JP2005137288A - Apparatus and method for screening microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening apparatus enabling an unknown microorganism to be isolated in high probability with high throughput of markedly improved efficiency, and to provide a method for screening such a microorganism using the apparatus. <P>SOLUTION: The apparatus for screening microorganism comprises a means for individually isolating and/or arranging a naturally occurring unknown or known microorganism. Preferably, the means is an extruder comprising a vessel for holding a liquid with the microorganism dispersed therein and a head communicating with the vessel and functioning to extrude the liquid as droplets or a continuous fluid onto a medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel microorganism screening apparatus and a microorganism screening method. More specifically, the present invention relates to a microorganism screening apparatus and a microorganism screening method capable of separating and identifying unknown microorganisms with high probability, with high throughput with greatly improved efficiency.

In nature, there are an enormous variety and number of unknown microorganisms, including microorganisms contained in soil. Among these microorganisms, search for useful microorganisms such as drug discovery materials, food and chemical products, environmental purification and environmental sensing materials, and the like has been conducted. The search (screening) process includes collection of microorganisms, pretreatment (impurity separation, concentration, dilution, dispersion in a culture solution), placement on a medium, culture, assay, and the like. These processes are mostly done manually and require a great deal of manpower, time and money. Specific examples of microbial screening procedures using existing technology are as follows.

Take a microbial sample from soil (generally containing 10 ⁵ to 10 ⁹ microorganisms / g), add water to prepare a suspension sample, and dilute (in a test tube with a volume of about 5 ml) Perform multi-step dilution). Thereafter, several ml of the suspension sample is applied onto a medium selected in advance according to the target microorganism, and cultured by being kept warm and moisturized. In addition, the container which put the agar culture medium is about 10 cm in diameter, and a culture | cultivation period is 1 to 7 days normally. After sufficiently culturing, the number of colonies is visually counted to isolate the strain. For this bacterial strain separation, the colony is brought into contact with the tip of a sterile needle, and the sample adhering to the tip of the sterile needle is suspended and stirred with the culture solution in the test tube, or the sample is applied to the surface of the agar medium by the same method as above. Culture. A pure strain is obtained through these steps, and then an evaluation test of the strain is performed.

  According to such a conventional method, a very large number and types of microorganisms are mixed in the screening sample. Therefore, the mixed microorganisms adversely affect each other's proliferation and growth, and the probability of separating (identifying) one microorganism is extremely small. And most of the process has to be done manually. In fact, according to the prior art, most of the separated microorganisms are known microorganisms (that is, unknown microorganisms can hardly be separated and identified), despite a great deal of labor, time and expense. Generally, it is said that 90% or more of microorganisms existing in nature fail to be separated and identified. In other words, it is said that only a few percent of the microorganisms that exist in nature have been separated and identified so far (that is, very many useful microorganisms exist in nature without being known. It is considered).

  By the way, in the field of new drug development, for example, processes such as culture / screening, synthesis and toxicity evaluation of a huge number of unknown microorganisms and their enzymes existing in nature such as bacteria and viruses are required. As described above, since the screening efficiency of the conventional technology is extremely low, it generally takes 15 to 20 years and development costs of 15 to 20 billion yen to develop one new drug. It is said. Furthermore, in this field, new drugs such as antibiotics that have been newly developed have been unable to sustain their effects due to the emergence of drug-resistant bacteria in a short period of time. The development of new antibiotics and competition with resistant bacteria will continue.

  As described above, there is a strong demand for the development of screening methods for innovative microorganism search with high efficiency and high probability.

  The present invention has been made to solve the above-described conventional problems, and the object of the present invention is high-throughput with greatly improved efficiency and capable of separating unknown microorganisms with high probability. A screening apparatus and a microorganism screening method are provided.

As a result of diligent studies to achieve the above object, the present inventor separated and / or arranged the collected microorganisms individually on a medium together with a culture solution, etc. The inventors have found that screening can be performed with high probability, and have completed the present invention.

  According to one aspect of the present invention, a microorganism screening apparatus is provided. This device comprises means for individually separating and / or arranging unknown or known microorganisms present in nature.

According to the above configuration, since the microorganisms can be individually placed on the medium, there is no adverse effect due to other microorganisms of the same type and toxins produced by other types of microorganisms, and there is no competition between the microorganisms for competing for nutrients. Can be cultured very purely. As a result, culture and / or screening under more favorable nutritional conditions and environmental conditions becomes possible, and the separation probability of unknown microorganisms is significantly increased as compared to the conventional case.

In a preferred embodiment, the means for separating and / or arranging the microorganisms comprises a holding container for holding a liquid in which microorganisms are dispersed, and the holding container communicated with the holding container, and the liquid is formed as a droplet or a continuous fluid on the medium. A discharger having a discharge head for discharging.

According to the above configuration, even when various microorganisms are dispersed in the liquid, each microorganism can be individually separated and / or arranged only by discharging the liquid onto the medium. .

In a preferred embodiment, the dispenser is an applicator selected from a dispenser device, an ink jet device and a spotter device, or an applicator selected from a spray, spray, nebulizer and atomizer.

According to the above configuration, it is possible to apply or apply a minute amount of liquid with high accuracy and high reproducibility, without causing damage such as impact to microorganisms, and efficiently at high speed. In addition, these devices can sterilize the wetted part relatively easily and are not easily affected by foreign bacteria, so that culture and / or screening under very favorable nutritional and environmental conditions is possible.

In a preferred embodiment, the device can control the volume of the droplet from 1 femtoliter to 1 microliter.

  According to the above configuration, by controlling the volume of the droplet, the number of microorganisms contained in the droplet can be controlled to be substantially one with much higher accuracy than before. As a result, culture and / or screening under highly favorable nutritional and environmental conditions is possible.

In a preferred embodiment, the means for separating and / or arranging the microorganisms has means for independently moving at least one of the discharge head and the medium in two orthogonal directions.

  According to said structure, it becomes possible to isolate | separate and / or arrange | position the microorganisms of many types and many in high speed individually by the arbitrary patterns in the arbitrary positions on a medium.

According to another aspect of the present invention, a method for screening a microorganism is provided. This method comprises the steps of dispersing unknown or known microorganisms existing in nature in a liquid, discharging the liquid as a droplet or continuous fluid onto a medium, and separating and / or arranging the microorganisms individually. Including.

According to the above configuration, since the microorganisms can be individually placed on the medium, there is no adverse effect due to other microorganisms of the same type and toxins produced by other types of microorganisms, and there is no competition between the microorganisms for competing for nutrients. Can be cultured very purely. As a result, culture and / or screening under more favorable nutritional conditions and environmental conditions becomes possible, and the separation probability of unknown microorganisms is significantly increased as compared to the conventional case.

  In a preferred embodiment, the concentration of the liquid is adjusted so that there is substantially one microorganism in the droplet.

  According to said structure, it becomes possible to isolate | separate for every microorganism only by apply | coating on a medium, without providing a special separation means. Furthermore, by changing the concentration, it is possible to arbitrarily change the number of microorganisms that can be separated and / or arranged on the medium.

In a preferred embodiment, the volume of the droplet is adjusted so that there is substantially one microorganism in the droplet.

  According to said structure, it becomes possible to isolate | separate for every microorganism only by apply | coating on a medium, without providing a special separation means. Furthermore, by changing the volume of the ejected droplets, it is possible to arbitrarily change the number of microorganisms that can be separated and / or arranged on the medium.

  In a preferred embodiment, the medium is a flat plate made of a material selected from glass, ceramics, metal, plastic, carbon agent, natural organic matter, fiber, fine cloth and non-woven cloth, and having irregularities on the surface.

  According to said structure, isolation | separation and / or arrangement | sequence of microorganisms on a medium are performed comparatively easily. Furthermore, such a medium is easy to maintain nutrient conditions and environmental conditions that are easy to culture for microorganisms, and culture and screening are relatively easy. In addition, by using a medium having irregularities on the surface, the microorganisms enter the recesses along with the flow of the liquid, so that the microorganisms can be well separated individually by the liquid crosslinking force. The liquid cross-linking force is also referred to as a liquid adhesion force, and in this case, the liquid that has been capillary-aggregated at the contact portion between the microorganism and the inner wall of the recess is a force that attracts both. The liquid crosslinking force is expressed by the surface tension of the liquid + the capillary negative pressure.

In a preferred embodiment, the medium has a hydrophobic region and a hydrophilic region.

  According to said structure, according to the state (for example, aqueous solution type | system | group, organic solvent type | system | group, oil type) of discharge liquid, a charged state (for example, positive, negative, charge amount) or physical property, or the charged state or physical property of microorganisms itself. Thus, the microorganisms can be well separated and / or arranged individually.

In a preferred embodiment, the medium has a charged area and an uncharged area.

  According to the above configuration, the microorganisms can be well separated and / or arranged in the charged area or the uncharged area on the medium according to the discharge state of the discharge liquid or the microorganism itself.

In a preferred embodiment, the medium has electrodes formed in a predetermined pattern.

According to the above configuration, by applying a predetermined voltage to the electrode pattern and controlling the electric field, the microorganisms are separated individually better according to the properties of the discharge liquid or the microorganism itself, such as the charge state and dielectric constant. And / or can be arranged.

In a preferred embodiment, the liquid is tailored to have an environment in which certain microorganisms can grow, grow, survive, degrade and / or synthesize.

  According to the above configuration, by using a liquid (typically a culture medium) adjusted to an optimum environment for the target microorganism species, the microorganisms can be propagated, grown and survived after being discharged onto the medium. Allows to control and screen.

  In a preferred embodiment, the medium is tailored to have an environment in which a particular microorganism can grow, grow, survive, degrade and / or synthesize.

  According to the above configuration, by using a medium adjusted to an optimum environment for the target microorganism species, it is possible to control and screen the proliferation, growth and survival of microorganisms after being discharged onto the medium. To do.

In a preferred embodiment, the method further includes a step of charging the liquid before the discharging step.

According to the above configuration, even when the discharge liquid and / or the microorganism itself has a small charge amount or a mixture of microorganisms having different charges, the microorganisms are well separated and / or arranged individually. It becomes possible.

  Preferred embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited to these embodiments.

A. Explanation of Terms Terms and expressions used in this specification have the following meanings.

“Individually” means substantially one by one. More specifically, even if a large number of various types of microorganisms are contained in the sample liquid, the microorganisms are substantially contained in each independent part (part corresponding to the droplet) of the liquid applied on the medium. This means that there is only one.

“Separation” means taking out one kind of microorganism from many kinds of microorganisms.

“Arrangement” refers to arranging sample liquids (resulting in microorganisms) in an arbitrary pattern at an arbitrary position on a medium.

“Coating” means that fine droplets are ejected and adhered to a medium in a spot shape.

“Applying” means that fine droplets are sprayed to adhere to a medium in a thin film shape so that the droplets do not substantially overlap each other.

“Microorganism” means any unknown and known minute organism (typically an organism having a size of 5 micrometers or less) existing in nature, and includes bacteria, fungi, protozoa, viruses, and the like. .

“Liquid” means any liquid that can be used for screening microorganisms, including water, culture solution, separation solution, dilution solution, organic solution, organic solvent, polymer gel solution, colloidal dispersion solution, aqueous electrolyte solution, inorganic solution. It includes an aqueous salt solution, an aqueous metal solution, an aqueous solution to which a surfactant for microbial dispersion is added in a small amount, an aqueous solution containing an osmotic pressure adjusting agent, and a mixed liquid thereof.

“Substantially one microorganism is present in the ejected droplet” means that the microorganism can proliferate and grow substantially without being affected by other microorganisms of the same type and / or other types of microorganisms. Means in the droplet.

B. Screening Device FIG. 1 is a schematic perspective view of a microorganism screening device according to a preferred embodiment of the present invention. This screening apparatus 100 includes means for individually separating and / or arranging microorganisms. In the embodiment of FIG. 1, the separating / arranging means is a discharger 20. Typical examples of the discharger 20 include applicators such as dispenser devices, ink jet devices, and spotter devices, and applicators such as atomizers, sprayers, nebulizers, and atomizers. Hereinafter, the case where the discharger 20 is an inkjet device will be described.

The discharger 20 communicates with the holding container 2 (syringe in the illustrated example) 2 that holds the sample liquid in which the microorganisms are dispersed and the holding container 2 through the needle unit 3, and the liquid in which the microorganisms are dispersed as droplets or And a discharge head 4 for discharging onto the medium 1 as a continuous fluid. The syringe 2 and the needle unit 3 are provided with any appropriate temperature mechanism (coil heater in the illustrated example) 5 and can appropriately adjust the temperature of the sample liquid according to the purpose.

  Any appropriate ink jet head can be adopted as the ejection head 4. An example of the ink jet head will be described with reference to FIGS. FIG. 2 is a schematic perspective view of the head 4, and a cross section is shown by cutting a part for explanation. The nozzle 210 is disposed through the nozzle forming substrate 212 at a position communicating with the liquid chamber 211. The pressure generating member 213 contacts and presses one of the walls of the liquid chamber 211 formed of the elastic plate 214. The pressure generating member 213 is typically a piezoelectric element in which a piezoelectric material 41 such as lead zirconate titanate and internal electrodes 42a and 42b such as silver palladium are laminated. The elastic plate 214 is disposed so as to cover the entire surface of the liquid chamber, and is configured by a film having a uniform thickness. Typical examples of the material of the elastic plate 214 include metals such as Ni electroforming and stainless steel, or polymer films such as polyimide and polysulfone.

  FIG. 3 is a perspective view for explaining a contact state between the drive unit 31 and the liquid flow path 32. A case 215 is joined to the pressure generating member 213, and an end surface 216 that contacts the elastic plate 214 of the pressure generating member 213 and an end surface 217 of the case 215 maintain a constant distance relationship of a distance g in the displacement direction. As shown in FIG. 2, the end face 217 of the case 215 and the surface 218 of the elastic plate 214 come into contact with each other at the time of joining, so that the pressure generating member 213 is uniformly pushed into the elastic plate 214. FIG. 4 is a cross-sectional view illustrating a liquid discharge state. The liquid 51 in which the microorganisms are dispersed has reached the nozzle 210, and a meniscus 53 is formed by the pressure of the liquid 51. When the pressure generating member 213 is uniformly pushed into the elastic plate 214, the elastic plate 214 is deformed, and the meniscus 53 protrudes due to the pressure. As a result, the liquid 51 becomes a droplet 54 and flies from the nozzle 210 toward the medium 1.

  The head 4 may have a single nozzle configuration or a configuration having a plurality of nozzles (so-called multihead) depending on the purpose. By configuring a multi-head, screening efficiency (speed) can be remarkably improved. The head 4 is preferably the piezo method as described above than the bubble jet method. This is because the bubble jet system instantaneously heats the liquid to a high temperature, which may adversely affect microorganisms dispersed in the liquid.

  The discharger 20 in the screening apparatus 100 of the present invention preferably has a volume of droplets in which the microorganisms are dispersed, preferably from 1 femtoliter (fl) to 1 microliter (μl), more preferably from 1 picoliter (pl). The discharge can be controlled at 100 nanoliters, most preferably from 10 picoliters to 10 nanoliters. By controlling the pressure applied to the liquid by the inner diameter of the nozzle and the pressure generating member, the volume (particle diameter) of the droplet can be controlled.

  Preferably, the microorganism separating and / or arranging means (discharger 20) includes means 7 for independently moving at least one of the discharge head 4 and the medium 1 in two orthogonal directions. The moving means 7 is typically a so-called XY plotter. In the embodiment of FIG. 1, the configuration in which the ejection head 4 is moved in the X direction and the medium 1 is moved in the Y direction is illustrated, but the ejection head 4 may be moved in the Y direction and the medium 1 may be moved in the X direction. Good. Of course, any one of the ejection head 4 and the medium 1 may be moved in two directions of X and Y. Since the configuration of the XY plotter is well known, detailed description thereof is omitted.

C. Screening Method Next, a microorganism screening method according to a preferred embodiment of the present invention will be described.

First, the microorganisms are dispersed in a predetermined liquid. A surfactant, a dispersant, and the like can be added to the liquid as necessary. This process is carried out according to the same procedure as before. An example is as follows. Collect a source (eg, soil). It is said that 10 ⁵ to 10 ⁹ microorganisms are present in 1 g of soil. This microbial concentration is the basis for determining the dispersion concentration and the volume of ejected droplets, which will be described later. The collected soil is suspended in water, and impurities are removed by physical means (typically, a filter). Next, the suspension from which impurities have been removed is diluted in multiple stages to a predetermined concentration using an appropriate liquid according to the purpose to prepare a discharge dispersion liquid (sample liquid).

  The predetermined concentration is preferably such a concentration that there is substantially one microorganism in the ejected droplets from the screening apparatus. By adjusting the concentration of the discharge dispersion calculated based on the microorganism concentration of the separation source and the volume of the discharge droplet, adjustment is made so that substantially one microorganism is present in the discharge droplet. Is possible.

  Further, the discharge dispersion liquid is adjusted so as to have an environment in which the target microorganism can proliferate, grow, survive, decompose and / or synthesize. For example, a dispersion liquid in which different environments are formed by systematically changing nutrients, temperature, pH, oxygen, water activity, pressure, specific compound content, etc. is prepared, and the dispersion liquid is prepared using the multi-head dispenser. By applying each of these to a medium, screening can be performed with a much higher efficiency than in the past, and the search for the target microorganisms is greatly facilitated.

If necessary, the discharging dispersion is subjected to a charging process by any appropriate means.

  Next, the obtained dispersion liquid for discharge is put into the holding container 2 of the screening apparatus 100 as shown in FIG. Temperature control is performed by the temperature adjustment mechanism 5 as necessary. Next, the dispersion liquid is ejected from the ejection head 4 to fly toward the medium 1 and is attached to the medium 1. As long as microorganisms adhere to the medium 1 individually, the dispersion may be discharged as droplets or may be discharged as a continuous fluid. From the viewpoint of individual separation, it is preferable to eject the droplets. FIG. 5 is a schematic diagram showing a state in which the droplets 54 of the dispersion liquid are attached to the medium 1. In each droplet 54, there is substantially one microorganism. Microorganisms can be finally separated and identified by performing biochemical reaction, material decomposition, energy conversion and / or growth on the medium 1. The medium is tailored to have an environment in which the microorganism of interest can proliferate, grow, survive, degrade and / or synthesize. For example, media having different environments formed by systematically changing nutrients, temperature, pH, oxygen, water activity, pressure, specific compound content, etc. are prepared, and the multi-head ejector is used for each medium. By applying the dispersion liquid, screening can be performed with much higher efficiency than in the past, and the search for the target microorganism is greatly promoted. If the environment of both the dispersion and the medium is systematically changed, the screening efficiency is further improved, and the search for microorganisms is further promoted.

  Typical examples of the material constituting the medium 1 include glass, ceramics, metal, plastic, carbon material, natural organic matter (for example, agar, cellulose), fiber, fine cloth, and non-woven cloth. Natural organics, plastics and glass are preferred. Natural organic substances can be easily and easily cultured for microorganisms. Plastics can be easily and inexpensively processed according to the purpose. Glass can be processed according to the purpose, microorganisms can be easily cultured with the processed product, and light transmittance can be secured.

  In one embodiment, the medium 1 is a flat plate having irregularities on the surface. Any appropriate shape can be adopted as the shape of the unevenness according to the purpose. For example, a hemispherical recess may be formed, a rectangular recess in plan view may be formed, or a number of grooves 66 as shown in FIG. 6 may be formed. Applying the dispersion liquid to each of the grooves as shown in FIG. 6 has the following advantages: (1) Liquid culture is possible instead of a solid medium such as agar. That is, it is possible to hold the culture solution in the grooves in advance, apply the microorganisms or a liquid containing the microorganisms to each groove (add dropwise), and perform culture. (2) By forming elongated grooves, microorganisms grown and cultured in the grooves flow in the longitudinal direction of the grooves due to capillary force and change in volume. Therefore, it is very easy to detect that the flowed part is a cultured part. it can. (3) Since each groove | channel is independent, the culture solution of each groove | channel does not mix. As a result, mutual adverse effects between microorganisms can be prevented, and culture is performed in a very good environment. (4) By systematically changing the culture conditions (eg, pH, dissolved oxygen content, nutrients) of each groove, it becomes possible to examine various culture conditions in parallel, and the screening efficiency is remarkably improved. improves.

  In another embodiment, the medium 1 has a hydrophobic (water-repellent) region 71 and a hydrophilic region 72 as shown in FIG. The hydrophobic region 71 and the hydrophilic region 72 can be arranged in an arbitrary pattern according to the purpose. For example, the hydrophobic region may be arranged in an island shape in the hydrophilic region on one surface as shown in the drawing, or both may be alternately arranged in a row or may be arranged in a checker shape. The hydrophobic region 71 and the hydrophilic region 72 can be formed by any appropriate means. For example, it may be processed with plasma gas using a semiconductor surface modification processing technique, or an organic silane-based or organic thiol-based self-assembled film may be applied and formed. In the case of using such a medium, very good results can be obtained even if the dispersion is discharged as a continuous fluid or a relatively large droplet instead of as a micro droplet. The hydrophilic region and the hydrophobic region use the fact that the electrostatic dispersion force of the charged dispersion or microorganisms acts to change the adhesion behavior, so just apply it to match the hydrophobic / hydrophilic pattern of the medium. This is because they can be separated. For example, when the liquid is water-based, it is selectively trapped in the hydrophilic region, and when the liquid is oil-based and organic solvent-based, it is selectively trapped in the hydrophobic region.

  In yet another embodiment, the medium 1 has a charged area and an uncharged area. In this case, separation and arrangement of microorganisms can be promoted by utilizing the charged state of the charged region. The charged area and the non-charged area can be formed with an arbitrary latent image pattern by any appropriate means. For example, as shown in FIG. 8, it may be formed by contact charging using an electrode needle 80, contact charging by contact of a conductive material pattern, or non-contact charging by electron beam irradiation. The contact of the conductive material pattern is to form a latent image pattern selectively only at the contact portion by bringing a conductive material molded in a predetermined pattern into contact with the medium and energizing it.

  In yet another embodiment, the medium 1 has electrodes 90 formed in a predetermined pattern. The electrode pattern 90 is typically formed by photolithography. The application condition of the voltage to the electrode pattern can be appropriately set according to the purpose. That is, direct current may be applied, alternating current may be applied, and the alternating current frequency may be changed. More details are as follows. When the charged state of the microorganism is larger than the charged state of the liquid, electrophoresis is used. In this case, the voltage application waveform is controlled so that the charged microorganisms move in the electric field. When the microorganism is not charged and the dielectric constant is larger than the dielectric constant of the liquid, positive dielectrophoresis is used to move the microorganism to the higher electric field strength. When the microorganism is not charged and the dielectric constant is smaller than the dielectric constant of the liquid, negative dielectrophoresis that moves the microorganism to the smaller electric field strength is used. Since the dielectric constant varies depending on the microorganism, it is possible to adapt to almost all microorganisms by appropriately selecting the AC voltage (especially its frequency).

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Isolate impurities from the soil using conventional methods, and use a microbial sample (Bacillus
subtilis). This sample is suspended in an aqueous solution (LB medium, 10 g of polypeptone in 1 L medium, 10 g of yeast extract and 5 g of sodium chloride), diluted with a culture solution, and the number of microorganisms applied on the medium is 0. The microorganisms were dispersed by adjusting the concentration within the range of 10 ³ to 10 ⁶ / ml so as to be 0.01 / drop to 23 / drop. This dispersion sample was filled in the syringe part of the applicator. The discharge amount from the syringe part of the coating machine was adjusted in advance to be 10 nl / droplet. As shown in FIG. 5, this dispersion sample was ejected drop by drop on the agar medium. Such a discharge pattern was formed at high speed using an XY plotter. The coated medium was allowed to stand and cultured in an environment of 30 ° C. and 50% RH. The relationship between the culture time and the appearance rate of colonies is shown in Table 1 and FIG. Further, Table 2 shows the relationship between the culture time and the colony diameter (measured average value) at a concentration of 1 / droplet.

  As is apparent from these results, when Bacillus subtilis was applied to the agar medium at a concentration of 1 / droplet, 92.6% colonies were formed after 54 hours of culture time. The colony diameter increases almost linearly up to 54 hours.

Isolate impurities from the soil using conventional methods and remove microbial samples (Rhodococcus
erythlopolis). This sample is suspended in an aqueous solution (LB medium, 10 g of polypeptone in 1 L medium, 10 g of yeast extract and 5 g of sodium chloride), diluted with a culture solution, and the number of microorganisms applied on the medium is 0. The microorganisms were dispersed by adjusting the concentration within the range of 10 ³ to 10 ⁷ / ml so as to be 0.01 / drop to 100 / drop. This dispersion sample was filled in the syringe part of the applicator. The discharge amount from the syringe part of the coating machine was adjusted in advance to be 10 nl / droplet. As shown in FIG. 5, this dispersion sample was ejected drop by drop on the agar medium. Such a discharge pattern was formed at high speed using an XY plotter. The coated medium was allowed to stand and cultured in an environment of 30 ° C. and 50% RH. The relationship between the culture time and the colony diameter is shown in Table 3 and FIG.

  As is clear from these results, in the case of pure culture, when the number of microorganisms in the sample droplet is 10 or less, an advantage is clearly recognized compared to the case of 100, and the smaller the number is, It turns out that nutrition does not antagonize and it grows up well.

Using conventional methods to separate impurities from soil, two microbial samples Bacillus
subtilis and Rhodococcus erythlopolis were collected. These samples are suspended in an aqueous solution (LB medium), diluted with a culture solution, and 10 times so that the number of the microorganisms applied on the medium becomes 0.04 / drop to 5 / drop. ^The concentration was adjusted within the range of ³ to 10 ⁵ cells / ml to disperse the microorganisms. This dispersion sample was filled in the syringe part of the applicator. The discharge amount from the syringe part of the coating machine was adjusted in advance to be 10 nl / droplet. As shown in FIG. 5, this dispersion sample was ejected drop by drop on the agar medium. Such a discharge pattern was formed at high speed using an XY plotter. The coated medium was allowed to stand and cultured in an environment of 30 ° C. and 50% RH. The relationship between the culture time and the colony appearance rate is shown in Table 4 and FIG.

Further, in the present example, the following matters were observed. All colonies that appeared at a concentration of 5 / droplet were Bacillus subtilis. In addition, colonies that appeared at a concentration of 1 / drop or less are Rhodococcus
The cases of erythlopolis and Bacillus subtilis were observed, but from the results of PCR-DGG measurement, only one type of microorganism was present in the colony, and the two types were not mixed. Also Rhodococcus
Compared with the case of Rhodococcus erythlopolis pure culture according to Example 2, the erythlopolis colony clearly had a smaller colony diameter. From these, Bacillus
It can be seen that the culture of Rhodococcus erythlopolis is inhibited by the secretion of subtilis or Bacillus subtilis. That is, Rhodococcus
When erythlopolis and Bacillus subtilis are mixed and applied to the same place on the medium (medium), Rhodococcus erythlopolis cannot be cultured and Bacillus
It is cultured with subtilis predominance. Thus, it can be seen that, by individually applying, separating, and arranging various types of microorganisms as in the present invention, the cells can be cultured with high probability without being adversely affected by other microorganisms.

In various media shown in Table 5, fine hydrophobic regions and hydrophilic regions as shown in FIG. 7 were formed. Specifically, the hydrophobic treatment was performed using an RIE (reactive ion etching) apparatus under the conditions of O ₂ gas = 50 sccm, power = 100 W, gas pressure = 0.05 Torr, and treatment time = 60 seconds. The hydrophilic treatment was performed using an RIE apparatus under the conditions of CHF ₃ gas = 20 sccm, power = 100 W, gas pressure = 0.02 Torr, and treatment time = 120 seconds. The contact angle was used as an index representing hydrophobicity and hydrophilicity. The contact angles of water and culture solution in various media were measured by ordinary methods. The results are shown in Table 5. From this result, it can be seen that since the original surface state varies greatly depending on the medium, it is preferable to properly use the hydrophobic treatment and the hydrophilic treatment.

  A fine charged pattern (latent image pattern) was created on an acrylic plastic plate as shown in FIG. Specifically, a voltage of 100 V was applied for several seconds between the surface of the acrylic plastic plate and the back surface of the acrylic plastic plate with a large number of fine needles, and a latent image pattern was created by contact charging. As the charged state (+ or −) of the latent image pattern, the voltage application electrode was appropriately selected so as to create a charged state opposite to the charged state of the liquid or microorganism to be applied.

  On the acrylic plastic substrate, as shown in FIG. 9, a fine comb electrode having a level of several μm or less, for example, is patterned in accordance with the size of the microorganism, and an AC voltage is applied. FIG. 13 is a schematic diagram for explaining how the applied microorganisms move on the medium based on the principle of electrophoresis. More specifically, when the microorganism 131 in the applied dispersion is charged to-, it moves to the + pole of the electrode 90, and when the microorganism is charged to +, it moves to the-pole. Separation and sequencing are performed. FIG. 14 is a schematic diagram for explaining how the applied microorganisms move on the medium based on the principle of dielectrophoresis. More specifically, when the dielectric constant of the applied dispersion is larger than the dielectric constant of the microorganism, the microorganism 141 moves to a region where the electric field strength of the electrode 90 is small by negative dielectrophoresis. When the dielectric constant of the applied dispersion is smaller than the dielectric constant of the microorganism, the microorganism 141 moves to a region where the electric field strength of the electrode 90 is high by positive dielectrophoresis. In this way, the microorganisms are separated and arranged.

  As described above, according to the present invention, there are provided a microorganism screening apparatus and a microorganism screening method that are capable of separating and identifying unknown microorganisms with high probability, with high throughput with greatly improved efficiency. Such an apparatus and method can be suitably used for searching for useful microorganisms such as materials for drug discovery, materials such as foods and chemical products, and environmental purification and environmental sensing materials.

1 is a schematic perspective view of a microorganism screening apparatus according to a preferred embodiment of the present invention. It is a schematic perspective view explaining an example of the inkjet head of the microorganism screening apparatus of this invention. It is a schematic perspective view explaining the contact state of the drive part and liquid flow-path part of the inkjet head of FIG. It is a schematic sectional drawing for demonstrating the liquid discharge state of the inkjet head of FIG. It is a schematic perspective view explaining the medium in one embodiment of this invention. It is a schematic perspective view explaining the medium in another embodiment of this invention. It is a schematic perspective view explaining the medium in further another embodiment of this invention. It is a schematic perspective view explaining the medium in further another embodiment of this invention. It is a schematic perspective view explaining the medium in further another embodiment of this invention. 2 is a graph showing the relationship between culture time and colony appearance rate in Example 1. It is a graph which shows the relationship between the culture | cultivation time in Example 2, and a colony diameter. It is a graph which shows the relationship between the culture | cultivation time in Example 3, and a colony appearance rate. In the embodiment of FIG. 9, it is a schematic diagram explaining how the applied microorganisms move on the medium based on the principle of electrophoresis. FIG. 10 is a schematic diagram illustrating how the applied microorganisms move on the medium based on the principle of dielectrophoresis in the embodiment of FIG. 9.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Medium 2 Holding container 4 Discharge head 5 Temperature control mechanism 7 XY plotter 20 Discharge machine 100 Microorganism screening apparatus

Claims (15)

A microorganism screening apparatus comprising means for individually separating and / or arranging unknown or known microorganisms existing in nature. The microorganism separation and / or arrangement means includes a holding container that holds a liquid in which microorganisms are dispersed, and a discharge head that communicates with the holding container and discharges the liquid as a droplet or a continuous fluid onto a medium. The microorganism screening apparatus according to claim 1, wherein the microorganism screening apparatus is a discharge machine. The microorganism screening according to claim 1 or 2, wherein the dispenser is a coating machine selected from a dispenser device, an inkjet device and a spotter device, or a coating machine selected from spraying, spraying, a nebulizer and an atomizer. apparatus. The microorganism screening apparatus according to claim 2 or 3, wherein the volume of the droplet can be controlled from 1 femtoliter to 1 microliter. The microorganism screening apparatus according to any one of claims 2 to 4, wherein the microorganism separation and / or arrangement means includes means for independently moving at least one of the ejection head and the medium in two orthogonal directions. Dispersing unknown or known microorganisms existing in nature in a liquid;
Discharging the liquid as droplets or as a continuous fluid onto a medium, and separating and / or arranging the microorganisms individually. The screening method according to claim 6, wherein the concentration of the liquid is adjusted so that there is substantially one microorganism in the droplet. The screening method according to claim 6 or 7, wherein the volume of the droplet is adjusted so that substantially one microorganism is present in the droplet. 9. The medium according to claim 6, wherein the medium is made of a material selected from glass, ceramics, metal, plastic, carbon agent, natural organic matter, fiber, fine cloth and non-woven cloth, and is a flat plate having irregularities on the surface. The screening method described. The screening method according to claim 6, wherein the medium has a hydrophobic region and a hydrophilic region. The screening method according to claim 6, wherein the medium has a charged area and an uncharged area. The screening method according to claim 6, wherein the medium has electrodes formed in a predetermined pattern. The screening method according to any one of claims 6 to 12, wherein the liquid is adjusted to have an environment in which a specific microorganism can proliferate, grow, survive, degrade and / or synthesize. The screening method according to any one of claims 6 to 12, wherein the medium is adjusted so as to have an environment in which a specific microorganism can proliferate, grow, survive, degrade and / or synthesize. The screening method according to claim 6, further comprising a step of charging the liquid before the ejection step.

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