JPH07170973A - Selective recovery of microorganism - Google Patents

Abstract

PURPOSE:To enable microorganisms living in soil, activated sludge, bottom mud to be selected and collected by fractionating and recovering the microorganisms according to their respective desities by the specific gravity ingredient centrifugation. CONSTITUTION:A suspension of soil, activated sludge or bottom mud is fed in on the uppermost part of the ultracentrifugation tube in which aqueous solutions of specific gravity changed stopwise or gradiently are piled up, to undergo centrifugal treatment. According to the specific gravity of the microorganism in the suspension, bands A, B, C or precipitation D are formed and the microorganisms separated in the target bands are selectively collected. The ingredient for specific gravity is adjusted by dissolving solutes such as sucrose, cesium chloride, Ficoll, etc., in various concentrations.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selecting and collecting a plurality of types of microorganisms by gravity gradient centrifugation. The present invention also relates to a method for selectively collecting and recovering a plurality of types of microorganisms by utilizing liquid phase electrophoresis.

[0002]

2. Description of the Related Art In recent years, with the development of genetic engineering, the possibility of producing useful substances using microorganisms and enzymes and decomposing harmful substances, which is different from conventional chemical methods, has been actively investigated. Came. In addition, in order to carry out genetic engineering production while maintaining the advantages of existing chemical engineering production,
Research on microorganisms that inhabit and operate in special environments such as low temperature, high alkali, and high water pressure has also become popular.

Activated sludge and rivers in soil, sewage and wastewater treatment tanks
It is considered that a large number of microorganisms that have such useful functions but have not yet been isolated inhabit the bottom mud of lakes and seas.Isolate such microorganisms and write information about their functions. The technology for extracting the obtained DNA has become very important not only for the basic research of genetic engineering but also for the further development of the applied technology field.

The method of obtaining microorganisms from soil or the like, which is mainly used at present, is to prepare a suspension of a sample of soil or the like, dilute it as needed, and then isolate the desired microorganism. Apply on an agar medium containing the matched components, leave the medium for a few days in an environment suitable for the growth of the target microorganism and culture it, select the colonies of the grown microorganisms, and further accumulate culture to determine the concentration. This is a method in which the product is raised and then precipitated by centrifugation or the like and collected.

However, most of a wide variety of microorganisms living in soil or the like are not known even in the conditions for their separation and culturing, and 99 to 100% of the microorganisms in the suspension prepared by the above-mentioned method can be obtained.
For 99.9%, it is said that it is difficult to grow and separate them on an agar medium. For this reason,
Most useful microorganisms in the sample suspension are not recoverable. In addition, even if activated sludge in a wastewater treatment tank that is relatively easy to estimate culture conditions, the ratio of microbial species is changed due to a subtle difference between the actual conditions inside the tank and the conditions inside the laboratory medium. There is a high possibility that the priority species in activated sludge cannot be recovered as the priority species in the medium.

[0006]

From the above-mentioned viewpoints, there is a need for a method for directly separating and recovering microorganisms from a suspension of soil, activated sludge, bottom mud and the like.

However, the suspension obtained by the above-mentioned conventional method contains various solid substances other than microorganisms, and most of them are
In many cases, it is difficult to separate the microorganisms from the microorganisms simply by using a method such as ordinary filtration or centrifugal precipitation. Separation of microorganisms from a sample mixed with such a solid substance requires complicated and labor-intensive operations such as setting delicate centrifugal conditions and repeating filtration with several kinds of filter papers having different pore sizes. Furthermore, when microorganisms adhere to particulate solids and live, it is extremely difficult to separate the microorganisms by filtering or centrifuging it as it is. It is necessary to agitate to try to separate the microorganisms from the solid matter, which further complicates the treatment process.

On the other hand, in obtaining DNA from the collected microorganisms, the quality and purity of the obtained DNA are very important factors for the subsequent treatments such as enzymatic digestion, PCR and hybridization. However, microparticles and immobilization carriers contain a large amount of organic substances such as humus as well as microorganisms, and if the microparticles and microparticles or immobilization carrier are not sufficiently separated, these organic substances will be collected together with the microbes. To be done. Therefore, it is important to separate the microorganism from other unnecessary substances even when the nucleic acid such as DNA is collected from the microorganism. Furthermore, even when the microorganisms and other unnecessary substances can be separated by making full use of the above-mentioned centrifugation and filtration techniques, when the desired microorganisms are obtained in a mixed state of many kinds of microorganisms, for example, , DNA is extracted from the obtained microorganism sample, PCR amplification is performed,
DNA from the target microorganism by the hybridization method
In the case of detecting (1), since DNA other than the target microorganism is also amplified and mixed, there is a problem that it becomes difficult to obtain an accurate analysis result. Such a problem can be solved by culturing for screening the target microorganism using an agar medium or the like. However, when the above-mentioned microorganism is one that does not grow in a medium such as agar medium, it is impossible to separate it from other microorganisms by such screening, and when the growth is slow. Is time consuming to screen and is not efficient.

Therefore, microorganisms are directly separated and recovered from various places such as soil, activated sludge, bottom mud of rivers, etc.
In order to investigate the information of A, it is required to establish a method for collecting the target microorganism with high purity, that is, in a state as close to a single strain as possible, and easily.

An object of the present invention is to provide a method for selectively collecting and recovering microorganisms inhabiting from a system containing various solid substances such as soil, activated sludge and bottom sludge.

[0011]

The method for selecting and recovering microorganisms of the present invention includes a method by specific gravity gradient centrifugation and an electrophoresis method by which microorganisms are moved in a liquid phase by a DC voltage. Hereinafter, each of these methods will be described in detail.

In the method using the specific gravity gradient centrifugation, paying attention to the fact that the specific gravity of microorganisms varies depending on the type, a high-concentration suspension in which a plurality of microorganisms are mixed has a specific gravity gradient within a predetermined range of 1 or more. Centrifugation operation after overlaying on the solution, by collecting only the microorganisms collected in the portion of the specific gravity of interest, it was made based on the new knowledge of the present inventors that the target microorganisms can be selectively collected It is a thing.

The method using the specific gravity gradient centrifugation method is a method for selecting a plurality of microorganisms mixed in a sample, and is characterized in that each microorganism is fractionated by specific gravity gradient centrifugation and collected.

Conventionally, it takes several days to several weeks until a mixture of microorganisms is appropriately diluted and applied on an agar medium to form colonies, and it is impossible to separate and collect microorganisms that do not form colonies. There was an inconvenience. On the other hand, in the method of the present invention, it is possible to directly select the microorganisms by using the specific gravity gradient centrifugation, it is possible to omit the culture for a long time on the agar medium, and it is also possible to select the microorganisms that do not grow on the agar medium. Become.

The microorganisms to be separated in this method are systems in which a plurality of microorganisms are mixed, such as soil, activated sludge in sewage / wastewater treatment tanks, bottom mud in rivers / lakes / sea, digestive tracts of animals, etc. The contents, etc. can be mentioned.

The microorganisms of the present invention can be selected and recovered by suspending the samples sampled from these locations in an appropriate solution and subjecting them to specific gravity gradient centrifugation. In addition, in the preparation of the sample suspension, a pretreatment for removing unnecessary solids and the like by various separation methods such as filtration and centrifugation can be performed as necessary.

The specific gravity gradient can be formed, for example, by stacking high-density aqueous solutions having different specific gravities obtained by adding solutes at various concentrations to a solution in a centrifuge tube or the like. The gradient of the specific gravity may change stepwise in the direction in which centrifugal force is applied during centrifugation, or may change continuously.

As the solute used at this time, sucrose, cesium chloride, cesium sulfate, etryzamide, ficoll, etc., which are used in centrifuge centrifugation of DNA and the like can be used, and are dissolved in water in a large amount from 1.1 to 1. A material having a specific gravity of about 5 is desirable. You may use these in combination of 2 or more types.

When preparing a stepwise specific gravity gradient, prepare several kinds of aqueous solutions having an appropriate specific gravity between 1.1 and 1.5, and be careful not to mix them in order from the heaviest one to make them uniform. While stacking on the centrifuge tube. In addition, for example, a commercially available gradient maker can be used as it is to create the continuous specific gravity gradient. That is, put the aqueous solutions with specific gravities of 1.1 and 1.5 into two cylinders of a commercially available gradient maker used when preparing a gradient gel for protein electrophoresis, and pump with stirring with a stirrer or the like. Fill the centrifuge tube with. According to this manufacturer, the specific gravity of 1.
No. 5, then the specific gravity continuously changes depending on the mixing ratio of the two solutions, and finally one with a specific gravity of 1.1 comes in.

Specific gravity gradient centrifugation can be performed by placing the suspension of the sample on the uppermost layer of the specific gravity gradient and centrifuging.

The centrifugation conditions in the centrifuge are selected such that the centrifugation time and the number of revolutions required for collecting the desired microorganisms further increase,
For example, centrifugation conditions of several tens of thousands of rotations for 1 to 2 hours can be used. In addition, it is advisable to preliminarily examine the centrifugation conditions and in which layer the target microorganisms are collected under some conditions. After centrifugation, based on the results of the preliminary examination, only the portion where the target microorganisms are forming a layer is collected with a pipette or the like, diluted with distilled water at a volume ratio of about 10 times to reduce the specific gravity, and then again. The target microorganism can be recovered by centrifugation for about 10 minutes.

On the other hand, in the method of selecting and recovering microorganisms by electrophoresis, a sample region to which a sample is added and a sample region of the sample region are added in a liquid phase to which a DC voltage for moving the microorganisms can be applied from a pair of electrodes. A collection area is provided which communicates with the moving direction of the microorganisms through a first filter for selecting microorganisms, and a recovery area having a second filter for capturing microorganisms is provided on the electrode side for attracting the microorganisms, and a plurality of kinds are provided in the sample area. In a state in which the sample in which the microorganisms are mixed is dispersed, a DC voltage is applied from the pair of electrodes into the liquid phase, and the microorganisms that can pass through the first filter are moved from the sample area to the recovery area. After that, the microorganisms left in the sample area or the microorganisms moved to the recovery area are recovered.

Since the surface of the microorganism is charged in this method by electrophoresis, when the electrode is inserted into the suspension liquid in which the microorganism is suspended and a voltage is applied, it moves to either pole. Focusing on the fact that the size of the microorganisms differs depending on the type, a filter for selecting the microorganisms is provided in the migration path of the microorganisms, and the size of the microorganisms that have passed through the filter is determined according to the pore size of the filter. It is a method of selecting and collecting by.

[0024] According to this method, the culture for a long time in the conventional method can be omitted, and the microorganisms that do not form colonies on the agar medium can be separated and recovered, as in the method using the specific gravity gradient. Becomes

An example of a method of utilizing this electrophoresis will be described below with reference to the drawings.

FIG. 1 is a sectional view showing a main part of an example of the apparatus for selecting microorganisms according to the present invention. This device has a sample area (sample suspension area) 6 partitioned by a first filter 4 for selecting microorganisms inside a water tank (electrophoresis tank) 1 provided with a pair of electrodes 2 and 3 for applying a DC voltage. And a recovery area 7 are provided. The sample area 6 and the recovery area 7 are in communication with each other through the first filter, and are arranged in the moving direction of the microorganisms that move when a DC voltage is applied to the electrodes 2 and 3 (direction from the electrodes 2 to 3). Has been done. In addition, in the case of FIG.
Although the electrode 3 is an electrode that attracts microorganisms, if there are microorganisms that move in the direction of the electrode 2, a second recovery region via a filter for microorganism selection may be provided on the electrode 2 side as well.

The first filter 4 is set to have a pore size that allows the target microorganisms to be recovered in the recovery area to pass therethrough, but does not allow larger impurities such as larger microorganisms and soil particles to pass through. The second filter is for capturing the microorganisms that have moved into the collection area 7 in the area and cannot pass the microorganisms, but can pass the electrolyte and minute impurities such as fine particles. Further, the filter 8 is also a filter which can prevent the passage of microorganisms but allows the passage of the electrolytic solution and the like, and separates the arrangement region 9 of the electrode 2 and the sample region 6. In the configuration of FIG. 1, each divided area of the water tank 1 communicates only with a filter.

The first filter 4 has a pore size that allows passage of desired microorganisms, maintains its shape even when immersed in water such as nitrocellulose, and becomes a good conductor of electricity when impregnated with an aqueous electrolyte solution. For example, a commercially available product having a pore size of 0.2 μm or more can be appropriately selected and used. Further, as the filters 5 and 8 provided on the electrodes 2 and 3, those having a pore size of less than 0.2 μm and gel thin films such as semipermeable membranes and agarose that do not allow passage of microorganism-sized substances are suitable. Further, since the filter 8 is for preventing the sample from mixing with the electrolytic solution in the region 9 on the side of the electrode 2, it may be an ordinary filter paper or a glass fiber filter paper which can achieve such an object.

First filter 4 and second filter 5
If the distance is too long, the separation efficiency decreases, so the shorter the distance, the better as long as there is no contact, for example, about 0.5 to 1 cm.

In the apparatus for selecting microorganisms of the present invention, the collection area can be further divided into two or more sections by one or more filters, and the selection according to the size of finer microorganisms can be performed. In the apparatus shown in FIG. 2A, the collection area 7 is divided into two sections 7-1 and 7-2 by a filter 10 for sectioning.
The classification filter 10 has a pore size smaller than that of the first filter 4, and the microorganisms that have moved to the recovery area 7 are classified into two types according to the size thereof. To be selected. That is,
Of the microorganisms that have moved to the collection area 7-1, microorganisms larger than the sorting filter 10 are left in the collection area 7-1.
Microorganisms that have passed through the sorting filter 10 are collected in the recovery area 7-
Captured by 2. As described above, the partitioning filter 10 has a function of selecting microorganisms according to their size, like the first filter 4, and maintains its shape even when immersed in water such as nitrocellulose, and has an electrolyte aqueous solution. A substance that becomes a good conductor of electricity when soaked is used, and for example, it can be appropriately selected and used from commercially available substances having a pore size of 0.2 μm or more. Further, if the spacing between the filters 4, 10 and 5 is too large, the separation efficiency will be poor, so it is better to be as small as possible, for example about 0.5 to 1 cm. In the apparatus of FIGS. 1 and 2, a portion having a sample area and a recovery area may be detachably provided in the water tank 1 as a rack. FIG. 2B shows an example of the case where the rack 11 for electrophoresis is configured.

Next, an example of a method for selecting and collecting microorganisms using the apparatus having the configuration shown in FIG. 2 will be described below.

First, a sample containing the target microorganism is suspended in the sample area 6. As the sample, for example, an aqueous solution or a solid substance in which a plurality of microorganism groups are present is used, and specifically, a medium in which a plurality of microorganism groups are growing, soil, activated sludge,
Examples include suspensions such as bottom mud and the contents of the digestive tract of animals. In addition, in the preparation of the sample suspension, a pretreatment for removing unnecessary solids and the like by various separation methods such as filtration and centrifugation can be performed as necessary.

This sample is suspended in an aqueous solution of an electrolyte that conducts electricity such as a buffer solution used in electrophoresis of DNA or protein, or in the case of a suspension originally, an appropriate electrolyte is added to conduct electricity. It is made into an aqueous solution and injected into the sample region 6. Alternatively, the sample region 6 may be filled with an electrolyte solution, and the sample may be added to and suspended in the sample region 6. As an electrolyte used at this time, a substance that lyses or deactivates microorganisms by extremely lowering or raising pH is not suitable.

The amount of microorganisms suspended in the sample area 6 is 1
If the amount is too small, such as 0 ⁴ pieces / ml or less, the efficiency of collection will be poor, but if it is too high, the filter will be clogged. Therefore, 10 per cm ^{2 of} filter
It is desirable that the number be ¹⁰ or less.

In a state where the sample area is filled with the electrolytic solution in which the sample is suspended, and the other area of the water tank is filled with the electrolyte solution, under the condition that the target microorganisms can be selectively collected.
A DC voltage is applied from the electrodes 2 and 3. The voltage is selected from the range of, for example, several tens to several hundreds of volts and is applied for several hours. At this time, the relationship between voltage and time is inversely proportional, but if the voltage is extremely high, the microorganisms will die or lyse due to the heat generated by the voltage and the suspension. It is advisable to preliminarily examine whether the activity of will be lost. If a large amount of negatively charged soil particles or other contaminants are present in the suspension,
If the suspension is allowed to stand in the sample area 6 for about 10 minutes to precipitate impurities and then electrophoresis is started, clogging of the filter can be prevented. After migration, the microorganisms are attached to each filter, so the surface of the negative electrode of the filter should be rubbed carefully,
After the reverse voltage is applied for several minutes to remove the deposits, the target microorganisms are recovered from the microorganisms left in the recovery area 6 and the microorganisms transferred to the recovery areas 7-1 and 7-2.

[0036]

EXAMPLES The present invention will be described below with reference to examples. Example 1 Escherichia coli was placed in an LB liquid medium having the composition shown in Table 1 below and shake-cultured overnight in a temperature-controlled room at 37 ° C. for growth, and then the culture was concentrated to prepare a milky white suspension. As shown in FIG. 3, the specific gravity is 1.20, 1.
Put the sucrose aqueous solution having a concentration of 25 and 1.30 in three steps at the top of the ultracentrifuge tube having a volume of 5 ml.
It was centrifuged at 10,000 rpm at 20 ° C. for 4 hours.

As a result, as shown in FIG. 3, E. A milky white suspended band of E. coli was formed.

[0038]

[Table 1] Example 2 Burkho in LB liquid medium at 37 ° C. as in Example 1.
lderia cepacia KK01 strain (FERM
A milky white suspension obtained by concentrating a culture obtained by culturing BP-4235) (previously classified into the genus Pseudomonas ) was subjected to sucrose gradient gravity centrifugation in the same manner as in Example 1.

As a result, as shown in FIG. 3, B. cepacia KK0
A concentrated milky white suspended band of 1 strain resulted. Example 3 Methyl was added to LB liquid medium at 37 ° C. as in Example 1.
The orange suspension obtained by concentrating the culture obtained by culturing osinus trichosporium was subjected to sucrose specific gravity gradient centrifugation in the same manner as in Example 1.

As a result, as shown in FIG. 3, M. An orange suspension band accumulating trichosporium resulted. Example 4 Saccharomyces cere grown by shaking culture at 37 ° C. for 2 days in a PYG liquid medium shown in Table 2 below.
The ocher suspension obtained by concentrating the culture of visiae (yeast) was subjected to sucrose gravity gradient centrifugation in the same manner as in Example 1.

As a result, as shown in FIG. 3, S. An ocher precipitate of cerevisiae formed.

[0042]

[Table 2] Example 5 E. coli (pUC19 (Takara Shuzo Co., Ltd. about 1k
(Introduced one into which a bp DNA fragment has been inserted), B. cepacia KK01 strain, M. tri
chosporium and S. cerevisiae was cultivated in each of the bacterial culture media described in Examples 1 to 4, and the obtained cultures were concentrated and mixed to prepare a microbial suspension, which was prepared in the same manner as in Example 1. Sucrose specific gravity gradient centrifugation was performed. As a result, three suspended zones A, B and C and a precipitate D were generated as shown in FIG.

Next, the zones A, B, and C were separately collected with a pipette, the sucrose concentration was reduced with distilled water, and the mixture was centrifuged to collect and collect microorganisms to obtain samples A, B, and C. Also,
After discarding all of the supernatant aqueous sucrose solution, the precipitate D was suspended in distilled water and centrifuged again to collect and collect the microorganism, which was designated as sample D.

Each sample was transferred to a microtube having a volume of 1.5 cc, suspended in 500 μl of distilled water, applied to an LB agar medium, and left in a thermostatic chamber at 37 ° C.

As a result, the agar medium coated with the sample A had an M. Only orange-colored colonies with the same shape as trichosporium were formed, and other colonies were not formed even when left to stand thereafter. Two days later the sample B to the applied agar medium B. cepacia KK
Only milky white colonies with the same shape as the 01 strain are formed,
After that, no other colony was formed even if left standing.
For the agar medium coated with sample C, E. coli
Only a milky white colony of the same shape was formed, and other colonies were not formed even if it was left to stand thereafter. For the agar medium coated with sample D, S. cerevis
Only the same ocher colonies as iae were formed. From these, it was confirmed that Samples A to D were all composed of a single microorganism.

Further, the same operations as above are repeated to prepare samples A to D, which further contain the above-mentioned plasmid.
E. E. coli cells were used as sample E. Transfer each sample to a microtube with a volume of 1.5 cc.
10 μl of a 10% SDS solution was added to the product suspended in 0 μl of distilled water and heated at 70 ° C. for 1 hour to lyse the microorganisms in the solution. Next, DNA was extracted from each solution using a phenol-chloroform solution, precipitated with ethanol and collected. This DNA was dissolved in 50 μl of distilled water to obtain microbial DNA solutions A to E.

These DNA solutions were diluted 100-fold and then annealed with this and the above plasmid to obtain about 1 kbp.
For amplifying the DNA fragment of M13 (M13 primer M4
And M13 primer RV: both Takara Shuzo Co., Ltd., PCR
After mixing the enzymes for use so as to have the composition shown in Table 3, PCR amplification was carried out under the temperature conditions shown in Table 4 (temperature cycles in the order of). After amplification, the reaction mixture was electrophoresed on an agarose gel, and only DNA solutions C and E had a D of about 1 kbp.
NA fragment was detected. That is, 4 of DNA solutions A to D
E., which has the same plasmid as E. E. coli is present only in sample C, and E. It was confirmed that E. coli was not mixed.

[0048]

[Table 3]

[0049]

[Table 4] Example 6 E. E. coli was placed in an LB liquid medium having the composition shown in Table 1 above, shake-cultured overnight in a constant temperature room at 37 ° C. to grow, then concentrated and suspended in 20 cc of the electrophoresis buffer solution having the composition shown in Table 5 below. Then, a milky white suspension was prepared. This suspension was put in the sample area (suspension area) 6 of the electrophoresis rack 11 shown in FIG. In this example, a nitrocellulose membrane filter (Advantech Toyo Co., Ltd.) having the following pore size was used.

First filter (4) ··· 5.0 μm Classification filter (10) ··· 0.8 μm Second filter (5) ··· 0.2 μm Electrode 2 side filter (8) · -0.2 μm 10 cc of buffer solution was injected into each of the collection areas 7-1 and 7-2, and the rack was placed in the electrophoresis tank 1 containing about 200 cc of electrophoresis buffer solution in advance as shown in FIG. 2 (a). 1
1 was set and a DC voltage of 200 V (about 50 mA) was applied (electrode 2 was a negative electrode and electrode 3 was a positive electrode).

After 1 hour, the microorganisms adhering to the left side of each of the three filters 4, 10 and 5 were thoroughly scraped off, and then 10 cc of liquid at the position of the filter 4 in the sample area 6 and the recovery area 7 About 10 cc of each of the buffer solutions of -1, 7-2 were collected and centrifuged.
A milky white precipitate was observed at the bottom of the centrifuge tube of the sample from E. It was confirmed that the E. coli was collected only in the collection area 7-1.

[0052]

[Table 5] Example 7 In the same manner as in Example 6, M. tri
The culture broth in which chosporium was cultivated was concentrated and suspended in 20 cc of the buffer for electrophoresis to prepare an orange suspension. This suspension was electrophoresed in the same manner as in Example 1.

After 1 hour, the solution was recovered and centrifuged in the same manner as in Example 1. As a result, an orange precipitate was observed at the bottom of the centrifuge tube of the sample from the recovery area 7-2, and M. trich
It was confirmed that osporium gathered only in the recovery area 7-2. Example 8 S. cerevisiae grown in the PYG liquid medium shown in Table 2 above by shaking culture at 37 ° C. for 2 days. cerevisiae (yeast) was concentrated, and 20 cc of electrophoresis buffer having the composition shown in Table 5 was used.
To produce an ocher suspension. This suspension was electrophoresed in the same manner as in Example 6.

[0054] After 1 hour in Example 1 and was centrifuged to recover the liquid in the same manner, the precipitate ocher is observed Centrifuge bottom of the sample from the sample region 6, S. cerevisia
It was confirmed that e gathered on the positive electrode side of the suspension tank A. Example 9 E. coli (pUC19 similar to that used in Example 5)
Which has a DNA fragment of about 1 kbp inserted thereinto), M. trichosporium and S. c
erevisiae was cultivated in each of the bacterial culture media described in Examples 6 to 8, and the obtained cultures were concentrated and mixed to prepare a microbial suspension, which was prepared in the same manner as in Example 6. Electrophoresed.

After 1 hour, the liquid was collected and centrifuged in the same manner as in Example 1, and the resulting precipitates were sample A (collected from sample area 6), B (collected from collection area 7-1), C (collection area 7).
-2).

Each of the samples A to C has a volume of 1.5 c.
After transferring to a microtube of c and suspending in 500 μl of distilled water, it was applied to LB agar medium and left in a constant temperature room at 37 ° C.

As a result, after 5 days, S. Only ocher colonies with the same shape as S. cerevisiae were formed. For the agar medium coated with sample B, E. Only a milky white colony having the same shape as E. coli was formed, and other colonies were not formed even if it was allowed to stand thereafter. The agar medium coated with Sample C had M. Only orange-colored colonies with the same shape as trichosporium were formed, and other colonies were not formed even when left to stand thereafter. From these, it was confirmed that A to C are all composed of a single microorganism.

Next, by repeating the above-mentioned operation, samples A to
C. was prepared and E. col
The microbial cell of i was used as sample D. Volume of each sample is 1.5
10 μl of a 10% SDS solution was added to the suspensions in 500 μl of distilled water and suspended in 500 μl of distilled water, and the mixture was heated at 70 ° C. for 1 hour to lyse microorganisms in the solution. Next, from each solution, use phenol-chloroform solution
Was extracted, precipitated with ethanol and collected. This DN
Microbial DNA solution A by dissolving A in 50 μl of distilled water
~ D.

After diluting these DNA solutions by 100 times, PCR enzymes were mixed to carry out PCR amplification.
The PCR conditions were the same as in Example 5. After amplification
When the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1 kbp was detected only in DNA solutions B and D.
That is, among the four samples A to D, E. coli having the same plasmid as D. Only sample B has coli .
A and C are E. It was confirmed that E. coli was not mixed.

[0060]

As described above, the following effects were obtained by selecting and collecting a plurality of microorganism groups by using the centrifugal gradient method or the electrophoresis method. 1) Suspension without the operation of preparing an agar medium under various expected or expected conditions, applying a microbial suspension and culturing for several days like the plate culture method Various microorganisms inside can be easily sorted and collected according to specific gravity in a few hours. 2) In the case of the plate culture method, colonies cannot be formed because the culture conditions are not suitable even though they are useful microorganisms, and as a result, microorganisms that could not be recovered can be selectively collected. 3) Unlike the suction filtration method, it is not necessary to frequently replace the filter paper due to clogging with solid matter, and once the filter is set, the microorganisms can be selectively collected by simply applying voltage. 4) Unlike the centrifugation method, it is not necessary to finely control the centrifuge by preliminarily investigating the slight difference in the centrifugation conditions due to the difference in the type of microorganisms. If a filter with an appropriate pore size is set, the voltage will be applied afterwards. Microorganisms can be selected and collected only by themselves. 5) D lysed and extracted from microorganisms recovered by this method
NA has such a purity that an enzymatic reaction such as a PCR amplification reaction can be performed without further treatment for removing impurities.

[Brief description of drawings]

FIG. 1 shows a schematic cross-sectional view of an example of a microorganism selection device of the present invention.

FIG. 2 shows the structure of another example of the microorganism selection device of the present invention, (a) is a schematic sectional view of the entire water tank, and (b) is a schematic sectional view of an electrophoresis rack.

FIG. 3 is a schematic cross-sectional view showing a state in a centrifuge tube for explaining an operation in the method of the present invention by a centrifugal gradient method.

[Explanation of symbols]

 1 Water tank (electrophoresis tank) 2,3 Electrode 4 First filter (for microorganism selection) 5 Second filter (for microorganism capture) 6 Sample area (suspension area) 7,7-1,7-2 Recovery Area 8 Filter 9 Electrode 2 side area 10 Classification filter (for microbial selection) 11 Electrophoresis rack

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[Procedure amendment]

[Submission date] January 31, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction content]

[0038]

[Table 1] Example 2 Pseudo was added to LB liquid medium at 37 ° C. as in Example 1.
monas cepacia KK01 strain (FERM
The milky white suspension obtained by concentrating the culture obtained by culturing BP-4235 ) was subjected to sucrose specific gravity gradient centrifugation in the same manner as in Example 1.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

As a result, as shown in FIG. 3, at the boundary between 1.20 and 1.25, P. cepacia KK0
A concentrated milky white suspended band of 1 strain resulted. Example 3 Methyl was added to LB liquid medium at 37 ° C. as in Example 1.
The orange suspension obtained by concentrating the culture obtained by culturing osinus trichosporium was subjected to sucrose specific gravity gradient centrifugation in the same manner as in Example 1.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction content]

[0042]

[Table 2] Example 5 E. coli (pUC19 (Takara Shuzo Co., Ltd. about 1k
bpDNA fragment inserted thereinto), P. cepacia KK01 strain, M. tri
chosporium and S. cerevisiae was cultivated in each of the bacterial culture media described in Examples 1 to 4, and the obtained cultures were concentrated and mixed to prepare a microbial suspension, which was prepared in the same manner as in Example 1. Sucrose specific gravity gradient centrifugation was performed. As a result, three suspended zones A, B and C and a precipitate D were generated as shown in FIG.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0045

[Correction method] Change

[Correction content]

As a result, the agar medium coated with the sample A had an M. Only orange-colored colonies with the same shape as trichosporium were formed, and other colonies were not formed even when left to stand thereafter. For the agar medium coated with sample B, P. cepacia KK
Only milky white colonies with the same shape as the 01 strain are formed,
After that, no other colony was formed even if left standing.
For the agar medium coated with sample C, E. coli
Only a milky white colony of the same shape was formed, and other colonies were not formed even if it was left to stand thereafter. For the agar medium coated with sample D, S. cerevis
Only the same ocher colonies as iae were formed. From these, it was confirmed that Samples A to D were all composed of a single microorganism.

Claims (20)

[Claims] 1. A method for selecting and collecting a plurality of microorganisms mixed in a sample, which comprises fractionating each microorganism according to specific gravity by specific gravity gradient centrifugation and collecting the microorganisms. 2. The method for selecting and recovering microorganisms according to claim 1, wherein the specific gravity gradient is prepared by dissolving a solute in water in a range of one or more specific gravities. 3. The method for selecting and collecting microorganisms according to claim 1, wherein the specific gravity gradient is such that the specific gravity changes continuously. 4. The method for selecting and collecting microorganisms according to claim 1, wherein the specific gravity gradient is such that the specific gravity changes stepwise. 5. The method according to claim 1, wherein the solute comprises at least one selected from sucrose, cesium chloride, cesium sulfate, metrizamide and ficoll. 6. The method for selecting and recovering a microorganism according to claim 1, wherein the sample is a mixed culture solution of a plurality of kinds of microorganisms. 7. The method according to claim 1, wherein the sample is a soil suspension.
5. The method for selecting and collecting microorganisms according to any one of 5 above. 8. The method for selecting and collecting microorganisms according to claim 1, wherein the sample is activated sludge in a sewage / wastewater treatment tank. 9. The method for selecting and recovering microorganisms according to claim 1, wherein the sample is bottom mud of a river, lake, sea or the like. 10. The method for selective collection of microorganisms according to claim 1, wherein the sample is the contents of the digestive tract of an animal. 11. A sample region to which a sample is added in a liquid phase to which a DC voltage for moving microorganisms from a pair of electrodes can be applied, and a first region for selecting microorganisms in the moving direction of the sample region and the microorganisms. A state in which a sample which is in communication with the first filter and which has a second filter for capturing microorganisms is provided on the electrode side for attracting microorganisms, and a sample in which a plurality of types of microorganisms are mixed is dispersed in the sample region. Then, a direct current voltage is applied to the liquid phase from the pair of electrodes, and the microorganisms that can pass through the first filter are moved from the sample area to the recovery area, and then the microorganisms left in the sample area. Alternatively, a method of selecting and recovering a microorganism is characterized by recovering the microorganism that has moved to the recovery area. 12. The recovery area is divided into two or more by providing one or more dividing filters between the first filter and the second filter, and the pore diameters of the first filter and the dividing filter are selected. The method for selecting and recovering microorganisms according to claim 11, wherein the microorganisms are sequentially reduced in size in the moving direction of the microorganisms according to the size of the microorganisms. 13. The method for selecting and collecting microorganisms according to claim 11, wherein the electrode for attracting the microorganisms is a positive electrode. 14. The method for selecting and collecting microorganisms according to claim 11, wherein the sample is a mixed culture solution of a plurality of kinds of microorganisms. 15. The sample according to claim 1, which is a soil suspension.
The method for selecting and collecting microorganisms according to any one of 1 to 13. 16. The method for separating and collecting microorganisms according to claim 11, wherein the sample is activated sludge in a sewage / wastewater treatment tank. 17. The method for selecting and recovering microorganisms according to claim 11, wherein the sample is sediment of river, lake, sea or the like. 18. The method for selective collection of microorganisms according to claim 11, wherein the sample is the contents of the digestive tract of an animal. 19. A tank capable of forming a liquid phase, a pair of electrodes for applying a DC voltage for moving microorganisms in the liquid phase, and a liquid containing a sample containing a plurality of types of microorganisms. A collection region that communicates with the sample region to be added in the moving direction of the microorganisms in the sample region through a first filter for selecting microorganisms, and has a second filter for capturing microorganisms on the side of an electrode that attracts microorganisms. A device for selecting a microorganism, comprising: 20. One or more dividing filters are provided between the first filter and the second filter to divide the recovery region into two or more, and the pore diameters of the first filter and the dividing filter are: 20. The apparatus for selecting microorganisms according to claim 19, which is set so as to become smaller in order in the moving direction of the microorganisms according to the size of the microorganisms to be selected.