JPH0467952B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides a method for transferring microorganism colonies produced by flat culture of microorganisms from a culture dish to a test dish or test tube. This invention relates to a colony transfer device that allows breeding colonies to be utilized as effectively as possible.

[Prior Art] In order to develop new antibiotics, methods are used to discover effective microorganisms from soil and the like.
For this purpose, soil is collected from various places around the world, diluted 10,000 to 100,000 times with sterilized water, and isolated and cultured on a suitable agar plate medium. To select effective microorganisms, this cultured colony must be transferred to further test dishes or test tubes.

In addition, research in genetic engineering, which has been rapidly progressing in recent years, involves selecting and culturing specific types of microorganisms, and hopes to create new organisms by recombining their genes. Colony translocation operations are also frequently performed during this process.

Conventionally, colony transplantation was carried out manually using a specially shaped "platinum loop" while observing the colony group with the naked eye or using an optical projection device. There are various methods of transplantation, but one example is to select colonies that have grown to a diameter of 1 to 2 mm from among the colonies growing on an agar medium in a cultured petri dish, and to contact them with a platinum loop. Collect the
It consists of arranging and transplanting in designated positions on the culture medium of a separate test dish. This work requires considerable skill and effort.

Next, to describe the aspect of the container to be transferred to, the test tube is suitable for transferring colonies of many types of bacteria, and the test tube is used to culture a large amount of a single type of bacteria. The method used is to disperse and transplant the species over a wide area.

It is important to prevent contamination from other germs during the translocation work, so the work must be carried out in a safety cabinet, and all equipment used must be disinfected and sterilized (hereinafter simply referred to as ``sterilization'') in a timely manner.

The above-mentioned points will be explained in more detail. In the case of research and development, the process of initially culturing a promising and useful new strain from among a mixture of various bacterial species and culturing it into a single variety as pure as possible. It is a repetition of In addition, if the product is already in the stage of industrial mass production, problematic bacteria and contaminating bacteria that should not have been present in the culture should be eliminated. In reality, thousands, tens of thousands, or more specimens often have to be handled every day.

Even though the colony transplantation process mentioned above seems simple at first glance, it is extremely tiring for the worker to repeatedly identify the target colony within the field of view of a microscope, accurately collect and transfer it over a long period of time. and
There were problems such as the need for a large number of workers with some level of skill.

In order to eliminate the above-mentioned disadvantages associated with colony transfer work, a colony transfer device that mechanically performs this work has been developed. This colony transfer device is disclosed in Japanese Patent Application Laid-Open No. 60-83597. The colony transfer device disclosed in this publication consists of two sets of (a) culture culture plates for colonies and test plates or test tubes each mounted separately on a common platform.
(b) a monitor section for selecting a specific colony on the surface of the medium in a culture dish; (c) collecting the selected colony and placing it on the surface of the medium in a test dish or test tube; (d) a pick-up processing section for treating the end of the pick-up section for transplantation; (e) a culture shear, test shear or test tube, and a pick-up processing section; , and a transfer section for transferring the pick-up section as specified. Each is configured to operate as specified.

The colony transfer device disclosed in the above-mentioned publication automatically and mechanically performs processes other than selecting specific colonies accurately, thereby significantly reducing the physical fatigue of workers involved in colony transfer work. Reduce to.

Colonies grow in dotted, circular, filamentous, irregular, rhizoidal, or spindle-like shapes. If a colony is collected and transplanted while being visually observed by humans, the shape of the colony does not matter. but,
When performing colony transplantation work mechanically, in practice it is necessary to transfer colonies with a specific shape (e.g. circular).
I can only handle it. For example, a device that automatically selects only colonies with a specific shape is disclosed in Japanese Patent Application No. 59-235021.
No. 59-235024 and the like.

The colony transfer apparatuses described above, which are equipped with or without a device for automatically selecting colonies of a specific shape, have the following problems. That is, the pick-up section is directed only to the same point in the selected colonies. Therefore, even if the pick-up section is lowered toward the same colony again after collecting a colony, it will only enter the part that has already been collected and is empty. As a result, other unharvested parts of the colony are left unused. This is a serious loss that cannot be overlooked in colony translocation work.

[Object of the Invention] Therefore, an object of the present invention is to provide a colony transfer device that allows selected colonies to be used without waste.

[Means for Solving the Problems] With the remarkable progress in so-called mechatronics in recent years, it is not easy to manufacture a device that can continuously repeat positioning with an accuracy of 1 to 2 μm. It is relatively easy to obtain a device that can perform positioning with much higher accuracy than humans, for example, on the order of 10 μm. Moreover, if a piezo actuator is used in conjunction with micro-movement control, positioning accuracy on the order of 1 μm is possible. Instead of looking directly into a microscope, a television camera can be connected to the microscope and the enlarged image can be reproduced on a large receiver. When selecting the position on this screen, instead of slightly moving the XY stage (mounting stage) and reading the scale so that the target colony position matches the hairline cross of the microscope,
For example, when a target object is specified on an enlarged reproduced image with a cursor, the position information of the target object (the position information itself can be on the order of μm) can now be immediately stored in the storage device.

Furthermore, a series of long and complex sequential controls that were impossible in the past when cams and the like were used can now be performed extremely easily and accurately using computers. Furthermore, the control means can be changed easily.

The present invention aims to dramatically improve the efficiency of colony transfer work by optimally combining the above-described high-precision mechatronics technology and computer control technology with a colony transfer device.

Therefore, the means in the colony transplantation apparatus of the present invention is to divide the region of the cultured colony to be transplanted into a plurality of regions, and to sequentially collect and transplant from each region.

[Effect] By collecting samples from divided areas in this way, it is possible to collect precisely selected colonies with much higher precision than by manual methods, and it is easier to pure culture a single bacterial species. The working time is greatly reduced, microbial research dealing with biological phenomena can be carried out accurately and efficiently, and the physical fatigue associated with conventional manual work is also greatly reduced.

[Example] The present invention will be described in more detail below with reference to the drawings. The shear plate in which specific colonies on the culture medium are selected and collected is called the culture shear plate, and the side in which the colonies are transferred is called the test shear plate.

FIG. 1 is a schematic diagram showing the state of colony collection by the colony transfer device of the present invention. 1st
In the figure, the position of the selected colony (1) 13 among the colonies of various strains distributed in the culture medium 12 of the cultured dish 11 is stored in the storage device at the same time as the selection as described above. Based on this stored information, the XY stage on which the culture shear dish 11 is placed is moved. If the colony (1) 13 is divided into four, for example, to have approximately equal areas, the center of the four divisions is stored in the storage device. An x-axis and a y-axis are drawn from the center of the division, and the sections defined by the axes are tentatively defined as a first quadrant, a second quadrant, a third quadrant, and a fourth quadrant.

As shown in FIG. 1a, the XY stage is moved based on the stored information regarding the division center, and the first part of the colony (1) 13 is placed directly under the tip ball 7 of the pick-up section 10 having the transfer needle 2. Position it approximately in the center of the quadrant. Thereafter, the ball 7 is lowered, and the first quadrant of the colony (1) 13 is attached to the ball and collected. At this time, the distance (height) of the ball 7 is controlled by measuring the distance (height) to the surface of the culture medium 12 using the principle of triangulation, for example, using a distance measuring means 14 equipped with an LED, an optical fiber, a photodiode, etc., and controlling the height of the ball 7. 7 and the colony. Next, the ball 7 in the pick-up section is pulled up, and the ball 7 in the pick-up section is brought to a designated position on the culture medium 12a of the test shear tray 15 as shown in FIG. Colony (1) 1st of 13
Perform quadrant transplantation.

After collecting the colonies in the first quadrant, the XY stage on which the culture shear plate 11 is placed is moved by, for example, a piezo actuator (PZT actuator) based on the memory information of the division center, and the ball 7
The approximate center of the second quadrant of colony (1) 13 is located directly below. Thereafter, the ball 7 is lowered, and the second quadrant of the colony (1) 13 is attached to the ball and collected. The collected colony is then transplanted to another designated position in the same test dish (or a similarly designated position in another test dish). Hereafter, repeat the same operation up to the 4th quadrant of colony (1) 13.

Note that the collection of the target colony may be performed multiple times as long as the same location (in the same quadrant) does not become vacant.

By doing this, colonies can be transplanted to up to four locations on a medium having the same composition, as shown in FIG. 1b. Also, Figure 1c
Divide one colony into four as shown in
In the four test dishes 15, it is possible to transfer to four types of media with different compositions. Therefore, the effective utilization of colonies is 4% compared to the conventional technology.
This means that I have improved greatly.

To adjust the relative position between the ball 7 and the culture shear dish and the test shear dish, a shear dish is installed respectively.
Move the XY stage side. Movement between the divided quadrants is performed by moving a PZT actuator arranged in the X-Y direction as a mechanism for fine movement with even higher precision. All positional control in the above series of transplanting operations is performed by computer control, and each X-Y drive mechanism is driven by an X-Y drive device or a drive circuit.

In addition to the circular colony shown by (1) 13 in FIG.
(2)13, Random filamentous colony (3)13, Spindle-like colony
(4)13 and rhizoid colonies (5)13 can also be applied. However, although it may not be applicable to punctate colonies (6)13, the efficiency will not be very high. If the shape of the colony is different, the type of microorganism forming the colony should also be different.

Therefore, when transferring colonies of a plurality of different bacterial strains, it is necessary to make a new ball 7 each time the bacterial strain is changed in order to prevent the culture medium from being contaminated. For example, as shown in FIG. 2(a), a method for manufacturing a new ball includes winding a thin wire 22 of gold, aluminum, glass fiber, etc. around a wire reel 21, and letting this thin wire 22 be fed out by a feeder 23. Fix it with a clamper 24 and cut off the tip with a cutter 25.

Next, as shown in Fig. 2b, the thin wire 2 is
The cut ends of 2 are heated and melted to form a ball 7. Using this ball 7, colonies are collected and transplanted as shown in FIG. 2c. If cleaning or sterilization is required, return to step a in FIG. 2, cut off the end including the ball 7 that was previously used, and heat and melt the cut end with a torch to make a new ball 7. In this way, the molten ball is sterilized at the same time as it is made, and washing is not necessary.

The process of creating the molten ball 7 is performed under computer control while moving the XY stage. As the torch flame 26, a discharge torch or an oxyhydrogen flame (direction controlled by leaving it on) may be used. Instead of using a molten ball, the tip is cut off using a cutting tool 28 as shown in FIG. 3a, and at the same time the end is bent and formed as shown in FIG. Heat sterilization is performed using an electric heating or high frequency heating type heater 29. In this case as well, the washing step is not necessary because the previously used portion is discarded.

FIG. 4 shows an example of a case where a colony of one bacterial species is transferred to a test tube 16. In this case, medium 1 in the tube
The surface 12' of tube 2 forms an inclined surface with respect to the tube axis, and it is necessary to disperse and transplant 13 colonies (1) of one bacterial species onto this surface 12'. The reason why the surface is sloped is that it is desirable for culture to widen the effective area of the medium surface.

In this case, the test tube 16 is rotated by the turntable 17, the height of the surface 12' is detected by the distance measuring means 14, and the test tube 16 is stopped at the lowest position, for example, and the height of the surface 12' is
2' is fixed in a certain direction. Thereafter, while using the distance measuring means 14, the ball 7 is moved to the surface 1 forming the slope.
2' to spread the bacteria.

FIG. 5 is a block diagram showing the entire configuration of an embodiment of the present invention, in which a culture shear 11 and a test shear 15 are shown.
Alternatively, the test tubes 16 are mounted on XY stages 39-1 and 39-2 installed near both ends of the common stage 8, and the pick-up section 10 is moved between the culture shear 11 and the test shear by the transfer mechanism 30. 15 or test tubes 16 under the control of a microprocessor 33 equipped with a memory 34 and an operation panel 35. And XY stage 39-
1 and 39-2 are equipped with X-PZT actuators 39a and Y-PZT actuators 39a and 39-2, respectively, in order to perform minute positioning for moving between the compartments of the collected colony.
An actuator 39b is mounted.

As already explained, the height to the culture medium surface is detected by the distance measuring means 14 attached to the pick-up section 10, and the height of the collecting and transplanting end of the pick-up section is controlled. XY stage 3
9-1 and 39-2 can be slightly moved in two orthogonal directions, X and Y, by driving a motor such as a pulse motor. And by voltage control, the X-PZT actuator 39a,
Y-PZT actuator 39b is controlled and
-The Y table will move slightly.

Now, in the middle of the XY stages 39-1 and 39-2, there is a pick-up processing section 31 for manufacturing the molten ball 7 as described in FIG. This processing is necessary when the colony to be translocated selected via the monitor section 32 (described later) is different from the one that has been handled up to that point, or when starting up after work has been interrupted. The processing work is performed under the control of the microprocessor 33 in conjunction with the movement of the XY stage. The collection and transplantation of colonies by the pick-up section is as already described in connection with FIGS. 1 to 4. The monitor section 32 consists of a television camera and a monitor television combined with a microscope if necessary, and the television camera is mounted on a common stand 38 so as to be able to clearly photograph the medium surface of the culture dish 11.

FIG. 6a is a plan view of the main parts of the embodiment of the present invention, centering on the transfer mechanism 30 described with reference to FIG.
The transfer is performed by rotating the rotary arm 30-1, causing the pick-up section 10 to make a circumferential movement as indicated by arrow D, and stopping at a predetermined position. FIG. 6b is a front (partially sectional) view of the above embodiment, in which a television camera 32-1 is attached above the culture dish 11, and a monitor television 32-2 is placed at an appropriate location where it can be easily seen by the operator. ing. A colony to be transplanted can be selected by operating the operation panel 35 and moving a cursor on the screen of the monitor television 32-2.
By eliminating the need for the worker to look directly into the microscope, the physical fatigue of the worker is greatly reduced. In addition, numeral 36 in the figure is a bactericidal and light-shielding cover.

By the way, in the example, 4 colonies were transplanted.
The process is divided into two parts, but the number of divisions is
The sampling area is not limited to four divisions, but may be divided into a plurality of parts.

[Effects of the Invention] As explained above, according to the colony transplantation device of the present invention, the region of the cultured colony to be transplanted is divided into a plurality of regions, and the transplantation is performed by sequentially collecting samples from each region. Compared to manual work,
It has become possible to collect accurately selected colonies with much higher precision, making it easier to pure culture a single bacterial species, greatly reducing work time, and allowing microbial research dealing with biological phenomena to be carried out accurately and efficiently. This greatly reduces the physical fatigue associated with traditional manual work.

[Brief explanation of the drawing]

Figures 1 a, b, and c are explanatory diagrams of balls formed by heating and melting the cut ends of thin wires used for colony collection and transplantation in the embodiments of the present invention, and Figures 2 a, b, and c.
3 is an explanatory diagram showing the end portion of a cut thin wire used for colony collection and transplantation in another embodiment of the present invention, which is bent, molded, and heat sterilized. FIG. diagram showing,
Figures 3b and 3c are diagrams showing the test shear and transfer method in the embodiment of the present invention, Figure 4 is an explanatory diagram of the method of transferring colonies to test tubes in the present invention, and Figure 5 is a diagram showing the entire procedure of the embodiment of the present invention. Block diagram showing the configuration, Figure 6a,
b is a plan view and a front (partially sectional) view of essential parts of an embodiment of the present invention, respectively. 2...Thin wire, 3...Feeder, 4...Clamba, 6...Torch flame, 7...Ball, 8...Cutting tool, 9...Heater, 10...Pickup section, 11...Culture shear, 12...Medium, 13
... Colony, 14... Distance measuring means, 15...
Test share, 16...Test tube, 28...Common stand, 30...Transfer mechanism, 31...Pickup processing section, 32...Monitor section, 33...Microcomputer, 34...Memory, 35...Operation panel , 39-1, 39-2...XY stage, 39
a...X-PZT actuator, 39b...Y
−PZT actuator.

Claims (1)

[Scope of Claims] 1. Two sets of XY stage parts each carrying a colony culture shear and a test shear or test tube separately, and a monitor part for selecting a specific colony on the surface of the medium in the culture shear. a pick-up section for collecting the selected colonies and transferring them to the surface of a medium in a test shear or test tube as specified, and a pick-up processing section for processing the end of the pick-up section for collection and transfer. In a colony transfer device equipped with a transfer unit for transferring a culture vial, a test vial or a test tube, and a pick-up processing unit as specified, the processor calculates the center of the selected colony and sets this center as the origin. The memory includes a program for dividing the colony into n parts (where n is an integer of 2 or more) and a program for positioning a translocation needle in each divided area and performing the translocation process. A colony transplantation device characterized by: