JPH10136973A - Isolation of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for isolating a specific microorganism such as a bacterium in a short time without carrying out the colony proliferation of the microorganism by observing the proliferation process of a microorganism group comprising plural microorganisms and subsequently isolating the specific microorganism. SOLUTION: This method for isolating a microorganism comprises disposing a stage 4a and a device for projecting image on a monitor 9a through an optical microscope 4 and a camera 8 in a box 1 equipped with a temperature controlling device 2 and a filter 3 of character clean air and used as a clean bench, disposing a laboratory dish 5 having a culture medium therein on the stage 4a, dropping a specimen containing a microorganism group comprising plural similar microorganisms such as bacteria belonging to the genus Bacillus on a culture medium 6 in the laboratory dish 5, observing the proliferation processes of the microorganisms with images projected on the monitor 9a through the optical microscope 4 and the camera 8, and subsequently isolating the microorganism from the microorganism group with a needle 7 during the initial to microcolony stage of the microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a microorganism isolation method for isolating microorganisms such as bacteria.

[0002]

2. Description of the Related Art Conventionally, microorganisms such as bacteria are generally isolated as follows.

[0003] First, a suspension (microorganism-containing liquid) is prepared by mixing microorganism-containing substances such as soil containing microorganisms with a solvent such as water or saline, and this suspension is added to a medium such as agar. The microorganism is transplanted to the medium by dropping.

[0004] Thereafter, differences in the size, shape and color of colonies composed of microorganisms formed by growth for one to several days are visually identified, and a part of the colonies determined as a single microorganism colony is identified as platinum. Separate by taking out with an instrument such as an ear.

[0005] Subsequently, each of the fractions collected from the colonies is confirmed to be a single microorganism by using a staining method or a biochemical test method, and the isolation of the microorganism is completed.

[0006]

However, in the above-described isolation method, it usually takes one day to several days to grow a colony to a size that can be determined as a single colony. There is a problem that time is very long.

[0007] In addition, when colonies are grown until they can be visually judged as described above, there is a problem that frequently adjacent colonies are mixed with each other. In this case, the above method is repeated again. .

[0008] In addition, a colony of a single microorganism is determined,
Even when isolated from the colony, staining or biochemical testing may reveal that the isolated organism contains a plurality of microorganisms. Has to be repeated.

[0009] In particular, the above method has a problem that it is very difficult to determine whether a similar microorganism is composed of a single microorganism from the appearance of the colony.

[0010] Further, in the above-mentioned isolation method, even when it is apparent in advance that the microorganism-containing liquid is composed of a plurality of predetermined microorganisms, there is a problem that the same growth time as described above is required, and the desired method is also required. Until microbes were found, it was necessary to sequentially examine colonies using staining and biochemical testing.

[0011] The present invention has been made in view of the above problems, and has as its object to provide a microorganism isolation method capable of isolating microorganisms in a short time.

[0012]

Means for Solving the Problems The microorganism isolation method of the present invention is characterized in that the microorganism is isolated while observing the growth process of the microorganism.

In the method of the present invention, the microorganism is to be isolated by utilizing the growth pattern and the like during the growth process of the microorganism. It is not necessary to form a large colony so that the cells can be distinguished from each other, and isolation in a short time is possible.

Moreover, in this method, since large colonies do not have to be formed, the possibility that different microorganisms are mixed can be reduced. That is, according to this method, it is possible to easily isolate the microcolonies before they come into contact with each other.

[0015] Further, in this method, the unity is not determined from the appearance of the colonies, but is determined based on the growth pattern. Therefore, the risk of contamination by other microorganisms is reduced. It is easy to isolate a specific microorganism.

In addition, in this method, by observing the growth process a plurality of times or continuously, it is possible to easily discriminate the microorganisms that grow by the growth from foreign substances such as dust that does not change over time.

Further, in this method, when it is known in advance that the predetermined microorganism is present, the identification work of the predetermined microorganism can be reduced without performing identification by a staining method or a biochemical inspection method.

The microorganism isolation method of the present invention is characterized in that a specific microorganism is isolated by observing the growth process of a microorganism group comprising a plurality of microorganisms.

In the method of the present invention, a specific microorganism is to be isolated by utilizing the difference in the growth pattern during the growth of the microorganism. Therefore, it is not necessary to form a large colony so that the colony can be distinguished by the shape and the like, and isolation in a short time is possible.

In addition, since this method does not require the formation of large colonies, it is possible to reduce the risk that the plurality of microorganisms are mixed. That is, according to this method, it is possible to easily isolate the microcolonies before they come into contact with each other.

Further, in this method, the unity is not determined from the appearance of the colonies, but the unity is determined based on the growth pattern. Therefore, a specific microorganism is isolated from a plurality of microorganisms. It is easy.

In addition, according to this method, by observing the growth process a plurality of times or continuously, it is possible to easily distinguish microorganisms that grow by growth from foreign substances such as dust that does not change over time.

Furthermore, in this method, when it is known in advance that a predetermined microorganism is present, identification of the predetermined microorganism is easy without performing identification by a staining method or a biochemical test method.

In particular, the microorganism group contains a plurality of similar microorganisms, and a specific microorganism is isolated from the plurality of similar microorganisms.

In this case, the difference in the growth patterns of similar microorganisms having different growth patterns can be utilized from the observation during the growth process, so that it is easy to isolate a specific microorganism from the similar microorganisms.

The similar microorganism may be a microorganism of the same species, for example, a bacterium of the same species.

[0027] In particular, the method is characterized in that the microorganism is isolated using a growth pattern of the microorganism.

In this case, it is possible to utilize the difference in the growth patterns of microorganisms having different growth patterns, so that it is easy to isolate a specific microorganism.

[0029] In particular, the isolation of the microorganism is carried out during the initial state of the microorganism during the micro-colony state.

In this case, since isolation is performed in a microcolony state without forming a large colony, the possibility that different microorganisms are mixed can be reduced.

In addition, colonies larger than microcolonies are more likely to be unable to determine the growth pattern due to the concentration of microorganisms. Therefore, isolation in a microcolony state is also preferable from this point.

In particular, the present invention is characterized in that the isolated microorganism is a microorganism belonging to the kingdom Fungi.

Further, the microorganisms belonging to the kingdom of the fungus are bacteria.

Further, the bacterium belongs to the genus Bacillus.

The microcolonies in the present invention are
It shows a colony in which the growth pattern of each microorganism can be identified, and is an aggregate of usually up to about several hundred, preferably several to tens of microorganisms, which can be identified by an optical microscope (for example, 100 to 2000 times).

[0036] The specific microorganism of the present invention refers to a microorganism to be isolated in advance if the microorganism to be isolated is a specific microorganism and if the microorganism to be isolated is not determined in advance. Refers to a microorganism to be isolated as a specific microorganism.

In addition, since the growth pattern of the microorganism changes depending on the temperature, the environmental atmosphere gas and the like, it is preferable to isolate the microorganism by controlling the temperature and / or the environmental atmosphere such as the environmental atmosphere gas.

Further, the observation during the growth process in the present invention includes:
The observation may be continuous or discontinuous.

As described above, of course, the method of observing the growth of a microorganism and identifying a specific microorganism from the growth pattern, which is used in the microorganism isolation method of the present invention as described above, is a method of identifying the specific microorganism. It is also useful as a method.

[0040]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The principle of the present invention will be described first. The invention of the present application has been made as a finding that, even in a similar microorganism, for example, a microorganism of the same species, in a growth process involving cell division leading to the formation of microcolonies, a phenomenon in which different growth patterns are observed has been found. .

As an example, of the genus Bacillus, which is a kind of bacteria, Bacillus subtilis I2 (Bacillus subtilis I2: hereinafter referred to as sample A) and the same species
Bacillus subtilis ATCC6051 strain (Bacillus subtilis ATCC6051 strain: hereinafter referred to as sample B)
1 to 3 and FIGS. 4 to 7 show the growth process over time. In addition, each figure is an enlarged view of the observation image of 400 times, and each growth time T is shown in the figure.

In this experiment, sample A was first prepared by adding 10 g of meat extract, 10 g of peptone, and 1 g of salt to distilled water.
After mixing with 10 ml of the mixture that had been sterilized at 121 ° C. for 15 minutes and dissolved at 00 ° C.
The sample A is grown by shaking culture for a time. Thereafter, the mixed solution that has undergone this growth step is mixed with 0.85% saline solution for 1 hour.
A microbial-containing solution A diluted to 1: ⁵ is prepared.

Similarly, sample B was prepared by adding 10 g of meat extract, 10 g of peptone, and 1 g of salt to 100 ml of distilled water.
After mixing with 10 ml of a sterilized mixture at 121 ° C. for 15 minutes dissolved in 0 ml, sample B is grown by shaking culture at 37 ° C. for 20 hours. Thereafter, the mixed solution that has undergone this growth step is diluted with 0.85% saline solution for 10 minutes.
A microorganism-containing liquid B diluted ⁵ times is prepared.

Thereafter, the microorganism-containing liquid A and the microorganism-containing liquid B were each mixed with 10 g of meat extract, 10 g of peptone, 1 g of salt, and 15 g of agar in distilled water in a clean air environment at a growth temperature of 30 ° C. Dissolve in 1000 ml,
Each meat extract bouillon agar medium which had been sterilized at 121 ° C. for 15 minutes and was prepared by adjusting the pH to 7 was dropped into the medium, and samples A and B were grown on the respective mediums.

First, as can be seen from FIGS. 1 to 3, the sample A has a proliferation pattern in which the cells proliferate in a microcolony state without fragmentation even when cell proliferation occurs due to cell division. You can see that.

Next, as can be seen from FIGS. 4 to 7, Sample B is different from Sample A, despite being of the same species as Sample A, after several cell divisions in the microcolony state. ,
It can be seen that the growth pattern is such that the cells are divided and proliferated in the form of a parallel string.

As can be seen from the experimental results, it is possible to discriminate that even in the same kind of bacteria, at least in a microcolony state, the growth pattern is different, and it is possible to isolate a specific bacterium.

Therefore, the present invention makes use of the fact that the growth patterns are different as described above, and observes the growth process of the microorganism, discriminates a specific microorganism from the growth pattern, and selectively selects the microorganism. To be taken out.

Hereinafter, an embodiment of the microorganism isolation method of the present invention will be described. FIG. 8 is a schematic configuration diagram of an apparatus used in the present embodiment.

First, the above device will be described.

In FIG. 8, reference numeral 1 denotes a box as a clean bench (a container for blocking dust) for maintaining a clean atmosphere, and 2 denotes a temperature for controlling the temperature inside the container 1 so that microorganisms can grow at a preferable growth rate. The temperature control device 3 is a filter for introducing clean air from the outside when performing in an air atmosphere. When breeding is carried out in an atmosphere other than an air atmosphere such as an atmosphere gas such as nitrogen, the filter is shut off, and a desired clean atmosphere gas is introduced from an introduction section (not shown).

Reference numeral 4 denotes an optical microscope (object enlarging means) for observing the growth pattern of the growing microorganisms. Reference numeral 5 denotes a three-dimensionally movable stage 4a incorporated in the optical microscope 4 in the box 1. A plastic or glass Petri dish (proliferation container), and 6 is a growth medium installed in the Petri dish 5.

The micromanipulator 7 is provided with a sharp needle (microorganism pick-up tool) 7a for selectively extracting a specific microorganism from the growth medium 6. Means).

Reference numeral 8 denotes a camera connected to the optical microscope 4;
A computer 10 having a monitor 9a for taking in information of an image captured by the camera 8 and projecting the image;
Is a controller (stage controller) for controlling the stage movement of the stage 4a and the micromanipulator 7 connected to the computer 9, and 11 performs image processing of the image information captured by the computer 9, and This is an image processing device that calculates position information (coordinates) where (microcolonies) exist.

According to such a method, first, the inside of the box 1 is controlled to a predetermined temperature by operating the temperature control device 2 while keeping the inside of the box 1 in a clean environment atmosphere.
a, a petri dish 5 containing the above-mentioned medium 6 made of agar or the like is installed.

Subsequently, a suspension (microorganism-containing liquid) is prepared by mixing a microorganism-containing substance such as soil containing microorganisms with a desired solvent such as water or saline in the same manner as in the prior art. The microorganism is transplanted to the culture medium 6 by dropping the suspension into the culture medium 6.

Thereafter, an image of the growth pattern of the microorganisms growing on the transplanted medium 6 projected onto the monitor 9a via the optical microscope 4 and the camera 8 is appropriately observed, and microcolonies (microorganisms) are identified from the growth patterns. After this identification, the controller 4 is operated to designate a desired specific microcolony, so that the stage 4a and the micromanipulator 7 move based on the position information of the microcolonies from the image processing apparatus 11. Then, a part of the microcolonies, that is, a specific microorganism is selectively taken out from the microcolonies using the needle 7a.

In this method, for example, when the above-mentioned suspension is a mixed solution (microorganism group) in which the above-mentioned sample A and the above-mentioned sample B are mixed, the sample A and the sample B are prepared several hours after transplantation. Since the growth patterns are different, the samples A and B can be easily isolated from the growth patterns.

As described above, according to this method, it is not necessary to grow the colony for one to several days and form a large colony so that it can be distinguished by its shape or the like, so that a specific microorganism can be isolated in a short time.

In addition, according to this method, isolation is performed in a microcolony state without forming a large colony, so that the possibility that different microorganisms are mixed can be reduced.

Further, according to this method, the unity is not judged from the appearance of the colonies, but the unity is judged based on the growth pattern. Therefore, it is easy to isolate a specific microorganism from similar microorganisms. is there.

Further, according to this method, if it is known in advance that the microorganism-containing liquid contains a predetermined microorganism, the identification of the predetermined microorganism can be performed without performing the identification by a staining method or a biochemical inspection method. Easy.

In the above-described embodiment, the isolation of the specific microorganism is performed automatically. However, the specific microorganism is taken out by operating the manipulator 7 and the like manually while confirming with an optical microscope. Alternatively, various methods may be employed as appropriate.

Although the method for isolating Bacillus bacterium has been described above, the method of the present invention can be appropriately used for isolating microorganisms such as other bacteria.

[0065]

According to the present invention, a microorganism isolation method capable of isolating a microorganism in a short time can be provided.

[Brief description of the drawings]

FIG. 1: T = 1.5 hours (hours) after transplantation of sample A,
FIG. 4 is an enlarged view showing a growth pattern when T = 2 hours, T = 2.3 hours, and T = 2.7 hours.

FIG. 2 is an enlarged view showing a proliferation pattern at T = 3 hours after transplantation of sample A.

FIG. 3 is an enlarged view showing a growth pattern at the time T = 3.3 hours after transplantation of sample A.

FIG. 4: T = 1.3 hours, T = 2.2 after implantation of sample B
FIG. 4 is an enlarged view showing a time and a growth pattern after T = 2.7 hours.

FIG. 5 is an enlarged view showing a growth pattern after T = 3.3 hours after transplantation of sample B.

FIG. 6 is an enlarged view showing a growth pattern at T = 3.7 hours after transplantation of Sample B.

FIG. 7 is an enlarged view showing a growth pattern after T = 4.1 hours after transplantation of Sample B.

FIG. 8 is a schematic configuration diagram of a microorganism isolation device according to one embodiment of the present invention.

[Explanation of symbols]

 4 Optical microscope (object enlargement means) 7 Manipulator (microorganism extraction means)

Claims (8)

[Claims] 1. A method for isolating a microorganism, comprising observing the microorganism during its growth process and isolating the microorganism. 2. A method for isolating a microorganism, comprising observing the growth process of a microorganism group comprising a plurality of microorganisms and isolating a specific microorganism. 3. The microorganism isolation method according to claim 2, wherein the microorganism group includes a plurality of similar microorganisms, and a specific microorganism is isolated from the plurality of similar microorganisms. 4. The method according to claim 1, wherein the isolation of the microorganism is performed using a growth pattern of the microorganism.
Or the microorganism isolation method according to 3. 5. The microorganism isolation method according to claim 1, wherein the isolation of the microorganism is performed during an initial stage of the microorganism and a microcolonial state. 6. The microorganism according to claim 1, wherein the microorganism to be isolated is a microorganism belonging to the kingdom of the genus Fungi.
Or the microorganism isolation method according to 5. 7. The microorganism isolation method according to claim 6, wherein the microorganism belonging to the kingdom Kingdom is a bacterium. 8. The method according to claim 7, wherein the bacterium belongs to the genus Bacillus.