JPH11346796A - Inspection of microbial specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection procedure that can properly form an isolated colony of a microorganism in the specimen with high reproducibility and gives exact inspection results. SOLUTION: A specimen (as an excrement) including microorganisms (bacteria or the like) is sampled with a spreader A and the spreader is slid on the surface of one corner of a solid flat culture medium under a prescribed pressure to adjust the state of the specimen sticking to the spreader A. The adjusted spreader B is moved to another corner of the culture medium, and slid under a prescribed pressure in a prescribed spreading pattern in one direction. This latter half sliding motion ensures the exact formation of properly separated colonies with high reproducibility. In a preferred embodiment, the pressure of the spreader B to the culture medium is higher in the first half adjusting motion than in the latter half sliding one.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for examining a microorganism sample for more reliably extracting microorganisms such as bacteria contained in a sample such as a stool test and performing an appropriate test.

[0002]

2. Description of the Related Art Generally, inspection of fresh foods (vegetables, fish and shellfish), including human stool, sputum, etc., refers to the presence or absence of malignant microorganisms, that is, the presence of pathogenic bacteria. In particular, in recent years, new pathogenic species have been discovered, and the frequency of the tests has become extremely high with the accuracy of the test.

[0003] In general, a test for a specimen containing a malignant microorganism (for example, a bacterium) is performed by smearing the specimen on a solid plate medium and culturing to isolate a colony based on the desired microorganism, that is, the microorganism to be examined (independent). It is a method of forming, collecting and examining the colony. The most important point here is how many colonies are surely formed in the smear.
In general, various microorganisms are often contained in a specimen, and therefore, it is not possible to accurately and quickly remove the microorganism of interest from a continuous colony. That is, proper inspection cannot be performed.

Regarding the specific method of the smear, regardless of whether it is manual or automatic, generally, a sample such as a stool is first collected and adhered to the tip of a smear stick (the tip is barbed, spoon-shaped, etc.) and secured. It is stored in a stick-shaped container containing a liquid or a calibrator medium. Then, the smear is taken out of the container, brought into contact with the plate-shaped medium hardened by agar or the like, and smeared thereon by sliding at a stroke according to a predetermined smear pattern. Here, when the smear is taken out of the container, an operation of rubbing against the edge of the container is performed to adjust the state of attachment of the sample (such as the amount or the place of attachment). This is to improve the conditions for smearing the medium surface in the next predetermined smear pattern and to improve the formation of appropriate isolated colonies. In addition, the isolated colony is formed on the trace of the pattern while drawing the smear pattern.

[0005]

However, the above-mentioned conventional method has the following disadvantages, and has not been sufficiently satisfied, and a solution has been demanded. In order to perform an appropriate test as described above, first, colonies are surely separated and formed in a larger state. Furthermore, a provisional judgment can be immediately made with the naked eye as to what microorganism the formed colony is due to, and it can be easily collected alone. It is important that a large number be formed within the narrowest possible range.

However, in the above-mentioned conventional method, the formation of the isolated colonies is first concentrated in the latter half of the smear pattern drawn (the former half is a thick continuous colony), so that the number is too small. Since the colonies are extremely small, it is difficult to visually determine which microorganism is caused by the microorganisms. Moreover, few of the colonies are formed in a completely separated (independent) state, and the colonies are connected by thin linear colonies. It is often formed in such a state. Further, even if an appropriate colony is formed, it is difficult to perform quantitative formation due to lack of reproducibility. In the colony in such a state, an extremely large obstacle is caused in the next confirmation test.

[0007] The present invention has been intensively studied and found to solve the above-mentioned four drawbacks at once, and can be easily achieved by taking the following means.

[0008]

That is, according to the present invention, a smear (A) to which a sample is attached is smeared and cultured on a solid plate medium as described in claim 1, and the microorganisms in the sample are examined. A method for testing a microbial specimen, which comprises sequentially performing the following steps (1) to (3). (1) First, smear the smear (A) using one corner of the medium,
An adjusting step for adjusting the adhesion state of the specimen by sliding the surface (2) Subsequently, the smear (B), which has been subjected to the above (1), is moved to another place of the medium, and a predetermined smear pattern is formed. Smearing step of smearing by sliding the surface in one direction (3) Inspection step of collecting and inspecting isolated colonies of microorganisms cultured and formed on a predetermined pattern in the smearing step.

[0009] Claims 1 and 2 are also provided as preferred embodiments. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, first, specimens of microorganisms to be examined are mainly gram-positive bacteria (staphylococci, pneumococci, diphtheria bacteria, etc.) and gram-negative bacteria (S. typhi), which are malignant bacteria in microorganisms. Stool, sputum, extracts from fresh or processed foods, etc., which contain Shigella, Shigella, Escherichia coli, pertussis, etc.), acid-fast bacteria (M. tuberculosis, etc.). However, without being specified in these, spread on a solid plate medium,
Form colonies and test (confirmation work, qualitative work)
All of the methods that can be performed are targets (eg, specimens containing yeasts, molds, mushrooms, and actinomycetes). However, among these, it is more effective for highly viscous specimens such as stools (humans and animals) which are generally inspected as stool tests.

The smear (A) is a tool that adheres and collects a required minimum amount of a sample at the tip thereof, appropriately (manually or automatically) grasps the other end (handle), and smears the culture medium.
Therefore, the shape of the tip portion can be exemplified by an arrowhead shape, a spoon shape, a spiral groove shape, a triangular shape, a V-shape, and the like, and may be appropriately selected according to the state of the sample. However, as long as the measures according to the present invention are taken, the shape of the tip of this smear is not selectable and any shape may be used. This is also one of the features of the present invention.

[0012] The culture medium is a so-called nutrient source that allows the microorganisms in the specimen to selectively propagate and facilitate detection. Therefore, the components constituting the medium differ depending on the type of microorganism. The state of the medium is generally a viscous liquid as a whole, or an elastic solid. In solid form, other components are hardened with agar or gelatin. In such a solid medium (plate), even if it is pressed to some extent by the smear (A) and slides on its surface, it is not damaged. In the present invention, it can be said that the present invention is more effective especially when isolated colonies are formed using this solid plate medium.

As described above, although the medium varies depending on the type of microorganism, there are few cases in which only one type selectively propagates, and in many cases, two or three types grow simultaneously. . However, even in such a breeding situation, it is easy to make a judgment because the separated colonies that are formed appear with differences in color, their arrival, shape, etc. However, this determination is not easy unless the colony is a separated colony formed by the method of the present invention. In a colony state as in the conventional method, the colony is not formed with a clear difference, so that it is difficult to perform a more appropriate test.

Next, the main steps of the present invention are steps (1) to (1).
(3) will be described. In the present invention, the adjusting step described in (1) is required prior to smearing the above-mentioned sample on the medium. In this adjustment step, the sample is attached to the tip of the smear (A), and the collected smear (A) is manually or automatically slid on the surface of the medium using one corner of a solid plate medium. In addition, the state of adhesion of the sample is adjusted. By taking this step, conditions are set such that colonies are completely separated in the next smearing step, and colonies of not only one kind but a plurality of microorganisms are formed in a size that is easy to collect. That you can do it. Therefore, since it is significant that this adjustment step is performed on the culture medium, the adjustment by a method in which the smear (A) is rubbed against the rim of the container containing the preservation solution or the like as in the conventional method means that There is a big difference in the effect.

Here, regarding the sliding of the smear (A) on the surface of the solid plate medium, the sliding pattern may be any one of left or right, left and right, circular, circular, polygonal, random, etc. . However, it is preferable not to slide on the same pattern twice.

The adjustment of the adhesion state of the sample is, as described above, in order to maximize the effect in the next smearing step, when the amount of the sample adhered to the tip of the smear is large, Is to be reduced, and in the case where the adhesion state is disordered, this is uniformly adjusted. Whether or not this adjustment is appropriate should be tested and checked in advance and then performed under that condition. Check items in this adjustment are mainly the pressure, number of times, and distance of sliding of the smear (A) with respect to the culture medium surface.

The smear (A) may be subjected to an adjusting step immediately after the sample is collected. However, the smear (A) is once stored in a container containing a preservation solution or a calibrator medium, which has been generally performed conventionally. May be used in the adjustment process, and such a use is more preferable because the state of the attached sample becomes more uniform. Of course, the colonies formed in this adjustment step are not substantially formed in a separated state, but are continuous colonies.

After the preparation step (1) has been completed, the smear is transferred to the next smearing step, where a full-scale smear for forming a proper separated colony is performed.

In the smearing step, first, the step is performed in another place different from the one place of the culture medium used in the adjusting step. Movement of the smear to another place, after finishing the adjustment process,
There are cases where the robot moves while continuing sliding, and cases where the robot moves once away from the surface of the culture medium. The latter case is preferable. This is because it is desirable to quickly form an isolated colony with better conditions.

The smear slides on the medium surface in one direction in a predetermined pattern. Since the above-mentioned separated colonies must be firmly formed on the pattern, at least the gap between the patterns must be such that the separated colonies when adjacent to each other do not come into contact with each other.
The shape of the smear pattern can be exemplified by the same pattern as in the adjustment step in the previous step. In addition, the pressure, the number of times of the sliding, and the distance are determined in advance by checking the relationship with these factors so that the above-mentioned appropriate separated colonies are formed. Pressure is also more important because it has a significant effect on proper segregation colony formation. The sliding pressure is preferably selected in the range of 0.3 to 3 times the pressure in the adjusting step.

Finally, microorganisms are confirmed (qualitatively and quantitatively) by an inspection step. For this purpose, a medium (generally prepared in a flat plate in a petri dish) that has been subjected to the adjustment step and the smearing step is insulated. And cultivate. When the cells are cultured, continuous colonies are mainly formed in one corner of the adjustment step, and proper isolated colonies are not formed. On the other hand, in the smearing process,
Since an appropriate isolated colony is formed with an appropriate set, the isolated colony formed in this smearing step is collected and examined visually or under a microscope. In some cases, the isolated colonies obtained by obtaining the first culture may be further cultured.

Since the composition (components) of the medium varies depending on the type of microorganism, the type of microorganism must be predicted and checked in advance to determine the type of microorganism in the sample. For example, for bacteria, gravy agar (or gelatin) medium, etc.
Examples of yeast include potato-glucose agar medium, and examples of actinomycetes include yeast-malt agar medium and oatmeal agar medium. Also, by mixing a small amount of chromosomes in this medium, the formed isolated colonies are colored or not (clear). That is, since the color tone changes depending on the type of microorganism, the type can also be known temporarily (visually).

The adjusting step and the smearing step described above are
Either manual operation or automatic operation is effective, but automatic operation is preferable for more quantitative and rapid operation. In the case of automation, the automatic chuck of the handle of the smear (A), when using a container containing a preservation solution, etc., when the smear is stored in and removed from the container, the sliding pressure of the smear on the culture medium Then, a mechanism such as control of the sliding speed, selection of the smear pattern, continuous or discontinuous switching from the adjustment process to the smear process, etc. is adopted, and an automatic apparatus is obtained.

[0024]

Next, the present invention will be described in more detail with reference to examples together with comparative examples. It goes without saying that the present invention is not limited by the examples.

(Example 1) First, a broth agar plate medium having the following composition was prepared in a Petri dish of 90 mm ¢.
8.0 g of peptone, 10.0 g of lactose, 8.5 g of sodium citrate, 5.0 g of sodium thiosulfate, 1.0 g of iron citrate, 0.33 g of brilliant green, 0.03 g of neutral red and 13.5 g of agar powder Heat and dissolve in 1 liter of water (PH = 7.2 ± 0.2)
The temperature was adjusted to 0 ° C., 25 ml of this was taken, dispensed into a horizontally held petri dish, and cooled and solidified. 1 in the plan view (1-C) of FIG. 1 is a solidified medium in a petri dish.

On the other hand, the stool of a person who is considered positive as a specimen (possibly dysentery) is collected with the tip 7a of a spoon-shaped smear stick A shown in the perspective branch diagram (1-D) of FIG. It was housed in a stick-shaped container into which a calibrea medium was injected.

Next, the handle 7 of the smear A was automatically chucked and pulled out of the stick-shaped container while rotating (three times) (referred to as smear B). Then, using one corner of the 培 地 medium 1, an adjustment step was first performed under the following conditions. In other words, the tip of smear B is set to start point 3, 2.5
mm with a dent pressure of 4 mm and spirally outward four times with a gap of 0.5 mm (rotation speed 180 rpm).
m) The surface was slid and stopped at the position 4 to complete the process.

Next, the smear stick B was lifted up, moved away from the position 4 and moved to the other corner, that is, the position 5 to perform the smearing process under the following conditions. That is, the smear bar B after the adjustment is pressed with its tip at the position of 5 with a depression pressure of 1 mm, and spirally with a gap of 4 mm.
The surface was slid 4.5 times outward (at a speed of 180 rpm) to stop at position 6, and the process was completed.

(Comparative Example 1) (Smearing by Spiral Pattern by Conventional Method) First, the medium solution prepared in Example 1 was dispensed into a Petri dish in the same manner as in this example, and cooled and solidified to prepare a solid medium. This is indicated by 8 in FIG. On the other hand, the same sample as in Example 1 was similarly collected with a smear stick A and temporarily stored in a stick-shaped container. Then, the smear A was taken out from the container three times in the same manner as in the example (the smear B was used). Immediately, the medium 8 was smeared outward in a spiral pattern. The smear conditions are as follows: first, the tip of the smear B
At a depression pressure of 1 mm, and spirally rotate 9 times at a speed of 180 rpm outward with a gap of 4 mm.
It ended at 0.

Next, the petri dishes that had been smeared in Example 1 and Comparative Example 1 were placed in a 45 ° C. sterilized heat insulation room and cultured. When taken out and visually judged, on the culture medium 1 of Example 1, black spiral continuous colonies were formed in the preparation step, and no isolated colonies were formed. However, in the smearing process, a number of appropriate separated colonies were formed from the starting point 5 to the end point 6 of the smear, and the colonies were of two types, black (black spots in the figure) and pink (white circles in the figure). The diameter of the black colony was about 0.8 mm, and that of the pink colony was about 1.1 mm.

On the other hand, in the medium 8 of Comparative Example 1, continuous black colonies were mostly formed, and isolated colonies were formed near the end point 10, and were small and small in number. This was not a proper isolated colony and was unsatisfactory for performing a sufficient inspection. The isolated colonies had a diameter of about 0.5 mm for black and about 0.7 mm for pink.

Then, the isolated colony in Example 1 was picked up (it was easy to pick up because it was large), and when observed with a magnifying microscope, it was found that the black colony was due to Shigella and the pink color was due to Escherichia coli.

(Example 2) First, the medium solution prepared in Example 1 was similarly dispensed into a petri dish and cooled and solidified.
This is indicated by 11 in the plan view branch diagram (3-E) in FIG.

On the other hand, using the same specimen as in the first embodiment, the same specimen 19a is provided with the tip (the vertical groove is provided on the inclined surface) of the arrow-shaped smear stick A1 in the perspective branch view (3-F) of FIG. The stool was collected, and this was temporarily stored in a stick-shaped container containing a calibrator medium. Then, it was extracted while rotating four times, and the following adjustment process was first performed.

That is, after the extraction, the tip 19a was brought into contact with the starting point 12 using the upper half 11a of the medium 11, and the medium surface was slid toward the end point 13 in an arc at a dent pressure of 3 mm. The adjustment of the circular arc here was about 25 mm on one side and 2.5 reciprocations.

Subsequently, the system was moved to the starting point of No. 13 and the smearing step was performed under the following conditions. In other words, the smear bar after the adjustment step is lifted and brought into contact with the position 14
Medium 1 in an arc shape with a recess thickness of 1 mm
a Slided on the surface. The arc is about 45mm on one side
Thus, three round trips were made.

(Comparative Example 2) (Conventional method in which a single point is pierced and then smeared) Under the same conditions as in Example 2, another barbed smear A1 taken out of the stool and pulled out of the stick-shaped container was used as a medium. 11
The lower half 11b was used to first touch the position 16 and pierce until it touched the bottom of the petri dish. Then, it was lifted, moved to the position of 17, and smeared up to 18 points in an arc shape under the smearing conditions performed in Example 2. However, the distance of one side of the arc was 45 mm and the number of reciprocations was about 4.3.

Finally, the petri dishes smeared in Example 2 and Comparative Example 2 were cultured in a greenhouse in the same manner as in Example 1. As a result, the state of the colonies formed on the culture media 11a and 11b was as shown in FIG. 3 (3-E). When the isolated colonies obtained in Example 2 were picked up and observed with a magnifying microscope, black colonies (black circles) were Shigella and pink (white circles) were Escherichia coli, as in Example 1.

In Example 2, as can be seen from Example 1, in the smearing step, a proper isolated colony was immediately formed from the starting point 14,
It can be seen that they are formed between many and short smear patterns. That is, in Comparative Example 2, as shown at 16, before the smear, the operation of once piercing one point of the medium, breaking the medium, and adjusting the state of the sample at the tip of the smear is performed. However, even if such a pre-process is performed, an isolated colony that can be inspected somehow is formed at the end of the smear, and is small and small in number, without forming an appropriate isolated colony. Also, as seen in Examples 1 and 2, the medium area required for both comparative examples may be smaller.
This has the possibility that many inspections can be performed simultaneously in one petri dish, which greatly contributes to speeding up the inspection.

[0040]

As described above, the present invention has the following effects.

The microorganisms in the specimen are formed as isolated (independent) colonies by themselves, without including other microorganisms, and are reliably formed in a state in which appropriate inspection (identification) is possible.

Variations (the size of colonies, formation positions, etc.) in the formed isolated colonies are small, and the colonies are formed with good reproducibility.

Due to the above effects, the smear area for forming the isolated colonies may be small. Therefore, many cultures can be performed in one petri dish. The above effects eventually lead to an increase in inspection speed.

[Brief description of the drawings]

FIG. 1 is a plan view in which colony formation states in Example 1 and Comparative Example 1 are imaged from above.

FIG. 2 is a plan view in which colony formation states in Example 2 and Comparative Example 2 are imaged from above.

[Description of numbering]

 1,8,11: Medium in Petri dish

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[Procedure amendment]

[Submission date] November 6, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 is a plan view in which colony formation states in Example 1 and Comparative Example 1 are imaged from above.

FIG. 2 is a plan view in which colony formation states in Example 2 and Comparative Example 2 are imaged from above.

FIG. 3 is a plan view showing a state of a colony formed in Comparative Example 2 from above.

[Explanation of symbols] Medium in 1, 8, 11 Petri dishes

Claims (3)

[Claims] 1. A method for testing a microorganism specimen in which a smear (A) to which a specimen is adhered is smeared on a solid plate medium and cultured to examine microorganisms in the specimen, the following (1) to (3): A method for testing a microbial sample, comprising sequentially performing the steps described in 1. (1) First, smear the smear (A) using one corner of the medium,
An adjusting step for adjusting the adhesion state of the specimen by sliding the surface (2) Subsequently, the smear (B), which has been subjected to the above (1), is moved to another place of the medium, and a predetermined smear pattern is formed. Smearing step of sliding the surface in one direction and smearing (3) Inspection step of collecting and inspecting isolated colonies of microorganisms cultured and formed on a predetermined pattern by the smearing step 2. The method according to claim 1, wherein the specimen is a highly viscous body containing bacteria. 3. The slide pressure according to claim 1, wherein the sliding pressure of the smear (B) on the culture medium surface in the smearing step is 0.3 to 3 times the sliding pressure in the adjusting step. Testing methods for microbial specimens.