2017079691A - Anaerobe diversity observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem in which there exists a device for observing individual bacteria but there does not exist a device for observing the diversity itself of various bacterial floras.SOLUTION: An anaerobe diversity observation device according to the present invention makes it possible to observe various conditions of an intestinal bacterial flora in the same environment as the intestinal environment.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an anaerobic bacteria diversity observation apparatus.

  There are devices for the culture and observation of anaerobic bacteria.

Japanese Patent Application No. 2007-167191

  The above-mentioned patent document relates to culture of anaerobic bacteria and a method for observing them, which is for observing bacteria individually and observing the diversity itself of various bacterial flora. Is different. About 100 types of anaerobic bacteria are said to exist in the intestine, and it is known that the state of the intestinal microflora (intestinal flora) affects the health of the human being, the host. Therefore, since the state of the intestinal flora is a barometer of human health, a device that can easily observe various states of the intestinal flora in an environment equivalent to the intestinal environment is desired. It was rare. By the way, in the intestinal environment, the intestinal atmosphere is important in addition to the intestinal retained matter. This is because intestinal bacteria live in a gaseous environment realized in the intestine, that is, in the intestinal atmosphere. On the other hand, the intestinal atmosphere has a large individual difference that realizes the intestinal environment, and cannot be uniformly reproduced by the apparatus. Accordingly, there is a need for a culture environment in which the intestinal environment of the individual can be reproduced by the intestinal bacteria themselves contained in the sample obtained by diluting the stool collected from the individual who wants to observe the intestinal environment.

  In order to solve the above problems, as a first invention, a well plate base and an upper surface opening provided with a culture medium that is disposed on the well plate base and has a consumption rate that varies depending on the type of anaerobic bacteria are provided. There is provided a well plate base structure comprising a plurality of wells and a cover that forms an upper space of the plurality of well plate bases and is disposed to cover the well plate base.

  As a second invention, there is provided the well plate base structure according to the first invention in which the upper space is in an oxygen-free state.

  As a third invention, there is provided the well plate base structure according to the first or second invention, wherein the cover is transparent.

  As a fourth invention, the well or the well plate base according to any one of the first to third inventions, wherein the cover or / and the well plate base is provided with a gas inlet for making the upper space oxygen-free. A plate base structure is provided.

  As a fifth invention, there is provided the well plate base structure according to any one of the first to fourth inventions, wherein the well plate base is a substrate type well plate base in which a culture medium is fixed to a transparent substrate.

  As a sixth invention, there is provided the well plate base structure according to any one of the first to fifth inventions, wherein the well plate base is a nonwoven fabric type well plate base in which a dry medium is fixed to a nonwoven fabric.

  According to a seventh aspect of the invention, a stool collection step for collecting animal stool, a sample preparation step for preparing a sample by diluting the collected stool, and dropping the prepared samples into the plurality of wells in approximately equal amounts Any one of the first to sixth inventions comprising: a dropping step; an atmosphere creating step in which the cover is disposed after dropping to make the upper space oxygen-free; and an observation step for observing the state of each well. A method for observing enteric bacteria using the well plate base structure described in 1) is provided.

  As an eighth aspect of the invention, there is provided the method for observing intestinal bacteria according to the seventh aspect, wherein the observing step is performed a plurality of times at intervals of time.

  As a ninth invention, the observation step provides the method for observing intestinal bacteria according to the seventh or eighth invention, which is color observation.

  As a tenth invention, the observing step provides the method for observing intestinal bacteria according to any one of the seventh to ninth inventions, which is the observation of color intensity.

The anaerobic bacteria diversity observation apparatus according to the present invention makes it possible to observe a wide variety of intestinal flora in an environment equivalent to the intestinal environment.

Conceptual diagram of well plate base structure of embodiment 1 AA sectional view of the well plate base structure of Embodiment 1 Conceptual diagram of well plate base structure according to fifth invention of embodiment 1 Conceptual diagram of well plate base structure according to fifth invention of embodiment 1 Conceptual diagram of well plate base structure according to fifth invention of embodiment 1 Conceptual diagram of well plate base structure relating to sixth invention of embodiment 1 Conceptual diagram of well plate base structure relating to sixth invention of embodiment 1 Conceptual diagram of well plate base structure relating to sixth invention of embodiment 1 Schematic diagram of oxygen adsorbent installed in well plate base structure of embodiment 1 Schematic diagram of gas discharge installed in well plate base structure of embodiment 1 Schematic diagram of gas inlet installed in well plate base structure of embodiment 1 Conceptual diagram of reaction in each well of Embodiment 1 Conceptual diagram of the gas environment produced by the bacteria of the first embodiment Conceptual diagram of bacteria group of Embodiment 1 Exemplary diagram of reaction list of fungal group of embodiment 1 Conceptual diagram of observation results of Embodiment 1 Flow chart of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2 Example of processing flow diagram of observation method of embodiment 2

0100 Well plate base structure 0101 Well plate base 0102 Well 0103 Medium 0104 Cover 0205 Upper space 0906 Oxygen adsorbent 1007 Valve 1008 Gas exhaust 1109 Gas inlet 1110 Gas exhaust 1211 Bacterial nutrient absorption 1212 Metabolite 1213 Respiratory respiration 1214 Metabolism Gas 1215 Color change 1316-1 Gas component A
1316-2 Gas component B
1316-3 Gas component C
1316-4 Gas component D
1317 Collected exhaust gas 1521 Well number 1522 Bacteria group 1523 Reactivity 1524-1 Reaction result (Yes)
1524-2 Reaction result (none)
1701 Stool collection step 1702 Specimen creation step 1703 Dropping step 1704 Atmosphere creation step 1705 Observation step

  Embodiments of the present invention will be described below with reference to the drawings. The first embodiment relates to claims 1 to 6, and the second embodiment relates to claims 7 to 10. In addition, this invention is not limited to these embodiments at all, and can be implemented in various modes without departing from the gist thereof.

Embodiment 1

<Overview>
The present embodiment relates to a well plate base structure.

<Functional configuration>
This embodiment will be described with reference to FIG. The well plate base structure (0100) of this embodiment includes a “well plate base”, a “well”, and a “cover”.

<Description of each configuration>
(Well plate base)
The “well plate base” (0101) is a portion that serves as a base for installing a plurality of wells to be described later. The material of the well plate base is not particularly limited, but plastic, silicon, paper, etc. are conceivable. This well plate base structure is for culturing anaerobic bacteria and observing the state thereof. Among the anaerobic bacteria, the obligate anaerobic bacteria refers to bacteria that can grow only where there is almost no free oxygen, and include clostridial, methane bacteria, and sulfate-reducing bacteria. Many so-called intestinal bacteria, which are bacteria that inhabit the intestines of humans and the like, are obligate anaerobes. Intestinal bacteria include those called good bacteria and bad bacteria. Specifically, many of the so-called good bacteria make organic acids such as lactic acid and butyric acid, such as Bifidobacterium genus represented by bifidobacteria and Lactobacillus genus bacteria called lactobacilli. The bad bacteria are often those that produce so-called spoilage substances that cause bad odor, such as Clostridium genus represented by C. perfringens and Escherichia coli. Using the well plate base structure of the present application, anaerobic conditions similar to those in the intestine are created, and anaerobic bacteria including intestinal bacteria are cultured and observed, and the state of the bacterial flora in the human intestine By examining it, it becomes possible to grasp the health state, mental state, etc. of the subject who is the host.

(Well)
The “well” (0102) is arranged on the well plate base and is configured to be an upper surface opening, with at least a portion of the medium having different consumption rates depending on the type of anaerobic bacteria. A plurality of wells are provided on the well plate base. The well is a cylindrical structure having a circular cross section and a bottom, and a culture medium to be described later and a specimen obtained by diluting the collected stool are placed therein. The number of types of obligate anaerobes is not necessarily required, and the number of types of media may be sufficiently smaller than the number of types of bacteria that can actually be detected. The culture medium is configured so that at least some of the respective culture media can reproduce certain bacterial species but not other bacterial species, but the specific bacterial species are included in it. There is no need to indicate it directly. It is only necessary to determine whether or not the intestinal flora has a healthy aspect when observed as a whole. The medium is prepared so that components such as acidity and contained minerals are different, and history of how each responded to healthy people or how to respond to people with health problems As a whole, it can be determined based on the database accumulated as So-called empirical rules are stored in this stored database, and individual judgments are made based on the stored empirical rules. Therefore, the pattern which a healthy body shows is not necessarily one type, and the pattern which an unhealthy body shows is not necessarily one type.

  The number of wells is preferably at least 6 or more because the present application observes the reaction of a plurality of bacterial groups. Moreover, it is preferable that the material of the well does not affect the medium and the specimen. The volume of the well can be determined according to the amount of medium and specimen. For example, it is 0.2 cc to 2 cc.

  The “medium” (0103) provided in the well may be any medium that can culture anaerobic bacteria. Specifically, agar may be considered, and glucose may be dropped in advance so as to nourish anaerobic bacteria. Furthermore, since the present application examines the presence / absence / amount of anaerobic bacteria by color, a color former that reacts with anaerobic bacteria may be provided in advance. An additive may be added to each well. Specific examples of the medium include materials based on brain heart infusion, tryptic soy broth, and the like. It is also conceivable that yeast extract or menadione is appropriately added to these, the acidity is controlled, or the blood of the sample provider is mixed in some cases. This is to reproduce the case where blood is mixed in the intestine. In addition, if there is a characteristic ingredient in the meal taken on the day or the day before (for example, a large amount of coffee or alcohol), add it to the medium. It is also possible to mix.

(cover)
A “cover” (0104) is configured to form an upper space of a plurality of well plate bases and to be disposed over the well plate bases. The term “upper space” as used herein refers to providing a space in the opening direction of each well with a certain interval, instead of covering each well with a cover without a gap. In this application, after creating an anaerobic state similar to that in the intestine, the substance produced by the fungus in the same way as in the intestine is used to observe changes to other fungi, so there is a common space above each well. Is provided.

  Since the reaction of the medium in each well is observed by a change in color, it is preferable that the cover is transparent so that it can be observed with the cover removed without removing the cover. Further, a colorless and transparent cover may be used in order to strictly observe the color difference as a reaction result. Further, a lens integrally formed on the cover may be provided for each well. Furthermore, an optical filter for cutting light of a specific frequency may be provided. This is convenient when light in a specific frequency band is a noise component.

  The cover only needs to be able to seal the well plate base structure. Therefore, a plastic bag that covers the well plate base may be used instead of a dedicated product. Further, a well plate base may be installed in a container that can be sealed, such as a “tapper”, and the lid of the tapper serving as a cover may be closed and sealed. Even if there is no visible space between the cover and each well, if there is a substantial gap between the cover and each well on each well, the upper space of each well is also the same for all wells. The cover of the present invention is configured when the cover and the well are not intentionally sealed.

  FIG. 2 is a cross-sectional view taken along the line AA in FIG. As can be seen from this figure, an upper space (0205) is provided in the upper part of each well (0202), which is in the same state as in the intestine. Thereby, the influence etc. with respect to other intestinal bacteria can be investigated with the gas which intestinal bacteria produce. In order to prevent oxygen (air) from entering so as not to kill the anaerobic bacteria, it is preferable to seal the well plate base structure by covering. The sealing may be performed by, for example, rubber packing, or may be configured to be sealed with a plastic zipper-like one. The upper space volume is designed according to the approximate measurement period (time), but the upper space volume is designed to be relatively large as the measurement period (time) becomes longer. For example, when the well volume is 0.5 cc, the upper space volume per well is preferably about 0.1 cc to 2.0 cc.

  FIG. 3 is a conceptual diagram of a base type well plate base structure in which a culture medium is fixed to a transparent substrate. FIG. 4 is a cross-sectional view taken along the line BB. The culture medium (0403) in each well (0402) is set up from the well plate base (0401). A cover (0504) as shown in FIG. 5 is installed and sealed. Since the well plate base structure of the present application observes the reaction caused by the anaerobic bacteria in each well by a change in color, it is preferable that it is transparent so that the color difference between the wells can be well recognized. Further, by making the well plate base transparent, it is possible to observe from the back side.

  The well plate base used for the well plate base structure may be a non-woven fabric type well plate base in which a dry medium is fixed to a non-woven fabric. FIG. 6 is a conceptual diagram of a non-woven type well plate base structure. As a specific example of the dry medium, dried agar can be considered. As will be described later, since a specimen made of stool collected in each well is dropped, the agar contains water due to the water, and anaerobic bacteria can continue. In addition, the non-woven type well plate base can be attached to the culture medium of each well using a printing machine or the like, so that it can be mass-produced at low cost and can be used to observe more specimens. Can be done.

  FIG. 7 is a cross-sectional view taken along the line CC. The culture medium (0703) in each well (0702) is set up from the well plate base (0701). A cover (0804) as shown in FIG. 8 is installed and sealed.

  The upper space of the well plate base structure needs to be oxygen-free as in the intestine. Since anaerobic bacteria are gradually killed by contact with oxygen, it is preferable to provide an oxygen adsorbent (0906) on the top of the cover (FIG. 9, 0904) to adsorb oxygen. The material and amount of the oxygen adsorbent can be determined as appropriate.

  In addition to using an oxygen adsorbent, a valve (1007) may be provided on the upper part of the cover (FIGS. 10 and 1004) to discharge the gas (lower the pressure, 1008) so as to remove oxygen.

  Alternatively, a gas inlet for making the upper space of the cover or / and well plate base oxygen-free may be provided. FIG. 11 is a conceptual diagram of a well plate base structure in which a gas inlet (1109) and a gas outlet (1110) are provided in a cover (1104). A valve provided at each inlet is designed to prevent gas backflow. It is preferable that the gas to be injected does not affect the fungus group that is a specimen, and is difficult to alter and react with the gas produced by the fungus group. Nitrogen, an inert gas, can be considered.

  FIG. 12 is a conceptual diagram of the reaction in each well. When enteric bacteria absorb nutrients (1211), they produce metabolites (1212) such as short chain fatty acids. Further, respiration (1213) produces metabolic gas (1214). In other words, it grows by fermenting the nutrients contained in the diet ingested by the host as the main nutrient source, and at the same time produces various metabolites. Produces gas produced by fermentation by intestinal bacteria. This changes the color tone of the reagent in the medium in the well (1215).

  FIG. 13 is a conceptual diagram of metabolic gas produced by anaerobic bacteria in each well. The intestinal environment is filled with metabolic gases produced by various anaerobic bacteria (gas component A · 1316-1, gas component B · 1316-2, gas component C · 1316-3, gas component D · 1316-4) Further, it is considered that this set of metabolic gases (1317) is further used for respiration of anaerobic bacteria and affects the distribution and survival rate of anaerobic bacteria.

  The mechanism of anaerobic bacteria production is as follows. Although the digestive tract of the human body cannot digest many polysaccharides, which are dietary fibers other than starch and glycogen, on its own, some of the short chains such as butyric acid and propionic acid are produced by anaerobic fermentation of enteric bacteria in the large intestine. It is converted into fatty acid and absorbed as an energy source. Dietary fiber produces energy by fermentative degradation by enteric bacteria. Butyric acid bacteria are anaerobic spore-forming gram-positive rods that produce butyric acid. It is also a type species of the genus Clostridium. Although it is widely present in the environment in the form of spores, it is particularly known as an resident bacteria in the digestive tract of animals.

  Vitamin K is supplied by multiple intestinal bacteria belonging to several types, along with intake from food. Biotin is also supplied by the intestinal flora. Pyridoxine (vitamin B6) is also supplied by enteric bacteria. In addition, enterobacteria also produce pantothenic acid (vitamin B5), folic acid (vitamin B9), riboflavin, niacin (vitamin B3), biotin (vitamin B7), vitamin B6, vitamin B12, and vitamin K.

  FIG. 14 is a conceptual diagram of a collection of enteric bacteria that are anaerobic bacteria. It is said that more than 100 types of enteric bacteria exist, and many fungal groups affect other fungal groups to generate an intestinal environment. The part indicated by (1) in the figure indicates that the well (1420) is affected by the bacteria group A and bacteria group B.

  FIG. 15 shows that there are eight types of anaerobic bacteria groups (1522, A to H) for each well (there are wells 1521 and (1) to (8) with different culture media). .) Is a list that indicates a reaction, and the rightmost column (1523) indicates the degree of reaction in a specific observation result as “Yes” (1524-1), “No” (1524-2). If the fungus group “C” exists in the intestine, the well containing the medium of (2) (3) (4) (5) (6) should react. In this example, Since there was no reaction with respect to 4) (see FIG. 16, the result is that the C fungal group did not exist).

<Effect>
With the well plate base structure having the above configuration, it is possible to observe various states of the intestinal bacterial flora, which is an anaerobic bacterium, in an environment equivalent to the intestinal environment.

<< Embodiment 2 >>

<Overview>
This embodiment relates to a method for observing enteric bacteria.

<Functional configuration>
The present invention will be described with reference to FIG. The observation method of the present embodiment includes a “stool collection step”, a “specimen creation step”, a “dropping step”, an “atmosphere creation step”, and an “observation step”.

<Description of each configuration>
(Stool collection step)
The “stool collection step” (1701) is a step of collecting animal stool.

(Sample preparation step)
The “sample preparation step” (1702) is a step of preparing a sample by diluting the stool of the animal collected in the stool collection step with water or the like.
The dilution factor can be determined as appropriate.
The liquid to be diluted may be any liquid that does not transform or kill intestinal bacteria present in the collected stool in addition to water.

(Drip step)
The “dropping step” (1703) is a step of dropping the specimen created in the specimen creating step into the plurality of wells in approximately equal amounts. The amount to be dropped is determined by adjusting the volume of the well and the amount of the already installed medium.

(Atmosphere creation step)
The “atmosphere creation step” (1704) is a step of placing the cover and placing the upper space in an oxygen-free state after dropping the specimen into the well. Enterobacteria are anaerobic bacteria that die when exposed to oxygen for a certain period of time. Therefore, it is desirable to install a cover as soon as the dripping is complete, and make oxygen-free by the action of an oxygen adsorbent, etc. .

(Observation step)
The “observation step” (1705) is a step of observing the state of each well.

  The observation step may be performed a plurality of times with a time interval.

  The observation step may be color observation.

  The observation step may be observation of color intensity. When the reaction is strong, that is, when the number of bacteria is large, if the color becomes dark, it can be determined that the number of bacteria is large in the well that is producing a dark color.

  The measurement method will be described more specifically.

   A specific example of a measurement method when the intestinal environment is expressed by, for example, one kind of anaerobic bacteria index will be described with reference to FIGS. Note that only one of the methods shown in these specific examples may be used, or two or more methods may be used in combination.

  1. Regression characteristic value averaging method (Figure 18)

  As shown in FIG. 18, the regression characteristic value averaging method performs “regression” analysis on the measurement result of cumulative consumption (1801a, 1801b), and then “averages” predetermined values based on the regression function (1803a, 1803b). Is the method. FIG. 18A shows a processing flow diagram of the normal regression characteristic value averaging method, and FIG. 18B shows a processing flow of the regression characteristic value averaging method using the average information amount.

  1-1. Normal regression characteristic value averaging method (Fig. 18 (a))

First, regression analysis of a [cumulative consumption (y _i ) −elapsed time (x)] plot is performed for each nutrient source (0301a). Here, i (= 1, 2,..., N) is an identification number of the nutrient source, and the number n of the nutrient sources to be used is the maximum value (hereinafter common). Therefore, n regression functions are obtained by this processing.

Next, a characteristic value (α _i ) is obtained based on the obtained regression function (1802a). That is, the characteristic value (α _i ) is calculated for each nutrient source by the processing. Here, the characteristic value (α _i ) is an estimated value of cumulative consumption obtained from the regression function at a specific time, an estimated value of consumption rate obtained from the slope of the regression function at a specific time, or a specific time after the start of culture. This corresponds to the time average of the cumulative consumption estimated value obtained by dividing the integral value of the regression function up to the elapsed time (hereinafter, simply referred to as the cumulative consumption estimated value time average). Here, the specific time is a time after a certain time has elapsed, or a time when the regression function reaches a geometric feature such as an inflection point, or the regression function is infinite. In the case of convergence after the lapse of a large time, the time after the infinite time may be used. The “certain time” is as described above.

Next, the characteristic value (α _i ) calculated for each nutrient source is averaged for all the n nutrient sources to obtain an average characteristic value (A) (1803a). That is, one average characteristic value (A) is obtained in one measured intestinal environment by the processing. Further, the average characteristic value (A) is used for converting the measured intestinal environment into an intestinal environment index (1804a). The calculation method in the conversion may be any method that can compare the superiority and inferiority between the measured intestinal environment and the reference intestinal environment with respect to the calculation result of the intestinal environment index. For example, sections of characteristic values may be divided based on comparison with a reference intestinal environment where diversity of anaerobic bacteria is clear, and converted into grades such as A, B, and C. If the comparison is possible, the characteristic value itself may be simply used. In this case, the characteristic value itself becomes the intestinal environment index.

  The type of regression function obtained in the regression analysis process (1801a) is not particularly limited, but a logistic curve or other growth curve is applicable. A sigmoid curve based on the Gompertz function is more preferable. It also includes linear regression. The same applies to regression functions described in calculation examples described later. The linear model is effective in that the calculation cost can be suppressed although the accuracy is low. The nonlinear model is effective in terms of higher accuracy, but the calculation cost increases accordingly. Moreover, the average performed in the average process (1803a) is not particularly limited as long as it can be calculated from the measurement result of the accumulated consumption, such as arithmetic average, geometric average, and harmonic average. The same is true for the averages described below.

  And, as described above, the average characteristic value A is a cumulative consumption estimated value based on a regression function, a consumption speed estimated value, a cumulative consumption estimated value time average, etc. at a specific time across all nutrient sources. The average is applicable.

  In this case, regardless of whether the average characteristic value A is an average of the cumulative consumption estimated value, the consumption speed estimated value, or the cumulative consumption estimated value time average, the larger the characteristic value is, the larger the average value for various nutrient sources is. Therefore, it can be said that it is a good intestinal environment for growing anaerobic bacteria having a higher activity of intestinal bacteria. Therefore, the average characteristic value A is compared with that of the standard intestinal environment. And On the contrary, when the average characteristic value A is smaller than the standard intestinal environment, the intestinal environment having a small intestinal environment index, that is, an intestinal environment that is not suitable for anaerobic bacteria growth. In this case, for example, the intestinal environment index is a value obtained by subtracting the average characteristic value A of the measured intestinal environment from the average characteristic value A of the reference intestinal environment, for example. At this time, the intestinal environment index of the intestinal environment that is not suitable for anaerobic bacteria growth is a negative value. In the case where comparison is made between two or more measured intestinal environments without setting the reference intestinal environment, the average characteristic value between the measured intestinal environments may be simply compared. That is, by converting the average characteristic value of the measured intestinal environment in a form that can be compared with the reference intestinal environment or other measured intestinal environment, or by directly using the average characteristic value, The intestinal environmental index is taken as the measurement result.

A specific example in the case where the regression function is a Gompertz curve represented by the following formula 1 is shown below.
[Formula 1]

y = ka ^ b ^ (x-d)
Where a, b, k, d are parameters determined by regression calculation

The derivative of Equation 1 is expressed by Equation 2 below, where x = d is the inflection point.
[Formula 2]

y ′ = k (lna) × a ^ b ^ (x−d) × (lnb) b ^ (x−d)
In this case, the slope at the inflection point, that is, the estimated consumption rate (e) (Equation 3), the estimated estimated consumption value (f) at the inflection point (Equation 4), and the accumulated consumption at the time after infinite time has elapsed. The estimated value (k) can be used as the characteristic value.
[Formula 3]

e = k (lna) × a × (lnb)
[Formula 4]

      f = ka

In such a case, the average characteristic value A is calculated by averaging the values of the characteristic values e _i , f _i , and k _i for each nutrient source i as characteristic values α _i over all nutrient sources. The average characteristic value (A) is converted in a form comparable to the reference intestinal environment or other measured intestinal environment to calculate the intestinal environment index of the measured intestinal environment, or the average characteristic value Is taken as the intestinal environment index, and the measurement result

      1-2. Regression characteristic value averaging method using average information (Figure 18 (b))

First, regression analysis of a [cumulative consumption (y _i ) −elapsed time (x)] plot is performed for each nutrient source i (1801b). Next, a characteristic value (α _i ) is obtained based on the obtained regression function (1802b), and the characteristic value (α _i ) is averaged over all nutrient sources to obtain an average characteristic value (A) (1803b). The steps so far are the same as the steps described in FIG. 18A (1801a, 1802a, 1803a).

Next, an average information amount (E) of the characteristic value (α _i ) calculated for each nutrient source in step 1802b is calculated (1804b). That is, the bias of the cumulative consumption for each nutrient source is obtained using the concept of average information amount. Note that either step 1803b or step 1804b may be performed first. A method for calculating the average information amount (E) will be described later.

  Next, the average characteristic value (A) calculated in step 1803b is multiplied by the average information amount (E) (1805b). Here, the value of the average information amount (E) is smaller as the bias of the accumulated consumption with respect to the nutrient source is larger, and the value of the average information amount (E) is larger as the bias is smaller. Moreover, since the bias of cumulative consumption can be said to be bias of assimilation with respect to each nutrient source, the average information amount (E) can be said to be a value reflecting the diversity of anaerobic bacteria inhabiting. Therefore, the value of the average information amount (E) is larger as the diversity of the anaerobic bacteria inhabiting is larger, and the value of the average information amount (E) is smaller as the diversity is smaller. Therefore, the multiplication value (AE) can be used for measuring the intestinal environment index of the measured intestinal environment as an index reflecting the diversity of anaerobic bacteria living in the measured intestinal environment. Therefore, as in the method described in section 1-1, the multiplication value (AE) can be converted in a form comparable to the reference intestinal environment or other measured intestinal environment, or the multiplication value can be used as it is. The intestinal environment index of the intestinal environment to be measured is taken as the measurement result (1806b).

The average information amount E is given by the following equation regarding the probability p _i of occurrence of the event i.
E = -Σp _i log p _i (where Σ is for i)
Now, for the characteristic value α _i corresponding to each nutrient source i, if the ratio to the total over all nutrient sources is p _i ,
p _i = α _i / Σα _j where Σ is performed for j.
It becomes. The average information amount is 1.0 when p for all nutrient sources is 1 / n (n is the number of nutrient sources) and takes the maximum value and sets the base of the logarithm to n.
At this time,
E = -Σ (α _i / Σα _j ) log _n p _i = Σα _i log _n p _i / Σα _j
It becomes. On the other hand, the arithmetic mean A is
A = Σα _j / n
So when E is multiplied by the arithmetic mean,
AE =-Σα _i log _n p _i / n
It becomes. That is, compared to the simple average, since only being applied the term of log _n p _i, can be regarded as a kind of weighted average.

    2. Mean data regression method

    As shown in FIG. 19, the average data regression method is to “average” the measurement results of accumulated consumption at each time over all nutrient sources by time (1901a, 1901b), and then to set the time as an explanatory variable and at each time. In this method, “regression” analysis is performed using the average value as a dependent variable (1902a, 1904b). FIG. 19A shows a processing flow diagram of a normal average data regression method, and FIG. 19B shows a processing flow of the average data regression method using the average information amount.

      2-1. Normal average data regression method (Fig. 19 (a))

First, the cumulative consumption value (y _{i, t} ) of each nutrient source is averaged over all nutrient sources at every predetermined time (Y _t ) (1901a). That is, from the [cumulative consumption value (y _{i, t} ) −elapsed time (x)] plot obtained for each nutrient source by the treatment, an average of [cumulative consumption value (Y _t ) −elapsed time ( x)] a plot is required. Hereinafter, the intestinal environment index of the intestinal environment to be measured is measured using the average one [cumulative consumption value (Y _t ) −elapsed time (x)] plot.

Next, regression analysis of the average [cumulative consumption value (Y _t ) −elapsed time (x)] plot is performed (1902a). The regression analysis is as described in “1. Regression characteristic value averaging method”.

  Next, a characteristic value (α) based on the regression function is calculated (1903a). The characteristic value (α) is as described in “1. Regression characteristic value averaging method”. The characteristic value (α) is converted into a form that can be compared with the reference intestinal environment and other measured intestinal environment, or the characteristic value is used as it is to obtain the intestinal environment index, and the measured intestinal environment. (1904a).

  2-2. Average data regression method using average amount of information (Fig. 19 (b))

First, the cumulative consumption value (y _{i, t} ) of each nutrient source is averaged over all nutrient sources at every predetermined time (Y _t ) (1901b). Next _, an average information amount (E _t ) of the cumulative consumption value (y _{i, t} ) is calculated every predetermined time (1902b). Note that the order of processing in step 1901b and step 1902b may be performed first. Then, the cumulative consumption average value (Y _t ) is multiplied by the average information amount (E _t ) to obtain a multiplication value (Y _t E _t ) reflecting the bias of the cumulative consumption amount for each nutrient source (1903b).

Next, using the multiplication value (A _t E _t ), regression analysis of a [Y _t E _t -elapsed time (x)] plot is performed (1904b). Then, a characteristic value (α) based on the regression function is calculated (1905b). The characteristic value (α) is as described in “1. Regression characteristic value averaging method”. By converting the characteristic value (α) in a form that can be compared with the standard intestinal environment and other measured intestinal environments, or by using the characteristic values as they are, the intestinal environment index is obtained. It is set as an intestinal environmental index measurement result (1906b).

  3. Other methods

  An example of a simpler method without performing regression analysis will be described below.

3-1. This will be described with reference to FIGS. 20 (a) and 20 (b). First, an average value of the cumulative consumption (y _{i, t} ) of each nutrient source is calculated for all nutrient sources at predetermined time intervals (Y _t ) (2001a). In other words, the average [accumulated consumption value (Y _t ) −elapsed time] from the [accumulated consumption value (y _{i, t} ) −elapsed time (x)] plot obtained for each nutrient source by the treatment. (X)] plot is required. Next, an integral value (S) from time 0 to a fixed time (T) is calculated for the [average cumulative consumption (Y _t ) −elapsed time (x)] plot (2001b). Next, the integrated value (S) is divided by the elapsed time (T) (2003a), and the divided value (M) is converted in a form that can be compared with the reference intestinal environment or other measured intestinal environment. By calculating the intestinal environment index or using the divided value (M) as it is, the intestinal environment index is obtained and the intestinal environment index measurement result of the measured intestinal environment is obtained (2004a). When the cumulative consumption is measured at equal time intervals, the method regresses to a linear function [y = b (b is a constant)] in the above “2-1. Normal Average Data Regression Method”. It is logically equivalent to the method.

In this method as well, the concept of average information amount may be used as shown in FIG. An average information amount (E _t ) of the cumulative consumption (y _{i, t} ) for each predetermined time is calculated (2002b), and this is multiplied by the average cumulative consumption value (Y _t ) calculated in step 2001b to accumulate A multiplication value (Y _t E _t ) reflecting the bias of the consumption with respect to the nutrient source is obtained (2003b). Then, [Y _t E _t - elapsed time (x)] is calculated integral value of plots (S) (2004b), divided by the integrated value (S) elapsed time (T) (2005b), the division value Convert (M) to a standard intestinal environment or other measured intestinal environment, etc., or use the divided value (M) as it is to obtain an intestinal environment index and measure It is set as the measurement result of the intestinal environment index of the intestinal environment (2006b). In addition, when the accumulated consumption is measured at equal time intervals, the method is the linear function [y = b (b is a constant) in “2-2. Average data regression method using average information amount”]. It is logically equivalent to the method of returning to].

3-2. This will be described with reference to FIGS. 21 (a) and 21 (b). First, an integrated value (s _i ) from time 0 to a fixed time (T) is calculated for the [cumulative consumption (y _i ) −elapsed time (x)] plot of each nutrient source (2101a). Next, the integral value (s _i ) is divided by the elapsed time (T) (2102a), and the division value (m _i ) is averaged over all nutrient sources (2103a). And the average value (M) is converted in a form that can be compared with the standard intestinal environment or other measured intestinal environment, or the average value (M) is used as it is as an intestinal environment index, The measurement result of the intestinal environment index of the intestinal environment to be measured is used (2104a). In addition, when the accumulated consumption is measured at equal time intervals, the method returns to a linear function [y = b (b is a constant)] in the above “1-1. Normal regression characteristic value averaging method”. It is logically equivalent to the method to do.

Also in this method, the concept of average information amount may be used as shown in FIG. The average information amount (E) of the division value (m _i ) calculated in Step 2101b and Step 2102b is calculated (2103b), and this is multiplied by the average value (M) of the division value to reflect the bias of the cumulative consumption amount. The multiplied value (ME) obtained is obtained (2105b). Then, the multiplication value (ME) is converted in a form comparable to the reference intestinal environment or other measured intestinal environment, or the multiplication value (ME) is used as it is to obtain an intestinal environment index. It is set as the measurement result of the intestinal environment index of the measured intestinal environment (2106b). When the cumulative consumption is measured at equal time intervals, the method is the linear function [y = b (b is a constant) in “1-2. Regression characteristic value averaging method using average information amount”. )] Is logically equivalent to the method of regression.

In summary, a sample generation step for generating a suspension of the measured gut environment as an anaerobic bacteria sample,
A dropping step in which a suspension of the generated anaerobic bacteria sample is dropped onto n nutrient sources (n is a natural number) whose consumption rate differs at least in part by anaerobic bacteria;
After dropping, an observation step of observing the cumulative consumption of each nutrient source by anaerobic bacteria within a certain period of time,
A regression function creating step for obtaining n regression functions representing the relationship between elapsed time and cumulative consumption based on the observed cumulative consumption of each nutrient source;
Based on each of n regression functions, the estimated value of cumulative consumption at a specific time, the estimated value of the consumption rate obtained from the slope of the regression function at a specific time, and the integrated value of the regression function from the start of culture to the specific time An estimated value acquisition step for obtaining one or more of time averages of cumulative consumption estimated values divided by time;
Average characteristic value averaged over all nutrient sources for each of one or more of the estimated value of cumulative consumption, estimated value of consumption rate, and time average of estimated value of cumulative consumption obtained in the estimated value measurement step. An intestinal environment index obtaining step for obtaining A as an intestinal environment index of a measured intestinal environment;
An intestinal environment index measuring method for intestinal environment including can be employed.

<Effect>
The number and type of anaerobic bacteria can be observed by the method for observing anaerobic bacteria of this embodiment, and the health condition of the subject can be examined.

Claims (10)

A well plate base,
A plurality of wells with a top opening with a medium disposed on a well plate base and at least partially comprising a medium with different consumption rates depending on the type of anaerobic bacteria;
A cover that forms an upper space of the plurality of well plate bases and is arranged to cover the well plate bases;
Well plate base structure consisting of   The well plate base structure according to claim 1, wherein the upper space is in an oxygen-free state.   The well plate base structure according to claim 1, wherein the cover is transparent.   The well plate base structure according to any one of claims 1 to 3, wherein the cover or / and the well plate base is provided with a gas inlet for making the upper space in an oxygen-free state.   The well plate base structure according to any one of claims 1 to 4, wherein the well plate base is a substrate type well plate base in which a medium is fixed to a transparent substrate.   The well plate base structure according to any one of claims 1 to 5, wherein the well plate base is a non-woven well plate base in which a dry medium is fixed to a non-woven fabric. A stool collection step for collecting animal stool;
A sample preparation step for preparing a sample by diluting the collected stool;
A dropping step of dropping the prepared specimen into the plurality of wells in approximately equal amounts;
An atmosphere creation step of placing the cover after dropping to make the upper space anoxic,
An observation step for observing the state of each well;
A method for observing enteric bacteria using the well plate base structure according to any one of claims 1 to 6.   The observation method of intestinal bacteria according to claim 7, wherein the observation step is performed a plurality of times at intervals of time.   The observation method for enteric bacteria according to claim 7 or 8, wherein the observation step is color observation. The observation method for enteric bacteria according to any one of claims 7 to 9, wherein the observation step is observation of color intensity.

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