JPH0533040B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to automation of screening work for extracting useful microorganisms with specific properties from a huge variety of microorganisms existing in nature, and further relates to In detail, by improving the fishing bacteria head part of the colony transplantation device and the cutter device, it is possible to securely remove the bacteria from the growing strain without the attachment of the growth medium, and to ensure the prevention of contamination with unintended colonies and other bacteria. This invention relates to a colony transplantation method that greatly improves the success rate of screening operations.

[Conventional technology] Conventionally, in screening work, an operator uses the naked eye or a microscope to identify colonies to be collected on a growing shear, collects the target colony using a platinum wire, etc., and transfers it to another shear. However, this work required a high degree of skill as it required the ability to identify colonies and the accuracy of positioning. In addition, the work of collecting colonies from a growth tray and transplanting them into a transplant tray (hereinafter referred to as "fishing work") requires repeating the work until a pure culture of the desired colony is possible, and requires a lot of labor from the operator. It was very tiring, and since it was a manual process, the data obtained could vary due to individual differences between workers, and microorganisms carried by the workers could confuse test results.

Under these circumstances, there was a desire for the development of a device that could perform the screening work without human intervention, and a recent application filed in an attempt to meet this demand is Japanese Patent Application No. 57-84011. There is a colony transfer device.

This device places a growing shear dish and a transplanting shear dish on two sets of XY stage sections, and moves both shears as appropriate under the control of a computer based on image information obtained from a monitor section. The general idea is to rotate and move the pick-up section, which corresponds to the bacterial head section, to carry out the bacterial fishing operation, and the pick-up section is made of gold, aluminum, or metal, just as in the past when it was done manually. A thin glass fiber wire is used, and the device is equipped with means for cutting and heat sterilizing the thin wire at appropriate locations, and the thin wire is appropriately fed out to collect the desired colony on the growth shear plate, transplanted to the transplanting shear tray, and then cut. At the same time, it is heat sterilized.

[Problem that the invention seeks to solve]

In this device, a hard material such as metal or glass fiber is used for the fishing rod that serves as the colony collecting part, but this is because it is easy to sequentially feed out the thin wire wound around the reel and form it into a straight line. This is because the tip of the thin wire does not swing even when the pick-up section is moved, so it is possible to accurately pick up a specific colony on the growing shear.However, on the other hand, the elasticity in the longitudinal direction Therefore, if the distance between the pick-up part and the surface layer of the growth medium is not precisely measured and the vertical movement of the pick-up part is controlled, the tip of the thin wire will penetrate into the medium when collecting colonies, and the growth medium itself will also be collected at the same time. That will happen. In reality, it is extremely difficult to measure the distance considering the thickness of each colony and to control the vertical movement of the pick-up part based on the measured value, so adhesion of the growth medium is unavoidable. In this case, there was a risk that the screening work itself, which aims to create pure cultures of bacteria based on their adaptability to foreign media, would become meaningless.

[Means for solving problems]

The present invention was made to solve these problems, and uses a rod-shaped viscous synthetic resin extruded from a thin wire for the fishing rod serving as the sampling part, so that the fishing rod flexibly bends according to the undulations of the surface layer of the growth medium. By absorbing the distance deviation between the head of the fishing bacteria and the surface layer of the growth medium, the aim is to ensure colony collection without the growth medium adhering, thereby significantly increasing the success rate of screening work. The gist of this is: a transport position for transporting the growing shear and the transplanting shear to a predetermined position; and having a thin tube at the tip that serves as an outflow path for the viscous synthetic resin, and extruding the viscous synthetic resin based on appropriate instructions. a fishing bacteria head having a mechanism for extruding fishing bacteria in a substantially quantitative amount; a transfer device capable of holding the fishing bacteria head and accurately transferring it to a predetermined position; A cutter device equipped with a heating means for cutting the collecting end of the head; a visual sensor and an image recognition device for identifying colonies on the growing and transplanting shears and controlling the position management of the fishing fungus head. and forming a fishing rod by extruding the viscous synthetic resin from the thin tube using a substantially quantitative extrusion mechanism, and cutting the tip of the fishing rod to form a collection end,
Next, under the control of the image recognition device, the head of the fished bacteria is moved onto a growth tray, stopped on a specific colony, and then lowered to collect a colony at the collecting end. The method is characterized in that the above process of moving and lowering the head portion to a suitable location in the shear tray for transplantation and transplanting the colony is repeated an appropriate number of times.

[Effect]

A colony transplantation method based on such a configuration will be explained with reference to the principle diagram shown in FIG. When the growing shears and transplanting shears transferred by the conveyance device stop at a predetermined position, a fixed amount of viscous synthetic resin j is extruded from the thin tube d at the tip of the fishing fungus head by a substantially quantitative extrusion mechanism, and the fishing rod e is extended. Subsequently, the fishing rod e is transferred to a suitable location in the cutter device l, and the tip of the fishing rod e is cut off, exposing the collecting end f. As a result, the sampling end f is kept sterile at all times, and it is possible to completely prevent unintended bacteria from being mixed into each fishing operation. In addition, since the fishing rod e pushed out from the thin tube e always hangs vertically due to its own weight, positioning when contacting a specific colony, which will be described later, is easy, and the accuracy of the amount of extrusion is also
Since the length of the fishing rod e is made uniform by cutting with the cutter device l, it does not require much precision.

Subsequently, the fishing bacteria head part b is transferred above the growth shear g by the transfer device e, and stops precisely above the colony i to be collected. This operation is monitored by a visual sensor c installed near the fishing bacteria head and controlled by an image recognition device connected to the visual sensor c, and is based on pre-registered colony selection data and manual input each time. This will be done according to the information.

Next, as shown in Figure 1 D, the fishing rod head b is lowered a certain distance and the collecting end f of the fishing rod e is brought into contact with colonies of different heights growing on the growth medium to collect the colonies. However, there is no need to measure the descending distance at this time, and it can always be constant.For example, if the collecting end f is slightly pressed against the growth medium h and just touches it, the fishing rod e will be able to move the colony. Even after contact with i, it is further pushed and curved, moderately buffering the pressing force caused by the descent of the fishing bacteria head b, and preventing the collecting end f from rushing into the growth medium h. It is possible to ensure the attachment of colony i to the cell. When the collection is completed, the head b of the fish is raised, transferred to a predetermined position in the shear k for transplantation as shown in FIG. 1E, and lowered again. At this time, since the fishing rod e is curved, it is necessary to make its descending distance a little longer than when collecting, or to place the shear k for transplantation at a slightly higher position than the shear g for growth. Yes, but
Since the horizontal position does not require as much precision as when collecting, there is no problem due to the curved fishing rod e.

Once the transplantation is complete, the fishing rod e is moved to its initial position to prepare for the next colony collection, but as mentioned above, the fishing rod e is first extended and the curved tip is Since the parts are cut and discarded, the fishing rod e is always kept vertical and straight during collection, and new colonies can be collected with high precision.

〔Example〕

Next, details of the colony transplantation method according to the present invention will be described based on a specific device. FIG. 2 is a block diagram showing an outline of an embodiment of the apparatus embodying the present invention, and in the figure, 1 indicates a bacterial fishing station, which is a predetermined position of a growing shear 2 and a transplanting shear 3, respectively, in the fishing bacteria work. 4. A conveying device for transferring to the transplant station 5, which is not shown in this embodiment, but is composed of tube-shaped rubber belts arranged in parallel at intervals narrower than the diameter of the shears;
The seedlings for growing and the seedlings for transplanting are placed facing each other. In the illustration, only one transplanting shear can be supplied at a time, but it is possible to use an appropriate mechanism to supply multiple transplants at the same time, and the transplant medium in each shear is a medium containing different nutrients. This makes it possible to test multiple growth conditions at once, greatly increasing work efficiency. In addition, a light source 6 that illuminates the growth shear plate 2 is installed at the bottom of the fishing bacteria station, realizing a bright field and dark field light source corresponding to the translucency of the colony. A light source may also be employed as appropriate.

In the figure, reference numeral 7 denotes a fishing head, which is a main part of the present invention, and its configuration is as described below. Information obtained from the visual sensor 8 is processed by an image recognition device 11 provided at an appropriate location by arranging an image sensor, etc., to control the fishing operation, for example, to select and collect colonies on a growth tray, and to select and collect uncollected colonies. Checking the colony.

The fishing bacteria head 7 and the visual sensor 8 are held by a transfer device and moved between the growing shear 2 and the transplanting shear 3, but in this embodiment, the movement in the X and Y axes is performed with an accuracy of several tens of μm. possible at the same time
XY stage 9 is used.

Reference numeral 10 in the figure is a cutter device, which is placed between the fishing rod station 2 and the transplanting station 3, and cuts off and discards the tip of the fishing rod immediately before each fishing operation to expose a new harvesting end. This is to keep the distance from the tip of the capillary tube to the collection end constant and to keep the collection end sterile at all times, and any device can be used, but for example, in this example, a horizontal reciprocating device is used as shown in Figure 5. A moving cutting blade 27 is used, a thin ceramic plate is used for the cutting blade 27, and a resistance pattern 28 is printed on the thin plate as a heating part to constantly heat sterilize the cutting blade 27. Further, the cutter device can be constructed separately from the cutting blade 29 and the heating unit 30 as shown in FIG. The heating section 30 is fixedly installed near the cutting blade 29 to constantly heat the cutting blade 29, and the peripheral area of the cutter device is insulated by an appropriate mechanism so that the heat supplied from the heating section 30 can be efficiently heated to the cutting blade 29. It is sufficient if the structure is such that the cutting blade 29 is sufficiently heated and sterilized. If such a structure is adopted, not only will the range of material selection for the cutting blade be expanded, there will be no need to worry about breakage of the lead wire connected to the heating section 30, and even if a failure occurs, parts replacement will be minimized. This means that maintenance can be done easily and at low cost. In the illustrated example, the cutting blades 29 are provided facing each other and are configured to reciprocate at the same time, but the cutting blades 29
It is also possible to use only one side of the cutting blade 9, or to fix one side of the cutting blade 29 so that there is only one movable part.

Next, we will discuss the surroundings of the fishing bacteria head section and the structure of the fishing bacteria head section 7. For example, as shown in FIG. It is connected to a base 15 and can be raised and lowered via a rod 17 using a cylinder 16 as a driving source. The fishing fungus head section 7 is constructed by arranging a thin tube 1 that serves as an outflow path for the viscous synthetic resin 13 at the bottom of a test tube-shaped cylinder, and houses the viscous synthetic resin 13 inside, and the upper surface of the viscous synthetic resin 13 By arranging a pushing plate 20 and supplying compressed air from above as appropriate, it is possible to push out the fishing rod 19 at intervals of a fixed length.

In addition, the viscous synthetic resin 13 has no nutrients so that its components do not affect the growth conditions of bacteria, and has an appropriate viscosity that allows the fishing rod 19 pushed out of the thin tube 18 to maintain its shape for a certain period of time. Although silicone resin is used in this embodiment, any viscous synthetic resin may be used as long as it satisfies the above conditions. Furthermore, in this embodiment, the housing section 24 for the viscous synthetic resin 13 and the thin tube 18 are integrally constructed, and the housing section 24 also moves up and down as the fishing rod 19 moves up and down.The thin tube 18 and the area around the thin tube are shown in FIG. The accommodating portion 21 is fixed to the pedestal 22 and arranged as a separate body, and the guide tube 2
If the mechanism is to feed a fixed amount of viscous synthetic resin to the thin tube 18 through the tube 3, the vertically moving portion will be the thin tube peripheral portion 27.
It is also possible to limit the weight of the lifting and lowering parts to only one part.

Each of the above-mentioned devices is housed in a sterilized airtight case, and an opening/closing device 12 is provided in close proximity to the transport device 1 to remove and attach the lid of each tray.
It is constructed so that contamination with germs can be eliminated as much as possible by installing an air purifier and the like as appropriate.

Colony transplantation using the device constructed in this manner is performed as follows. The growing shear 2 and the transplanting shear 3 placed on the end of the conveying device from the outside of the sealed case by appropriate means are used as a fishing bacteria station 4 and a transplanting station 5, respectively.
It will be transported to the point where it will stop. The conveying device 1 consists only of rubber belts arranged in parallel, and does not stir up unnecessary bacteria in the sealed case due to vibrations during movement. Next, the opening/closing device 12 provided close to the conveying device 1 is driven to remove the lid of each tray, and a certain amount of compressed air is supplied from the upper part of the fishing bacteria head to the presser plate 24 in the storage section 24. When pressed, the viscous synthetic resin 13 is pushed out by a certain amount from the thin tube 18 and hangs down in the vertical direction due to its own weight. The length of the fishing rod 19 formed by extrusion at this time is
Since the cutting operation of the subsequent cutter device 10 is adjusted to be almost constant, it can be seen that even if the precision of the quantitative extrusion device is not so high, it does not pose an obstacle to the fishing operation. Needless to say, high precision is desirable from an economical point of view, as the parts will be discarded.

Next, the tip of the fishing rod 19 is cut off by the cutter device 10 to expose a new collection end 26 in preparation for colony collection, but this cutting ensures that the length of the fishing rod 19 is always kept constant. At the same time, the sampling end 26 is made sterile to prevent unintended bacteria from being mixed into the fishing operation. In particular, in this embodiment, the cutting blade 27 is heat sterilized, so that the cutting blade 27 is always maintained in a sterile state, and the cutting blade 27 does not carry bacteria. In addition, if the cutting position is set so that when the fishing rod 19 descends, it will slightly press the surface layer of the growth medium of standard thickness, the fishing rod 19 will be able to bend flexibly even if the thickness of the growth medium varies slightly. Since it absorbs distance differences, it can be used with growth media of any thickness.

Once the fishing rod 19 is cut to a predetermined length, the XY stage 9 is moved to stop moving the fishing bacteria head 7 and the visual sensor 8 onto the growing shear tray 2.
An image of the growing shear 2 is captured. At this time, whether the light source 6 is bright field or dark field is appropriately determined depending on the type of colony. The captured image is processed by the image recognition device 11 to identify the colony to be collected, and the XY stage 9 is simultaneously moved along the X and Y axes to accurately stop the fishing head 7 above the target colony. However, since this process of identifying colonies is controlled by software, the conditions for identifying colonies can be changed and set as desired. For example, you can extract only those of a certain size and shape, or By combining with the filters that are placed, it is also possible to add color to the conditions.

Once a colony to be collected is identified, the fishing head 7 is lowered by the lifting mechanism 25 to bring the collection end 26 into contact with the colony and attach bacteria, but this lowering distance may always be a constant distance. .
That is, the heights of individual colonies from the surface of the culture medium differ, but as mentioned above, the fishing rod 19 flexibly curves according to the height of each colony to absorb the distance difference, so that the fishing rod 19 It will not rush into. If bacteria adhere to the fishing rod 19, the fishing rod head 7
At the same time, the XY stage 9 moves the fishing bacteria head 7 to the upper part of the predetermined array position of the transplanting shear dish 3 and stops it, and then lowers it again to remove the bacteria attached to the collecting end 26 into the transplant medium. port to. Although the fishing rod 19 is curved at this time, the position of contact of the harvesting end 26 with the transplant medium does not require much precision, so there is no problem in being curved. In consideration of this curvature, a vertical step is provided in the mounting position of the growing shear 2 and the transplanting shear 3. After transplantation, the fishing bacteria head 7 is moved to a predetermined position, and then the fishing rod 19 is extended and cut again to remove the curved part of the fishing rod 19 and sterilize the collection end 26, and then the next colony is harvested. The collection work will begin.

The above process is repeated under the control of the image recognition device 11, and the end point is when certain conditions are met, such as when all bacteria of a specific shape and size have been transplanted. After the opening/closing device 12 is driven to cover the transplant tray 3, the transfer device 1 transfers it to the outside of the sealed case. As shown in FIG. 4, if the fishing rod head section is constructed separately from the viscous synthetic resin storage section 21 and the thin tube peripheral section 31, the load on the lifting mechanism 25 can be reduced due to weight reduction, and the load on the lifting mechanism 25 can be reduced during lifting and lowering. It is possible to eliminate the negative influence of inertia on the extrusion operation, and it is also possible to increase the storage volume of the viscous synthetic resin, making it possible to work continuously for a long time without replenishing it.

In addition, when multiple transplanting dishes are arranged, the adaptability of the same colony to a different type of culture medium can be investigated by distributing the colonies collected in one fishing operation to multiple transplanting dishes in succession. This makes it possible to further improve the efficiency of screening work.

〔Effect of the invention〕

According to the present invention, screening work, which conventionally required a large amount of time to be performed by skilled workers, can be automated without human intervention, making screening work faster and more efficient. Not only can this greatly reduce the burden on workers, it can completely eliminate bacteria transmitted by workers by eliminating manual labor, and it can also improve the accuracy of screening work by eliminating variations in data due to individual differences between workers. It is something that can increase the

In addition, in the present invention, since the fishing rod is made of viscous synthetic resin, it can flexibly respond to the surface condition of any growth medium. It is possible to reliably collect only those colonies that will Furthermore, the collecting end is supplied immediately before each fishing operation, and this supply is achieved by cutting the tip of the fishing rod with a cutter device kept in a sterile state in a heating section. The success rate of screening work can be greatly improved without contamination with bacteria other than the intended one.

[Brief explanation of the drawing]

Figure 1 A, B, C, D, and Ho are simplified explanatory diagrams showing an overview of the colony transplantation method according to the present invention;
The figure is a block diagram showing an outline of an embodiment of the present invention, FIG. 3 is a simplified diagram showing the structure of the area around the fishing rod head in the same embodiment, and FIG. 4 is a diagram showing the vicinity of another fishing rod head in the same embodiment. FIG. 5 is a simplified explanatory diagram of the cutter device in the same embodiment.
FIG. 6 is a simplified explanatory diagram of another cutter device in the same embodiment. a... Transfer device, b... Fishing bacteria head, c...
Visual sensor, d...tube, e...fishing rod, f...
Harvesting end, g...Shear for growth, h...Growing medium, i...Colony, j...Viscous synthetic resin, k...
...Shear for transplantation, l...Cutter device, 1...
Conveying device, 2... Shear tray for growth, 3... Shear tray for transplantation, 4... Fishing bacteria station, 5... Transplanting station, 6... Light source, 7... Fishing bacteria head section, 8... Visual sensor, 9 ...XY stage,
10... cutter device, 11... image processing device,
12... Switching device, 13... Viscous synthetic resin, 14
... Lifting holding part, 15 ... Pedestal, 16 ... Cylinder, 17 ... Rod, 18 ... Thin tube, 19 ...
Fishing rod, 20... Pressing plate, 21... Accommodation section, 22...
... Pedestal, 23 ... Guide tube, 24 ... Accommodation section, 25
... Lifting mechanism, 26 ... Collection end, 27 ... Cutting blade, 28 ... Resistance pattern, 29 ... Cutting blade, 3
0... Heating section, 31... Tube periphery.

Claims (1)

[Scope of Claims] 1. A conveying device for transporting the seedlings for growing and the seedlings for transplantation to predetermined positions; A substantially quantitative transporting device having a thin tube at the tip serving as an outflow path for the viscous synthetic resin and extruding the viscous synthetic resin in accordance with an appropriate command. a fishing bacteria head having a push-out mechanism; a transfer device capable of holding the fishing bacteria head and accurately transferring it to a predetermined position; and a transfer device that is disposed at an appropriate position within the movement range of the fishing bacteria head and moving the fishing bacteria head. a cutter device equipped with a heating means for cutting the collecting end of the fish; and a visual sensor and an image recognition device for identifying colonies on the growing petri dish and the transplanting petri dish and controlling the position management of the fishing bacteria head. A fishing rod is formed by extruding the viscous synthetic resin from a thin tube using a substantially quantitative extrusion mechanism, and the tip of the fishing rod is cut to form a collection end, and then a fishing bacteria head is removed under the control of an image recognition device. is moved onto the growth shear plate and stopped on a specific colony, and then lowered to collect a colony at the collecting end, and then the fishing fungus head is moved to an appropriate location on the transplantation shear plate and lowered. A method of transplanting a colony by repeating the above process an appropriate number of times. 2. The colony according to claim 1, characterized in that the cutting blade of the cutter device is made of ceramics, and the cutting blade is also provided with a heating section to disinfect and sterilize the surface layer of the cutting blade. How to transplant. 3. The method of transplanting a colony according to claim 1, wherein the cutter device is constructed separately from a cutting blade that reciprocates horizontally and a fixed heating section. 4 Claims 1 and 2 characterized in that silicone resin is used as the viscous synthetic resin.
A method for transplanting a colony according to paragraphs 1 and 3. 5. Claims 1 and 2 characterized in that a plurality of shears for transplantation are supplied at the same time.
The method for transplanting a colony according to paragraphs 3 and 4.