JPS6265700A - Transplantation of colony - Google Patents

Abstract

PURPOSE:To enable the sure collection of a colony without accompanying a culture medium, by using a fishing rod composed of a viscous synthetic resin rod extruded from a capillary tube. CONSTITUTION:A fishing rod (e) is formed by extruding a viscous synthetic resin (j) from the capillary tube (d) of a finishing head (b) and the tip of the rod (e) is cut off to form a collecting end (f). The fishing head (b) is transferred above the culture Petri dish (g) under the control with an image recognizing device, stopped above a specific colony (i) and lowered to collect the colony (i) with the collecting end (f). Thereafter, the fishing head (b) is transferred to another Petri dish (k) for transplantation and lowered to effect the transplantation of the colony.

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 For details, by improving the fishing bacteria head and cutter device of the colony transplantation device, we are able to reliably collect bacteria from the growth petri dish without the attachment of the growth medium, and also ensure the prevention of contamination with unintended colonies and other bacteria. This invention relates to a method for transplanting colonies that greatly improves the success rate of star-leaning operations.

[Conventional technology]

Traditionally, in screening work, a worker used the naked eye or a microscope to identify the target colony on a growth petri dish, collected the target colony using a platinum wire, etc., and transplanted it to another petri dish. However, this work required a high level 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 petri dish and transplanting them to a transplantation dish (hereinafter referred to as bacterial fishing work) must be repeated until a pure culture of the desired colony is possible, which requires a lot of effort from the operator. It is very tiring, and since it is manual work, the data obtained may vary due to individual differences among workers.
Microorganisms transmitted by workers could also confuse test results.

In such a situation, there was a strong demand for the development of a device that could perform the screening work without human intervention, and a patent application filed in 1983 was recently filed in an attempt to meet this demand. There is a YIrJ knee transfer device of Fi, No. 1011.

This device is 2! The 1F cultivation petri dish and the transplanting petri dish are placed on the XY stage part of lJ1, and both dishes are moved appropriately under the control of a computer based on the image information obtained from the monitor part. The general idea is that the picking section, which corresponds to the bacterial head section, is rotated to perform the bacterial fishing operation, and the collecting section of the pickup section is made of gold, aluminum, or glass, similar to the conventional manual method. Using a thin fiber wire, and providing means for cutting and heat sterilizing the thin wire at appropriate locations in the device,
The thin wire is drawn out as appropriate to collect the desired colony on the growth petri dish and transplanted to the transplantation petri dish, which is then cut and sterilized by heat.

[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 collection 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 oscillate even when the pink amplifier part is moved, so it can be expected to have the effect of accurately collecting a specific colony on a growth dish. However, on the other hand, the elasticity in the longitudinal direction Therefore, if the distance between the pickup part and the surface layer of the growth medium is not precisely measured and the vertical movement of the pink amplifier part is controlled, the tip of the thin wire will penetrate into the medium when collecting colonies and destroy the growth medium itself at the same time. It will be collected. In reality, it is extremely difficult to measure the distance considering the thickness of each colony and to control the vertical movement of the pink-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 tube for the fishing rod serving as the collection 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 fishing bacteria head and the surface layer of the growth medium, it is possible to reliably collect colonies without the growth medium adhering, thereby significantly increasing the success rate of screening work. The gist of this is that it includes a conveying device that transports the growth petri dish and the transplanting petri dish to a predetermined position; it has a thin tube at the tip that serves as an outflow path for the viscous synthetic resin, and extrudes the viscous synthetic resin based on appropriate instructions. a fishing bacteria head having a fixed view pushing mechanism; 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 collection end of the head; a visual sensor and an image recognition device for identifying colonies on the growth and transplanting petri dishes and controlling the position management of the fishing bacteria head. A fishing rod is formed by extruding 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 the fishing bacteria are collected under the control of an image recognition device. Move the head part onto a petri dish for growth and prepare for a specific colony.
Second, after stopping, it is lowered and a colony is collected at the collecting end, and then the fished bacteria navel F part is moved to an appropriate place in a petri dish for transplantation, and the colony is transplanted by lowering it.
It is characterized by repeating the process described above 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 growth petri dish and the transplanting petri dish transferred by the conveyance device stop at a predetermined position, a fixed amount of viscous synthetic resin j is pushed out from the thin tube d at the tip of the fishing bacteria head by a substantially quantitative extrusion mechanism, and the fishing rod e is extended.

Next, the fishing rod e is transferred to the cutter device e and the fishing rod e is
Cut off the tip of the sample to expose the collection end r. 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, it is easy to position the fishing rod e when it comes into contact with a specific colony, which will be described later. The length of e is uniform.
, so it does not require much rigor.

Subsequently, the fishing bacteria head is transferred above the growth petri dish g by the transfer device a, 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. It is done according to the information.

Next, as shown in Fig. 1 (d), the fishing head part is lowered a certain distance to bring the collecting end f of the fishing rod e into contact with colonies of different heights growing on the growth medium. Colonies are collected, but there is no need to measure the descending distance at this time; it should always be constant (for example, if the collecting end f is slightly pushed into contact with the growth medium,
After the fishing rod e comes into contact with the colony i, it is further pushed and curved, and the pressing force caused by the descent of the fishing rod head is moderately buffered to prevent the collection end f from rushing into the growth medium. At the same time, it is possible to ensure the attachment of the colony i to the sampling end f. When the collection is completed, the fishing rod head is raised, transferred to a predetermined position in the transplantation dish as shown in Figure 1 (e), and lowered again.At this time, the fishing rod e is bent and Therefore, it is necessary to make the descending distance a little longer than when collecting, or to place it on the transplanting petri dish a little higher than the growing petri dish g, but the horizontal position is I don't need any precision, so I used a fishing rod e.
There are no obstacles due to the curved surface.

Once the transplantation is complete, move the fishing fungus head to its initial position.
Preparations are made again for the next colony collection, but as mentioned above, the fishing rod e is extended before this, and
Since the curved tip is cut off 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 overview of an embodiment of the apparatus embodying the present invention, and 1 in the figure indicates a fishing culture station where a growth Petri dish 2 and a transplanting Petri dish 3 are respectively predetermined positions in the fishing bacteria operation. 4,
This is a conveying device for transferring to the transplant station 5, and although not shown in this embodiment, it is constructed by arranging tube-shaped rubber healds in parallel at intervals narrower than the diameter of the petri dish. They are placed facing each other. In the illustration, only one transplanting dish can be supplied at a time, but if an appropriate mechanism is used to supply a plurality of dishes at the same time, and the transplanting medium in each petri dish 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 is installed at the bottom of the fishing bacteria station to illuminate the growth petri dish 2.
Although bright-field and dark-field light sources corresponding to the translucency of colonies are realized, a reflective light source that illuminates from the top surface of the Petri dish may also be used as the light source.

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 later.
A visual sensor 8, such as an image pickup tube or a solid-state image sensor, which moves along with the horizontal movement of the camera, is arranged, and the information obtained from the visual sensor 8 is processed by an image recognition device 11 installed at an appropriate place to control the fishing operation. For example, we select and collect collected colonies on a petri dish for growth, and we perform cheening of uncollected colonies.

The fishing bacteria head part 7 and the visual sensor 8 are held by a transfer device and moved between the growth petri dish 2 and the transplantation petri dish 3, but in this embodiment, the movement in the X and Y axes is performed with an accuracy of about several tens of μm. An XY stage 9 that can be used simultaneously is used.

In the figure, reference numeral 10 denotes a cutter device, which is placed between the fishing bacteria station 2 and the transplanting station 3, and cuts and discards the tip of the fishing rod just before each fishing bacteria 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. Although any device can be used, for example, in this example, the L-shaped horizontal tube shown in Figure 5 is used. Reciprocating cutting blade 27
using a thin ceramic plate for the cutting blade 27,
A resistance pattern 28 is printed on the thin plate as a heating part, and the cutting blade 27 is constantly heated and sterilized. Further, the cutter device can be constructed separately from a cutting blade 29 and a heating section 30 as shown in FIG.
The heating section 30 is fixedly installed near the horizontal reciprocating motion of the cutting blade 29 to constantly heat the cutting blade 29. It is sufficient if the structure is such that the heat is efficiently transmitted to the cutting blade 29 and the cutting blade 29 is sufficiently sterilized by heating.

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 there is a failure, 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 move back and forth at the same time. It is also possible to use only one part as appropriate.

Next, the vicinity of the fishing bacteria head section and the structure of the fishing bacteria head section 7 will be described.For example, the fishing bacteria head section 7 is supported by an elevating holding section 14 and extended from the XY stage 9, as shown in FIG. It is connected to the base 15 and can be moved up and down via a rod 17 using a cylinder 16 as a driving source. In addition, the fishing bacteria head section 7 is constructed by arranging a thin tube 18 that serves as an outflow path for the viscous synthetic resin 13 at the bottom of a test tube-shaped cylindrical body. 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 accommodation section 24 for the viscous synthetic resin 13 and the thin tube 18 are integrally constructed, and the accommodation section 24 also moves up and down as the fishing rod 19 moves up and down.
As shown in the figure, if the housing section 21 is configured as a separate body, the accommodating section 21 is fixed to the pedestal 22, and the viscous synthetic resin is fed in a fixed amount to the thin tube 1B via the guide tube 23, the vertically moving part can be replaced with the thin tube. It can also be limited to only the peripheral portion 27, and the weight of the elevating portion can be reduced.

Each of the above-mentioned devices is housed in a sterilized airtight case, and an opening/closing device 12 for removing and attaching the lids of each petri dish is installed adjacent to the transport device 1, and an air purifying device or the like is installed as appropriate. The structure is such that contamination with germs can be eliminated as much as possible.

Colony transplantation using the device constructed in this manner is performed as follows. The growth petri dish 2 and the transplanting petri dish 3 placed on the end of the transport device from the outside of the sealed case are transferred to the fishing bacteria station 4 and the transplanting station 5, respectively, by appropriate means, and then stopped. The transport device 1 consists only of rubber healds arranged in parallel, and vibrations during movement will not stir up unnecessary bacteria inside the sealed case. Next, the opening/closing device 12 provided close to the conveying device 1 is driven to remove the lid of each petri dish, and a fixed amount of compressed air is supplied from the upper part of the fishing bacteria head to the holding plate 24 in the storage section. When 20 is 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. Fishing rod 1 formed by extruding at this time
Since the length of 9 is adjusted to be almost constant by the subsequent cutting operation of the cutter device 10, 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 bacterial fishing operation. Since the tip of the fishing rod 19 will be discarded, it goes without saying that a highly accurate one is desirable from an economic standpoint.

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 as the dripping hole, the collecting 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 slightly presses the surface layer of the growth medium of standard thickness, the fishing rod 19 will be able to bend flexibly even if there is some variation in the thickness of the growth medium. 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, the fishing bacteria head section 7 and the visual sensor 8 are moved and stopped above the growth petri dish 2, and an image of the growth petri dish 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 place them in front of the light source. It is also possible to add color to the conditions by combining it with a filter.

Once a colony to be collected is identified, the fishing bacteria head 7 is lowered by the elevating mechanism 25 and the collecting end 26 is brought into contact with the rod 1- to attach the bacteria, but this descending distance is always A certain distance is fine. 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 19 is lifted up, and at the same time, the fishing rod 19 is moved to the upper part of the predetermined arrangement position of the transplanting petri dish 3 by the XY stage 9, and then stopped again. The bacteria attached to the collecting end 26 are transplanted to a transplant medium. Although the fishing rod 19 is curved at this time, the contact position of the collection end 26 with the transplant medium is as follows.
Since it does not require much precision, there is no problem with being curved. In consideration of this curvature, the growth petri dish 2 and the transplanting petri dish 3 are provided with a vertical step at their mounting positions. 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 portion 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,
The end is when certain conditions are met, for example when all bacteria of a particular shape and size have been transplanted, and the opening/closing device 12 is driven to cover the transplantation dish 3. , and is transferred to the outside of the sealed case by the transfer device 1. As shown in FIG. 4, if the fishing head section is configured separately into 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 Petri dishes are arranged, the adaptability of the same colony to a different type of culture medium can be investigated by successively distributing the colonies collected during the -th fishing operation to multiple transplanting Petri dishes. This makes it possible to further improve the efficiency of screening work.

(Effects of the Invention) According to the present invention, screening work that conventionally required a large amount of time to be performed by skilled workers can be automated without human intervention, resulting in faster and more efficient screening work. This not only greatly reduces the negative IFl of workers, but also eliminates the bacteria transmitted by workers by eliminating manual labor, and also eliminates the dispersion of data due to individual differences among workers. This makes it possible to improve the accuracy of the lost item screening process.

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 the heating section. Furthermore, the success rate of screening operations can be greatly improved without contamination with extraneous bacteria.

[Brief explanation of the drawing]

Figures 1 (a), (b), (c), (d), and (e) are simplified explanatory diagrams showing an overview of the colony transplantation method according to the present invention, and Figure 2 is an outline of an embodiment of the present invention. 3 is a simplified diagram showing the structure of the surrounding area of the fishing bacteria head in the same embodiment. FIG. 4 is a simplified diagram showing the structure of the surrounding area of the fishing bacteria head in the same embodiment. FIG. 5 is a simplified explanatory diagram of a cutter device in the same embodiment, and 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: Thin tube e: Fishing rod f: Collection end g: Petri dish for growth h: Growth medium i: Colony j: Viscous synthetic resin: Petri dish for transplantation l: Cassotar device 1: Transport device 2: Petri dish for growth 3: Petri dish for transplantation 4: Fishing bacteria station 5: Transplanting station 6: Light source 7: Fishing bacteria head section 8: Visual sensor 9: XY stage 10: Cassoter device 11: Image processing Device 12
: Opening/closing device 13: Viscous synthetic resin 14: Lifting holding part 1
5: Pedestal 16: Cylinder 17: Rosodo
18: Thin tube 19: Fishing rod 20: Holding plate 21: Accommodating part 22: Pedestal 23: Guide tube 24: Accommodating part 25: Lifting mechanism 26: Collection end 27: Cutting blade
28: Resistance pattern 29: Cutting blade 3
0: Heating section 31: Tube periphery Patent applicant Duck Engineering Co., Ltd. g1 Kaisho 62-65700 (7)

Claims (1)

[Scope of Claims] 1) A conveying device for transporting a petri dish for growth and a petri dish for transplantation to a predetermined position; A device having a thin tube at the tip as an outflow path for the viscous synthetic resin and extruding the viscous synthetic resin based on an appropriate command. a fishing bacteria head having a fixed quantity extrusion mechanism; a transfer device capable of holding and accurately transferring the fishing bacteria head to a predetermined position; a cutter device equipped with a heating means for cutting the collecting end of the fish; a visual sensor and an image recognition device for identifying colonies on the growth petri dish and the transplanting petri dish and controlling the position management of the fishing bacteria head part; A fishing rod is formed by extruding 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. The part is moved onto a growth petri dish, stopped on a specific colony, and then lowered to collect a colony at the collecting end,
Next, move the fishing bacteria head to a suitable place in a Petri dish for transplantation.
A method for transplanting colonies 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) A method for transplanting a colony according to claims 1, 2, and 3, characterized in that a silicone resin is used as the viscous synthetic resin. 5) A method for transplanting a colony according to claims 1, 2, 3, and 4, characterized in that a plurality of petri dishes for transplantation are supplied at the same time.