KR102384408B1 - Microorganism population counting method and system for the same - Google Patents

Abstract

An embodiment of the present invention provides a sample arrangement unit having a plurality of divided cells each containing a culture material, distributing a sample to be measured to the plurality of divided cells, sequentially to the plurality of divided cells irradiating a wave, sequentially detecting individual wave information emitted from samples accommodated in each divided cell in connection with the sequentially irradiated wave, the presence of microorganisms in each divided cell using the individual wave information It provides a method for counting the number of microorganisms, comprising the steps of determining the presence or absence and calculating the number of microorganisms in the sample by using the number of divided cells in which the microorganisms are present.

Description

Microorganism population counting method and system for the same}

Embodiments of the present invention relate to a method for counting microbial populations and a system for counting microbial populations.

In the field of the food industry, unintentional microorganisms are frequently generated in the manufacturing process, which is often a problem. In order to check whether such microorganisms proliferate, conventionally, a culture-type counting method using a medium has been used as a cell detection system. For example, as a microorganism counting method, the method of counting the number of colonies of the microorganisms propagated using an agar medium is used. On the other hand, instead of the method of visually counting the number of colonies generated on an agar medium, etc., a method of counting the number of colonies by data processing the image data of the medium to be counted recently photographed using a CCD camera or the like has also been proposed.

However, the above-described counting methods do not directly count the number of microorganisms, and have no choice but to count by culturing them in a colony state that can be confirmed with the naked eye.

In order to solve the above problems and/or limitations, an object of the present invention is to provide a method for counting the number of microorganisms and a system for counting the number of microorganisms that can reduce the counting time using a chaotic wave sensor.

An embodiment of the present invention provides a sample arrangement unit having a plurality of divided cells each containing a culture material, distributing a sample to be measured to the plurality of divided cells, sequentially to the plurality of divided cells irradiating a wave, sequentially detecting individual wave information emitted from samples accommodated in each divided cell in connection with the sequentially irradiated wave, the presence of microorganisms in each divided cell using the individual wave information It provides a method for counting the number of microorganisms, comprising the steps of determining the presence or absence and calculating the number of microorganisms in the sample by using the number of divided cells in which the microorganisms are present.

In an embodiment of the present invention, the individual wave information may be information obtained by detecting a laser speckle generated by multiple scattering from a sample accommodated in each of the plurality of divided cells at each preset time point. there is.

In one embodiment of the present invention, the determining of the presence or absence of the microorganism includes obtaining a temporal correlation of the detected laser speckle using the detected laser speckle, and It may be a step of determining the presence or absence of microorganisms in the corresponding divided cell based on the time correlation.

In an embodiment of the present invention, the individual wave information includes first image information of the laser speckle detected at a first point in time among laser speckles emitted from the divided cell, and the laser spec detected at a second point in time. It includes the second image information of the cleavage and the third image information of the laser speckle detected at a third time point, wherein the first to the third time points are different from each other, and to determine the presence or absence of the microorganism In the step, the presence or absence of the microorganism may be determined using a difference between the first image information and the third image information.

In an embodiment of the present invention, the plurality of divided cells are arranged in a matrix form, and the number of the plurality of divided cells may be greater than the expected number of microorganisms included in the sample.

An embodiment of the present invention, each containing a culture material, a sample arrangement unit having a plurality of divided cells for distributing and accommodating a sample to be measured, a wave source for sequentially irradiating waves to the plurality of divided cells, A detection unit for sequentially detecting individual wave information emitted from a sample accommodated in each divided cell in connection with the sequentially irradiated wave and the detected individual wave information to determine the presence or absence of microorganisms in each divided cell, It provides a system for counting the number of microorganisms, including a control unit for calculating the number of microorganisms in the sample by using the number of divided cells in which the microorganisms are present.

In an embodiment of the present invention, the individual wave information may be information obtained by detecting a laser speckle generated by multiple scattering from a sample accommodated in each of the plurality of divided cells at each preset time point. there is.

In one embodiment of the present invention, the control unit obtains a temporal correlation of the detected laser speckle using the fraudulently detected laser speckle, and based on the acquired temporal correlation, microorganisms in the divided cell existence can be determined.

In an embodiment of the present invention, the individual wave information includes first image information of the laser speckle detected at a first point in time among laser speckles emitted from the divided cell, and the laser spec detected at a second point in time. second image information of the image and third image information of the laser speckle detected at a third viewpoint, wherein the first to the third viewpoints are different viewpoints, and the control unit includes the first image information to the third image information may be used to determine the presence or absence of the microorganism.

In an embodiment of the present invention, the plurality of divided cells are arranged in a matrix form, and the number of the plurality of divided cells may be greater than the expected number of microorganisms included in the sample.

Other aspects, features and advantages other than those described above will become apparent from the following drawings, claims, and detailed description of the invention.

The microbial population counting system and the microbial population counting method according to embodiments of the present invention can accurately detect microorganisms in units of one population by using a chaotic wave sensor. Through this, embodiments of the present invention detect and count the presence or absence of microorganisms in each of the plurality of divided cells, thereby rapidly calculating the number of microorganisms included in the entire sample. The microbial population counting system and the microbial population counting method according to embodiments of the present invention can detect a microorganism without culturing a colony as a bar, and thus the incubation time is significantly reduced, thereby shortening the time for counting the population.

1 is a conceptual diagram schematically illustrating a system for counting the number of microorganisms according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view taken along line III-III of FIG. 1 .
3 is a flowchart sequentially illustrating a method for counting the number of microorganisms according to an embodiment of the present invention.
4 and 5 are diagrams for explaining a method of counting the number of microorganisms.
6 is a conceptual diagram schematically illustrating a microbial population counting system according to another embodiment of the present invention.
7 is a conceptual diagram schematically illustrating a sample arrangement unit according to another embodiment of the present invention.
8 is a view for explaining the principle of the chaos wave sensor according to an embodiment of the present invention.
9 and 10 are conceptual views schematically illustrating a microbial individual counting system according to another embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the following embodiments will be described in detail with reference to the accompanying drawings, and when described with reference to the drawings, the same or corresponding components are given the same reference numerals, and the overlapping description thereof will be omitted.

Since the present embodiments can apply various transformations, specific embodiments are illustrated in the drawings and described in detail in the detailed description. Effects and features of the present embodiments, and a method of achieving them will become clear with reference to the details described later in conjunction with the drawings. However, the present embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

In the following embodiments, terms such as first, second, etc. are used for the purpose of distinguishing one component from other components without limiting meaning.

In the following examples, the singular expression includes the plural expression unless the context clearly dictates otherwise.

In the following embodiments, terms such as include or have means that the features or components described in the specification are present, and the possibility that one or more other features or components will be added is not excluded in advance.

In the following embodiments, when it is said that a part, such as a unit, region, or component, is on or on another part, not only when it is directly on the other part, but also other units, regions, components, etc. are interposed therebetween. cases are included.

In the following examples, terms such as connect or couple do not necessarily mean direct and/or fixed connection or coupling of two members unless the context clearly indicates otherwise, and does not necessarily mean that another member is interposed between two members. It's not about exclusion.

It means that a feature or element described in the specification is present, and does not preclude the possibility that one or more other features or elements may be added.

In the drawings, the size of the components may be exaggerated or reduced for convenience of description. For example, since the size and thickness of each component shown in the drawings are arbitrarily indicated for convenience of description, the following embodiment is not necessarily limited to the illustrated bar.

1 is a conceptual diagram schematically illustrating a system for counting the number of microorganisms according to an embodiment of the present invention. FIG. 2 is a cross-sectional view taken along line III-III of FIG. 1 .

1 to 2 , the system 10 for counting the number of microorganisms according to an embodiment of the present invention includes a sample arrangement unit 100 , a wave source 200 , a detection unit 300 , and a control unit 400 . can do.

As used herein, the term “microorganism” refers to a prokaryotic or eukaryotic microorganism having the ability to produce a useful target substance such as L-amino acid. For example, microorganisms having an increased intracellular ATP concentration include Escherichia sp., Erwinia sp., Serratia sp., Providencia sp. ), Corynebacteria sp., Pseudomonas sp., Leptospira, Salmonellar sp., Brevibacteria sp., Hypomononas sp.), Chromobacterium sp., Norcardia or a microorganism belonging to a fungus or yeast. Specifically, the microorganism may be a microorganism of the genus Escherichia.

Or, the microorganism is Staphylococcus (Staphylococcus), coagulase-negative Staph Coagulase (staph Coagulase negative), Staphylococcus aureus (Staph. aureus), Streptococcus spp. Streptococcus viridans group), Enterococcus spp., Corynebacterium spp., Aerococcus spp., Micrococcus spp., Peptostreptococcus spp. , Lactococcus spp., Leuconostoc spp., Tothia spp., Gemella spp., Alcaligenes spp., Alternaria spp. , Flavobacterium spp., Bacillus spp., Achromobacter spp., Acinetobacter spp., Actinobacillus spp., Alcaligenes spp., Campylobacter spp., Edwardsiella spp.; Ehrlichia spp., Enterobacter spp. (Ewingella spp.), Flavobacteria, Hafnia spp., Klebsiella spp., Klebsiella spp., Kluyvera spp., Legionella species spp.), Morxella spp., Morganella spp., Neisseria strains ( Neisseria spp.), Pasteurella spp., Prevotella spp., Proteus spp., Providencia spp., Pseudomonas spp. , Rahnella spp., Salmonella spp., Serratia serratia spp., Shigella spp., Sphingobacterium spp., Vibrio Vibrio spp., Yersinia spp., Neisseria spp., Kingella spp., Cardiobacterium, non-tuberculous anti-acid It may include bacteria selected from the group consisting of non-Tuberculosis mycobacteria (NTB), Mycobacterium tuberculosis, and Mycobacterium avium. More specifically, the microorganism may be E. coli. However, the technical spirit of the present invention is not limited thereto, and of course, it may further include other microorganisms.

The sample arrangement unit 100 may include a plurality of divided cells 110 for distributing and accommodating a sample to be measured. At this time, each of the plurality of divided cells 110 may include a culture material 120 for culturing microorganisms. The culture material 120 corresponds to the type of microorganism to be counted, and may include a material capable of effectively culturing the microorganism.

The medium containing the culture material 120 used for culture should suitably satisfy the requirements of specific microorganisms. Various microbial culture media are described, for example, in the literature ("Manual of Methods for General Bacteriology" by the American Society for Bacteriology, Washington D.C., USA, 1981.). These media contain various carbon sources, nitrogen sources and trace element components. Carbon sources include carbohydrates such as glucose, lactose, sucrose, fructose, maltose, starch and fiber; fats such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; Alcohols such as glycerol and ethanol and organic acids such as acetic acid may be included, and these carbon sources may be used alone or in combination, but are not limited thereto. Nitrogen sources include organic nitrogen sources and urea such as peptone, yeast extract, grain, malt extract, corn steep liquor (CSL) and bean flour; Inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be included, and these nitrogen sources may be used alone or in combination, but are not limited thereto. The medium may additionally contain potassium dihydrogen phosphate, potassium dihydrogen phosphate, and corresponding sodium-containing salts as a phosphoric acid source, but is not limited thereto. . Also, the medium may contain a metal such as magnesium sulfate or iron sulfate, and amino acids, vitamins and suitable precursors may be added thereto.

In addition, in order to maintain the aerobic condition of the culture medium, oxygen or a gas containing oxygen (eg, air) may be injected into the culture medium. The temperature of the culture may be generally 20-45 °C, specifically 25-40 °C.

Here, the sample (S) may be prepared by being diluted according to the dilution factor for counting the number of colonies. In general, the number known as the number of bacteria that can be spread on a plate and can be separated and counted is about 1000 E. coli colonies in the case of a plate with a diameter of 10 cm. As will be described later, in the present invention, the number of countable bacteria is determined by the dilution factor of the sample (S) and / or depending on the number of divided cells 110, more bacteria than the above-mentioned number of bacteria can be counted.

Specifically, the plurality of divided cells 110 may be provided in the form of a (n, m) matrix, and the number may be greater than the expected number of microorganisms included in the sample S. As will be described later, the sample (S) is uniformly accommodated in the plurality of divided cells (110), and the sample (S) may be prepared by being diluted according to a dilution factor for counting the number of colonies in general. For example, when the plurality of divided cells 110 are formed of a matrix of (100, 100), the sample S may be diluted so that the number of microorganisms does not exceed 10 ⁴ . Alternatively, when the plurality of divided cells 110 are formed of a matrix of (1000, 1000), the sample S may be diluted so that the number of microorganisms does not exceed 10 ⁶ .

Meanwhile, the sample arrangement unit 100 may include a multiple scattering amplification region for amplifying the number of times that a wave incident to the dividing cell 110 is multiple scattering within the sample S. For example, the sample arrangement unit 100 may be formed to include a multiple scattering material in at least a lower region. For example, the multi-scattering material may include titanium oxide (TiO 2 ) and may reflect at least a portion of a wave passing through the sample S and incident to the sample arrangement unit 100 . The multi-scattering amplification region may allow a wave emitted by being multi-scattered from the sample S to reciprocate at least once in a space between the sample S and the multi-scattering amplification region.

As another embodiment, the microbial population counting system 10 may be configured such that the multi-scattering material T is included in the culture material 120 . 2 shows that the culture material 120 and the sample (S) are separated, when the sample (S) is accommodated in the divided cell 110, it may be mixed with the culture material (120). At this time, the multiple scattering material T may be disposed to surround the microorganism M to scatter the incident wave to increase the number of multiple scattering.

Meanwhile, as another embodiment, the microbial population counting system 10 may further include a separate multiple scattering amplifier (not shown). The multiple scattering amplification unit (not shown) is provided on the wave movement path between the wave source 200 and the sample arrangement unit 100 and/or between the sample arrangement unit 100 and the detection unit 300, so that multiple scattering of waves frequency can be amplified. When the multiple scattering amplification unit (not shown) is disposed between the wave source 200 and the sample disposing unit 100 , it may be formed in a detachable structure to distribute the sample S to the sample disposing unit 100 . can For example, the multiple scattering amplifier (not shown) may have a structure such as a lid. In addition, the multi-scattering amplifier (not shown) may be provided to correspond to each of the divided cells 110 , or may be formed to cover the entirety of the plurality of divided cells 110 .

On the other hand, the sample arrangement unit 100 is provided with a blocking area (110A) on the side of each divided cell (110), so that the wave incident on each divided cell (110) does not flow into the other divided cell (110) can do it The microbial population counting system 10 according to an embodiment of the present invention is characterized in that it sequentially irradiates waves to the plurality of divided cells 110 and detects a laser speckle emitted in response thereto. As will be described later, in this process, it is necessary to accurately detect whether microorganisms are present in each of the plurality of divided cells 110 , due to the laser speckle emitted from the divided cells 110 other than the corresponding divided cells 110 during the rapid detection process. Its accuracy may be reduced. In order to prevent this, the sample arrangement unit 100 is provided with a blocking area 110A on the side of each divided cell 110, so that the wave incident on the corresponding divided cell 110 flows into the other divided cell 110 and interferes. can be prevented from causing As an embodiment, the blocking region 110A may include a metal material having a reflective characteristic.

Meanwhile, in FIG. 2 , the blocking area 110A is illustrated only as being disposed on the side of the divided cell 110 , but the technical spirit of the present invention is not limited thereto. As another embodiment, the blocking region 110A may also be disposed on the lower surface of the divided cell 110 . In this case, the detection unit 300 may detect the laser speckle that is disposed on the sample disposition unit 100 and is emitted differently from that shown in FIG. 2 . Alternatively, as shown in FIG. 1 , the detection unit 300 is disposed to face the wave source 200 with respect to the sample arrangement unit 100 , and a blocking region is not formed on the lower surface of the sample arrangement unit 100 . A laser speckle emitted through a predetermined penetration region may be detected.

Although not shown, the sample arrangement unit 100 is formed in the same shape as the plurality of divided cells 110, and may further include a reference cell (not shown) positioned on the wave irradiation path. In this case, the reference cell (not shown) may have the same size as the plurality of divided cells 110 and may be provided with a culture material of the same type and capacity. However, the reference cell (not shown) does not accommodate the sample S, unlike the divided cells 110 .

The culture material has a certain degree of fluidity, which may act as noise in detecting laser speckle according to the microscopic movement of microorganisms. At this time, since the reference cell (not shown) provided in the sample arrangement unit 100 together with the plurality of divided cells 110 does not contain the sample S but contains a culture material, if the sample arrangement unit 100 When the culture material of the plurality of divided cells 110 has fluidity due to the occurrence of minute vibrations, the culture material of the reference cell (not shown) also has the same fluidity. The microbial population counting system 10 also irradiates a wave to the reference cell (not shown) in the same way and sets the laser speckle detected therefrom as a reference value, thereby removing noise and the presence of microorganisms in other divided cells 110 . can be accurately distinguished.

Here, first, with reference to FIG. 8, the principle of the chaotic wave sensor of the present invention will be described.

8 is a diagram for explaining the principle of a chaos wave sensor according to an embodiment of the present invention.

In the case of a material having a homogeneous internal refractive index, such as glass, refraction occurs in a certain direction when irradiated with light. However, when coherent light such as a laser is irradiated to an object having a non-uniform internal refractive index, very complex multiple scattering occurs inside the material.

Referring to FIG. 8 , among light or waves (hereinafter, referred to as waves for simplicity) irradiated from a wave source, some of the waves scattered through a complex path through multiple scattering pass through the inspection target surface. Waves passing through several points on the surface to be inspected cause constructive interference or destructive interference with each other, and constructive/destructive interference of these waves causes grain-shaped patterns (speckle) to occur. do.

In the present specification, the waves scattered by such a complicated path are called "chaotic waves", and the chaotic waves can be detected through laser speckle.

Again, the left view of FIG. 8 is a view showing when a stable medium is irradiated with a laser. When irradiated with interference light (for example, laser) for a stable daily without movement of internal components, a stable speckle pattern with no change can be observed.

However, as shown in the right diagram of FIG. 8 , when an unstable medium having movement among internal constituent materials such as bacteria is included therein, the speckle pattern is changed.

That is, due to microscopic biological activities of living things (eg, intracellular movement, movement of microorganisms, movement of mites, etc.), the optical path may change minutely with time. Since the speckle pattern is a phenomenon that occurs due to wave interference, a slight change in the optical path may cause a change in the speckle pattern. Accordingly, by measuring the temporal change of the speckle pattern, the movement of the organism can be quickly measured. As such, in the case of measuring the change over time of the speckle pattern, the presence or concentration of microorganisms can be known, and furthermore, the type of organism can be known.

In the present specification, a configuration for measuring a change in such a speckle pattern is defined as a chaotic wave sensor.

Referring back to FIGS. 1 and 2 , the wave source 200 may sequentially irradiate waves to the plurality of divided cells 110 of the sample arrangement unit 100 . All types of source devices capable of generating a wave may be applied to the wave source 200 , and may be, for example, a laser capable of irradiating light of a specific wavelength band. Meanwhile, the wave source 200 may be connected to a driving device such as a motor or an actuator to sequentially irradiate waves toward each divided cell 110 according to a preset time interval. The present invention is not limited in the type of the wave source, however, hereinafter, for convenience of description, the laser will be mainly described.

For example, a laser having good coherence may be used as the wave source 200 to form a speckle in the sample S accommodated in the plurality of divided cells 110 . In this case, as the spectral bandwidth of the wave source that determines the coherence of the laser wave source is shorter, the measurement accuracy may increase. That is, as the coherence length increases, the measurement accuracy may increase. Accordingly, laser light having a spectral bandwidth of the wave source that is less than a predefined reference bandwidth may be used as the wave source 200, and measurement accuracy may increase as the spectral bandwidth of the wave source is shorter than the reference bandwidth. For example, the spectral bandwidth of the wave source 200 may be set so that the condition of Equation 1 below is maintained.

[Equation 1]

According to Equation 1, in order to measure the pattern change of the laser speckle, the spectral bandwidth of the wave source 200 may be maintained to be less than 1 nm when light is irradiated to each divided cell 110 every reference time.

Meanwhile, the detector 300 may sequentially detect individual wave information emitted from the samples S accommodated in each divided cell 110 in connection with the sequentially irradiated waves. In this case, the individual wave information may be information obtained by detecting laser speckle generated by multiple scattering from the sample S accommodated in each of the plurality of divided cells 110 at preset time points. Specifically, the individual wave information includes first image information of a laser speckle detected at a first time point among laser speckles emitted from the divided cell 110 and second image information of a laser speckle detected at a second time point. and third image information of the laser speckle detected at the third time point, wherein the first to third viewpoints may be different from each other. The detector 300 may provide information about the laser speckle detected at the first to third time points for one divided cell 110 to the controller 400 . In this case, the first to third time points are the minimum number of detections for the controller 400 to analyze the temporal correlation of the laser speckle, and the detector 300 may detect the laser speckle at a plurality of time points greater than this. is of course

Here, the time refers to any one moment in the continuous flow of time, and the time points may be set in advance at the same time interval, but are not necessarily limited thereto, and are set in advance at an arbitrary time interval. it might be The detection unit 300 may include a detection means corresponding to the type of the wave source 200, for example, when a light source of a visible light wavelength band is used, a CCD camera, which is a photographing device for photographing an image, is used. can be

Specifically, when a wave is irradiated to the sample S accommodated in one dividing cell 110 , the incident wave may form a laser speckle by multiple scattering. Since the laser speckle is generated by the interference of light, if there are no microorganisms in the sample S, a constant interference pattern can always be displayed according to time by the multiple scattering amplification region. In comparison, when the microorganism M is present in the sample S, the laser speckle may change with time due to the movement of the microorganism M. The detector 300 may detect the laser speckle that changes according to time at each preset time point and provide it to the controller 400 . The detector 300 may detect the laser speckle at a speed sufficient to detect the movement of the microorganism M, for example, at a speed of 25 to 30 frames per second.

Meanwhile, when an image sensor is used as the detector 300 , the image sensor may be disposed such that the size d of one pixel of the image sensor is smaller than or equal to the grain size of the speckle pattern. For example, an image sensor may be disposed in the optical system included in the detector 300 to satisfy the condition of Equation 2 below.

[Equation 2]

As shown in Equation 2, the size d of one pixel of the image sensor should be less than or equal to the grain size of the speckle pattern, but if the size of the pixel becomes too small, undersampling occurs and the pixel resolution There may be difficulties in using Accordingly, the image sensor may be disposed such that a maximum of 5 pixels or less are located at a speckle grain size in order to achieve an effective signal to noise ratio (SNR).

The control unit 400 determines the presence or absence of the microorganism (M) in each divided cell 110 using the detected individual wave information, and uses the number of the divided cells 110 in which the microorganism (M) is present. It is possible to calculate the number of microorganisms in the sample (S). Specifically, the controller 400 obtains a temporal correlation of the detected laser speckle using the detected laser speckle, and based on the obtained temporal correlation, the microorganism in the divided cell 110 . The existence of (M) can be judged. The control unit 400 may estimate in real-time whether or not the microorganisms exist in each divided cell 110 based on the obtained temporal correlation. In the present specification, real-time means estimating the presence or absence of the microorganism (M) within 3 seconds, and preferably means estimating the presence or absence of the microorganism (M) within 1 second.

As an embodiment, the control unit 400 may estimate the presence or absence of microorganisms using laser speckles emitted from the sample S at different points in time by a wave irradiated to one divided cell 110 . For example, when the first wave S1 is irradiated to the (1,1) split cell in FIG. 1 , the time correlation between the first laser speckles P1 multi-scattered and emitted by the first wave S1 By obtaining (1,1), it is possible to estimate the presence or absence of microorganisms (M) in the divided cell. In this case, the control unit 400 includes the first image information of the first laser speckle P1 detected at the first time point, the second image information of the first laser speckle P2 detected at the second time point, and the second image information of the first laser speckle P2 detected at the second time point. The presence or absence of the microorganism M may be estimated by using the difference in the third image information of the first laser speckle P3 detected at three points in time.

Here, the first image information to the third image information may be at least one of laser speckle pattern information and wave intensity information. On the other hand, according to an embodiment of the present invention, at least a difference between three image information of laser speckles detected from different viewpoints is used, or by extending this, image information of a plurality of laser speckles can be used from a plurality of viewpoints. The control unit 400 may calculate a time correlation coefficient between images using image information of laser speckle generated for a plurality of preset viewpoints, and based on the time correlation coefficient, the microorganism (M) in the divided cell 110 . ) can be inferred. The temporal correlation of the detected laser speckle image may be calculated using Equation 3 below.

[Equation 3]

은 시간 상관 관계 계수,

은 표준화된 빛 세기, (x,y)는 카메라의 픽셀 좌표, t는 측정된 시간, T는 총 측정 시간,

는 타임래그(time lag)를 나타낸다. in Equation 3

is the time correlation coefficient,

is the normalized light intensity, (x,y) is the pixel coordinates of the camera, t is the measured time, T is the total measurement time,

denotes a time lag.

A time correlation coefficient may be calculated according to Equation 3, and as an embodiment, the existence of a microorganism may be estimated through analysis in which the time correlation coefficient falls below a preset reference value. Specifically, the existence of microorganisms can be estimated when the time correlation coefficient falls below a reference value beyond a preset error range.

Hereinafter, a method of counting the number of microorganisms using the above-described system for counting the number of microorganisms 10 will be described in detail.

3 is a flowchart sequentially illustrating a method for counting the number of microorganisms according to an embodiment of the present invention, and FIGS. 4 and 5 are views for explaining the method for counting the number of microorganisms.

3 to 5 , in the method for counting the number of microorganisms according to an embodiment of the present invention, first, the above-described sample arrangement unit 100 may be prepared ( S100 ). The sample arrangement unit 100 may include a plurality of divided cells 110 each containing a culture material.

Thereafter, the sample (S) to be measured is diluted by a predetermined dilution ratio and distributed to a plurality of divided cells (110) (S200). In this case, the size of the plurality of divided cells 110 is configured to be the same, and the sample S may be evenly distributed. Since the sample (S) is diluted so that the expected number of microorganisms (M) is less than the number of the plurality of divided cells 110, when evenly distributed, each divided cell 110 contains microorganisms ( M) may or may not be present.

Thereafter, waves are sequentially irradiated to the plurality of divided cells 110 by the wave source 200 , and individual waves emitted from the samples S accommodated in each divided cell 110 in connection with the sequentially irradiated waves Information can be sequentially detected (S300). For example, when a plurality of divided cells 110 are arranged in a (n,m) matrix form as shown, waves may be irradiated by sequentially scanning along each column. At this time, in connection with the wave to be irradiated, the detector 300 detects individual wave information emitted from each divided cell 110 . As described above, the individual wave information may be a plurality of image information of laser speckles detected at different viewpoints in the corresponding divided cell 110 .

The control unit 400 may determine the presence or absence of the microorganism (M) in each divided cell 110 by using the individual wave information (S400). For example, as shown in FIG. 4, when the microorganism (M) is present in the divided cell (1, 1) and the microorganism (M) is not present in the divided cell (2,1), the control unit 400 can define (1,1) divided cell as 1 and (2,1) as 0. Determination of the existence of these microorganisms (M) can be made from (1, 1) divided cells to (n, m) divided cells according to the sequential wave irradiation path.

When the determination of the presence or absence of microorganisms in all divided cells 110 is completed, the control unit 400 may calculate the number of microorganisms in the entire sample S by using the number of divided cells in which the microorganisms M are present. . In other words, by calculating the number of divided cells defined as 1 among all divided cells 110 , the number of microorganisms included in the total sample S can be calculated.

6 is a conceptual diagram schematically illustrating a microbial population counting system 10-1 according to another embodiment of the present invention.

Referring to FIG. 6 , the microbial population counting system 10-1 according to another embodiment of the present invention sequentially irradiates a wave irradiated from the wave source 200 to a plurality of divided cells 110, a wave path A change unit 210 may be further provided. In other words, the wave may be irradiated to the plurality of divided cells 110 by using the wave path changing unit 210, rather than being directly irradiated from the wave source 200 to the plurality of divided cells 110 . In another embodiment of the present invention, except for the wave irradiation method, the remaining components are the same as those of the embodiment, and overlapping descriptions will be omitted for convenience of description.

The wave path changing unit 210 may receive a wave from the wave source 200 . The wave path changing unit 210 may be formed of a micro mirror. The wave path changing unit 210 may have a reflective surface to reflect the incident wave toward the plurality of divided cells 110 . Although the reflective surface is illustrated as a flat surface having no refractive power, the present invention is not limited thereto. The wave path changing unit 210 may be driven finely by a driving control unit (not shown).

As another embodiment, the wave path changing unit 210 is finely driven by the controller 400 , and accordingly, the wave may be sequentially irradiated to the plurality of divided cells 110 . In this case, the control unit 400 may also control the detection unit 300 to detect the laser speckle emitted from the divided cell 110 in connection with the order of the waves irradiated by the wave path change unit 210 .

The micro-mirror constituting the wave path changing unit 210 may have various configurations in which a mechanical displacement of the reflective surface may occur according to electrical control, for example, a generally known micro electromechanical system (MEMS) mirror. , a digital micromirror device (DMD) device, etc. may be employed. Although the wave path changing unit 210 is illustrated as a single micro-mirror, this is exemplary, and a plurality of micro-mirrors may be two-dimensionally arrayed.

Meanwhile, the wave path changing unit 210 may further include a mirror 215 disposed on the wave path to adjust the angle of the wave irradiated to the plurality of divided cells 110 to be constant. In other words, when the wave path changing unit 210 irradiates a wave to the plurality of divided cells 110 using a micromirror, the angle of incidence of the wave may vary according to the location of the divided cell 110 . Accordingly, since the degree of multi-scattering in the sample S may vary depending on the positions of the plurality of divided cells 110 , it may be difficult to accurately compare and evaluate the divided cells 110 by further disposing a mirror 215 . It can be adjusted so that the angle of the wave incident to the beams can be uniform. Although only one mirror 215 disposed on the (1,1) divided cell is shown in the drawing, it is schematically illustrated for convenience of explanation, and a mirror is disposed on the upper end of each of the divided cells 110 so that the divided cells ( 110) to adjust the angle of the irradiated wave.

7 is a conceptual diagram schematically illustrating a sample arrangement unit 100-1 according to another embodiment of the present invention.

Referring to FIG. 7 , the sample disposing unit 100-1 according to another embodiment is different from the sample disposing unit 100 of one embodiment in the form of a petri dish such as an agar plate, fine It may be formed in the form of a multi-well-based microfluidics chip (microfluidics chip) connected to the fluid channel 135 .

Specifically, the sample arrangement unit 100-1 includes a plurality of divided cells 110 in the form of a multi-well and an input unit 130 capable of inputting the sample S, and includes a plurality of divided cells 110 . The input unit 130 may communicate with the microfluidic channel 135 . In the sample arrangement unit 100 - 1 , when the sample S is input to the input unit 130 , the sample S may be equally distributed to each divided cell 110 through the microfluidic channel 135 .

The sample arrangement unit 100-1 according to another embodiment may include a culture material for culturing microorganisms in each of the plurality of divided cells 110, as in the embodiment. In addition, the upper surface of the sample arrangement unit 100-1 may be made of a transparent material for optical imaging. However, the present invention is not limited thereto, and as described above, a multi-scattering amplifier may be disposed on the upper surface for multi-scattering amplification.

On the other hand, the sample arrangement unit 100-1 is provided with a blocking area on at least the side of each divided cell 110, so as to block the wave incident to each divided cell 110 from flowing into the other divided cell (110). can do it Through the blocking region, the sample arrangement unit 100 - 1 minimizes wave interference between the divided cells 110 to improve the accuracy of counting the population. In addition, the above-described sample arrangement unit 100-1 may be provided as a disposable kit.

Meanwhile, FIGS. 9 and 10 are conceptual views schematically illustrating a microbial individual counting system according to another embodiment of the present invention.

9 and 10 , the microbial individual counting system transmits the first wave signal scattered from the sample (S) accommodated in the plurality of divided cells (110), the wave of the wave source (200) is scattered to the sample (S) It may further include an optical unit 550 that modulates to restore the previous second wave signal. In this case, the optical unit 550 may include a spatial light modulator (SLM) 551 and a lens 552 . When the wave scattered from the measurement target is incident, the optical unit 550 may control the wavefront of the scattered wave to restore the wave (light) before being scattered again and provide it to the detector 300 .

The spatial light modulator 551 may receive a wave (light) scattered from the sample. The spatial light modulator 551 may control the wavefront of the wave scattered from the sample and provide it to the lens 552 . The lens 552 may focus the light and provide it back to the detector 300 . The detector 300 may detect the wave collected by the lens 552 and restore it as a wave output from the original wave source before being scattered and output it.

Here, the optical unit 550 may restore the first wave signal scattered from the sample S to the wave before scattering when there is no microorganism in the stable medium, that is, the sample S. However, when the microorganism M is present in the sample S, the first wave signal is changed due to the movement of the microorganism, and thus the phase control wavefront cannot be detected. cannot be tampered with The microorganism population counting system 10 including the above-described optical unit 550 more accurately estimates the presence or absence of the microorganism M in each of the plurality of divided cells 110 using the difference in the second wave signal. can do.

As described above, the microbial population counting system and the microbial population counting method according to embodiments of the present invention can accurately detect microorganisms in units of one population by using a chaotic wave sensor. Through this, the embodiments of the present invention detect and count the presence or absence of microorganisms in each of the plurality of divided cells, thereby rapidly calculating the number of microorganisms included in the entire sample. The microbial population counting system and the microbial population counting method according to embodiments of the present invention can detect a microorganism without culturing a colony, and thus the culture time is significantly reduced, thereby shortening the time for counting the population.

So far, the present invention has been focused on preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive point of view. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

10: microbial population counting system
100: sample arrangement part
110: split cell
200 : wave source
300: detection unit
400: control unit

Claims (1)

a sample arrangement unit comprising a plurality of multi-well-shaped divided cells, an input unit capable of inputting a sample, and a microfluidic channel communicating the plurality of divided cells and the input unit;
a wave source that sequentially irradiates waves to the plurality of divided cells;
a detector for sequentially detecting individual wave information emitted from the samples accommodated in each divided cell in connection with the sequentially irradiated waves; and
A control unit that determines the presence or absence of microorganisms in each divided cell using the detected individual wave information, and calculates the number of microorganisms in the sample by using the number of divided cells in which the microorganisms exist; includes,
The individual wave information is
It is information obtained by detecting a laser speckle generated by multiple scattering from a sample accommodated in each of the plurality of divided cells at preset time points,
The sample arrangement unit is provided with a blocking area on the side of each divided cell so that the wave incident to each divided cell does not flow into the other divided cell, the number of microorganisms counting system.

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