JPS6225348B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention enables the work of collecting specified colonies (bacterial species) on a culture Shearé medium and transplanting them to a specified position on a test Shearé medium or into a test tube to be carried out in a manner similar to that of the colony to be collected and transplanted. This article relates to a colony transplant mechanism that automatically performs operations other than designated operations.

Conventionally, in order to research and develop new types of antibiotics, methods have been used to find effective bacteria in soil, and bacterial species are collected and cultured from a wide variety of heterogeneous soils. In these cases, it is necessary to transplant the bacterial species of colonies between bottles or test tubes. In addition, colony transplantation operations are frequently performed in genetic engineering, which is currently being researched.

Conventional colony transplantation operations are carried out manually using earpick-shaped "platinum loops" while observing colonies of cultured sheares with the naked eye or with an optical projection device.

There are various aspects of porting work, but to give an example:
For the culture shear, colonies are grown in a medium made of agar, and those that have grown to a diameter of 1 to 2 mm are selected, and the bacterial species are collected by contacting them with a platinum loop. It is transplanted to a designated location, and this work requires considerable skill and effort.

Next, looking at the characteristics depending on the container to which the transplant is made,
The above-mentioned test tube is suitable for transplanting many types of bacteria, whereas test tubes are used when culturing a large amount of a single type of bacteria. In the case of test tubes, in addition to using agar as the medium, a culture solution may be used.

During transplantation work, it is important to prevent contamination by other germs, so the work must be done in a safety cabinet, and all instruments used must be disinfected and sterilized (hereinafter simply referred to as sterilization) in a timely manner.

To further summarize the points mentioned above, in the case of research and development, initially, a variety of miscellaneous bacterial species are cultivated, and then promising and useful new bacterial strains are cultivated as pure as possible. This is a repeated process of extracting the bacteria and culturing it as pure as possible, and if the process is already in the industrial mass production stage, it is a process of removing problematic bacteria and contaminant bacteria from the cultured species. In reality, there are many cases where thousands or more specimens must be handled every day, and it can be very stressful for workers to repeat the above-mentioned, seemingly not difficult, colony transplantation work over a long period of time. There were problems such as it was tiring and required a large number of workers with some level of skill.

In order to solve this problem, the present inventor has already established that, except for the operation of selecting and specifying specific things in the cultured colonies, which at present, humans are forced to perform,
We proposed a ``colony transfer device'' that automatically and mechanically performs all work processes (patent application 1984-084011).
issue).

This apparatus will first be explained with reference to FIG. For convenience, the side to which the colonies are supplied is designated as the culture shear dish 1, and the colony transplant destination is designated as the test shear dish 2. However, there are cases where the transplant destination is test tube 3. These culture plates 1 and test plates 2 or test tubes 3
are the first XY installed on the common stand 4, respectively.
It is mounted on stage 5-1 and second XY stage 5-2. A monitor unit 9 having a television camera is placed above the cultured dish 1, and the colony distribution state of the cultured dish 1 is projected onto the monitor television. When a desired colony is selected from the colony group by observing this projected image, its position (scale position of the XY stage) is determined by the microcomputer 10.
The data is stored in the memory 11 via. The pick-up part 6 has a pick-up 6-1 in a shape suitable for collecting and transplanting colonies made by processing thin wires of gold, aluminum, etc., such as a ball or earpick shape.
is provided, and the selected colonies are collected by moving the XY stage 5-1 and moving the pick-up 6-1 up and down. Next, transportation means 7
The pick-up section 6 is moved above the test shear 2 (or test tube 3), and by the reverse operation of the above-mentioned picking, the bacterial species of the colony is transplanted to the medium or culture solution. At this time, the distance between the pick-up 6-1 and the medium surface or culture solution surface is controlled via the height detector 6-2, so that the transplanting operation is performed smoothly. In the above, the horizontal movement of the XY stages 5-1 and 5-2, the vertical movement of the pick-up 6-1, and the signal processing and interval control of the height detector 6-2 are all regulated and sequentially controlled by the microcomputer 10. be done. Furthermore, in the pick-up processing section 8, the pick-up 6-1 from the thin wire such as gold is processed.
Works such as preparation, cleaning, and sterilization are performed.
These are also controlled by the computer 10.

The previously proposed colony transfer device places great importance on the cleanliness of the pick-up, and adopts a disposable method in which a new pick-up is made each time it is used from fine wire made of expensive material such as gold.
However, platinum loops, which have been used for many years, can be sterilized and reused without causing any problems.

An object of the present invention is to provide a colony transplantation mechanism in which the pick-up part of the colony transfer device can be reused by simple sterilization and has means suitable for mass culture of a single bacterial species. There is a particular thing.

In order to achieve the above object, in the present invention,
As a means for transferring the pick-up for collecting and transplanting bacterial species, the index unit has a rotary arm whose ends are positioned and stopped at four predetermined positions on the circumference on a common table, and which can move up and down there.
A platinum loop or a self-heating pick-up is attached to the end of this arm, and the 1st XY stage for culture shear dish, 2nd XY stage for test shear dish or test tube, and heating for sterilization are installed at the above four fixed positions. and cooling means were installed. Reusable pickups generally have a larger heat capacity than disposable ones, so we specially installed a cooling means in a separate location.

The present invention will be explained in more detail below with reference to the drawings.

The structure of the first pick-up according to the present invention is that a platinum loop, which has conventionally been used for manual colony transplantation, is attached to a transfer arm using a holder (collect), and cleaning and sterilization are performed by high-frequency heating. . In other words, when the tip of the platinum loop becomes red-hot using high-frequency heating, a sterilizing effect can be obtained, and the agar medium material attached to it will not burn and leave no residue.
Therefore, washing is not necessary. However, since it is desirable to quickly cool the red-hot end, a cooling pit filled with sterile water was provided.

FIG. 2 is a schematic perspective view of an embodiment using a platinum lug according to the present invention. It is constructed by attaching a collet 6-3 for holding a height detector 6-2 and a height detector 6-2. The height detector 6-2 may be either an optical type or a mechanical contact type. The transfer means 7 uses an index unit 7-1 that can stop the end of the arm 7-2 with high precision at four predetermined positions every 90 degrees on the circumference. A first XY stage 5-1 on which the culture shear 1 was placed, a second XY stage 5-2 on which the test shear 2 was placed,
A high frequency heating furnace 12 and a cooling pit 13 are provided. The arm 7-2 moves up and down at these four locations, and colonies are collected and transplanted by the pick-up 6-1, and if necessary, the pick-up is heat sterilized and cooled. If the transplant destination is the test tube 3, consideration must be given to lowering the mounting height of the XY stage 5-2 and making the test tube 3 stand upright, but this point will be described later.

The plan view is shown in Figure 3a as Pickup 6-1.
A device that sterilizes by self-heating by energizing is also used, as shown in the front view in b. The material used is a heat-resistant resistance wire, such as a nichrome wire 14, which is folded in half. An insulator 15 is interposed between the two reciprocating parts, and the tip 14-
Polish 1 to create a thin earpick shape. When this is energized, for example, as shown by the arrow, the tip 14-1 becomes red hot, allowing sterilization and incineration of deposits. FIG. 3c shows the applied current i, and FIG. 3d shows the heating temperature T caused by it. The advantages of this method are that the temperature T can be accurately controlled by adjusting the current i, and that the heating furnace 12 is not required, making the apparatus simple.

Next, regarding the method of transplanting to a test tube, there are two methods: using a culture solution and using an agar medium. In the case of a culture solution, transplantation can be performed simply by dipping the tip of the pick-up 6-1 into the culture solution. In the case of an agar medium, as shown in Figure 4a, the medium side 1
6' is tilted with respect to the test tube 3 to increase the effective area, and when transplanting, it is necessary to disperse the bacterial species as widely as possible and transplant it in a meandering manner, as shown in Figure b, for example. . After a predetermined period of time, the thus transplanted bacterial species grows fairly evenly on the medium surface 16'.

The first method of distributed transplantation in the present invention is the fourth
As shown in FIG.
Attach it to the end of 2. The operation of the transfer means 7 causes the tip of the pick-up to stop within the test tube 3 at a certain height close to the tube wall which is offset from the center. When the test tube 3 is rotated in the direction of arrow R by the rotation mechanism, the tip of the pick-up contacts the culture medium surface 16', which is detected by the strain gauge of the arm 17 and rotation is stopped, and the arm 7-2 is lowered and the XY stage 5- 2
As a result of the horizontal movement of , the tip of the pick-up comes into contact with the lower side of the culture medium surface 16' as shown by the dotted line in the figure. In this case, under program control of a microcomputer, the tip of the pickup can take a meandering trajectory as shown in FIG. 4b. FIG. 4c is a perspective view showing the method of mounting and rotating the test tube 3 on the XY stage 5-2 in this embodiment.
Using the auxiliary plate 4', remove the test tube 3 from the common stand 4.
A rotating table 18 having a test tube stand (not shown) for vertically holding test tubes is provided on this XY stage. In addition, so that the inclination direction of the culture medium surface 16' is constant,
If the test tube 3 is set in advance, the rotating table 18 is of course unnecessary.

The second method of distributed transplantation in the present invention uses a short plate, and this will be explained with reference to a side view of FIG. 5A and a perspective view of FIG. 5B. 19 is a short plate, ball bearing 20
It is attached to the tip of the rod 21 so that it can freely tilt and rotate, and when pressed against an (inclined) plane in any direction, it can tilt and rotate in relation to this plane and come into close contact. If a bacterial species is attached to the surface of this small plate, it can be transplanted over a wide area in one motion by sliding it on the inclined culture medium surface 16'. The procedure of transplantation using the short plate 19 will be explained with reference to the drawings. The rod 21 is held through the holding mechanism 22, and the vertical position of the rod 21 with respect to the holding mechanism 22 can be freely controlled. This holding mechanism 22 is attached to the XY stage 5-2 using a support 23 so that it can be rotated 180 degrees as shown by arrow E.
First, with the holding mechanism 22 placed at the position where the tall plate is at the upper end of the rod as shown by the broken line, the bacterial species collected from the designated colony of the cultured shear using a pick-up 6-1 such as a platinum loop is collected. It is attached to the surface of the plate 19. In this case, it goes without saying that the movement of the XY stage 5-2 is also used to spread the adhesive over this surface. Note that the spring 24 is used to hold the short plate 19 horizontally at this time. Next, the holding mechanism 22 is reversed so that the tall plate is at the lower end of the rod as shown by the solid line, and when the rod 21 is moved downward, the tall plate 19 is inserted into the test tube 3, and the medium surface 16' Regardless of the direction, the surface of the stiff plate 19 is in close contact with the double base 16'. Then stick 2
1, the small plate 19 slides on the culture medium surface 16' and the bacterial species are dispersed and transplanted. In this case, if necessary, the plate 19 is moved upwardly from below the medium surface 16'.
However, if the direction of the culture medium surface 16' is uncertain, it is not possible to insert the stiff plate 19 into the lower part of the test tube 3 first.
The direction of the medium surface 16' can be set by aligning it in a certain direction, or by detecting the direction of the culture medium surface 16' with an appropriate sensor.
2) and the test tube (held on a common stand) must be controlled by a computer. Note that since the culture medium surface 16' is long, the vertical stroke of the rod 21 may be insufficient. Also stick 21
If the stroke is insufficient, the short plate will remain in the test tube even if the short plate is pulled up the entire stroke from the lowest contact position with the culture medium surface 16', and the rod and short plate system cannot be reversed. Therefore, if the stroke of the rod 21 is insufficient, move the holder 25 of the test tube 3 into the slide groove 26.
It is attached to the common stand 4 via a vertical sliding mechanism.

Naturally, even in the case of the short plate method, cleaning and sterilization of the short plate 19 may be necessary. The board type is the first
Since it is fixed to the 2XY stage, the sterilization heating means 12 for picking up platinum loops etc. cannot be used, so a dedicated sterilization means is installed. Figure 5a
As shown in the upper right corner, a high-frequency heating coil 27 is provided above the holding mechanism 22, and the plate 19 is raised until it enters this coil for heat sterilization, or as shown in Figures a and b, it is heated using a laser beam. A laser beam 28' from a source 28 is focused by a focusing lens 29, the direction of the beam is appropriately bent by a mirror 30, and the plate 19 is irradiated with a sufficiently strong laser beam to instantaneously heat only the plate surface. It may also be sterilized.
To irradiate the entire surface of the board 19 with the laser, use XY
This is done by moving the stage 5-2 or controlling the direction of the mirror 30.

In addition to the above, there are the following modifications to the embodiment of the colony transplantation mechanism. One method is to use a small plate attached to the pick-up part 6, which has a large contact area, so it can be used as a stamp type to transplant many bacterial species at the same time while keeping the same colony distribution pattern. The second method is to place a test tube instead of a cultured shear on the supply side of the bacterial seeds, and then transplant the seeds into a test shear or test tube. Since these are merely applications of the present invention which have already been explained in detail, their explanation will be omitted.
Next, the relationship and control between the microcomputer 10 and its related parts will be explained with reference to the block diagram shown in FIG.

The microcomputer 10 includes a microprocessor 50 connected to a bus 56 and receiving data from the monitor section 9 and the index unit 7-1 via an interface 53, an image memory 51 connected to the bus 56, and an image memory 51 connected to the bus 56. Image memory 51
It consists of a CRT display 52 connected to a computer, a keyboard 54 connected to a microprocessor 50 via an interface 53, and a bus 56, and exchanges data with the memory 11 that stores various control programs and data.

Here, the memory 11 includes an image processing/display program 11a and an XY stage control program 1.
1b, a pick-up control program 11c, an arm rotation control program 11d, various data areas 11e for recording transplant targets input from the keyboard 54, coordinates of transplant positions, etc., and other data/program areas 11f. By starting each of these programs, the arm 7-2 is rotated and positioned at a predetermined position under the control of the microprocessor 50, and the first or second XY stage 5-1, 5- 2 is controlled, and the pickup 6-1 moves up and down.
The arm 7-2 then moves to the next predetermined position. As a result, the pick-up section 6 is sequentially positioned as the arm 7-2 rotates, and the work of collecting colonies from the cultured dish and transplanting the collected colonies to the test dish is performed, followed by heat sterilization and cooling. Processing is done and this is repeated.

On the other hand, information obtained from the camera of the monitor unit 9 is sent to the microprocessor 50 via the interface 53, and the microprocessor 50 executes the image processing/display program 11a according to this information.
starts up and performs predetermined image processing, and the predetermined image is stored in the image memory 51 and displayed on the CRT display 5.
Displayed by 2.

The specific control of the positioning of the first XY stage 5-1 is performed by the microprocessor 50 starting the XY stage control program 11b, and the microprocessor 50 controls the interface according to the information in the various data areas 11f. The position control information is sent to the first XY stage control circuit 5 via the Ace 53.
5a. The first XY stage control circuit 55a drives the first XY stage drive motor 55b according to position control information from the microprocessor 50. Note that this XY stage drive motor 55b usually consists of a pair of a stepping motor in the X direction and a stepping motor in the Y direction.

The positioning control of the second XY stage 5-2 is also the same as above, and the microprocessor 50 controls the positioning of the second XY stage 5-2.
This is done by activating the XY stage control program 11b, and the microprocessor 50 sends position control information to the second XY stage control circuit 56a via the interface 53 in accordance with the information in the various data areas 11f. And the second
The XY stage control circuit 56a controls the second XY stage according to the position control information from the microprocessor 50.
The stage drive motor 56b is driven.

The vertical position control of the pick-up section 6 is similar to that described above, and is performed by the microprocessor 50 starting the pick-up control program 11c. The position control information is sent to the pickup section vertical movement control circuit 57a via the pickup section. The pickup vertical movement control circuit 57a drives the pickup vertical movement drive motor 57b in accordance with the vertical position control information from the microprocessor 50.

The rotational position control of the arm 7-2 is also the same as described above, and is performed by the microprocessor 50 starting the arm rotation control program 11d.
The microprocessor 50 has various data areas 11
According to the information f, rotation control information is sent to the arm circuit control circuit 58a via the interface 53. The arm circuit control circuit 58a drives the arm drive motor 58b according to the rotation control information from the microprocessor 50, and controls the first XY stage 5-1, the first The XY stage 5-2, the heating furnace 12, and the cooling pit 13 are positioned and stopped, respectively. In this case, the arm drive motor 58b may be a stepping motor or a normal AC or DC motor.

As described above, move arm 7-2 to the first XY position.
Stage 5-1, second XY stage 5-2, heating furnace 12, and cooling pit 13 can be positioned at four specific rotational positions according to index unit 7-1. In this case, if the motor is a stepping motor, the motor can also serve as an index unit.

Further, the computer control method using each program stored in the memory 11, a control circuit for each motor drive, and a drive motor is a general drive control configuration normally performed in computer control. The method is not limited to this, and various known drive control methods can be employed.

As explained above, according to the present invention, a pick-up can be repeatedly used many times with a relatively simple configuration, which is economical, and dispersion and transplantation into test tube culture can be performed efficiently.

[Brief explanation of the drawing]

Fig. 1 is a block diagram of a colony transfer device, Fig. 2 is a schematic perspective view of an embodiment using a platinum loop of the present invention, Fig. 3a is a plan view of a self-heating pick-up according to the present invention, and b is a Its front view, c is a current diagram, d is a heating temperature diagram, Figures 4a, b, and c are explanatory diagrams of the structure and the method of dispersing and transplanting into a slanted culture medium in a test tube by pick-up according to the present invention, Figure 5a , b is an explanatory diagram of the structure and operation of the method of dispersing transplantation into test tubes using a fragile plate system according to the present invention, and FIG.
FIG. 2 is a block diagram of an example of the configuration of a computer in the colony transplantation mechanism. 1...Culture shear, 2...Test shear, 3
...Test tube, 4...Common stand, 5-1...1st XY stage, 5-2...2nd XY stage, 6...Pickup section, 6-1...Pickup, 6-2...
...Height detector, 6-3...Collection, 7...Transfer means, 7-1...Index unit, 7-2
... Arm, 9 ... Monitor section, 10 ... Microcomputer, 12 ... Heating furnace, 13 ... Cooling pit, 14 ... Nichrome wire, 14-1 ... Tip,
16... Culture medium, 16'... Culture medium surface, 17... Arm equipped with a strain gauge, 18... Rotating table,
19...Failure board, 21...Bar, 22...Holding mechanism, 23...Support, 25...Holder, 26...
Slide groove, 27...High frequency heating coil, 28...
...Laser source, 29...Focusing lens.

Claims (1)

[Scope of Claims] 1. A rotating arm, a pick-up section provided at the tip of the rotating arm that moves up and down, and an index indicating at least four specific positions relative to the rotation of the rotating arm. a first XY stage provided corresponding to the position of the pickup section at the first specific position among the four, and a position of the pickup section at the second specific position among the four. a second XY stage provided correspondingly; a heating section provided corresponding to the position of the pickup section at the third specific position of the four; and a fourth of the four
A cooling unit is provided corresponding to the position of the pick-up unit at a specific position, and a control device having a computer is provided. A test container is placed thereon, and the pick-up section has a pick-up for collecting colonies or bacterial species from a culture medium or solution, and the control device controls the rotating arm according to the position indicated by the index unit. positioning control, and when the rotating arm is positioned at a first specific position, a first
The XY stage and the pick-up section are controlled to collect colonies or bacterial species from the culture medium or solution in the culture container, and when the rotating arm is positioned at a second specific position, the second XY stage and the pick-up section are controlled. control to plant colonies or bacterial species in the culture medium or solution in the test container, and when the rotating arm is positioned at a third specific position, control the pick-up section to control the heating section to plant the colony or bacterial species in the medium or solution in the test container. Colony transplantation, characterized in that the pick-up is heated and sterilized, and when the rotating arm is positioned at a fourth specific position, the pick-up section is controlled to cool the pick-up using the cooling section. mechanism. 2 The heating section is removed by self-heating the pick-up in place of the heating section at the third specific position, and the indexing unit is configured to use the rotating arm to indicate at least three specific positions excluding the heating section. A colony transplantation mechanism according to claim 1, characterized in that: 3 Pick-up is a patent characterized in that a heat-resistant resistance wire with an insulating layer on the surface is folded in half with the insulating layer inside, the diameter of the resistance wire at the bent portion is made thinner, and the shape near the bent portion is shaped like an earpick. A colony transplantation mechanism according to claim 1 or 2. 4. The colony transplantation mechanism according to claim 1, wherein the culture container or the test container is a shear plate. 5. Claim 1, characterized in that at least one of the culture vessels or test vessels is a test tube.
Colony transplantation mechanism as described in Section. 6. The colony transplantation mechanism according to claim 1, wherein a test container is placed on the second XY stage via a tracing plate system.