JPH03292880A - Method for preparing set of plural and different diluting liquid, it's device and equipments suitable for the same method - Google Patents

Abstract

PURPOSE:To automate operation of inoculation of diluting liquid and microorganism in microbial test and observation of cultured result by carrying out a specific process under a control means previously programmed and preparing a set of the plural and different diluting liquids. CONSTITUTION:A diluting liquid of prescribed amount is each poured and sealed into each diluting vessel 1a to 1b of aggregate of diluting vessel to make it possible to feed each diluting vessel to test site. In the test site, each diluting liquid is weighted and poured into the aggregate of diluting vessels and may be arranged in prescribed position in a clean room. When used, either one of grip parts 2a and 2b of lid closing means is packed by man power or machine or fixed and at least partially released. In this time, form of inflating part 1e has effect capable of gradually decreasing release resistance as the release progresses. Therefore, it can be prevented that liquid in the aggregate of diluting vessels is spilt without shaking the aggregate when released.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates generally to a method for preparing a set of different diluents, an apparatus thereof and a diluent container suitable for use in the method and apparatus, and in particular to microbial testing. A method for automating tasks such as adjusting the dilution solution, pouring medium, and incubator containing the medium, inoculating microorganisms, culturing, and observing culture results;
This article relates to the devices and containers used therefor.

Definition of Terms In this specification, the following terms are used with the following meanings.

Clean room: means a room that is clean with respect to the intended use of the diluent; if the diluent is used for chemical testing, it only needs to be chemically clean; if the diluent is used for microbial testing, it must be chemically clean;
It is fine as long as it is microbiologically clean.

Liquid to be diluted: A liquid containing a substance to be diluted or a liquid containing microorganisms to be diluted.

Diluent: A liquid used to dilute a liquid to be diluted, and includes not only distilled water but also physiological saline, buffer, liquid medium, and molten solid medium. When the diluted liquid is used for microbial testing, it goes without saying that it is sterile.

Diluent: Refers to a liquid obtained by diluting a given liquid to be diluted with another liquid, and also includes a pouring medium in which a liquid containing a given substance, a suspension of microorganisms, etc. is diluted with a medium.

Microbial testing: Tests related to the presence or absence of microorganisms, one type of microorganisms, and their characteristics, such as viable count tests, microbial identification, microbial screening, microbial resistance tests, etc., as well as tests for microorganisms such as drugs, drugs, etc. Tests that use microorganisms as a means, such as searching for biologically active substances, determining effectiveness or effective concentration, etc., such as antibacterial spectrum testing of drugs, minimum inhibition circle measurements, etc.

This refers to various tests involving microorganisms, including tests for the activity of growth-promoting substances.

Specimen: means a liquid containing a microorganism to be tested or a liquid containing a substance to be tested.

Microorganisms.

It is used to include not only unicellular organisms but also cultured cells of multicellular organisms or higher organisms.

Technical Background and Problems Generally, in chemical testing, it is the task of preparing sets of multiple dilutions of a specimen, and in microbial testing in particular, it is the task of preparing sets of multiple dilutions of various specimens that are compatible with the testing purpose. It necessarily involves operations such as adjustment, preparation of a culture medium, measuring and dispensing a predetermined amount of culture medium into an incubator, inoculating the culture medium with microorganisms, and culturing.

Particularly in microbial testing, dilution series of one type of specimen, dilution solutions of multiple specimens, etc. are prepared in large quantities as a set.
Alternatively, repeated preparations may be necessary.

These operations are often performed manually by skilled engineers and are extremely inefficient.

Especially in microbiological tests, these dilutions.

In many cases, operations such as preparing the culture medium, measuring and dispensing 1, etc. must be performed manually in a sterile room, which is inefficient.

Cleaning and sterilization of pipettes, petri dishes, petri dishes, Erlenmeyer flasks, etc. used in these operations also required a great deal of manpower and labor.

Furthermore, if the microorganisms to be cultured are or are likely to be pathogenic, there is a risk of infection to workers.

In particular, dilution, preparation of culture medium, measuring and dispensing, cleaning and sterilizing used instruments require an extremely large amount of time and manpower in these tests.

In addition, in these tests, observations and judgments were made by experts with the naked eye, with or without using a microscope or the like.

Therefore, it is desirable to consistently automate a portion of these operations, preferably a continuous portion thereof, or to automate all of them.

Conventionally, there have been automatic dispensing devices, mixing and dispensing devices, microbial inoculation devices, measuring and dispensing devices, etc. that dispense culture media. The process involves mixing ingredients, measuring and dispensing prepared diluted solutions, culture media, etc. to prepare multiple sets of different culture media, inoculating microorganisms into the resulting set of culture media, culturing them, and making observation decisions. No device was known that could do several things consistently.

Furthermore, there has been no dilution container that allows at least a portion of the dilution work that should be performed in a sterile room to be performed in a normal environment.

In addition, methods and devices for easily conducting certain types of microbial tests and instruments suitable for use therein have not been known.

Means for Solving the Problem In view of the current state of the art, the present inventors prepared a dilution series of one type of specimen, a set of different dilutions of many types of specimens in large quantities, or prepared them repeatedly. We have been conducting intensive research on automated methods and devices, as well as various instruments suitable for use in these processes.

The present inventors also used dilution in microbiological tests.

We have conducted extensive research into methods, devices, and instruments suitable for use that can consistently perform several tasks, including preparation of culture media, preparation of culture vessels containing culture media, inoculation of microorganisms, cultivation, and observation/judgment. . As a result, the present invention was completed.

In the most basic method of the present invention, in one clean room, (a) at least one diluted liquid container containing a diluted liquid (sample liquid) is placed at a predetermined position; (b) dilution (c) disposing at a predetermined position at least one of a diluent container assembly in which a plurality of dilution containers containing a diluent and sealed by a closing means are integrally molded in a predetermined arrangement; (c) the above-mentioned dilution; (d) exchanging at least one negative and positive pressure generating means to which a bipenototinob can be removably attached, and a pipette tip attached to said negative and positive pressure generating means; and a moving means for moving the at least one negative and positive pressure generating means relative to the pipette tip changing means and the liquid to be diluted container and the dilution container assembly; lining up each of the pipette tips attached to the negative and positive pressure generating means and each of the liquid to be diluted containers, and sucking a predetermined amount of the liquid to be diluted into the pipette tip; (e) the measuring and dispensing device; relatively move each of the pipette tips attached to the dilution container assembly to align each of the pipette tips with each of the corresponding number of dilution containers in the dilution container assembly, and discharging the liquids to be diluted sucked into each of the chips into the arranged dilution containers and mixing them uniformly to prepare at least one dilution liquid; (f) the above steps (d) and (e); ) is repeated a predetermined number of times as necessary, and (g) the following two steps are repeated a predetermined number of times (including 0 times) as necessary; (i) the diluted solution prepared in the previous step; (ij) aspirating a predetermined amount of each of the above into a corresponding number of pipette tips attached to the measuring and dispensing device; (ij) each of the pipette tips attached to the measuring and dispensing device and the dilution container assembly; relatively moving to align with a corresponding number of other dilution containers in the dilution container assembly, and discharging each of the aspirated dilution liquids into the aligned dilution containers and mixing uniformly; Ch) exchanging at least one pipette tip attached to the metering and dispensing device at a predetermined timing using a pipette tip changing device; preparing a plurality of sets of different dilutions, characterized in that the set of plurality of different dilutions is prepared under controlled control.

In the above method, the clean room is a sterile room when the diluted solution to be prepared is used for microbial testing.

The liquid to be diluted is contained in at least one container and placed at a predetermined position within the clean room.

In the method of the present invention, a diluent is contained.

A dilution container assembly in which a plurality of dilution containers sealed by a closing means are integrally formed in a predetermined arrangement is placed at a predetermined position in a clean room. It is desirable that the container assembly is made of an inexpensive material such as plastic and is disposable.

The plurality of dilution containers in the dilution container assembly may be arranged in any arrangement. For example, a plurality of dilution containers may be arranged in a straight line, in a matrix, or in a ring. These sequences include the number of types of dilution liquids that should be prepared in a set according to the test purpose, and
The movements of the moving parts of automated equipment are determined with simplification considerations. The dilution liquid stored in the dilution container assembly may be (sterile) distilled water for dilution, (sterile) physiological saline for dilution, (sterile) buffered water for dilution, (sterile) medium for dilution, etc. depending on the specimen. It's good.

When the dilution medium is a solid medium, it is necessary to set the temperature in the clean room to a temperature exceeding the gelation temperature of the solid medium. Furthermore, when microorganisms are handled within the clean room, it is further necessary to maintain the temperature within the clean room at a temperature at which the microorganisms involved can survive. For example, in an agar medium, the temperature can be about 45°C. Since a predetermined amount of diluent or medium for mixing is stored in the dilution container set in advance, work at the inspection site becomes efficient. It is also possible to use one or more dilution containers in the dilution container assembly as a container for the liquid to be diluted, and in this case, it is sufficient to leave one or more dilution containers in the dilution container assembly empty. . If necessary, it is also possible to provide means for sterilizing the external surface of such a dilution container assembly. Furthermore, means may be provided for stocking a plurality of such dilution container assemblies at a predetermined location and supplying them from there to a predetermined location. If necessary, a sterilization chamber may be provided adjacent to the sterile chamber to supply sterilized dilution container assemblies to the stock location. Multiple sets of diluents and culture media required for a given test can be prepared as a set, and they are formed into an integrated dilution container assembly, making subsequent handling convenient and eliminating the possibility of mixing up diluents. Mistakes can also be avoided.

The closing means of the dilution container assembly may be of any type as long as the closing means is opened manually. When producing, storing, and transporting products, especially when opening is performed by a machine, it is desirable to heat-seal or adhesive-seal the sealing sheet. The mechanical seal may be opened by piercing it with the tip of a pipette tip, which will be described later, or may be opened by cutting means, heating and melting means, etc. prior to insertion of the pipette tip. Furthermore, a means for mechanically locking the closing means may be provided, and the sealing sheet may be peeled off by relatively moving the locking means and the dilution container assembly.

The measuring and dispensing means includes at least one negative-positive pressure generating means to which a pipette tip can be attached, a pipette changing means for exchanging the pipette tip attached to the negative-positive pressure generating means, and at least one negative-positive pressure generating means attached to the negative-positive pressure generating means. It has a pipette tip, a pipette exchange means, a moving means for relatively moving the diluent container and the dilution container assembly. The pipette tip itself may be a pipette tip that has a bag-like part made of an elastic material for generating negative and positive pressure like a general pipette, and a cylindrical part for containing liquid, or it may simply be a pipette tip. A pipette tip having only a cylindrical portion may also be used. In the former case, the means for generating negative and positive pressure is provided with a means for compressing and relaxing the bag-like portion of the pipette tip, and in the latter case, the means for generating negative and positive pressure is provided with a means for compressing and relaxing the bag-shaped portion of the pipette tip.
For example, a cylinder whose stroke can be adjusted by an electromagnetic nozzle or an air cylinder may be provided. The pipette tip may be attached to the negative and positive pressure generating means in any manner, for example, by push-fitting. The pipette exchange means may be of a type in which a large number of pipette tips are stocked, a used pipette tip is removed, and a new pipette is pushed in and fitted. The pipette tip attached to the negative and positive pressure generating means is moved relative to the liquid to be diluted container by the moving means, and a predetermined amount of liquid to be diluted is aspirated into the pipette tip. The pipette tip that has completed suction is moved relative to the dilution container assembly by the moving means, aligned with the corresponding number of dilution containers, and the liquid to be diluted is discharged. Any method may be used to uniformly mix the (sterile) dilution liquid and the liquid to be diluted that have been stored in advance. However, if the volume of the dilution container is small, positive pressure can be alternately applied within the pipette tip to aspirate and mix the liquid mixture. Other methods include shaking or shaking the dilution container assembly, and inserting a stirring means into the dilution container for stirring.In this case, a rolling object is placed in the dilution container in advance. You can also leave it there.

Thus, at least one dilution is prepared.

Specifically, when preparing a set of diluted solutions of multiple different diluents, one set of diluted solutions is prepared simultaneously or in parallel using pipette tips corresponding to the number of diluted solutions. Alternatively, a single pipette tip can be used to perform multiple dilutions serially.

In the latter case, the pipette tip can be replaced every time one type of dilution is prepared, if necessary.

When preparing a multi-step dilution series of one type of liquid to be diluted, prepare one dilution liquid in the first dilution container, then aspirate a predetermined amount of the dilution liquid and transfer it to the next dilution container. After injecting and mixing uniformly, the same operation may be repeated to prepare one set of dilution series. At this time, if multiple pipettes are used, multiple sets of diluted solutions of the same dilution series can be prepared at the same time.

Pipette tips may be replaced according to the sequence of dilution steps.

In the above process, at least the relative movement and alignment of the pipette tip and the diluted liquid container in the measuring and dispensing means, the suction of a predetermined amount of the diluted liquid, and the relative movement, alignment, and suction of the pipette tip and the dilution container are performed. Dispensing the diluted liquid into the dilution liquid, stirring, aspirating a predetermined amount of one diluent, moving the pipette tip relative to another dilution container, and diluting the aspirated diluent to another. The steps of dispensing the liquid, stirring and changing the pipette tip are carried out under the control of a programmable control means.

Thus, according to the basic method of the invention it is possible to prepare a set of different dilutions of the specimen, ie a liquid containing the microorganism to be tested, or a liquid containing the substance to be tested.

It is also possible to arrange one or more containers containing the third liquid at a predetermined position in the clean room and mix it with the liquid to be diluted and the liquid for dilution. The third liquid may be mixed with the diluent using a metering and dispensing means, or each container containing the third liquid may be individually provided with its own metering and dispensing means. The latter method has the advantage of reducing the frequency of pipette tip changes when it is desired to accommodate a relatively large amount of material compared to the liquid to be diluted.

The diluted solution thus obtained is then dispensed using the above-mentioned measuring and dispensing means.
Alternatively, it is also possible to dispense into the diluent container using other similar metering and dispensing means.

Furthermore, it is also possible to add a microorganism inoculation means to inoculate the prepared diluted medium with microorganisms.

The thus obtained pour medium and medium inoculated with microorganisms are
It is also possible to be transported to another sterile room by a transport means and cultured for a predetermined period of time. Furthermore, observation and judgment of the cultured medium can also be automated.

In the simplified method of the invention, instead of a Petri dish or Petri dish, a special diluent plate according to the invention (
culture plate). In this example,
A diluent plate having a predetermined number of diluent drop areas in a predetermined arrangement and a separating area surrounding them is placed at a predetermined position on a glass or plastic plate surface. Instead of dispensing the diluent into a diluent container, a small amount of the diluent is dripped onto the drop area of the diluent plate and spread as thinly as possible over as wide an area as possible. When the diluent is a culture medium, grown colonies will not overlap in the thin culture medium formed in the area where the diluted solution is dropped, so the determination can be automated, for example, in viable bacteria count tests, etc. It is effective for Moreover, in this method, the amount of the medium used is extremely reduced, so the medium raw materials can be saved, the apparatus can be downsized, and if the culture plate is disposable, the cost of the culture plate can also be reduced. Multiple media required for a given test can be prepared as a set.
As they are integrated and dripped onto the diluent plate,
Subsequent handling is convenient, and mistakes such as mixing up the culture medium can be avoided.

In another simple testing method of the present invention, a cylindrical diluent container is used. This diluted liquid container has a suction port at one end, a mounting part for attaching to the negative and positive pressure generating means at the other end, and a hollow part in the middle part, and the X, Y, and Z axes orthogonal coordinates define the internal volume of the hollow part. At least one axial dimension of the axial direction is formed to be as small as possible. Thus, when the diluent is sucked into the cylindrical diluent container, the diluent is spread as thinly as possible over as wide an area as possible, providing the same advantages as the diluent plate described above.

A general outline of the apparatus of the invention and of the apparatus suitable for the method and apparatus of the invention is clear from the above description.

The method, apparatus, and various instruments of the present invention have been outlined above, and specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

DESCRIPTION OF EMBODIMENTS Since the method and apparatus of the present invention also relate to improvements in the instruments used therein, embodiments of these instruments will first be described.

FIG. 1a is a plan view of a dilution vessel assembly suitable for use in the method and apparatus of the present invention, and FIG. 1a is a right side view thereof. The dilution container assembly includes a container portion 1 in which a predetermined number of dilution containers are arranged in a predetermined arrangement and is integrally molded from a material such as plastics, and a closing means for sealing the openings of the dilution containers. 2. In this example, the container section l is shown as having four dilution containers 1a=ld aligned in a straight line. Each dilution container generally has a truncated cone shape and is provided with an outwardly bulging portion 1e in its upper portion.

This bulging portion 1e is formed by partially peeling off the closing lid means 2.
Accepts injection means such as pipettes. Each dilution container 1a
-1d openings are connected to each other by a connecting plate 3. The connecting plate 3 has a generally rectangular planar outline and is provided with at least one notch in its peripheral edge. In this embodiment, cutouts are provided at both corners of one of a pair of opposing short sides of the rectangular shape of the closing means 2, and a trapezoidal cutout is provided at the center of the other short side. has been done. In this embodiment, the closing means 2 is a rectangular adhesive sheet having an adhesive surface, and when the adhesive surface is attached to the top surface of the connecting plate 3, the closing means 2 is a rectangular adhesive sheet having an adhesive surface. The portion 2a protrudes from the cut-off portion of the connecting plate 3, and the other central portion 2b protrudes from the trapezoidal cut-off portion of the connecting plate 3. These protruding portions serve as grip portions when the closing means 2 is opened manually or mechanically.

The present invention contemplates dispensing a predetermined amount (for example, 9 m0) of diluent into each dilution container 1a to 1d of the dilution container assembly, sealing the container, and supplying the diluent to the inspection site. Of course, the dilution liquid is measured into the dilution container assembly at the inspection site.

It may be dispensed and placed at a predetermined position in the clean room. In use, either one of the grip portions 2a, 2b of the closing means 2 is pinched or locked manually or mechanically, and is at least partially peeled off. At this time, the shape of the bulging portion 1e has the effect of gradually reducing the peeling resistance as the peeling progresses, so that the dilution container assembly is not shaken during peeling, and the content liquid can be prevented from spilling. .

FIG. 2 is a plan view showing a modification of the embodiment shown in FIG. 1. Similar parts have been given similar reference numerals and will not be described in order to avoid duplication, and only the different parts will be described below. In this example,
The connecting plate 3 is provided with a plurality of notches 3a on one of a pair of opposing long sides, corresponding to the dilution containers. Further, the closing means 2 has a notch 2c extending from one side of a pair of opposing long sides of the rectangle to an area between adjacent dilution containers.
are formed, and the openings of the individual dilution containers 1a to ld can be individually and partially opened and closed.

FIG. 3 is a perspective view showing a modification of the dilution container assembly of FIG. 1. Similar parts have been given similar reference numerals and will not be described in order to avoid duplication, and only the different parts will be described below. In this embodiment, the container portion 1 is formed as a structure in which the container walls of a predetermined number (six in this embodiment) of dilution containers 1a-1f having a chevron-shaped cross-sectional shape are fused together. Therefore, there is no connecting plate 3, and the closing means (adhesive sheet) 2 having an adhesive surface is adhered to the upper end surface of the container wall.

The closing means 2 is shown as having an apron-like shape corresponding to the shape of the upper end of the container portion 1, and provided with a knob portion 2b having a hole 2c at one end thereof. The longitudinal direction of all the contour lines constituting the chevron-shaped figure of the container portion 1 is the peeling direction of the closing means 2 (arrow A).
arranged diagonally or parallel to the Thus, when the closing means 2 is peeled and opened manually or mechanically, there is no extreme change in resistance, and it is possible to prevent the contents from spilling out due to agitation of the dilution container.

FIG. 4 is a perspective view showing a modification of the dilution container assembly of FIG. 3. Similar parts have been given similar reference numerals and will not be described in order to avoid duplication, and only the different parts will be described below. In this embodiment, each of the dilution containers 1a to 1d has a substantially square cross-sectional shape, their respective sides are partially fused, and their diagonals are aligned parallel to each other. The grip portion 2b of the lid closing means (adhesive sheet) 2 is bent upward. By bending the knob, the dilution container assembly is fed in the direction opposite to arrow A, and while moving, the knob part 2b
The closure means 2 can be peeled off by simply holding it on a fixed part of the device. Furthermore, the side wall of the dilution container assembly has a serrated shape, which is an integral structure with the means for reliably holding and moving the dilution container assembly in the feeding mechanism of the supply means of the dilution container assembly. It is available as.

Furthermore, another advantage of this dilution container assembly is that, for example, after preparing a pouring medium of a predetermined dilution series in each of these dilution containers 1a-1d; By tilting the dilution container at an angle, it is possible to pour the medium from the corner of the opening of each dilution container into a plurality of culture medium containers arranged at corresponding intervals.

As a variation of the above, each dilution container has a square opening;
It is also possible to arrange one side of the square openings of adjacent dilution containers completely overlapping each other, in other words, it is also possible to have a structure in which a rectangular container is divided into a plurality of compartments each having a plurality of square openings. be. In this case as well, the same effect can be expected if the peeling direction of the closing means 2 is made oblique to each side of the square. Either a separate mechanism must be provided or the supply direction of the dilution container assembly must be arranged obliquely to each side of the square.

Such a design is undesirable because it requires extra equipment and/or space. However, if the lid closing means 2 is peeled off,
If it is done manually, such a dilution container assembly can also be used.

FIG. 5 is a plan view of a modification of the embodiment shown in FIG.
In this embodiment, there are n rows of dilution container assemblies.

This is the same as the embodiment shown in FIG. 2, except that it is formed as a set of nXm dilution containers (12 in 3 columns and 4 rows in this embodiment) arranged in a matrix of m rows. . For simplicity, each dilution container is not given a reference code, but each column and row is given a code, and the ranking numbers in each column and row are connected with a hyphen to indicate each dilution container. Make it.

This embodiment is suitable when (one set of) a multi-step dilution series is required, or when multiple sets of a small number of step dilutions and dilutions are required. For example, in the case of performing multi-stage (i2-stage) dilution and dispensing, how to use it will be described. Each column nl.

n2. Each dilution container 1-1.2-13-1 in row n3 ml
The diluting containers 1-1. can be filled with different amounts of diluting liquid in advance and dispensing different amounts of the liquid to be diluted, or each diluting container 1-1.
2-1.3-1 contains an equal amount of diluent in advance and dispenses different amounts of the diluted liquid, or each dilution container 1-
1.2-1.3=1, different amounts of diluent are sealed, and a fixed amount of the undiluted solution is dispensed to prepare a mixed solution at a dilution step that is a jump from the desired dilution step.

A predetermined amount of these dilution containers 1-1.2-1.3-1 is dispensed into the dilution containers 1-2.2-2゜3-2 in the next row of each column and stored in advance. Mix with the same or different predetermined amount of diluent and follow the same steps in sequence from 1-2 to 1-4.2-2.
~2-4. In each of the dilution containers from 3-2 to 3-4, prepare a diluted mixture that complements the intermediate concentration of the above-mentioned jumped dilution steps.

The amount of diluent stored in each dilution container in advance, the amount of liquid to be diluted to be dispensed into each dilution container in the first row, and the amount dispensed from each dilution container in the previous row to each dilution container in the next row. By appropriately adjusting the amount of the mixed liquid to be mixed, a desired multi-stage dilution series can be prepared in a shorter time than when serial dilution operations are performed.

When preparing 3 sets of 4-stage dilution series at the same time,
For example, 9 mQ of diluent is stored in each dilution container in advance, and 1 m+1 is stored in each container 1-1.2-1°3-1 of row m1.
Dispense the liquid to be diluted and transfer the mixed liquid to the next row m2.
It can be prepared by dispensing into each dilution container 1-2.2-2.3-2 and repeating the same operation up to dilution container 1-4.2-4.3-4 in the last row m4. be understood.

In each of the above embodiments, the shape of each dilution container is shown as circular, chevron-shaped, or cylindrical with a square cross section.
The cross-sectional shape is not particularly limited and may be any shape.

FIG. 6 is a cross-sectional view of a simple diluent plate according to one embodiment of the present invention. This diluent plate is suitable for use in place of traditional culture vessels (eg Petri dishes). In this embodiment, a predetermined number (four in this embodiment) of diluent drop areas 10 separated from each other are placed on the surface of a plate 10 made of a material such as glass or plastic.
a~] Od and an isolation region 10e surrounding them are formed. Each dropping area has a bottom area and a depth (shallowness) to spread the dropped diluent over as wide an area as possible.

It is formed as a recess. Conventionally, culture has been carried out in test tubes, Petri dishes, Erlenmeyer flasks, Sakaro flasks, and the like.

However, since these volumes are large, not only do they require a large amount of media, but if their handling is to be mechanized, storage space for media containers, a supply mechanism for the media containers, a clean room to house them, or The sterile constant temperature room will also be larger. In this embodiment, for example, when testing the number of viable bacteria in a solid medium, a predetermined amount of the above-mentioned diluent is dropped onto the drop area.

For example, by dropping 1 m12 of a mixed medium and spreading it over as wide an area as possible, the thickness of the medium can be reduced, and the volume occupied by one medium can also be reduced. Therefore,
The space for stocking plates, the plate supply mechanism, the clean room that houses them, and the sterile constant temperature room can be downsized. Moreover, since related media are placed on one plate, they can be handled together, and samples (medium)
Mistakes such as mix-ups can be prevented. Furthermore, since the thickness of the medium is thin, colonies do not overlap at a proper colony density, and therefore it is even possible to mechanize the counting of viable bacteria.

FIG. 7 shows a schematic sectional view of a modification of FIG. 6. Similar parts have been given similar reference numerals and will not be described in order to avoid duplication, and only the different parts will be described below. In this embodiment, affinity is given to the medium dropped onto the surface of the dropping area 10a''lOd.

This imparts non-affinity to the culture medium dropped onto the surface of the separation region 10e. For example, the plate 10 is made of glass, and the separation region 10e is covered with silicone resin. Although the thickness of the silicone resin coating is exaggerated in the figure, it can actually be on the order of μm. The diluted solution (medium) dropped in this manner can be spread over a sufficiently wide area to form an extremely thin medium. Furthermore, it is possible to form the contour of the dropped medium into a desired shape. This is extremely convenient for mechanical colony counting. In this embodiment, if there is a risk that the medium may dry out during culture, a cover may be provided to separately cover each dripping area or to cover the entire dripping area. Although the shape of such a cover is obvious without needing to illustrate it, FIG. 8 shows an example of the diluent plate IO and cover 11 in a perspective view, and FIG. 9 shows them in cross section. .

FIG. 10 is a schematic cross-sectional view showing a modification of the embodiment shown in FIG. 7. Similar parts have been given similar reference numerals and will not be described in order to avoid duplication, and only the different parts will be described below. In this embodiment, the drip areas 10a to 10d are provided on the back side of the plate IO.
A lens-shaped convex surface 10f is formed in an arrangement corresponding to. In this way, colonies can be seen magnified when viewed from the rear, and counting can be performed reliably both visually and mechanically.

FIG. 11 is a perspective view of a simple cylindrical diluent container 20 according to another embodiment of the present invention. The container 20 is shown as having a cylindrical structure with openings 20a, 20b at both ends and a cavity 20c in the middle. In this embodiment, the cavity 20c has a cylindrical structure with a flat rectangular cross section, the opening 20a is shown as a structure in which the end of the cylindrical structure is opened, and the opening 20b has a structure as described below. It is shown as having an enlarged cylindrical structure section attached to the negative and positive pressure generating means and a funnel structure section as a transition to the cylindrical structure.

More specifically, X defines the internal volume of the cavity 20c.

The dimension in at least one axis direction (in this embodiment, the Z-axis direction) of the Y and Z-axis orthogonal coordinates is formed to be as small as possible. When negative pressure is applied to the opening 20b by the negative and positive pressure generating means, the culture medium is sucked through the opening 20a. In the cavity, a pair of boundary lines 20d are displayed in the shape of a cylindrical headband, indicating that a predetermined amount of culture medium is present between them. Therefore, it is sufficient to count the number of colonies during that time. Instead of the boundary line, both ends of the cylindrical cavity may be made opaque to limit the observation area. Also, X, Y
, the dimensions in the two directions in the Z direction may be made as small as possible to form a thin tube shape.

Having described various device embodiments of the present invention above, the method and apparatus of the present invention will now be described through some embodiments.

The most basic method of the present invention has been fully explained above in connection with the dilution container assembly in the "Means for Solving the Problems" section. Therefore, the apparatus for carrying out the basic method of the present invention will now be described in detail.

FIG. 12 is a layout diagram schematically showing an embodiment of a device for carrying out the above-mentioned basic method. The diluter of this embodiment has at least one clean chamber 50 and control means C. Inside the clean room 50, there is a diluent container arrangement position 60 for arranging at least one diluent container containing a diluent, a (sterile) diluent containing a diluent,
A dilution container assembly arrangement position 70 for arranging at least one dilution container assembly in which a plurality of dilution containers sealed by a closing means are integrally molded in a predetermined arrangement, and a measuring and dispensing means 8
0.

The clean room 50 may be any clean room suitable for the purpose of use of the diluted solution to be prepared by the apparatus of the present invention, for example, when the purpose is to prepare a diluted solution for microbial testing. It may be a sterile room. In addition, when preparing a pour medium by melting a solid medium, a sterile thermostatic chamber is used, and the solid medium in the sterile constant temperature chamber is thereby maintained in a molten state. Of course, an air circulation device 51 equipped with a sterilization filter and a gas supply means 52 for maintaining the room in an anaerobic state are provided.
, a thermostat 53 for maintaining a constant indoor temperature, a heat exchange device 54, and the like. The equipment itself for these sterile rooms is well known, so detailed explanation thereof will be omitted.

It is essential that the diluent container arrangement position 60 is capable of arranging at least one diluent container containing the diluent to be tested at a predetermined position. It is shown as a test tube rack in which liquid containers 61, 62, 63, 64 are arranged in a row.

The liquid to be diluted is appropriately selected depending on the purpose of the test.

For example, if there is only one type of liquid to be diluted, only one liquid to be diluted container is required. When there are multiple types of liquids to be diluted, it is necessary to arrange the same number of liquid containers to be diluted in a predetermined arrangement at a predetermined position.

It is essential that the dilution container assembly arrangement position 70 be capable of arranging at least one dilution container assembly l described above at a predetermined position, but in this embodiment, a large number of dilution container assemblies are stocked, It is shown as a means for supplying dilution container aggregates from which they are placed one by one at a predetermined position (dilution station DS). However, the simplest device may be simply a positioning stand on which one or more of the dilution container assemblies 1 are placed in a predetermined position simply by hand. In this case, the closing means 2 of the dilution container assembly is opened manually.

Although any of the dilution vessel assemblies described above may be used, this embodiment is shown using the dilution vessel assembly shown in FIG. The number of dilution containers in the dilution container assembly 1 may be appropriately selected depending on the purpose of the test.

Although the dilution containers may be arranged in any form, the linear arrangement shown in FIGS. 1 to 4 is preferable for an assembly consisting of a relatively small number of dilution containers. Thereby, aspects of various operations within the clean room 50 can be simplified. In addition, each dilution container la, lb of the dilution container assembly l
The type and amount of the diluent stored in advance can be selected as appropriate depending on the purpose of the test. By supplying the dilution container assembly 1 containing the diluent in advance, the dilution work becomes more efficient, and related diluents can always be handled as a set depending on the inspection purpose, so that the dilution container assembly 1 containing the diluent can be handled in an integrated manner at all times during the inspection. This makes handling easier and avoids mistakes caused by mixing up dilution containers.

FIG. 13 shows a schematic layout of the dilution container assembly supply means 70. This supply means 70 includes a stock means 71 for stocking a large number of dilution container assemblies l, and a transfer means 7 for transporting the dilution container assemblies l one by one from the stock means 71 to a predetermined position (for example, dilution station DS).
2.

In mechanical technology, various types of stocking means for stocking a plurality of articles are known, and the stocking means 71 may be of any type, but in this embodiment, the stocking means 71 is stored in a cylindrical case 71a. A table 71 that moves left and right (direction perpendicular to the plane of the paper in FIG. 13) at
A large number of dilution container assemblies are stacked on top b to accommodate multiple rows (only one row is visible in FIG. 13), and the dilution containers are assembled from the opening lower 1c provided below the case 71a in the manner of a Daruma drop. It is shown as being pushed out one by one from the bottom of a stack of bodies. The extrusion of the dilution container assembly l is shown as a pusher means having a pusher 71d arranged below the table 71b and rotating along an elliptical trajectory. Butsu Super 71
d appears above and below the table surface through a slit 71e provided in the table 71b, moves in the pushing direction (arrow B) when protruding above the table surface, and sinks downward from the table surface when retreating. do. The table 71b is -
After discharging the stack of dilution container assemblies, it moves left and right by a predetermined position and pushes out the dilution container assemblies from the next stack in a similar manner.

Various types of transport means for transporting an object along a transport path are also known, and any type may be used, but in this embodiment, the transport means shown schematically in FIG. As shown in the figure, the transfer path 72 has a bottom surface 72b having a slit 72a in the transfer direction, and a side guide means 72c for guiding the side surface of the dilution container set in the transfer direction. It is shown as bushing means 72d guided along. The pusher means 72d transfers the dilution container assembly pushed out from the stock means 71 to the dilution station DS.

The measuring and dispensing means 80 includes at least one negative and positive pressure generating means 81 to which a pipette tip can be detachably attached, a pipette tip changing means 90 for attaching and detaching the pipette tip to and from the negative and positive pressure generating means 81, and at least one negative and positive pressure generating means. Means 8
1 as pipette tip exchange means 90 and diluted liquid container 61
- 64 and a moving means 82 for relative movement with respect to the dilution container assembly; and a pipette changing means 9 for attaching and detaching a pipette tip to and from the negative and positive pressure generating means.
0 and are provided.

In FIG. 12, the positive and negative pressure generating means 81 is shown as a cylinder, which is preferably of a type whose stroke can be changed by an electromagnetic solenoid (not shown). In this embodiment, the pipette tip 81a is
It is shown schematically as a mere cylindrical body with a tapered portion, without itself having an elastic sac.

The moving means 82 can use any known mechanism known in the robot technology, but in this embodiment, the first
As shown in FIG. 2, a U-shaped bridging member 82b that reciprocates in the direction of the arrow by an appropriate driving means (not shown) on a pair of site rails 82a, and the bridging member 8
A movable member 82 that moves in the direction of arrow E on the crossbeam member 2b.
c.

A cylinder 82d that can be moved up and down by a linear motor (not shown) is supported by the movable member 82c, and the cylinder 82d is an electromagnetic solenoid or an air cylinder (not shown).
It is shown as being driven by yin and yang to generate positive and negative pressure.

However, if the pipette tip 81a itself has a structure capable of generating negative and positive pressure, such as an elastic water injection bag, the negative and positive pressure generating means 81 compresses the holding means for holding the pipette tip and the elastic water injection bag 9. , and a simple pressing means for restoring.

As seen in FIG. 12, the pipette tip exchanging means 90 has a plurality of pipette tips 81a with the attachment ports for the negative and positive pressure generating means facing upward.

It is shown as having containers 91 arranged in a matrix, means for moving the containers vertically and horizontally on a horizontal plane, and a waste container 92 for receiving used pipette tips 81a. The container 91 is moved back and forth and left and right by a suitable drive means so that the unused pipette tip 81a is positioned at the pipette tip exchange position. Such drive mechanisms themselves are well known in mechanical technology and are therefore not shown and will not be described further. Of course, the yin-positive pressure generating means 8
1 is fixed to form a diluted liquid container and a dilution container assembly.

The pipette tip exchange means may be moved.

As is clear, the measuring and dispensing means 80 moves relative to the liquid to be diluted containers 61 to 64, one dilution container in the dilution container assembly containing a predetermined amount of the sterile dilution liquid, and the liquid to be diluted. The solution can be added to a sterile dilution solution. It is necessary to uniformly mix these liquids, but in this embodiment, the tip of the pipette 91 is dipped into the mixed liquid and suction and ejection are repeated multiple times.

It has been found that this mixing method can achieve sufficiently uniform mixing by suctioning and discharging about three times to mix a relatively small amount of liquid of about 10 to 20 r12. Aspirate to mix even larger amounts of liquid.

It is estimated that it will be necessary to slightly increase the number of discharges.

In order to access the pipette tip 81a into each dilution container in the dilution container assembly, it is necessary to at least partially unseal the closure means 2 of the dilution container assembly l. In a simple device, the closing means 2 is opened manually, but in this embodiment, it is shown as being opened by puncturing with the tip of a pipette 81a.

However, the applicant's separate application (Japanese Patent Application No. 63-28)
The perforation means disclosed in No. 7117, entitled: Apparatus for dispensing samples from sealed packages and sample collection tubes used therein, can be advantageously used.

When using multiple types of liquids to be diluted, they may be individually (or serially) measured and dispensed seven times, or multiple negative and positive pressure generating means 81b may be used in parallel to simultaneously (or in parallel) weighing).

It may also be dispensed.

In the above apparatus, only the liquid to be diluted and the dilution liquid dispensed in advance into the dilution container assembly are used, but if it is necessary to prepare a dilution liquid containing a third liquid, There are many. For example, the liquid to be diluted is a suspension of microorganisms, the dilution liquid is a sterile buffered dilution liquid, and one or more solid medium materials are mixed with the dilution series prepared using these to prepare a pour medium. in which case the melt of the solid medium is contained as a third liquid; one or more containers 1
01 can be placed inside a clean room. In this case, the culture medium melt can be dispensed by the above-mentioned measuring and dispensing means, but if necessary, dedicated measuring and dispensing means can be provided in each of the third liquid containers. As such a dedicated measuring and dispensing means, a tube 10 connected to each container is used.
A dispensing pump 104 is known in which a plurality of pressure rollers are integrally rotated to dispense 2 through a nozzle 103 by compressing and releasing a tube.

Some implementations of the method of the invention using the above-described apparatus are described below.

[Method for preparing one set of a dilution series with multiple dilution steps] A sample liquid containing microorganisms to be tested (for example, a homogenized suspension of food) is placed in a 6-l container for the diluted liquid, and Place it at 60.

A plurality of dilution container assemblies having dilution containers 1a to 1d each containing a predetermined amount (for example, 9 mQ) of sterile buffered dilution water and sealed by a sealing sheet (closing means 2) are arranged in the stock means 71.

One dilution container assembly is transferred to a predetermined distance of 1 m (dilution station DS).

The pipette 81a is attached to the negative and positive pressure generating means 81 of the metering and dispensing means by the pipette changing means 90.

The pipette tip 81a and the container 61 are relatively moved by the moving means 82 and aligned.

A predetermined amount of the diluted liquid in the diluted container 61 (for example, 1m+1
) is attracted.

By the moving means 82, the pipette tip 81a is moved relative to the dilution container assembly l positioned on the dilution stay/con DS, is aligned with one dilution container (for example, la), and the pipette tip is pressed down to seal the sealing sheet 2. The pipette tip 81a is punctured to open the package, and the negative and positive pressure generating means 81 generates positive pressure, thereby discharging the liquid to be diluted that has been sucked into the pipette tip 81a.

The tip of the pipette tip 81a is immersed below the liquid surface of the dilution container 1a, and suction and ejection are repeated several times to mix evenly, thereby preparing the first stage diluted liquid.

A predetermined amount (for example, lr+J) of the prepared diluted solution is aspirated into the pipette tip 81a by the negative and positive pressure generating means 81.

The dilution container assembly l and the pipette 81 are moved by the moving means 82.
The dilution container 1b and the pipette tip 81a are aligned by relatively moving the dilution container 1b and the pipette tip 81a, the sealing sheet 2 is opened at the tip of the pipette tip 81a, and the diluted liquid sucked by the negative and positive pressure generating means 81 is discharged.

The tip of the pipette tip 81a is immersed below the liquid surface of the dilution container 1b, and suction and ejection are repeated several times to mix uniformly, thereby preparing a second-stage diluted liquid.

Repeat the same operation to fill the third dilution container 1c, la. Prepare the fourth stage dilution.

The dilution series thus obtained is inoculated into a separately prepared medium and cultured. When the inoculation is to be sprayed onto the culture medium, the inoculation is dispensed onto the medium in the culture container using the above-mentioned metering and dispensing means, and spread on the surface of the culture medium by, for example, shaking the culture container by an appropriate means. be able to. Such rocking means are disclosed in a separate application of the present applicant.

In order to simultaneously prepare a plurality of sets of the above-mentioned dilution series, a plurality of dilution container assemblies are placed in the dilution station DS, a plurality of negative and positive pressure generating means 81 are connected to the measuring and dispensing means 80, and the above-mentioned dilution series is prepared by the moving means 82. You can perform similar operations at the same time.

[1 of multiple types of diluting liquids obtained by diluting multiple types of liquids to be diluted
Method for preparing a set 1 Prepare 4 sample solutions each containing multiple antibiotics to be tested.
The liquid to be diluted is stored in the containers 61 to 64 of the liquid to be diluted, and placed at the position 60.

A plurality of dilution container assemblies having dilution containers 1a to 1d each containing a predetermined amount (for example, 9 m+2) of sterile buffered dilution water and sealed by a sealing sheet (closing means 2) are arranged in the stock means 71.

One dilution container assembly is in a predetermined position (dilution station D).
S).

The pipette tip 81a is attached to the negative and positive pressure generating means 81 of the metering and dispensing means by the pipette changing means 90.

The pipette tip 81a and the container 61 are relatively moved and aligned by the moving means 82.

A predetermined amount (for example, 1 ml2) of the liquid to be diluted is aspirated.

By the moving means 82, the pipette tip 81a is moved relative to the dilution container assembly l positioned at the dilution station DS, aligned with one dilution container (for example, la), and punctures the sealing sheet 2 with the tip of the pipette tip. The container is opened, the positive pressure is generated in the negative and positive pressure generating means 81, and the liquid to be diluted sucked into the pipette tip sla is discharged.

The tip of the pipette tip 81a is immersed below the liquid surface of the dilution container la, and suction and ejection are repeated several times to mix uniformly, thereby preparing the first stage diluted liquid.

The pipette tip 81a attached to the negative and positive pressure generating means 81 of the metering and dispensing means is replaced by the pipette changing means 90.

The pipette tip 81a and the container 62 are relatively moved by the moving means 82 and aligned.

A predetermined amount (for example, 1 mα) of the second liquid to be diluted is aspirated.

The moving means 82 moves the pipette tip 81a relative to the dilution container assembly 1 positioned at the dilution station DS, aligns it with one dilution container (for example, lb), and seals the seal 7-t 2 with the tip of the pipette tip 81a. The pipette tip 81a is punctured and opened, the positive pressure is generated in the negative and positive pressure generating means 81, and the liquid to be diluted sucked into the pipette tip 81a is discharged.

The tip of the pipette tip 81a is immersed below the liquid surface of the dilution container 1b, and suction and discharge are repeated several times to uniformly mix and prepare the first stage diluted liquid.

Similar operations are repeated to prepare first to fourth antibiotic dilutions in each of the dilution containers 1a-1d.

Each set of diluted solutions thus obtained is used, for example, to prepare a set of media containing different antibiotics in a separately prepared media conditioning device.

In order to simultaneously prepare a plurality of sets of the plurality of diluents described above, a plurality of dilution container assemblies are arranged in the dilution station DS, a plurality of negative and positive pressure generating means 81 are connected to the measuring and dispensing means 80, and a plurality of negative and positive pressure generating means 81 are connected to the measuring and dispensing means 80, The same operations as above may be performed at the same time.

When performing the above-mentioned parallel dilution and parallel dispensing, the separation distance between the plurality of pipette tips 818 and the plurality of diluted liquid containers 61 to 6
If the distances between the four dilution containers or the distances between the dilution containers la to ld in the dilution container assembly are not equal, some preparation means is required. One method is to provide means for changing the spacing between pipettes, but another method is to provide a means for changing the spacing between pipettes, but another method is to provide a means for changing the spacing between the pipettes, but as shown in FIG.
The intervals between the pipettes are adjusted to a distance Q between 1 and 64, and the plurality of dilution container assemblies I are staggered from each other,
The distance between corresponding dilution containers of adjacent assemblies (for example, dilution containers 1a of each assemblage) may be adjusted. In this case, a matrix arrangement such as the dilution container assembly shown in FIG. 5 can be used.

Although not described in detail, it will be understood that by combining the methods described above, one or more sets of dilution series of different types of diluents can be prepared simultaneously.

In the above-described apparatus, the dilution container assembly supply means 70, the metering and dispensing means 80. The operations of the pipette changing means 90 and the maintenance of the conditions within the sterile chamber 50 are under the control of a programmable control means C.

Such control means are well known in robotics technology and will therefore not be described in detail.

When preparing a dilution solution for microbial testing, the dilution solution stored in the dilution container assembly in advance may be sterile physiological saline, sterile buffered dilution water, sterile distilled water for dilution, or sterile dilution medium. It's good. Further, the medium may be a liquid medium or a solid medium.

In the case of a solid medium, it is best to melt it in an autoclave or the like before placing it in a clean room.

On the other hand, the liquid to be diluted may be a liquid containing microorganisms to be tested, or a solution containing various substances to be tested. When a solid medium is prepared, the temperature inside the clean room 50 is
The temperature must exceed the gelation temperature of the solid medium.
Furthermore, when the liquid to be diluted or the third liquid is a specimen containing microorganisms, it is necessary to maintain the temperature within a range in which the microorganisms can survive. It will be clear to those skilled in the art that such a temperature range can be achieved by appropriately selecting the solid medium material depending on the microorganism. When the diluted liquid thus obtained is a medium containing microorganisms (pour-in medium), the container portion of the dilution container assembly 1 can be used as it is as an incubator.

Of course, it is also possible to dispense it into another culture container and culture it. When the obtained diluted solution is a medium containing a diluted solution of the diluted solution, the dilution container assembly can be used as an incubator to immediately inoculate and culture microorganisms. Alternatively, it can be dispensed into other culture containers, inoculated with microorganisms, and then cultured. Further, when the obtained diluted solution is a diluted solution of a substance, the diluted solution is mixed with medium components, and then inoculated with microorganisms and cultured.

Dispensing into culture containers can also be performed using other medium dispensing devices. Furthermore, the operation of inoculating microorganisms into the prepared culture medium-containing culture container can be carried out using the inoculation device disclosed in Japanese Patent Application No. 1983-225800 (Japanese Patent Application Laid-open No. 2-72898) filed separately from the present application. Furthermore, the second
It is also possible to dispense the medium into a sterile room or a sterile thermostatic chamber, inoculate or mix the microorganisms, and transport the culture medium containing the culture medium for a predetermined period of time. Such an inoculation device is built into the sterile room 50, or a second sterile room,
It is clear that the sterile constant temperature chambers may be arranged adjacent to each other and the transport means provided between them, and that the relative movement of the associated equipment and associated containers may be achieved by means of the above-mentioned movement means or their equivalents.

However, following the work described above, the present invention provides a special diluent (culture) plate according to the invention and a cylindrical diluent (
The present invention also relates to a method and apparatus for dispensing a culture medium and culturing using a culture container.

This method and its apparatus will be explained with reference to FIGS. 15 and 16. The sterile chamber 50 includes all the means shown in FIGS. 12 and 13, but most of them are omitted to simplify the drawings.

Conventionally, an Erlenmeyer flask, a petri dish, a petri dish, or the like has been used as a culture vessel containing a culture medium. In the method and apparatus of the present invention, it is of course possible to supply such an incubator set and dispense the diluted solution. At this time, it is advantageous to use a culture vessel assembly in which a plurality of culture vessels are integrated, similar to the dilution vessel assembly.

In a particular embodiment of the method and apparatus of the present invention, a special incubator is used to simplify and make the above-described method and apparatus more efficient.

A first embodiment of the simplified method is characterized by the use of a diluent plate having a set of a plurality of diluent drop areas, as shown in FIGS. 6-8. The use of such diluent plates saves the volume of medium used for the same test purpose, thus also miniaturizing the culture vessel itself, saving its material, and also miniaturizing the equipment that handles it, thus also reducing its This saves on materials, thereby making the culture vessels disposable, and also flattens the colony formation, thereby shortening the culture time, saving the energy required for it, and also making observation easier or easier to observe. It also makes mechanization possible.

The second embodiment of the simplified method is characterized by the use of a cylindrical diluent container 20 as illustrated in FIG. By using such a cylindrical diluent container, there are advantages similar to those of the first simplified method and apparatus.

To be more specific, in the first simplified method and apparatus, as shown in FIG. The drip area 10a of the plate IO is supplied using the above-mentioned metering and dispensing means 80.
-10d, drop a predetermined amount of diluent (medium). When a solid medium is used as the medium, the above operations are performed in a sterile room 50 maintained at the melting temperature of the solid medium. Plate 1 with diluted solution (medium) dropped onto a predetermined dropping area
0 is a transfer means 1 similar to or equivalent to the above-mentioned movement means 72 after coating the floor by the above-mentioned smearing means, if necessary.
20 to the second sterile room 130. The second sterile chamber 130 is maintained at a temperature below the melting temperature of the solid medium and at a desired culture temperature. Thus, when the plate 10 is transferred to the second sterile room 130, the solid medium on the plate 10 is solidified and cultured for a predetermined period of time. If necessary, when the culture medium is transferred to the second sterile room 130, it may be passed through a cooling room or exposed to cold air in order to cool and solidify the culture medium in a short time. The steps in the second sterile room can also be carried out under the preprogrammed control of the control means C.

When using the cylindrical diluent container 20 shown in FIG. 11, as shown in FIG. The opening 20b may be attached in place of the pipette tip 81a to aspirate the culture medium.

It is clear that the same or equivalent means as for the pipette tip 81a can be used for supplying and attaching the cylindrical diluent container. Therefore, a container corresponding to container 91 is shown as 141, and means for moving the container 14'l vertically and horizontally on a horizontal plane are not shown, and explanations of their operations will be omitted. Diluent container 20 into which the diluent (medium) has been sucked
The cells are transferred to the second sterile room 130 and cultured for a predetermined period of time in the same manner as in the above method.

Furthermore, observation 9 judgment of the culture results is done by counting the number of colonies,
Minimum inhibition circle measurement etc. can be easily automated using optical equipment, and standard data such as characteristics related to colonies of various microorganisms, such as the shape of the colony or its surroundings, light reflectance, absorption wavelength 1 reflected light wavelength, etc., can be accumulated. Automation may also be possible by automatically comparing data from the optical device. At this time, it is possible to add a substance for determination to the surface of the medium to facilitate determination.

Although some embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments, and those skilled in the art will be able to make various modifications and changes without departing from the technical idea of the present invention. It will be clear that variations are possible.

[Brief explanation of drawings]

FIG. 1a is a plan view of a dilution container assembly used in the method and apparatus of the present invention, FIG. 1 is a right side view of the dilution container assembly, and FIG.
FIG. 3 is a plan view of a modification of the dilution container assembly, FIG. 3 is a perspective view of a modification of the dilution container assembly, FIG. 81!4 is a perspective view of a modification of the dilution container assembly, and FIG. 5 is a dilution container assembly. 6 is a sectional view of a diluent plate, FIG. 7 is a sectional view of a modified diluent plate, FIG. 8 is a perspective view of a modified diluent plate, and FIG. 9 is a sectional view of a modified diluent plate. 8 is a sectional view of the diluent plate of FIG. 8, FIG. 10 is a sectional view of a modification of the diluent plate of FIG. 7, FIG. 11 is a perspective view of a cylindrical diluent container according to the present invention, and FIG. FIG. 13 is a schematic layout of a device for carrying out the basic method of the present invention; FIG. 13 is a schematic layout of a dilution container assembly of the present invention; FIG. 14 is a diagram showing the relative arrangement of the liquid container and the dilution container assembly. Layout diagram: FIG. 15 is a schematic layout diagram of a diluting device according to another embodiment of the present invention; FIG. 16 is a schematic layout diagram of a diluting device according to still another modification of the present invention. Explanation of symbols 1: Container portion, 1a-1f: Dilution container, le: Swelling portion, 2: Closing means, 2a: Both corner portions, 2b = Center portion, 2
c: Notch, 3: Connecting plate, 3a: Notch part, 10
Nibrate, 10a-10d: diluent dropping area, loe
: separation area, 10f = convex surface, 11: cover, 20: cylindrical diluent container, 20a-20b: opening, 20c: cavity, 20d: boundary line, 50: clean room, 51: air circulation device, 52: gas Supply means, 53: Thermostat, 5
4: heat exchange device, C: control means, 60: liquid to be diluted container arrangement position, 61 to 64: liquid to be diluted container, 70: dilution container assembly arrangement position, 80: measuring and dispensing means, 7I varnish docking means, 71a :Case, 71b:Table, 71c:Opening, 71d:Pu'i Yuba-171e Nislit, 72
: Transfer means, 72a Nislit, 72b: Bottom surface, 72c
: Side guide means, 72d: Pusher means, DS=diluting stainless steel, 80: Measuring and dispensing means, 8I: Positive and negative pressure generation means, 81a: Pipette tip, 82: Moving means, 82a:
Side rail, 82b = bridging means, 82C: movable member,
82d Niji Linda, 90: Pipette tip exchange means, 9
1: Pipette tip container, 92: Waste container, 101: Third liquid container, 102: Tube, 103: Nozzle, 104
: Dispensing pump, J'0 varnish docking means,] 20: Transfer means, 130: Second clean room, 141: Container, engraving of drawings (no changes to the contents) Fig. 1q Coffin 2 Helmet 1 Fig.

Claims (1)

[Scope of Claims] [1] In one clean room, (a) at least one liquid to be diluted container containing a liquid to be diluted is arranged at a predetermined position; (b) a liquid to be diluted is contained; (c) placing at a predetermined position at least one diluent container assembly in which a plurality of dilution containers sealed by a closing means are integrally molded in a predetermined arrangement; (c) closing the lid of the dilution container assembly; (d) at least one negative and positive pressure generating means to which a pipette tip can be removably attached; and means for exchanging the pipette tip attached to the negative and positive pressure generating means; A metering and dispensing means having a pipette tip exchange means and a moving means for moving one negative and positive pressure generating means relatively with respect to the liquid to be diluted container and the dilution container assembly is provided, and the negative and positive pressure generating means is provided. (e) aligning each of the pipette tips attached to the pipette tip with each of the liquid to be diluted containers and aspirating a predetermined amount of the liquid to be diluted into the pipette tip; (e) the pipette attached to the measuring and dispensing attachment; Each of the tips and the dilution container assembly are moved relative to each other to align each of the pipette tips with each of the corresponding number of dilution containers in the dilution container assembly, and aspiration is applied to each of the pipette tips. discharging the diluted liquids into the aligned dilution containers and mixing them uniformly to prepare at least one diluent; (f) performing steps (d) and (e) above as necessary; (g) repeating the following two steps a predetermined number of times (including 0 times) as necessary; (i) using a predetermined amount of each of the diluted solutions prepared in the previous step; (ii) relatively moving each of the pipette tips attached to the measuring and dispensing device and the dilution container assembly; (h) aligning the dilution containers with a corresponding number of other dilution containers in the dilution container assembly, discharging each of the aspirated dilution liquids into the aligned dilution containers and mixing uniformly; exchanging at least one pipette tip attached to the dispensing device at a predetermined timing with a pipette tip changing device; (i) performing at least the above steps (d) to (h) under preprogrammed control of a control means; 1. A method for preparing a set of different dilutions, the method comprising: preparing a set of different dilutions. [2] A method for preparing a set of a plurality of different dilution liquids in the method according to claim 1, further comprising the following steps: (a) storing a third liquid in the clean chamber; a third liquid container, a pump means for pumping a predetermined amount of the third liquid from the container, and a nozzle for discharging the pumped third liquid.
relatively moving at least one of the liquid supply means, the at least one nozzle, and the dilution container assembly;
A relative moving means for aligning the corresponding number of dilution containers is further provided, and when the at least one nozzle and the corresponding number of dilution containers are aligned, a predetermined amount of the third liquid is dispensed into each dilution container. (b) When the at least one third liquid is a solid medium component, the temperature in the clean chamber is maintained at a temperature exceeding the gelling temperature of the solid medium component, and microorganisms are not present in the clean chamber. (c) performing steps (a) and (b) in the clean room under pre-programmed control of the control means; things to do [3] The method according to claim 1 or 2,
A method for preparing a diluent plate onto which a plurality of sets of different diluents are dropped, further comprising the following steps: (a) providing a predetermined number of mutually separated diluent drop regions on the plate surface; (b) placing in place at least one diluent plate having a set of diluent drop areas arranged in an array and a separation area surrounding them; (b) at least one pipette tip in the metering and dispensing means; (c) aspirating a predetermined amount of the prepared diluent by relatively moving and aligning the corresponding number of dilution containers in the dilution container assembly; (c) the at least one pipette tip in the metering and dispensing means; and a corresponding number of diluent dropping areas on the diluent plate, and aligning them by relative movement, and dropping the diluent sucked into the pipette, respectively; (d) if necessary, step (b) above; and (c) repeating a predetermined number of times; and (e) performing the steps (b) to (d) in the clean room under preprogrammed control of the control means. [4] The method according to claim 1 or 2,
A method for preparing a set of cylindrical diluent containers containing a plurality of different diluents, characterized by further adding the following steps: (a) Attaching a suction port at one end to a negative and positive pressure generating means at the other end; A cylindrical diluted liquid container having a hollow part in the middle part and having a dimension as small as possible in the direction of at least one of the X, Y, and Z axes orthogonal coordinates that define the internal volume of the hollow part. (b) relatively moving at least one negative and positive pressure generating means in the metering and dispensing means and the set of cylindrical diluent containers to form at least one cylindrical diluent container; (c) relatively moving at least one cylindrical diluent container attached to the negative and positive pressure generating means and the corresponding number of dilution containers in the dilution container assembly; (d) detaching the cylindrical diluent container into which the diluent has been sucked from the suction means; (e) the step (b) ~ (d) under the pre-programmed control of said control means. [5] An apparatus for preparing a set of a plurality of different diluting solutions, characterized by the following features: having one clean chamber and one control means, the clean chamber (a) containing a liquid to be diluted; (b) A diluent container arrangement position in which at least one container for a diluent is arranged, and (b) a diluent solution in which a plurality of dilution containers containing a diluent and sealed by a closing means are integrally molded in a predetermined arrangement. (c) at least one negative-positive pressure generating means to which a pipette tip can be removably attached; and (c) at least one negative-positive pressure generating means to which a pipette tip can be removably attached, and attached to the negative-positive pressure generating means. and means for moving the at least one negative and positive pressure generating means relative to the pipette tip changing means and the liquid to be diluted container and the dilution container assembly. and a dispensing means, and the control means is a programmable control means for controlling the operations of various means in the clean room. [6] The apparatus according to claim 5, further comprising means for unsealing at least a portion of the closing means of the dilution container assembly, for preparing a set of a plurality of different diluents. Device. [7] In the device according to claim 5 or 6,
Further, an apparatus for preparing a set of a plurality of different diluting liquids, etc., characterized by: (a) a third liquid arrangement position for arranging at least one third liquid container containing a third liquid at a predetermined position; (b) The moving means in the measuring and dispensing means relatively moves the negative and positive pressure generating means and the third liquid container arranged at the third liquid arrangement position. [8] The apparatus according to any one of claims 5 to 7, further characterized in that: (a) at least one of the containers containing the third liquid; (b) a third liquid having a pump means for pumping a predetermined amount of the third liquid from the third liquid container, and a nozzle for discharging the pumped third liquid; and (c) a second moving means for relatively moving the at least one nozzle and the dilution container assembly. [9] The apparatus according to any one of claims 5 to 8, further characterized by: (a) a predetermined number of diluents on the plate surface; The method further includes a diluent plate arrangement position for arranging at least one diluent plate at a predetermined position, the diluent plate having at least one diluent plate having a set of mutually separated diluent drop regions arranged in a predetermined arrangement and a separation region surrounding them. (b)
The moving means in the measuring and dispensing means includes at least one pipette tip attached to the negative and positive pressure generating means;
It has a second moving means for relatively moving the corresponding number of diluent plates arranged at the diluent plate arrangement positions. [10] The apparatus according to any one of claims 5 to 8, which further comprises: (a) having a suction port at one end; ,
The other end has a mounting part that is attached to the negative and positive pressure generating means, and the intermediate part has a hollow part, and the dimension in the axial direction of at least one of the X, Y, and Z-axis orthogonal coordinates that determines the internal volume of the hollow part is possible. a cylindrical diluent container arrangement position in which a set of cylindrical diluent containers formed to be relatively small are arranged in a predetermined arrangement, and (b) a moving means in the measuring and dispensing means is attached to the negative and positive pressure generating means The method further comprises means for relatively moving at least one pipette and a corresponding number of cylindrical diluent containers disposed at the cylindrical diluent container arrangement positions to exchange them. [11] A predetermined number of mutually separated diluent drop areas and a separation area surrounding them are formed on the plate surface, and each of the drop areas spreads the dropped diluent over as wide an area as possible. A diluent plate having a bottom area and a depth (shallowness). [12] The diluent plate according to claim 11, wherein the dropping region is a recess having a large area and a shallow depth. [13] In the diluent plate according to claim 11 or 12, the surface of the dropping region has an affinity for the diluent to be dropped, and the surface of the separation region has an affinity for the diluent to be dropped. A culture plate characterized by being endowed with affinity. [14] In the diluent plate according to any one of claims 11 to 13, a lens-shaped convex surface is formed on the back surface of the plate in an arrangement corresponding to the dropping area. Features a diluent plate. [15] A mounting part for mounting a suction port on one end and a negative/positive pressure generating means on the other end, having a hollow part in the middle part, and at least one of the X, Y, and Z axes orthogonal coordinates defining the internal volume of the hollow part. 1. A diluted liquid container, characterized in that dimensions in two axial directions are formed as small as possible, and when the diluted liquid is sucked and filled into the cavity, the diluted liquid is spread thinly. [16] The diluent container according to claim 15, further comprising a boundary mark indicating a predetermined amount of the diluent in the cavity.