WO2011030526A1 - Method for detecting microorganism - Google Patents

Abstract

Disclosed is a method whereby a microorganism can be quickly detected at a high sensitivity. The method for detecting a microorganism comprises: a step for removing a liquid, which contains at least one kind of a microorganism and has been reserved in a liquid reservoir, from the bottom of a membrane filter (130) and thus concentrating the microorganism on the membrane filter (130); a step for introducing a liquid medium containing a physiologically active substance into the liquid reservoir and culturing the microorganism suspended in the liquid medium; a step for removing the liquid medium from the bottom of the membrane filter (130) and capturing said microorganism on the membrane filter (130); a step for introducing a reagent for determining the viability of the microorganism into the liquid reservoir; and a step for observing the microorganism in the chip (sensor chip (100)).

Description

Microorganism detection method

The present invention relates to a microorganism detection method.

A culture method is generally used as a method for detecting microorganisms. In this culturing method, for example, it is known that it takes a very long time to perform culturing for 24 hours.

Patent Document 1 describes a microorganism detection method that improves the point of individually detecting microorganisms. According to this document, the microorganism is propagated in a state where the microorganism is bound on the detection surface. Thereby, it is described that microorganisms can be detected individually.

Special table 2008-523820

As described above, in a general culture method, it took time to detect microorganisms. Therefore, a method for quickly detecting microorganisms is desired.

Further, as in Patent Document 1, when culturing in a state where a part of the microorganism is bound on a certain surface, the culture speed may be slow because there is a part where the culture solution and the microorganism are not in contact with each other. . Furthermore, since the composition concentration in the liquid varies in the vicinity of the surface, for example, it has been difficult to obtain the results of the growth of microorganisms when an antibacterial agent is added with good reproducibility. In addition, since no shaking mechanism is provided, culturing was difficult depending on the bacterial species.

According to the present invention,
A reservoir for storing a liquid for testing containing at least one type of microorganism;
An opening provided on the lower surface of the liquid reservoir,
Prepare a chip including a detection unit having a membrane filter provided so as to cover the opening,
Removing the test liquid in the liquid reservoir from below the membrane filter, and concentrating the microorganisms on the membrane filter;
Introducing a liquid medium containing a physiologically active substance into the liquid reservoir, and culturing the microorganisms floating in the liquid medium;
Removing the liquid medium from below the membrane filter and collecting the microorganisms on the membrane filter;
Introducing a reagent for determining the life and death of the microorganism into the liquid reservoir;
And observing the microorganism in the chip.
However, the order of this process may be changed depending on the situation.

According to the present invention, microorganisms can be detected quickly and with high sensitivity.

The above-described object and other objects, features, and advantages will be further clarified by a preferred embodiment described below and the following drawings attached thereto.

It is a flowchart which shows the method of detecting the microorganisms of embodiment of this invention. It is process sectional drawing which shows the detection method of the drug sensitivity of embodiment of this invention. It is process sectional drawing which shows the detection method of the drug sensitivity of embodiment of this invention. It is a top view which shows typically the sensor chip of embodiment of this invention. 1 is a schematic cross-sectional view taken along a line AA of a sensor chip according to an embodiment of the present invention. FIG. 4 is a schematic cross-sectional view taken along line BB of the sensor chip according to the embodiment of the present invention. It is a schematic cross section which shows the concentration apparatus of embodiment of this invention. It is a schematic cross section which shows the suction mechanism of embodiment of this invention. It is a schematic cross section which shows the detection apparatus of embodiment of this invention. It is a figure which shows the time change of the viable count of colon_bacillus | E._coli. It is a figure which shows the time change of the viable count of colon_bacillus | E._coli at the time of adding antibiotics. It is a figure which shows the viable count ratio of colon_bacillus | E._coli when the density | concentration of the added antibiotics is compared. It is a figure which shows the time change of the viable count of colon_bacillus | E._coli. It is a figure which shows the time change of the viable count of colon_bacillus | E._coli at the time of adding antibiotics. It is a figure which shows the viable count ratio of colon_bacillus | E._coli when the presence or absence of addition of an antibiotic is compared. It is a figure which shows the result of the fluorescence observation of a detection part. It is a figure which shows the result of having measured MIC by the conventional method (dilution method).

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

(First embodiment)
FIG. 1 is a flowchart showing a method of detecting a microorganism according to an embodiment of the present invention. FIG. 5 is a schematic cross-sectional view showing the sensor chip 100 according to the present embodiment.
In the method for detecting microorganisms according to the present embodiment, first, a liquid reservoir for storing a liquid for testing containing at least one kind of microorganism, an opening provided on the lower surface of the liquid reservoir, and covering the opening A chip (sensor chip 100) including a detection unit 140 having a membrane filter 130 provided in this manner is prepared. Then, a series of steps shown on the chip is performed.
Moreover, the method for detecting a microorganism of the present embodiment includes the following steps.
Step (1) A step of removing the liquid for testing containing at least one kind of microorganisms in the liquid reservoir from the lower side of the membrane filter 130 to concentrate the microorganisms on the membrane filter 130 (S100, S102).
Step (2) A step of introducing a liquid medium containing a physiologically active substance into the liquid reservoir and culturing microorganisms floating in the liquid medium (S104).
Step (3) A step of removing the liquid medium from below the membrane filter 130 and collecting the cultured microorganisms on the membrane filter 130 (S106).
Step (4) A step of introducing a reagent for determining the viability of the microorganism into the liquid reservoir (S108).
Step (5) A step of observing microorganisms in the chip (sensor chip 100) (S110).

An outline of a method for detecting a microorganism according to the present embodiment will be described.
In the method for detecting a microorganism of the present embodiment, a series of steps of the method can be performed on the sensor chip 100, particularly the detection unit 140 (S100 to S110). For this reason, according to the present invention, microorganisms can be detected quickly. And according to this invention, the whole detection time can be shortened.
In the present embodiment, the detection unit 140 that cultures microorganisms in a liquid medium containing a physiologically active substance is used as the detection unit 140 to be detected, while the microorganisms are cultured in a liquid medium that does not contain a physiologically active substance. The detection unit 140 is referred to as a reference target detection unit 140. At this time, the action of the physiologically active substance is enhanced in the detection target, while the growth rate of the microorganism is increased in the reference target. Furthermore, such effects can be enhanced by performing shaking culture. For this reason, in the present invention, the difference in the viable cell count ratio between the detection target and the reference target becomes clear, and the sensitivity of microorganisms can be detected with high sensitivity and speed. In addition, after culturing, microorganisms are collected in a very small area, and the microorganisms are fluorescently observed and counted, so a culture method for observing colonies that are a very large group of microorganisms, and the conventional method for detecting turbidity of samples Compared with the method, it is possible to detect a very small change in the number of microorganisms. Thus, in the present invention, the sensitivity of microorganisms can be detected with high sensitivity and speed.

In the present embodiment, for example, an antibiotic can be used as the physiologically active substance. Moreover, as a reagent which discriminate | determines the life and death of microorganisms, a commercially available fluorescent dyeing reagent can be used, for example. When antibiotics are used, the drug sensitivity of the detection target bacteria can be examined. Here, in this embodiment, examples of the microorganism include bacteria and fungi.

Next, a microorganism detection system used in the microorganism detection method of the present embodiment will be described.
In the present embodiment, a microorganism detection system including a liquid feeding mechanism, a suction mechanism, a shaking mechanism, a constant temperature mechanism, a washing mechanism, a moving mechanism, and a detection mechanism is used to perform a series of this process on the sensor chip 100. be able to. For example, in this embodiment, a microorganism detecting system including the concentrating device 160 and the detecting device 190 shown in FIGS. 7 and 9 is used.
In accordance with each process, the concentration device 160 and the detection device 190 are used with the sensor chip 100 set. Thereby, microorganisms can be detected quickly on the sensor chip 100.

First, as a premise of the microorganism detection system, the sensor chip 100 according to the present embodiment will be described.
FIG. 4 shows a sensor chip 100 according to the present embodiment. FIG. 5 is a cross-sectional view taken along the line AA of the sensor chip 100 in FIG. FIG. 6 is a cross-sectional view taken along line BB of the sensor chip 100 in FIG.

The sensor chip 100 includes a liquid reservoir for storing a liquid (sample) to be tested containing at least one type of microorganism, an opening provided on the lower surface of the liquid reservoir, and a membrane filter provided so as to cover the opening 130, and a chip (sensor chip 100) including the detection unit 140 is prepared. In one sensor chip 100, a plurality of detection units 140 are arranged in an array. The number of detection units 140 is not particularly limited. By using a sensor chip array provided with a plurality of detection units 140, it is possible to detect susceptibility for a plurality of types of antibiotics and a plurality of bacterial species in the same step.

The sensor chip 100 can be made of resin, for example. The sensor chip 100 is obtained by, for example, laser molding, injection molding, or the like. The shape of the detection unit 140 in the planar direction of the sensor chip 100 is not particularly limited, but may be a circle, for example. In addition, the cross-sectional shape of the detection unit 140 in the direction perpendicular to the planar direction of the sensor chip 100 is not particularly limited, but at least a part thereof can be tapered. The diameter (effective filter diameter) of the detection unit 140 is not particularly limited, but may be, for example, 0.1 mm or more and 5 mm or less (because it is much smaller than the normal filter diameter, and thus microorganisms are collected in a minute region). The height of the detection unit 140 is determined from the viewpoint of the focal length of the objective lens during fluorescence observation.

Further, the pore diameter of the membrane filter 130 is not particularly limited, but for example, 0.1 μm or more and 1 μm or less. Furthermore, the membrane filter 130 may be coated with a metal such as titanium. Alternatively, an aluminum oxide filter may be used. Thereby, the self-light emission of the membrane filter 130 can be suppressed, and noise at the time of detection can be reduced.

Further, as shown in FIG. 5, the sensor chip 100 includes an upper plate 110 provided with a liquid reservoir cylinder member (liquid reservoir) and a lower plate 120 provided with holes. The upper plate 110 and the lower plate 120 are joined so that the membrane filter 130 is sandwiched therebetween. As the joining means, for example, clamping means for clamping the upper surface of the upper plate 110 and the lower surface of the lower plate 120, means for screwing the upper plate 110 and the lower plate 120, and the like can be used. Alternatively, the injection-molded plastic can be bonded with a solvent. In addition, examples of the bonding means include an adhesive and ultrasonic bonding.

Here, as an example of the clamping means, as shown in FIG. 6, a groove is provided on the inner wall of the chip base 150, and the ends of the upper plate 110 and the lower plate 120 are inserted into the groove. Thereby, an edge part is clamped and the upper board 110 and the lower board 120 are joined. Therefore, the sensor chip 100 is mounted on the chip base 150 in an integrated state. Furthermore, as shown in FIG. 6, when viewed from the direction perpendicular to the planar direction of the sensor chip 100, the opening of the upper plate 110 (inner hole of the cylindrical member), the hole of the membrane filter 130, the lower plate 120, and the chip base It arrange | positions so that the opening part 152 of 150 may overlap.

Next, the concentration apparatus 160 according to the present embodiment will be described. FIG. 7A schematically shows the concentrator 160. FIG. 7B schematically shows a part of the concentrating device 160 shown in FIG. FIG. 8 schematically shows a part of the suction mechanism.
As shown in FIG. 7A, the concentration device 160 includes a culture mechanism 164 and a suction mechanism 162. The culture mechanism 164 includes a heater 180, fins 178, a temperature sensor (not shown), and a shaker 182. The suction mechanism 162 includes a vacuum pump 166, a waste liquid tank 168, a liquid feed pump 172, a cleaning liquid tank 170, a liquid feed tube 184, and a waste liquid tube 186. Further, the concentration device 160 may include a culture medium tank 174 and a fluorescent sample tank 176. Such a concentrating device 160 is provided in one housing. Further, as shown in FIG. 7A, a partition plate 208 is provided between the culture mechanism 164 and the suction mechanism 162 in the housing. In the present embodiment, the culture mechanism 164 is provided on the partition plate 208, but the present invention is not limited to this mode.

In the suction mechanism 162 shown in FIG. 7A, the vacuum pump 166 sucks the liquid such as the sample, the liquid medium, or the fluorescent reagent introduced into the liquid reservoir of the sensor chip 100. These liquids are introduced into the detection unit 140 from the cleaning liquid tank 170, the culture liquid tank 174, the fluorescent sample tank 176, or the like using a pump (not shown). For example, the liquid feeding pump 172 introduces the cleaning liquid in the cleaning liquid tank 170 into the liquid reservoir through the liquid feeding tube 184. Examples of the cleaning liquid include physiological saline and phosphate buffer. In this case, the position of the sensor chip 100 on the shaker 182 moves relative to the liquid feeding tube 184. Thereby, the liquid feeding tube 184 introduces the liquid into the plurality of detection units 140. Moreover, the liquid may be introduced into the liquid reservoir by a pipette or the like.
The sucked liquid passes through the membrane filter 130 from the liquid reservoir, and is removed from the opening 152 of the chip base 150 to the waste liquid tank 168 through the waste liquid tube 186.
In this way, the liquid in the reservoir can be removed by suction from below the membrane filter 130, leaving the microorganisms cultured on the membrane filter 130.

Further, as shown in FIG. 8, the suction mechanism 162 includes a suction plug 188 that is provided below the detection unit 140 and sucks liquid. The pressure of the suction plug 188 is controlled by a first control unit (not shown) in the suction mechanism 162. The control unit detects the pressure of the suction plug 188 and feedback corrects the pressure value of the suction plug 188 according to the detected value. At this time, in the example illustrated in FIG. 8A, the suction plug 188 is provided in each of the detection units 140. The suction plug 188 sucks the liquid in the detection unit 140 individually by the vacuum pump 166 by being separated from each other. In the example shown in FIG. 8B, one suction plug 188 is provided for the plurality of detection units 140. The suction plug 188 simultaneously sucks the liquid in the plurality of detection units 140 by the vacuum pump 166. Alternatively, the chip is moved using a slide mechanism and suction is sequentially performed for each detection unit.

In the culture mechanism 164, the culture temperature is adjusted by the heater 180 and the fins 178. At this time, when the fin 178 is not used, the temperature can be adjusted only by turning the heater 180 on and off. The culture temperature is adjusted according to the type of microorganism, the type of antibiotic, etc. while measuring with a temperature sensor (not shown). The culture temperature can be, for example, from room temperature (about 25 ° C.) to about 45 ° C. Here, the control temperature of the culture mechanism 164 can be, for example, from room temperature (about 25 ° C.) to about 60 ° C. The shaker 182 shakes the chip base 150 on which the sensor chip 100 is placed. For example, the tip base 150 is shaken by an eccentric motor. Thus, the microorganisms in the detection unit 140 of the sensor chip 100 can be shake-cultured in a liquid medium under a constant temperature condition.
At this time, in the concentrating device 160 shown in FIG. 7B, the sensor chip 100 is provided in an internal space (sealed space) constituted by the cover 210 and the partition plate 208 that can be freely opened and closed. When the cover 210 is opened, the upper part of the sensor chip 100 is exposed. For this reason, the operability of the sensor chip 100 is improved. That is, the movement of the sensor chip 100, the shaking of the sensor chip 100, the introduction of the liquid into the sensor chip 100, and the like can be performed simply and efficiently.

As described above, the concentration device 160 performs concentration, culture, washing, and fluorescent staining of microorganisms on the sensor chip 100. That is, the concentrating device 160 first concentrates the microorganisms on the membrane filter 130 of the detection unit 140, and then cultures the microorganisms in a liquid medium and captures them on the membrane filter 130. Thereafter, the microorganisms are washed. And perform fluorescent staining.

Next, the detection device 190 according to the embodiment of the present invention will be described. FIG. 9 schematically shows the detection device 190.
The detection device 190 performs fluorescence observation of microorganisms in the detection unit 140 on the sensor chip 100 that is fluorescently stained by the concentration device 160. The detection device 190 is not particularly limited, and for example, a general-purpose fluorescence microscope can be used.
The detection device 190 includes a moving stage 192, an excitation light source 194, an excitation light optical filter 195 (when the light source is a white light source including many wavelengths, an excitation filter needs to be used), an objective lens 196, a dichroic. A mirror 198, a radiation absorbing optical filter 200, an imaging lens 202, a detection mechanism 204, and a PC 206 are provided.
The moving stage 192 can freely move the chip table 150 on which the sensor chip 100 is placed in the X-axis direction, the Y-axis direction, and the Z-axis direction (three-dimensional direction). Thus, the position and height of the detection unit 140 on the sensor chip 100 are adjusted with respect to the objective lens 196.

Further, in the detection device 190 shown in FIG. 9, the excitation light source 194 emits excitation light. The dichroic mirror 198 splits the excitation light emitted from the excitation light source 194. The objective lens 196 collects the split excitation light. Subsequently, when the condensed excitation light irradiates the detection unit 140 on the sensor chip 100, a microorganism or the like emits fluorescence. The optical filter 200 transmits the fluorescence via the objective lens 196 and the dichroic mirror 198. The imaging lens 202 forms an image of the transmitted fluorescent light flux. The imaging mechanism is converted into electronic data by the detection mechanism 204. As the excitation light source 194, an LED or the like (other laser) can be used. As the detection mechanism 204, a CCD camera (Charge Coupled Device (CCD)) or the like (other CMOS) is used. As a result, image analysis using the PC 206 connected to the CCD camera can also be performed. Also, image data indicating the luminescence of the living microorganisms in each detection unit 140 is obtained. As the detection mechanism 204, an eyepiece may be used instead of the CCD camera. Thereby, visual observation can be performed.

Next, each step of the present embodiment using the microorganism detection system will be described below.
2 and 3 show the procedure of the drug sensitivity detection method according to the embodiment of the present invention.
FIG. 2 shows a detection unit 140 (first detection unit) for a reference target (negative control), and FIG. 3 shows a detection unit 140 (second detection unit) for a detection target. In FIG. 2, a liquid medium 310 containing no antibiotics is used, while in FIG. 3, a liquid medium 320 containing antibiotics is used.

[Step (1)]
First, using the sensor chip 100, the sample 300 (liquid to be tested including microorganisms) is separately introduced into the liquid reservoir in the reference target and detection target detection unit 140 (S100, FIG. 2A). FIG. 3 (a)).
In this step, the same sample 300 is used as the liquid to be detected for the reference object and the detection object. Therefore, the initial concentration of microorganisms in the reference object 300 and the detection object sample 300 is substantially the same. Further, as a premise, the sample 300 includes a target bacterium 340 (microorganism) that is sterilized by an antibiotic added in a later-described step or that inhibits bacterial growth.

The sample 300 is obtained by diffusing microorganisms collected from every place in a solvent. Examples of the collection location include living environments such as water, beverages, foods, livestock animals, plants, and beds, blood, skin, saliva, sputum, wounds, stomach contents, urine, feces, and other human bodies. As the solvent, for example, physiological saline, phosphate buffer, or the like can be used. The sample 300 may be a microorganism that has been cultured in advance by a normal culture method.

The initial concentration of the microorganism in the sample 300 may be very low. For example, the initial concentration can be about 1 / ml to 10E5 / ml.

Subsequently, the sample 300 in the detection unit 140 is removed, and the target bacteria 340 are left on the membrane filter 130 (S102, FIG. 2 (b), FIG. 3 (b)). Thereby, the density | concentration in the liquid of the object microbe 340 is concentrated. At this time, the liquid in the sample 300 is sucked from below the detection unit 140 using the suction mechanism 162 shown in FIG.

[Step (2)]
Subsequently, the liquid medium 310 is introduced into the detection unit 140 to be referenced, and the liquid medium 320 containing antibiotics is introduced into the detection unit 140 to be detected. Then, using the culture mechanism 164 shown in FIG. 7, each sensor chip 100 is shaken with a shaker 182 and the target bacteria 340 in each detection unit 140 is shake-cultured (S104, FIG. 2 (c), FIG. 3 ( c)).
As described above, in the main culturing step, the microorganisms are separated from the membrane filter 130 and float in the liquid medium by an action such as convection of the liquid medium generated when introduced into the detection unit 140. During the main culture process, any microorganisms are suspended in the liquid medium. Further, by shaking, the microorganisms can be suspended and the density of the microorganisms in the liquid medium can be made uniform. By culturing in such a floating state, the growth rate of microorganisms can be increased, and the growth inhibitory action of antibiotics can be increased. Furthermore, by performing shaking culture, oxygen, medium components and growth inhibitory substances necessary for the growth of microorganisms come into contact with the microorganisms efficiently, so that such an effect can be enhanced. Can be obtained with good reproducibility.

Here, in the reference target detection unit 140, the target bacteria 340 grow. As shown in FIG. 2 (c), the propagated microorganism is designated as a proliferating bacterium 370. On the other hand, in the detection unit 140 to be detected, the growth of the target bacteria 340 is inhibited by the bactericidal action of the antibiotic. As shown in FIG. 3C, a microorganism whose growth is inhibited is referred to as a growth-inhibiting bacterium 380.

Antibiotics according to the present embodiment include, for example, penicillins, cephams, β-lactams, aminoglycosides, peptides, tetracyclines, macrolides, new quinolones, chloramphenicol, aminoglycosides, etc. Can be used.
As the liquid medium, for example, an LB medium or a Muller Hinton medium can be used although it depends on the type of microorganism.
Although the culture time depends on the type of microorganism, it is not particularly limited and can be, for example, about 15 minutes or more and 180 minutes or less. The culture temperature is not particularly limited, although it depends on the type of microorganism and the type of antibiotic, and can be about 30 ° C. or higher and 45 ° C. or lower.

[Step (3)]
Subsequently, the liquid medium 310 in the reference target detection unit 140 is removed, and the target bacteria 340 and the proliferating bacteria 370 are left on the membrane filter 130. Further, the liquid medium 320 containing the antibiotic in the detection unit 140 to be detected is removed, and the target bacteria 340 and the growth-inhibiting bacteria 380 are left on the membrane filter 130 (S106, FIG. 2 (d), FIG. 3 (d). )). At this time, using the suction mechanism 162 shown in FIG. 7, the liquid in the liquid medium 310 and the liquid medium 320 containing antibiotics is sucked from below the detection unit 140.
In this way, the target bacteria 340 and the growth bacteria 370, the target bacteria 340, and the growth-inhibiting bacteria 380 are collected on the membrane filters 130 of the reference object and the detection object. In addition, since the cell membrane of the dead cell is weakened, the dead bacteria can be separated by passing through the membrane (membrane filter 130) by controlling the suction force. By such a microorganism collecting step, the microorganism and the liquid medium can be separated from the liquid medium containing the microorganism, and the microorganism (particularly, live bacteria) can be collected in a significantly small area.

[Step (4)]
Subsequently, a reagent (fluorescent reagent 330) for determining whether microorganisms are alive or dead is introduced into the liquid reservoir (S108, FIG. 2 (e), FIG. 3 (e)). As the fluorescent reagent 330, for example, a reagent that fluorescently stains only viable bacteria is used.
The introduction amount of the fluorescent reagent 330 is not particularly limited as long as the microorganisms (target bacteria 340, proliferating bacteria 370, and growth-suppressing bacteria 380) are in contact with the fluorescent reagent 330.

Here, the method for discriminating the viability of microorganisms is not limited to this, and for example, a method of discriminating viable and dead bacteria using a commercially available fluorescent staining reagent can be used. As a method for discriminating between live and dead bacteria, there are (a) a combined fluorescent staining method, and (b) a combined method of a live bacterial staining method and a whole bacterial staining method.
In the case of the method shown in (a), a fluorescent reagent having a different luminescent color is used as a fluorescent staining reagent. Specifically, a composite fluorescent staining reagent containing a green fluorescent SYTO9 dye and a red fluorescent propidium iodide dye can be used. In addition, YOYO-1, 7ADD can be used as a label for dead bacteria, and a fluorescent staining reagent including, for example, Ethidium homodimer-1, FDA, Calcein AM, LDS751, etc. can be used as a label for live bacteria.
In the case of the method shown in (b), a total bacterial staining method such as DAPI staining method, EB staining method, SYBR Green1 staining method, and viable bacterial staining methods such as CTC staining method, CFDA staining method can be used in combination. . For example, viable and dead bacteria are discriminated by double staining of the DAPI staining method and the CFDA staining method.

[Step (5)]
Subsequently, before the fluorescence observation, the inside of each detection unit 140 is washed. For cleaning, for example, a cleaning liquid is introduced into the detection unit 140, and this cleaning liquid is absorbed and removed. Thereafter, the sensor chip 100 is moved from the concentration device 160 to the detection device 190. Then, using the detection device 190, the microorganisms in the detection unit 140 of the sensor chip 100 are fluorescently observed (S110, FIG. 2 (f), FIG. 3 (f)). In this step, for example, fluorescence observation is performed by multiplying the optical magnification.
The fluorescent reagent 330, for example, fluorescently stains only live bacteria 350. Therefore, as shown in FIGS. 2 (f) and 3 (f), in the reference target detection unit 140, the target bacteria 340 and the proliferating bacteria 370 are detected as viable bacteria 350. On the other hand, in the detection unit 140 to be detected, the growth-suppressing bacteria 380 are not detected because they are dead bacteria 360, and only the target bacteria 340 are detected as live bacteria 350. Then, the number of microorganisms (for example, the number of viable bacteria) collected in the minute area is counted, and the ratio of the viable cell count of the detection target 140 to the reference target detection unit 140 is calculated.

Here, the viable cell count ratio obtained by the microorganism detection method of the present embodiment will be described.
In the present embodiment, the viable cell count ratio is a parameter indicating the sensitivity of microorganisms.
The definition of this viable count ratio is shown below.
(I) If the viable cell count ratio is within 1.0 ± α (logarithmic conversion), the detection target microorganism should be a standard that does not show sensitivity to antibiotics (that is, if the microorganism is resistant) The ratio of viable bacteria is about 1 or more).
(Ii) In the range where the viable cell count ratio is smaller than 1.0-α (logarithmic conversion), the detection target microorganism is used as a standard indicating sensitivity to antibiotics.
Here, α is determined by the culture time, culture temperature, antibiotic concentration, and the like. For example, α can be set to 0.5 or less, and further 0.2 or less.

A method for calculating the viable count ratio will be described. In this embodiment, in order to calculate the viable cell count ratio, for example, (1) the viable cell count may be calculated by counting the viable cell count, or (2) the image data indicating the viable cell count is calculated. The ratio of viable bacteria may be calculated by converting the ratio of the fluorescence areas.
(1) A method for calculating the viable cell count ratio from the viable cell count will be described. First, the number of viable bacteria per field of view is counted and multiplied by the opening area (filtration area) of the detection unit 140 / the area per field of view. That is, the viable cell count is calculated by the following formula. Number of viable bacteria = (average number of viable bacteria per visual field) × (filtration area) / area per visual field. Then, the ratio of the viable cell count of the second viable cell count (viable cell count in the detection unit 140 to be detected) to the first viable cell count (viable cell count in the reference target detection unit 140) is calculated.
(2) A method of calculating the viable cell count ratio by converting the ratio of the fluorescence areas will be described. First, fluorescence image data indicating the live bacteria 350 of each detection unit 140 is obtained. Then, the fluorescence area per field of view is calculated and multiplied by the opening area (filtering area) of the detection unit 140 / area per field of view. That is, the fluorescence area is calculated by the following equation. Fluorescence area = (average value of fluorescence area per field) × (filtration area) / area per field. Then, the ratio of the fluorescence area of the second fluorescence area (fluorescence area in the detection unit 140 to be detected) to the first fluorescence area (fluorescence area in the detection unit 140 to be referred to) is calculated, and this fluorescence area ratio is calculated. Convert to the viable count ratio.

As described above, by calculating the ratio of the number of viable bacteria of the detection unit 140 to be detected with respect to the detection unit 140 to be referred to, the sensitivity of the microorganism can be detected with high sensitivity and speed.

Here, the case of using the ratio of the second viable cell count to the first viable cell count after the culturing process (viable cell count ratio) has been described. The sensitivity of microorganisms can be detected quickly. For example, a method for determining sensitivity based on whether or not the difference between the first viable cell count and the second viable cell count after the culturing step is a predetermined value or more, or a fluctuation value of the first viable cell count (before the culturing step) A method for judging sensitivity based on whether or not the fluctuation value (the same as the left) of the second viable cell count relative to the viable cell count and the viable cell count after the culturing step is equal to or less than a predetermined value; Examples include a method of judging sensitivity by comparing the fluctuation tendency of the fluctuation value of the viable cell count and the fluctuation value of the second viable cell count.

The effect of this embodiment will be described.
In the microorganism detection method of the present embodiment, a series of steps such as concentration, culture, and fluorescence observation can be performed on the sensor chip 100. For this reason, microorganisms can be detected quickly. Therefore, the entire detection time can be shortened.
In addition, according to the present embodiment, the concentration process and the process of collecting the microorganisms in the micro area and performing the fluorescence observation are performed, so that the change in the number of micro microorganisms can be detected very accurately due to the concentration effect. .

In the present embodiment, in the detection unit 140 to be detected, the microorganism is shake-cultured in a liquid medium containing an antibiotic. Thereby, the growth inhibitory action of the antibiotic is strengthened, and the number of viable bacteria in the reference target detection unit 140 is reduced. Therefore, in the detection unit 140 to be detected, the difference in the viable cell count ratio before and after the culture becomes clear, and microorganisms that are sensitive to antibiotics can be rapidly detected.
Further, in the reference target detection unit 140, the microorganism is shake-cultured in a liquid medium not containing antibiotics. Thereby, the growth rate of microorganisms is increased, and the number of viable bacteria in the reference target detection unit 140 is increased. Therefore, after culturing, the difference in the viable cell count ratio between the detection target and the reference target becomes clear, and the sensitivity of the microorganism can be detected with higher sensitivity and speed.

Thus, on the premise of the same sample (liquid containing microorganisms), the viable cell count ratio between the first viable cell count under the condition not containing antibiotics and the second viable cell count under conditions containing antibiotics By calculating, the sensitivity of the microorganism to the antibiotic can be detected with high sensitivity.

In the present embodiment, the microorganisms are concentrated on the membrane filter 130 in a minute region before culturing. For this reason, even if the concentration of the microorganism in the sample is very low, the sensitivity of the microorganism can be detected. Furthermore, the difference in the viable cell count ratio becomes clear by the culturing process of the present embodiment, and the sensitivity of the microorganism can be detected with high sensitivity. Therefore, the sensitivity of the microorganism can be detected without depending on the concentration of the microorganism in the sample. For example, the microorganisms can be detected by the detection device 190 when the initial concentration of the microorganisms in the sample is about 10 cells / ml or more, more preferably about 1 cell / ml or more.

In this embodiment, it is possible to integrate culture, concentration, and fluorescence observation on the sensor chip 100 using the concentration device 160 and the detection device 190. Therefore, in the present embodiment, it is possible to simplify the process, increase the efficiency of work, and reduce the risk of contamination and infection. In addition, since all processes can be performed in the sensor chip 100, there is little risk of contamination and the safety of the experimenter can be achieved.

Also, in the drug sensitivity detection method of the present embodiment, microorganisms that have come into contact with antibiotics are collected, and by directly detecting the death or death due to the bactericidal action of the collected microorganisms, the drug sensitivity of the microorganisms can be quickly examined. can do. Compared with the culture method, the PCR method, and the like, the number of sample processing steps required for the inspection is small, so it is very simple. Furthermore, since the amount of reagents used is small compared to other inspection methods other than the culture method, it is possible to keep running costs low. This method for detecting drug sensitivity can also be applied to detection of nosocomial infection-causing bacteria, diagnosis of infectious diseases, rapid test for antibacterial agents, and the like.

(Second Embodiment)
The microorganism detection method of the second embodiment will be described below.
The microorganism detection method of the second embodiment is a method of calculating the minimum inhibitory concentration (MIC). That is, the minimum growth inhibitory concentration (MIC) is calculated from the result of the data indicating the viable cell count ratio obtained by the microorganism detection method of the first embodiment.

In the microorganism detection method of the present embodiment, in the step of concentrating microorganisms using a chip (sensor chip 100) including a plurality of first detection unit groups including a first detection unit and a second detection unit, In the step of introducing the same liquid to be detected separately into the detection unit and the second detection unit and culturing the microorganism, the physiologically active substance (first In the step of observing microorganisms with different concentrations of (antibiotics), the ratio of viable cell counts of the second viable cell count to the first viable cell count corresponding to the concentration of the physiologically active substance (first antibiotic) The step of calculating is included.
At this time, the first detection unit represents the reference target detection unit 140, and the second detection unit represents the detection target detection unit 140. The first viable cell count represents the viable cell count in the first detection unit, and the second viable cell count represents the viable cell count in the second detection unit.

Furthermore, the microorganism detection method of the present embodiment uses a chip (sensor chip 100) further including a third detection unit, and in the step of concentrating microorganisms, the first detection unit, the second detection unit, and the third In the step of introducing the same liquid for testing separately into the detection unit and culturing the microorganism, the third detection unit is different from the second detection unit in the third detection unit (second antibiotic). In the step of culturing microorganisms floating in the liquid medium and observing the microorganisms, and the first viable count of microorganisms in the first detector and the first Measuring a third viable cell count of the microorganism in the three detection units, and calculating a ratio of the viable cell count of the third viable cell count to the first viable cell count.
At this time, the third detection unit represents a detection unit 140 to be detected, which is different from the second detection unit. Moreover, the 3rd viable cell count represents the viable cell count in the 3rd detection part. In addition, a second detection unit group includes the first detection unit, the second detection unit, and the third detection unit.

In the present embodiment, a sensor chip 100 in which the number of vertical and horizontal detection units 140 shown in FIG. 4 is 3 × 6 can be used as a chip including a plurality of detection units.
In the detection unit 140 in the direction of arrows BB shown in FIG. 4, a liquid medium not containing an antibiotic, a liquid medium containing a first antibiotic, and a liquid medium containing a second antibiotic are sequentially introduced. Further, in the detection unit 140 in the direction of arrows AA shown in FIG. 4, the concentration of the first antibiotic and the concentration of the second antibiotic are set to six different concentrations.
In this way, for the two types of first antibiotic and second antibiotic, the concentration gradually decreases from the first concentration to the sixth concentration (from the first concentration to the sixth concentration). At this time, the interval of each concentration can be selected as appropriate.) A plurality of viable cell count data can be obtained.

Subsequently, in the same manner as in the first embodiment, the first viable cell number under the condition not containing the antibiotic and the second viable cell number under the condition containing the first antibiotic are the first The viable cell count ratio is calculated. Further, a first viable cell count ratio corresponding to the first concentration to the sixth concentration is calculated. These viable count ratios are premised on (samples with the same initial concentration of microorganisms in the sample).

Subsequently, in order to calculate the minimum growth inhibitory concentration (MIC), the presence or absence of the susceptibility of the microorganism to the first antibiotic is determined from the result of the data indicating the obtained viable cell count ratio.
For example, in the first viable cell count ratio, the first concentration to the fourth concentration are reference values indicating sensitivity, while the fifth concentration and the sixth concentration are reference values indicating no sensitivity. It is assumed that In this case, the first MIC of the microorganism for the first antibiotic can be determined as the fourth concentration. Further, the first viable cell count ratio is calculated by varying the concentration between the fourth concentration and the fifth concentration, and the first MIC is calculated more accurately from the result of the presence or absence of sensitivity. be able to.

Further, in addition to the above-mentioned first viable cell count ratio, a second viable cell count of the first viable cell count under the condition containing no antibiotic and the third viable cell count under the condition containing the second antibiotic. The viable cell count ratio is calculated. Further, the second viable cell count ratio corresponding to the first concentration to the sixth concentration is calculated. From the result of the second viable cell count ratio, the second MIC of the microorganism with respect to the second antibiotic can be calculated. Here, these viable count ratios are premised on (samples having the same initial concentration of microorganisms in the sample).
As described above, according to the second embodiment, two types of MICs can be calculated simultaneously. That is, the first MIC of the microorganism for the first antibiotic and the second MIC of the microorganism for the second antibiotic can be calculated.

As described above, by performing the microorganism detection method of the present embodiment using the sensor chip 100 including the plurality of detection units 140, the MIC for antibiotics can be determined quickly and with high sensitivity. Moreover, according to this Embodiment, MIC with respect to multiple types of antibiotics can be detected simultaneously.

In addition, when the method for examining drug sensitivity according to the present embodiment is used, if the type of microorganism is unknown, the presence or absence of drug-resistant bacteria can be determined for the prepared sample, and the MIC can also be examined. . Even when the type of microorganism is unknown, the bacterial species of the unknown microorganism can be roughly predicted by using a sensitive reaction of a plurality of types of antibiotics or using a selective medium. On the other hand, when the type of microorganism is known, the drug sensitivity and MIC of the antibiotic can be examined as described above.

Next, data is generated by evaluating the presence or absence of resistance of the microorganism to the physiologically active substance (antibiotic) from the obtained MIC result by the microorganism detection method of the present embodiment.
First, the minimum growth inhibitory concentration (MIC) is calculated from the result of data indicating the viable cell count ratio by the microorganism detection method of the second embodiment. When the minimum growth inhibitory concentration of the physiologically active substance (antibiotic) is equal to or higher than a predetermined concentration, it is determined that the microorganism exhibits resistance to the physiologically active substance (antibiotic). At this time, data indicating the presence or absence of resistance of the microorganism to the physiologically active substance (antibiotic) is generated.

Here, the criteria of the minimum growth inhibitory concentration (MIC) for judging to show tolerance are shown in the following (1) and (2).
(1) When MIC is R [μg / ml] or more, it is determined that the microorganism is resistant to the antibiotic.
(2) If the MIC is less than R [μg / ml], it is determined that the microorganism is not resistant to the antibiotic.

In addition, when it is R [μg / ml] or more, the degree of resistance may be evaluated for each stage. For example, when R is 1 or more and less than 10 [μg / ml], it is evaluated as having a low degree of resistance (LR (Low Resistant)), and when R is 10 or more and less than 25 [μg / ml] Is evaluated as having moderate resistance (MR (Middle Resistant)), and when R is 25 [μg / ml], it is evaluated as having high resistance (HR (High Resistant)).

Furthermore, in the present embodiment, it is possible to select a physiologically active substance (antibiotic) to which the microorganism does not exhibit resistance from data indicating the presence or absence of such microorganism resistance.
In practice, such antibiotics may be used even if the microorganisms are resistant. In this case, in the present embodiment, an antibiotic that exhibits low to moderate resistance can be selected.

As described above, in the present embodiment, test results on the sensitivity, MIC, and presence / absence of resistance of microorganisms can be obtained simply and quickly with respect to a plurality of types of antibiotics. For this reason, it is possible to quickly and accurately determine the antibiotic administration at the clinical site. Therefore, the risk of nosocomial infections due to postoperative infections, sepsis, drug resistant bacteria (MRSA, etc.) is reduced.

From the viewpoint of such a medical environment, it is preferable that the number of detection units 140 of the sensor chip 100 is large. For example, by providing 92 detection units 140 (12 (11 types of antibiotics + negative control) × 8 (8 types of concentrations)), the sensitivity of multiple types of antibiotics can be increased simultaneously and quickly. Sensitivity can be detected.

In the present embodiment, the microorganisms are shake-cultured in a liquid medium. Therefore, as a result of the concentration of the antibiotic in the liquid medium being uniform, the effect of the antibiotic on the microorganism in the detection target can be obtained with good reproducibility. For this reason, according to the present embodiment, the variation in the viable cell count and the viable cell count ratio is reduced, and the results of microbial sensitivity, MIC, etc. are obtained with good reproducibility.

Generally, in the case of postoperative infection or nosocomial infection, an appropriate antibiotic is selected by a drug sensitivity test using a culture method, and this antibiotic is treated in the patient. However, since the culture method usually takes more than one day, it is difficult to quickly select antibiotics. For this reason, from the viewpoint of rapidity, if various types of antibiotics are used, it causes the appearance of new resistant bacteria and causes a burden on the patient such as side effects.

In contrast, in the present embodiment, it is possible to detect the sensitivity of a plurality of types of antibiotics simultaneously and rapidly with high sensitivity. Therefore, when nosocomial infections due to drug-resistant bacteria occur, the same sensitivity test can be performed quickly. In addition, after surgery, patients are exposed to the threat of postoperative infections, leading to fatal sepsis, and clinicians can find and treat the most effective antibiotics as soon as possible. it can. The microorganism detection method of the present embodiment can also be used for applications in which a clinician wants to urgently conduct a sensitivity test for one patient.

(Third embodiment)
A screening method for a substance exhibiting antibacterial activity using the microorganism detection method of the present embodiment will be described. This rapid screening can be used for pharmaceuticals or production of antibacterial substances.
In the third embodiment, an unknown substance whose presence or absence of antibacterial activity has not been confirmed is used as the physiologically active substance. Examples of the unknown substance include actinomycete extracts, known compounds, derivatives of compounds exhibiting antibacterial activity, proteins, and the like.

The method for examining the antibacterial activity of an unknown substance according to the third embodiment includes the following steps. First, a sample containing at least one known microorganism is introduced into the plurality of detection units 140. Subsequently, in the step of culturing the microorganism, in the case of only the liquid medium as the negative control, in the case of the liquid medium containing the known antibiotic as the positive control, the three liquid mediums in the case of the liquid medium containing the unknown substance as the detection target The microorganisms are introduced into each of the three detectors 140 and cultured with shaking. Subsequently, a third viable cell ratio of the positive control to the negative control and a fourth viable cell ratio of the detection target to the negative control are calculated.
At this time, the reference indicating the antibacterial activity is the third viable count ratio. And the presence or absence of the antibacterial activity of an unknown substance can be judged with the reference | standard shown below.
(1) When the fourth viable cell number ratio is about the same as or greater than the third viable cell number ratio, the unknown substance has antibacterial activity.
(2) When the fourth viable cell count ratio is smaller than the third viable cell count ratio, the unknown substance has no antibacterial activity.

Moreover, the detection result obtained between the known antibiotic and the unknown substance is compared, and data indicating the bactericidal properties of the unknown substance is generated. Thus, the bactericidal action Y ¹ of the known antibiotics R ¹ for a known microorganism D ^1, an unknown substance believed to have bactericidal action Y ¹ homogeneous can be screened. Further, if the known microorganism D ² is different, the known antibiotic R ¹ may have a bactericidal action Y ² . In this case, the unknown material is screened as having a bactericidal action Y ² homogeneous.

This example shows a specific example of detection of susceptibility of microorganisms and MIC (minimum growth inhibitory concentration) of microorganisms of the present embodiment.
Example 1
In Example 1, as shown in FIGS. 10 to 12, the sensitivity of microorganisms was detected and the concentration of detectable microorganisms was measured.
Component to be detected: Escherichia coli Detection method: Fluorescence microscope observation Fluorescent staining: Compound fluorescent staining reagent In Example 1, the sensor chip 100 shown in FIG. 4, the concentrator 160 shown in FIG. 7, and the detector 190 shown in FIG. 9 are used. It was.
Viable count: Viable count = (average number of viable counts per field) x (filter area) / (area of 1 field)
Detection accuracy: The correlation coefficient between the filter method (pieces / ml) and the pour plate method (CFU / ml) of the present invention is 0.95.
Reservoir capacity: 1 ml
Filter diameter: 3mm
Temperature: 37 ° C (± 1 ° C)
Objective lens: × 10

First, six test liquid samples having different concentrations of Escherichia coli are prepared. Then, liquid samples for test having the same initial concentration of Escherichia coli are divided and introduced into the detection units 140 of the sensor chip 100 to be referenced and the detection target. Depending on the initial concentrations of the six types of E. coli, six sets of reference objects and detection objects are prepared. Subsequently, Escherichia coli in the sample was concentrated on the membrane filter 130 using the suction mechanism 162 of the concentrator 160. Subsequently, 800 μl of a liquid medium and 100 μl of physiological saline were introduced into the reference target detection unit 140. On the other hand, 800 μl of liquid medium and 100 μl of antibiotics (cephalum antibiotic preparation “pansporin” manufactured by Takeda Pharmaceutical Company Limited, concentration: 10 g / L) were introduced into the detection unit 140 to be detected. Subsequently, using the culture mechanism 164 of the concentrator 160, the Escherichia coli in the sensor chip 100 was shaken and cultured at a culture temperature of 37 ° C., changing the culture time conditions to 0 min, 30 min, and 60 min.
Subsequently, Escherichia coli was captured on the membrane filter 130 using the suction mechanism 162. Subsequently, a staining solution (composite fluorescent staining reagent) was introduced into each detection unit 140.
Here, LIVE / DEAD BacLight (trademark) Bacterial Viability Kits (manufactured by Invitrogen) was used as a composite fluorescent staining reagent. The composite fluorescent staining reagent contains a green fluorescent SYTO9 dye and a red fluorescent propidium iodide dye, which are different in membrane permeability. SYTO9 permeates the membranes of both live and dead bacteria and stains green. On the other hand, propidium iodide permeates only the membrane damaged by dead bacteria and stains red. Also, if both dyes are present in the bacteria, the fluorescence of SYTO9 is attenuated. Therefore, if the bacteria are alive, the composite fluorescent reagent fluoresces green, and if the bacteria are dead, the composite fluorescent reagent fluoresces red. The molecular chemical reaction took 10 minutes.
Thereafter, using the suction mechanism 162, a cleaning liquid (PBS) was introduced to clean the inside of the detection unit 140. Thereafter, using the detection device 190, each detection unit 140 of the sensor chip 100 was observed with a fluorescence microscope. At this time, the viable cell count of Escherichia coli in each detection unit 140 was calculated, and the viable cell count ratio between the reference object and the detection object was calculated from the result.

FIG. 10 shows the time change of the viable cell count of the reference object (without antibiotics). The horizontal axis represents time (min), and the vertical axis represents the viable count (logarithm) of E. coli measured by the filter method. Here, as for the initial concentration of E. coli (concentration in the sample), the black circle represents 9.4 × 10E4 (CFU / ml) and the black triangle represents 2.0 × 10E4 (CFU) when measured by the pour plate method. Black diamond represents 3.5 × 10E3 (CFU / ml), white circle represents 1.2 × 10E3 (CFU / ml), white triangle 6.2 × 10E2 (CFU / ml) The white square represents 6.0 × 10E1 (CFU / ml).
FIG. 11 shows the time change of the viable cell count of the detection target (including antibiotics). The symbols in the figure are the same as those in FIG.
FIG. 12 shows the time change of the viable cell count ratio between the reference object and the detection object. The symbols in the figure are the same as those in FIG. 10 except that the vertical axis in the figure represents the viable cell count ratio.

Thus, at all initial concentrations, the culture time is about 30 min and the viable cell count ratio is considerably smaller than 1. Therefore, according to the microorganism detection method of the present invention, it was found that the sensitivity of microorganisms can be detected with high sensitivity when the initial concentration of the sample is at least about 1 / ml to 10E5 / ml. Moreover, according to the microorganism detection method of the present invention, it has been found that the susceptibility of microorganisms can be rapidly detected in a culture time of about 30 to 60 minutes.

(Example 2)
In Example 2, as shown in FIG. 13 to FIG. 15, the MIC of the microorganism was detected.
Example 2 was carried out in the same manner as in Example 1 except that the conditions in which the concentration of antibiotics was changed in place of the conditions in which the initial concentration of Escherichia coli in the sample in Example 1 was changed.

FIG. 13 shows the change over time of the viable cell count of the reference object (without antibiotics). The horizontal axis represents time (min), and the vertical axis represents the viable count (logarithm) of E. coli measured by the filter method. Here, the concentrations of antibiotics to be detected are black circles representing 10E3 (μg / ml), black triangles representing 10E2 (μg / ml), black squares representing 10E1 (μg / ml), and black diamonds. 10E0 (μg / ml) is represented, white circles represent 10E-1 (μg / ml), and white triangles represent 10E-2 (μg / ml).
FIG. 14 shows the change over time of the viable cell count of the detection target (including antibiotics). The symbols in the figure are the same as those in FIG.
FIG. 15 shows the time change of the viable cell count ratio between the reference object and the detection object. The symbols in the figure are the same as in FIG. 13 except that the vertical axis in the figure represents the viable cell count ratio.
FIG. 16 is a diagram showing the observation results in the reference target and detection target detection unit 140 where the antibiotic concentration is 10E1 (μg / ml).

From the results shown in FIG. 15, when the antibiotic concentrations are 10E-1 and 10E-2 (μg / ml), the viable cell count ratio is approximately 1. On the other hand, when the antibiotic concentration is 10E0 (μg / ml) or more, the viable count ratio is considerably smaller than 1. Therefore, it was found that the MIC of Escherichia coli for pansporin is about 10E0 (μg / ml) or less. In this range, it was also found that accurate MIC was detected by detecting again with the concentration of antibiotics. Further, it was found that the MIC of microorganisms can be rapidly detected because the difference in the viable cell count ratio becomes remarkable when the culture time is about 30 to 60 minutes.

(Comparative example)
The MIC of E. coli against pansporin was measured by a conventional dilution method. A 2 × dilution series of the test drug is prepared using LB liquid medium, and then inoculated with 10E5 or 10E6 (cfu / ml) bacteria (E. coli) and cultured at 37 ° C. overnight. Thereafter, the minimum concentration of a drug (pansporin) in which bacterial growth inhibition was observed was defined as MIC.
The result is shown in FIG. The MIC was 310 (ng / ml) for the first time and 630 (ng / ml) for the second time. The MIC by this dilution method was found to be almost the same as the result according to the present invention. However, in the conventional dilution method, it took 18 hours or more from sample preparation to determination of MIC. On the other hand, in the present invention, the time required for the sample preparation step and the fluorescence observation step is about 1.5 hours before the MIC is determined.

(Reference example)
In the reference example, the liquid medium of Example 1 was not used for culture, but pansporin was introduced into the sample, followed by fluorescence observation. As a result, there were variations in the results such as the viable cell count and the viable cell count ratio. Moreover, in order to obtain the viable cell count ratio showing sensitivity, it was found that the time for introducing pansporin into the sample required at least 60 minutes.
On the other hand, since the microorganism detection method according to the present invention includes a culture process using a liquid medium, the difference in the viable cell count ratio between the reference object and the detection object becomes clear, and the microorganism can be rapidly and highly sensitively. It was possible to detect the sensitivity.

The embodiments of the present invention have been described above with reference to the drawings, but these are exemplifications of the present invention, and various configurations other than those described above can be adopted.
For example, the suction mechanism 162 may adjust the water level of the liquid in the detection unit 140 so that microorganisms on the membrane filter 130 of the sensor chip 100 are not dried. In addition, the suction mechanism 162 may add a liquid (a liquid medium, a sample, a cleaning solution, or the like) at a predetermined interval so that the microorganisms are not dried.

Further, the sensor chip 100 may be made of a heat resistant material. Thereby, the sensor chip 100 can be repeatedly used by performing autoclave or the like on the sensor chip 100. The sensor chip 100 may be disposable from the viewpoint of safety.

Further, an O-ring or the like may be provided between the membrane filter 130 and the lower plate 120. Thereby, the liquid leakage prevention function can be further improved. Alternatively, the membrane filter and the chip material may be directly bonded by an adhesive or ultrasonic bonding. Moreover, as shown in FIG. 5, the cross-sectional shape of the detection unit 140 is tapered. For this reason, microorganisms are efficiently collected on the membrane filter 130 in the lower surface opening portion of the detection unit 140.

Further, the physiologically active substance according to the present embodiment is not limited to the above-described antibiotics or screening samples, and may be, for example, phages. By calculating the viable cell count ratio due to lysis of microorganisms in contact with the phage, the species of microorganisms can be quickly identified. The detection method of specific bacteria using phage can be applied to food hygiene inspection, environmental hygiene analysis, infectious disease diagnosis and the like.
This application claims priority based on Japanese Patent Application No. 2009-210759 filed on Sep. 11, 2009, the entire disclosure of which is incorporated herein.

Claims (9)

1. A reservoir for storing a liquid for testing containing at least one type of microorganism;
   An opening provided on the lower surface of the liquid reservoir,
   Prepare a chip including a detection unit having a membrane filter provided so as to cover the opening,
   Removing the test liquid in the liquid reservoir from below the membrane filter, and concentrating the microorganisms on the membrane filter;
   Introducing a liquid medium containing a physiologically active substance into the liquid reservoir, and culturing the microorganisms floating in the liquid medium;
   Removing the liquid medium from below the membrane filter and collecting the microorganisms on the membrane filter;
   Introducing a reagent for determining the life and death of the microorganism into the liquid reservoir;
   Observing the microorganism in the chip.
2. The microorganism detection method according to claim 1, wherein the chip including a plurality of the detection units is used.
3. In the step of concentrating the microorganism, the same liquid for testing is introduced separately into the first detection unit and the second detection unit,
   In the step of culturing the microorganism, the first detection unit introduces a liquid medium containing no physiologically active substance into the liquid reservoir, and the second detection unit includes a liquid medium containing the physiologically active substance. Is introduced into the liquid reservoir, and the microorganisms suspended in the liquid medium are cultured,
   In the step of observing the microorganism, the method further includes calculating a first viable cell count of the microorganism in the first detection unit and a second viable cell count of the microorganism in the second detection unit. The microorganism detection method according to claim 2.
4. The method for detecting a microorganism according to any one of claims 1 to 3, wherein the physiologically active substance is an antibiotic.
5. Using the chip including a plurality of first detection unit groups including the first detection unit and the second detection unit,
   In the step of culturing the microorganism, the concentration of the physiologically active substance in the liquid medium is varied between the first detection unit groups,
   The step of observing the microorganism further includes a step of calculating a viable cell count ratio of the second viable cell count to the first viable cell count according to the concentration of the physiologically active substance. 4. The microorganism detection method according to 4.
6. Using the chip further comprising the third detection unit,
   In the step of concentrating the microorganisms, the same liquid for testing is introduced separately into the first detection unit, the second detection unit, and the third detection unit,
   In the step of culturing the microorganism, the third detection unit introduces a liquid medium containing a different type of physiologically active substance from the second detection unit into the liquid reservoir, and floats in the liquid medium. Cultivating said microorganisms,
   In the step of observing the microorganism, the first viable count of the microorganism in the first detection unit and the third viable count of the microorganism in the third detection unit are measured, and the first The microorganism detection method according to any one of claims 3 to 5, further comprising a step of calculating a viable cell count ratio of the third viable cell count to a viable cell count.
7. The method for detecting a microorganism according to claim 5 or 6 is a method for calculating a minimum inhibitory concentration (MIC) from a result of data indicating the viable cell count ratio,
   When the minimum growth inhibitory concentration of the physiologically active substance is not less than a predetermined concentration, it is determined that the microorganism is resistant to the physiologically active substance, and whether or not the microorganism is resistant to the physiologically active substance is determined. A method for detecting microorganisms that produces data to be shown.
8. The microorganism detection method according to claim 7, further comprising a step of selecting the physiologically active substance that the microorganism does not exhibit resistance from data indicating whether the microorganism is resistant to the physiologically active substance.
9. The microorganism detection method according to any one of claims 1 to 8, wherein in the step of culturing the microorganism, the microorganism is cultured while the chip is shaken.

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