JPH11253190A - Cell culture bottle and method for detecting bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently carrying out detection and identification of bacteria in good accuracy by measuring the nuclear magnetic resonance absorption of a material produced by metabolism by culturing the bacteria in a medium containing a stable isotope. SOLUTION: This bottle 1A for culturing bacteria is a tube with different diameter made by connecting a tube having a larger diameter and a tube having smaller diameter, sealing its either one end and installing a removable cap 4 on the other end. This method for detecting bacteria comprises culturing the bacteria by putting a medium containing at least one stable isotope selected from the group consisting of heavy hydrogen,<13> C and<15> N and a specimen, charging a magnetic field to the tube having a smaller diameter of the bottle, measuring a nuclear magnetic resonance absorption and detecting the bacteria from the presence of metabolites containing the stable isotope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bottle for culturing a bacterium and a method for detecting the bacterium used for culturing and testing the bacterium. It is intended to efficiently detect and identify bacteria by measuring magnetic resonance absorption.

[0002]

2. Description of the Related Art A method of culturing a specimen such as blood in a medium containing a stable isotope and measuring the nuclear magnetic resonance absorption of a substance produced by metabolism at that time to detect and identify bacteria in the specimen (hereinafter referred to as a method) , SI-NMR method). And as a method of culturing bacteria to carry out this method, conventionally,
There are the following two methods (A) and (B).

(A) A method of culturing in a culture bottle.
Culture bottles are the most popular bottles for bacterial culture. The bottle has a bottomed cylindrical shape, and a screw cap is screwed into the mouth of the bottle. What is usually marketed is 20-30m in thickness
m, and the depth is about 50 to 70 mm. Although this bottle is inexpensive and has the advantage of easy handling, the diameter of this bottle cannot be loaded directly into the measurement room of the nuclear magnetic resonance absorption apparatus for analysis because of its large diameter. Therefore SI
-When performing the NMR method, the culture bottle is used only for culturing, and in order to measure the nuclear magnetic resonance absorption, the culture material is transferred to a dedicated nuclear magnetic resonance absorption tube (hereinafter referred to as NMR tube). Was needed. As a result, there is a drawback that the number of necessary operations increases by one, and the risk of secondary infection increases because the worker has more opportunities to come into contact with bacteria.

(B) A method of culturing in an NMR tube. The NMR tube is a thin glass tube in which a uniform magnetic field is applied to a sample (culture solution). Usually, outer diameter is about 5mm, length is 1
30 to 150 mm. The bottom is sealed off and the top is sealed with a Teflon stopper.
If the bacteria are directly cultured in the NMR tube, the bacteria can be directly loaded into the measurement room of the nuclear magnetic resonance absorption apparatus and analyzed. Therefore, compared with the method (A) using a culture bottle, the operation of transferring the culture solution is not required,
The measurement can be simplified, and the worker does not come into contact with bacteria, thereby avoiding the risk of secondary infection. But N
Since the MR tube is a thin tube as described above, the amount of the medium put in the tube is limited, and accordingly, the amount of the nutrient source in the medium is also limited. Moreover, the contact area between the air in the tube and the culture medium must be reduced. Since the growth of general aerobic bacteria depends on dissolved oxygen and nutrient sources, the use of NMR tubes as culture bottles slows the growth rate of bacteria. Therefore, S
It takes a long time to detect and identify bacteria by the I-NMR method. Further, since the volume in the NMR tube is small, the injectable amount of a sample such as blood is also limited. When the bacteria present in the sample sample are at an extremely low concentration, the probability of injecting the bacteria into the sample sample decreases, and the detection rate decreases. Alternatively, a drawback such as a longer time until detection occurs.

[0005]

SUMMARY OF THE INVENTION The present invention provides a method for efficiently detecting and identifying bacteria by culturing bacteria in a medium containing a stable isotope and measuring the nuclear magnetic resonance absorption of a substance produced by metabolism. It is an object to provide means for performing the method with high accuracy.

[0006]

According to the present invention, there is provided a bottle for culturing bacteria, wherein one end of a different-diameter tube having a large-diameter cylindrical portion and a small-diameter cylindrical portion connected to each other is sealed and the other end is closed. With a removable cap attached to it. In this bottle, at least a part of the cap may be air-permeable.
Preferably, the small-diameter cylindrical portion serves as a nuclear magnetic resonance absorption tube, and the large-diameter cylindrical portion serves as a holder that engages with a sample tube holding portion of the nuclear magnetic resonance absorption apparatus. According to the method for detecting bacteria of the present invention, a culture medium containing at least one stable isotope selected from the group consisting of deuterium, ¹³ C, and ¹⁵ N, and a specimen are placed in the above-described bottle for bacterial culture of the present invention. Culture,
A magnetic field is applied to the small-diameter cylindrical portion of the bottle, nuclear magnetic resonance absorption is measured, and bacteria are detected from the presence of metabolites containing the stable isotope. In this method, after culturing in the large-diameter cylindrical portion of the bottle for bacterial culture, the culture solution may be transferred to the small-diameter cylindrical portion, and nuclear magnetic resonance absorption may be measured.

[0007]

FIG. 1 shows a first embodiment of a bottle for culturing bacteria (hereinafter referred to as a bottle) according to the present invention. This bottle 1A is a different-diameter pipe formed by connecting an upper large-diameter cylindrical portion 2 and a lower small-diameter cylindrical portion 3 to each other. The lower end of the small diameter cylindrical portion 3 is sealed off. The upper end of the large-diameter cylindrical portion 2 is open, and a screw portion (not shown) is formed on the outer periphery of the upper end portion. A screw cap 4 is screwed into the screw portion.

FIG. 2 shows a second embodiment of the bottle according to the present invention. This bottle 1B is also a different-diameter tube in which the large-diameter cylindrical portion 2 and the small-diameter cylindrical portion 3 are connected. However, contrary to the bottle 1A according to the first embodiment, the large-diameter cylindrical portion 2
Of the small-diameter cylindrical portion 3 is set as an upper opening, a screw portion is provided at an upper end portion of the small-diameter cylindrical portion 3, and a small-diameter screw cap 4 is screw-fitted. ing.

In these bottles 1A and 1B, the connecting portion between the large-diameter cylindrical portion 2 and the small-diameter cylindrical portion 3 may have any shape as long as the medium or the sample as a content can smoothly move between the two portions. There is no particular limitation. Further, the material of the bottles 1A and 1B is not limited, and may be any material usually used in an NMR tube. For example, quartz glass is suitably used. The thickness may be about 0.1 to 1.0 mm.

Although the inner diameter (X) of the large-diameter cylindrical portion 2 is not particularly limited, it usually has an inner diameter of about 10 to 50 mm.
It is preferably about 20 to 40 mm. The length (Y1) is about 30 to 100 mm, preferably 50 to 70 m
m. If the inner diameter and the length of the large diameter cylindrical portion 2 are too large, not only is it unnecessary for culturing bacteria, but also the workability is impaired. If it is too small, there are disadvantages in that the capacity of the medium and the specimen is reduced, and the growth of bacteria is slowed down due to insufficient contact area with air.

The small-diameter cylindrical portion 3 functions as an NMR tube used in nuclear magnetic resonance absorption analysis, and its inner diameter (Z) is not particularly limited.
mm, preferably about 2 to 20 mm.
The length (Y2) is about 50 to 250 mm, preferably about 100 to 200 mm. The inner diameter is too thick,
If the length is too long, it will be difficult to set it in a commonly used nuclear magnetic resonance absorption apparatus. Also, too thin,
If the length is too short, the capacity of the sample required for the analysis will be insufficient, and if it is too small, it will be difficult for the medium and the contents of the sample to move in the tubule.

FIG. 3 illustrates the screw cap 4 mounted on the bottles 1A and 1B. The cap 4 has a disk-shaped top surface portion and a cylindrical portion hanging down from the periphery thereof. On the inner peripheral surface of the cylindrical portion, screws to be screwed to screw portions provided at the bottle mouths of the bottles 1A and 1B are provided. Is formed. The main body 7 of the cap 4 is made of a synthetic resin, and its material is not particularly limited, but a fluororesin or a silicone resin is preferable. The center of the top surface of the cap 4 is a gas permeable portion 6 made of a gas permeable membrane, while the contents do not leak out.
It has become. The permeable portion 6 is made of a permeable resin film such as a silicone resin or a foam. In addition,
In this example, the screw cap 4 is attached to the bottles 1A and 1B.
However, if the bottle mouth can be closed so that the contents do not leak out, other closing means, for example, a push-in stopper or a combination of a forced-fitting cap and a bottle mouth are used. May be. When the bacterium to be detected is an anaerobic bacterium, a cultivation is performed by using a completely airtight cap instead of the air-permeable portion 6 or by placing the bottles 1A and 1B together in an anaerobic atmosphere.

In order to detect and identify bacteria in a sample using these bottles 1A and 1B, first, a medium and a sample (culture solution 5) are poured into sterilized bottles 1A and 1B, and the bottle is opened. Attach cap 4 to seal bottles 1A and 1B,
Culture the bacteria. Here, as the medium, an appropriate basic medium is selected and used according to the type of bacteria to be detected, and one of the nutrients, for example, D-glucose-1- ¹³ C is added as glucose as a carbon source. Use the stable medium containing stable isotopes. The stable isotope to be used is not limited to ¹³ C, and a nutrient containing deuterium and ¹⁵ N may be added to the sample for analysis. The amount of the culture solution 5 is determined so that the gas phase space remains in each of the bottles 1A and 1B. The culture temperature can be appropriately set according to the type of bacteria to be detected.
４０40 ° C.

After culturing, the bottles 1A and 1B are turned upside down (upside down) to stir the culture, the uniform culture is transferred to the small-diameter cylindrical section 3, and the bottles 1A and 1B are transferred to the nuclear magnetic resonance absorption analyzer. After loading, a magnetic field is applied to the small-diameter cylindrical portion 3 functioning as an NMR tube, and nuclear magnetic resonance absorption is measured.
If bacteria are present in the specimen, along with their growth, the bacteria take up and metabolize nutrients containing stable isotopes, and various metabolites containing stable isotopes depending on the type of bacteria, such as lactic acid, ethyl alcohol, Organic acids such as acetic acid and carbon dioxide are produced. By nuclear magnetic resonance absorption analysis,
Analysis of various metabolites enables detection and identification of bacteria in a sample.

The bottles 1A and 1B are provided with a large-diameter cylindrical portion 2 capable of accommodating a sufficient amount of a culture medium and a specimen, and a small-diameter cylindrical portion 3 usable as an NMR tube. In addition to being capable of culturing in a short time, the culture solution can be loaded into the measurement room of the nuclear magnetic resonance absorption apparatus of the culture solution in a state of being placed in the small-diameter cylindrical portion 3, and the metabolite containing stable isotopes Since the bacterial species can be detected and identified from the absorption peak of, the bacterial detection operation can be performed efficiently. Further, since the operation of transferring the culture solution 5 to the NMR tube is unnecessary, the risk of secondary infection during the operation can be avoided. Further, when loading the NMR tube into the nuclear magnetic resonance absorption analyzer, the NMR tube must be inserted into a dedicated holder having a diameter larger than that of the NMR tube and loaded into the measurement chamber. If so, the large-diameter cylindrical portion 2 can have a function as a holder, so that the operation of inserting a tube into the holder becomes unnecessary, and the analysis operation can be further simplified.

FIGS. 4 and 5 show a third embodiment of the bottle of the present invention. This bottle 1C has the same components as the bottle 1A according to the first embodiment shown in FIG. 1, and has a plurality of blades 8 provided below the large-diameter cylindrical portion 2 (upper portion of the small-diameter cylindrical portion 3). It is characterized by that. The blades 8 may be provided integrally when the bottle 1C is molded, or may be a member made of a synthetic resin in which the blades 8 are radially formed on a ring having a size fitted to the outer periphery of the small-diameter cylindrical portion 3.
It may be attached to the small diameter cylindrical portion 3. By providing the blades 8, when measuring nuclear magnetic resonance absorption, the bottle can be rotated at a high speed by applying an air flow to the blades so that a magnetic field can be uniformly applied.

This function is conventionally provided with a holder dedicated to an NMR tube having a large diameter. When performing an NMR measurement, the NMR tube is inserted into the center of the holder and the NM is attached.
What had to be loaded into the R analyzer was added to the large-diameter cylindrical portion 2 in this bottle 1C. Thereby, the operation of inserting the NMR tube into the dedicated holder can be omitted, so that the analysis operation can be simplified.

[0018]

EXAMPLES (Example 1) Culture rate of bacteria Two types of aerobic bacteria, Escherichia coli (ATCC 29522) and Staphylococcus aureus (ATCC 29213), were cultured in bottles of various shapes. The turbidity associated with the growth of the bacteria was determined by measuring the absorbance (O
D) and the growth rates were compared. In this case, the absorbance (OD) is an index indicating turbidity, and an increase in the OD value indicates that the bacterium has proliferated, and accordingly, glucose, which is a nutrient source, has been consumed, and more metabolites have been produced. .

The bottles used were a conventional culture bottle and an NMR tube as a comparative example, and the bottle of the present invention shown in FIG.
(Referred to as bottle (a) of the present invention) and FIG. 2 (referred to as bottle (b) of the present invention). Table 1 shows the dimensions of each bottle and the injection amount of the culture solution (total of the medium and the bacteria). In Table 1, "large diameter" and "small diameter" represent the inner diameter, depth, and internal volume of the large diameter cylindrical portion and the small diameter cylindrical portion, respectively. The contact area is the area where the culture solution and the gas phase (air) are in contact.

[0020]

[Table 1]

[0021] medium, the Brain Heart Fu John (Eiken Chemical Co., Ltd.) in culture medium containing 5.8 wt%, ^{D-glucose-1-13} C a material obtained by adding (USA ISOTEC Co., Ltd.) 1 wt% Using. The bacteria were inoculated into the sterilized medium. The concentration of the bacterium was adjusted to 10 ⁵ cells / ml. This culture solution was poured into each bottle in the amount shown in Table 1, and cultured at 37 ° C. with shaking (about 20 times per minute). After an appropriate time, the culture solution was taken into a spectroscopic cell, and the absorbance (OD) at a wavelength of 660 nm was measured. As a spectrometer, Ubest-30 manufactured by JASCO Corporation was used.

FIG. 6 shows the time course of the OD of Escherichia coli, and FIG. 7 shows the time course of the OD of Staphylococcus aureus. For both Escherichia coli and Staphylococcus aureus, cultivation with the bottles (a) and (b) of the present invention shows the same cell growth rate as cultivation with the conventional culture bottle, and it can be seen that culturing with the NMR tube is slower than these. . In other words, it is shown that the cultivation under a condition having a large contact area with air, a large amount of medium, and abundant nutrients results in faster growth of the bacteria. Therefore, SI-NM
In the R method, using the bottles (a) and (b) of the present invention rather than using an NMR tube as a culture bottle,
It was found that the bacteria could be cultured quickly.

Example 2 Measurement of Nuclear Magnetic Resonance Absorption of Product by Bacteria Escherichia coli was cultured in the same four kinds of bottles under the same conditions as in Example 1 for 8 hours. After the culture, dioxane was added as an internal standard at a rate of 1 μl / ml culture solution. The bottles (a) and (b) and the NMR tube of the present invention were turned upside down, and the contents were agitated so that the culture solution was evenly distributed in the narrow tube portion. When the cells were cultured in the culture bottle and the NMR tube of the present invention, they were directly loaded into a measurement room of a nuclear magnetic resonance absorption apparatus (GSX-270, manufactured by JEOL Ltd.).

FIG. 8 shows the results obtained by culturing in the bottle (a) of the present invention and measuring the nuclear magnetic resonance absorption. Of the metabolite peaks, about 17 ppm is ethanol, about 21 ppm is lactic acid,
Approximately 23 ppm are the peaks of acetic acid, which are substances produced with the growth of E. coli. 67 ppm is the dioxane peak of the internal standard. About 93 ppm and about 9
7 ppm is the glucose peak. Table 2 shows the ratio of the height of each peak cultured in the other bottles when the peak height when cultured in the bottle (a) of the present invention is set to 1 (the peak of dioxane is omitted).

[0025]

[Table 2]

FIG. 8 and Table 2 show that the bottle (a),
It can be seen that in the culture according to (b), a product peak having almost the same height as that in the culture using the culture bottle is obtained. In contrast, when cultured in an NMR tube, the peak of the same metabolite is obtained, but the height of the peak is low. That is, even if the initial bacterial concentration is the same, NM
In the culture using the R tube, the detection accuracy is low because the proliferation of bacteria is small. Attempting to increase the peak requires a long time for cultivation, resulting in poor working efficiency. Alternatively, if the initial bacterial concentration, and thus the number of bacteria in the sample, is low, then the cultivation and detection of the bacteria becomes substantially impossible. In other words, according to the culture bottle of the present invention, it is possible to detect bacteria from a specimen containing a trace amount of bacteria, as compared with culture using an NMR tube.

As described above, in Example 1, it was revealed that the culture bottle of the present invention can culture bacteria at the same speed as a conventionally used culture bottle. In Example 2, without performing the work of transferring the culture solution,
It has been proved that nuclear magnetic resonance absorption can be measured as it is using a small-diameter cylindrical portion as an NMR tube. According to the second embodiment,
Compared to culture using an NMR tube that does not require transfer of the culture solution, it is possible to culture and detect a small amount of bacteria, and
It became clear that detection was possible in a short culture time.

In Example 2, the nuclear magnetic resonance absorption of metabolites of Escherichia coli was measured. However, since metabolites produced by bacteria are unique to each microorganism, the culture bottle containing a stable isotope of the present invention was used. By culturing the bacterium and measuring the nuclear magnetic resonance absorption of a substance produced by metabolism, it is possible to detect the bacterium and identify the species.

[0029]

According to the present invention, there is provided a bottle for culturing a bacterium, comprising a large-diameter cylindrical portion capable of accommodating a sufficient amount of a medium and a specimen,
Since it has a small-diameter cylindrical part that can be used as a tube, it is possible to culture a trace amount of bacteria in a short time, and a nuclear magnetic resonance absorption device of the culture solution in a state where the liquid after culture is put in the small-diameter cylindrical part And the bacterial species can be detected and identified from the absorption peaks of metabolites containing stable isotopes, so that the bacteria detection operation can be performed efficiently. In addition, since the operation of transferring the culture solution to the NMR tube is unnecessary, the risk of secondary infection of the worker can be avoided. Furthermore, when loading the NMR tube into the nuclear magnetic resonance absorption analyzer, the NMR tube must be inserted into a dedicated holder whose diameter is larger than the NMR tube and loaded into the measurement chamber. Since the large-diameter cylindrical portion can have a function as a holder, there is no need to insert a tube into the holder, and the analysis operation can be further simplified.

[Brief description of the drawings]

FIG. 1 is a front view showing a first embodiment of a bottle for culturing bacteria of the present invention.

FIG. 2 is a front view showing a second embodiment of the bottle for culturing bacteria of the present invention.

FIG. 3 is a perspective view of a cap attached to the same bottle.

FIG. 4 is a sectional view showing a third embodiment of the bottle for culturing bacteria of the present invention.

FIG. 5 is a bottom view of the same bottle.

FIG. 6 is a graph showing the results of a proliferation test of Example 1 according to the present invention.

FIG. 7 is a graph showing another result of Example 1 according to the present invention.

FIG. 8 is a graph showing the results of Example 2 according to the present invention.

[Explanation of symbols]

 1A, 1B, 1C Bottle (bacteria culture bottle) 2 Large-diameter cylindrical part 3 Small-diameter cylindrical part 4 Cap

Claims (5)

[Claims] 1. A bacteria culture bottle in which one end of a different-diameter tube in which a large-diameter cylindrical portion and a small-diameter cylindrical portion are connected is sealed, and a detachable cap is attached to the other end. 2. The bottle for culturing bacteria according to claim 1, wherein a permeable portion is provided on at least a part of the cap. 3. The small-diameter cylindrical portion is configured to be a nuclear magnetic resonance absorption tube, and the large-diameter cylindrical portion is configured to have a size and shape to be a holder to be engaged with a sample tube holding portion of a nuclear magnetic resonance absorption apparatus. The bottle for culturing bacteria according to claim 1, wherein 4. A culture medium containing at least one stable isotope selected from the group consisting of deuterium, ¹³ C, and ¹⁵ N in the bottle for bacterial culture according to claim 1. A bacterium, comprising: culturing a sample and a sample; applying a magnetic field to a small-diameter cylindrical portion of the bottle; measuring nuclear magnetic resonance absorption; and detecting the bacterium from the presence of metabolites containing the stable isotope. Detection method. 5. The bacterium according to claim 4, wherein after the culturing is performed in the large-diameter cylindrical portion of the bacterial culture bottle, the culture solution is transferred to the small-diameter cylindrical portion and nuclear magnetic resonance absorption is measured. Detection method.