KR102113311B1 - Microorganism population counting method and system for the same - Google Patents

Abstract

In one embodiment of the present invention, each step of preparing a sample arrangement unit comprising a plurality of divided cells containing a culture material, distributing a sample to be measured to the plurality of divided cells, sequentially in the plurality of divided cells Investigating a wave, sequentially detecting individual wave information emitted from samples accommodated in each divided cell in connection with the sequentially irradiated wave, and the presence of microorganisms in each divided cell using the individual wave information It provides a method for counting the number of microorganisms, including determining the presence or absence and calculating the number of microorganisms in the sample by using the number of division 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 food industry, unintended microorganisms are generated in the manufacturing process, which often becomes a problem. In order to confirm whether or not such microorganisms proliferate, a cell counting system has conventionally used a culture-type counting method using a medium. For example, as a method for counting microorganisms, a method for measuring the number of colonies of microorganisms grown using agar medium is used. On the other hand, instead of visually counting the number of colonies generated on agar medium or the like, a method of counting the number of colonies by data processing the image data of the counting target medium photographed using a CCD camera has recently been proposed.

However, the above-described counting methods have a problem in that they cannot directly count the number of microorganisms, and inevitably count them by culturing in a colony state that can be visually confirmed.

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

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

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

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

In one embodiment of the present invention, the individual wave information includes first image information of the laser speckle detected at a first time point among laser speckles emitted from a corresponding split cell, and the laser spec detected at a second time point. The second image information of the claw and the third image information of the laser speckle detected at the third time point, wherein the first to third time points are different time points, and determining 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 one embodiment of the present invention, the plurality of divided cells are arranged in a matrix, and the number of the divided cells may be greater than the expected number of microorganisms included in the sample.

One embodiment of the present invention, each of which contains a culture material, a sample arrangement unit having a plurality of divided cells to receive and distribute the sample to be measured, a wave source for sequentially irradiating waves to the plurality of divided cells, The presence or absence of microorganisms in each divided cell is determined by using a detection unit 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, It provides a counting system for the number of microorganisms, including a control unit for calculating the number of microorganisms in the sample by using the number of division cells in which the microorganisms are present.

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

In one embodiment of the present invention, the control unit acquires a time correlation of the detected laser speckle using a fraud-detected laser speckle, and based on the obtained time correlation, microorganisms in a corresponding divided cell You can judge the existence of

In one embodiment of the present invention, the individual wave information includes first image information of the laser speckle detected at a first time point among laser speckles emitted from a corresponding split cell, and the laser spec detected at a second time point. The second image information of the claw, and the third image information of the laser speckle detected at the third time point, wherein the first to third times are different time points, and the controller is the first image information. The presence or absence of the microorganism may be determined using the difference between the third image information.

In one embodiment of the present invention, the plurality of divided cells are arranged in a matrix, and the number of the 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 as a unit of population using a chaotic wave sensor. Through this, embodiments of the present invention can quickly calculate the number of microorganisms contained in the entire sample by detecting and counting the presence or absence of microorganisms in each of the plurality of divided cells. The microbial population counting system and the microbial population counting method according to embodiments of the present invention can be detected without culturing microorganisms with colonies, and thus the culture time can be significantly reduced to shorten the time for population counting.

1 is a conceptual diagram schematically illustrating a counting system for microbial populations 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 showing a method for counting microbial populations according to an embodiment of the present invention.
4 and 5 are diagrams for explaining the method of counting the number of microorganisms.
6 is a conceptual diagram schematically showing a microbial population counting system according to another embodiment of the present invention.
7 is a conceptual diagram schematically showing a sample placement 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 is a conceptual diagram schematically showing a microbial counting system according to another embodiment of the present invention.

Hereinafter, the following embodiments will be described in detail with reference to the accompanying drawings, and the same or corresponding components will be given the same reference numerals when described with reference to the drawings, and redundant description thereof will be omitted.

Since the present embodiments can apply various conversions, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. Effects and features of the present embodiments and methods for achieving them will be clarified with reference to the contents described below in detail together 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 examples, terms such as first and second are not limited, but are used for the purpose of distinguishing one component from other components.

In the following examples, singular expressions include plural expressions, unless the context clearly indicates otherwise.

In the following examples, terms such as include or have, mean that the features or components described in the specification exist, and do not exclude the possibility of adding one or more other features or components in advance.

In the following embodiments, when a part of a unit, area, component, etc. is said to be on or on another part, other units, areas, components, etc. are interposed therebetween, as well as directly above the other part. Also includes cases.

In the following examples, terms such as connect or join do not necessarily mean direct and / or fixed connection or joining of two members, unless the context clearly indicates otherwise, that another member is interposed between the two members. It is not excluded.

It means that the features or components described in the specification exist, and does not preclude the possibility of adding one or more other features or components in advance.

In the drawings, the size of 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 shown for convenience of description, the following embodiments are not necessarily limited to those illustrated.

1 is a conceptual diagram schematically illustrating a counting system for microbial populations 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 counting system 10 for microbial populations according to an embodiment of the present invention includes a sample placement 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 a production capacity of a useful target substance such as L-amino acid. For example, microorganisms with increased intracellular ATP concentrations include Escherichia sp., Erwinia sp., Serratia sp., Providencia sp. ), Corynebacteria sp., Pseudomonas sp., Leptospira, Salmonellar sp., Brevibacteria sp., Hypomononas sp.), Chromobacterium sp., Norcardia or fungi or yeast. Specifically, the microorganism may be a microorganism of the genus Escherichia.

Alternatively, microorganisms include Staphylococcus, coagulase negative staph Coagulase negative, Staph. Aureus, Streptococcus spp., 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., Actinobacillus spp. Alcaligene s spp.), Campylobacter spp., Edwardsiella spp .; Ehrlichia spp., Enterobacter spp., Ewingera spp. , Flavobacteria, Hafnia spp., Klebsiella spp., Kluyvera spp., Legionella spp., Moroccan Cellar (Morxella spp.), Morganella spp., Neisseria spp., Pasteurella spp., Prevotella spp., And Proteus spp. Proteus spp.), Providencia spp., Pseudomonas spp., Rellaella spp., Salmonella spp., Serratia spp. ), Shigella spp., Sphingobacterium spp., Vibrio spp., Yesini (yersinia) fungus (Yersinia spp.), Neisseria spp., Kingella spp., Cardiobacterium, non-tuberculosis mycobacteria (NTB) , Mycobacterium tuberculosis and mycobacterium avium may include bacteria selected from the group consisting of. More specifically, the microorganism may be E. coli. However, the technical spirit of the present invention is not limited to this, 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 receiving samples to be measured. In this case, 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 cultivation must adequately satisfy the requirements of a specific microorganism. Various microbial culture media are described, for example, in "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. Examples of nitrogen sources include organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean flour, Inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and these nitrogen sources may be used alone or in combination, but are not limited thereto. The medium may additionally include, but is not limited to, potassium dihydrogen phosphate, potassium dihydrogen phosphate, and corresponding sodium-containing salts as a phosphate source. . In addition, the medium may include metals such as magnesium sulfate or iron sulfate, and amino acids, vitamins, and suitable precursors may be added.

In addition, in order to maintain the aerobic conditions 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 generally be 20-45 ° C, specifically 25-40 ° C.

Here, the sample S may be prepared by being diluted according to the dilution factor for the Colini's count. In general, the number known as the number of bacteria that can be deployed on a plate and can be separated and measured is approximately 1000 in the number of colonies of E. coli in the case of a plate having a diameter of 10 cm. As will be described later, in the present invention, after the microorganisms are developed on a plate, the number of microorganisms that can be counted is the dilution factor of the sample (S) and the number of microorganisms that can be counted because it is possible to detect each of the microorganisms accommodated in the plurality of division cells 110 without forming colonies. / Or depending on the number of division cells 110, it is possible to count more bacteria than the number of bacteria described above.

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 colony counting. For example, when the plurality of divided cells 110 is 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 is made of a matrix of (1000, 1000), the sample S may be diluted so that the number of microorganisms does not exceed 10 ⁶ .

On the other hand, the sample placement unit 100 may include a multi-scattering amplification region for amplifying the number of times the waves incident on the split cell 110 are multiple scattered in the sample S. For example, the sample placement unit 100 may be formed by including a multiple scattering material in at least a lower region. For example, the multi-scattering material may include titanium oxide (TiO2), and may reflect at least a portion of the wave incident on the sample placement unit 100 through the sample S. The multi-scattering amplification region may allow a wave that is emitted by being multi-scattered from the sample S to reciprocate the space between the sample S and the multi-scattering amplification region at least once.

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. In FIG. 2, the culture material 120 and the sample S are shown as separated, but when the sample S is accommodated in the split cell 110, it may be mixed with the culture material 120. At this time, the multi-scattering material (T) is arranged to surround the microorganism (M) to scatter the incident wave can increase the number of multi-scattering.

Meanwhile, as another embodiment, the microbial population counting system 10 may further include a separate multi-scattering amplification unit (not shown). The multi-scattering amplification unit (not shown) is provided on the wave movement path between the wave source 200 and the sample placement unit 100 and / or between the sample placement unit 100 and the detection unit 300, thereby allowing multiple scattering of waves. The number of times can be amplified. When a multi-scattering amplification unit (not shown) is disposed between the wave source 200 and the sample placement unit 100, a detachable structure may be formed to distribute the sample S to the sample placement unit 100. Can be. For example, the multi-scattering amplification unit (not shown) may be configured as a lid-like structure. In addition, the multi-scattering amplification unit (not shown) may be provided corresponding to each of the divided cells 110, or may be formed in a structure that covers the entire plurality of divided cells 110.

On the other hand, the sample placement unit 100 is provided with a blocking area 110A on the side of each division cell 110, so that the wave incident to each division cell 110 does not flow into the other division cell 110. I can do it. The microbial population counting system 10 according to an embodiment of the present invention is characterized by sequentially irradiating waves to a plurality of divided cells 110 and detecting a laser speckle emitted in response thereto. As will be described later, this process requires accurate detection of the presence of microorganisms in each of the plurality of divided cells 110. Due to the rapid detection process, due to the laser spectrocle emitted from the other divided cells 110 other than the corresponding divided cells 110, Its accuracy can be reduced. To prevent this, the sample placement unit 100 is provided with a blocking area 110A on the side of each division cell 110, and the wave incident to the division cell 110 flows into another division cell 110 and interferes. It can be prevented from causing. As an embodiment, the blocking region 110A may include a metal material having reflective properties.

Meanwhile, in FIG. 2, the blocking area 110A is only shown as being disposed on the side surface of the division cell 110, but the technical spirit of the present invention is not limited thereto. As another embodiment, the blocking area 110A may be disposed on the lower surface of the division cell 110. At this time, unlike the one shown in FIG. 2, the detector 300 may detect the laser speckle that is disposed on the sample placement unit 100 and is emitted. Alternatively, as shown in FIG. 1, the detection unit 300 is disposed to face the wave source 200 based on the sample placement unit 100, but a blocking area is not formed on the lower surface of the sample placement unit 100. It is also possible to detect a laser speckle emitted through a predetermined through region.

Although the sample placement unit 100 is not illustrated, it is formed in the same form as the plurality of division 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 be provided with a culture material having the same size and capacity as the plurality of divided cells 110 and the same size. However, unlike the divided cells 110, the sample cell S is not accommodated in the reference cell (not shown).

The culture material has a certain degree of fluidity, which can act as noise in detecting laser speckles due to microscopic movements of microorganisms. At this time, the reference cell (not shown) provided in the sample placement unit 100 together with the plurality of division cells 110 does not accommodate the sample S, but contains a culture material, so if the sample placement unit 100 When a fine vibration occurs and the culture material of the plurality of split cells 110 has fluidity, the culture material of the reference cell (not shown) also has the same fluidity. The microbial population counting system 10 irradiates waves to the reference cell (not shown) and sets the laser speckle detected therefrom as a reference value, thereby removing noise and presence or absence of microorganisms in the other split cells 110. Can be accurately distinguished.

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

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

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

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

In this specification, waves scattered by such a complex path are referred to as "chaotic waves," and chaotic waves can be detected through a laser speckle.

8 is a view showing when a stable medium is irradiated with a laser. Stable speckle pattern with no change can be observed when a stable day with no movement of internal components is irradiated with interference light (for example, a laser).

However, as shown in the right figure of FIG. 8, when the inside contains an unstable medium with movement among internal constituent materials such as bacteria, the speckle pattern changes.

That is, due to the microscopic life activities of the organism (eg, intracellular movement, microbial movement, mite movement, etc.), the optical path may change minutely with time. Since the speckle pattern is a phenomenon that occurs due to the interference of waves, changes in the fine optical path may cause a change in the speckle pattern. Accordingly, by measuring the temporal change of the speckle pattern, it is possible to quickly measure the movement of the organism. As described above, when the change over time of the speckle pattern is measured, the presence or concentration of microorganisms can be known, and furthermore, the type of organism can also be known.

In this specification, a configuration for measuring changes in the speckle pattern is defined as a chaotic wave sensor.

Referring again to FIGS. 1 and 2, the wave source 200 may sequentially irradiate the plurality of divided cells 110 of the sample placement unit 100. The wave source 200 may be applied to all kinds of source devices capable of generating waves, and may be, for example, a laser capable of irradiating light in a specific wavelength band. On the other hand, the wave source 200 is connected to a driving device such as a motor (motor) or an actuator (actuator), it is possible to sequentially investigate the wave toward each divided cell 110 according to a predetermined time interval. The present invention is not limited to the type of the wave source, however, hereinafter, for convenience of explanation, the case will be mainly described as a laser.

For example, a laser having good coherence may be used as the wave source 200 to form a speckle on a sample S accommodated in a plurality of split cells 110. At this time, the measurement accuracy may increase as the spectral bandwidth of the wave source, which determines the coherence of the laser wave source, is shorter. That is, as the coherence length is longer, measurement accuracy may increase. Accordingly, laser light having a spectral bandwidth of a wave source less than a predetermined reference bandwidth may be used as the wave source 200, and measurement accuracy may increase as the bandwidth is shorter. 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 at less than 1 nm when irradiating light to each divided cell 110 for each reference time.

Meanwhile, the detector 300 may sequentially detect individual wave information emitted from the sample S accommodated in each divided cell 110 in connection with the sequentially irradiated waves. At this time, the individual wave information may be information obtained by detecting a laser speckle generated by multiple scattering from a sample S accommodated in each of the plurality of divided cells 110 at predetermined time points. Specifically, the individual wave information includes the first image information of the laser speckle detected at the first time point and the second image information of the laser speckle detected at the second time point among the laser speckles emitted from the divided cell 110. And, third image information of the laser speckle detected at the third time point, wherein the first to third time points may be different time points. The detection unit 300 may provide the control unit 400 with information on the laser speckle detected at the first to third time points described above for one division cell 110. At this time, the first to third time points are the minimum number of detections for the control unit 400 to analyze the time correlation of the laser speckle, and the detector 300 can detect the laser speckle at a plurality of time points. Of course.

Here, the time (time) means any one moment in the continuous flow of time, the time (time) may be set in advance at the same time interval, but is not necessarily limited to this, set in advance at any time interval It may be. The detection unit 300 may include a detection means corresponding to the type of the wave source 200, for example, when using a light source in the visible wavelength band, a CCD camera (camera), which is an image capturing device, is used Can be.

Specifically, when a wave is irradiated to the sample S accommodated in one division cell 110, the incident wave may form a laser speckle by multiple scattering. Since the laser speckle is caused by an interference phenomenon of light, if there are no microorganisms in the sample S, a constant interference fringe can be exhibited over time by the multi-scattering amplification region. In comparison, when the microorganism (M) is present in the sample (S), the laser speckle may change over time by the movement of the microorganism (M). The detection unit 300 may detect a laser speckle that changes with time at a predetermined time point and provide it to the control unit 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 rate of 25 to 30 frames per second.

On the other hand, when the image sensor is used as the detection unit 300, the image sensor may be arranged 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, resulting in pixel resolution. Difficulties may exist. Accordingly, the image sensor may be arranged such that up to 5 pixels are positioned in the speckle grain size to achieve an effective signal to noise ratio (SNR).

The control unit 400 determines the presence or absence of microorganisms M in each division cell 110 using the detected individual wave information, and uses the number of division cells 110 in which the microorganisms M exist. The number of microorganisms in the sample S can be calculated. Specifically, the control unit 400 acquires a temporal correlation of the detected laser speckle using the detected laser speckle, and based on the obtained time correlation, the microorganisms in the divided cell 110 The existence of (M) can be judged. The controller 400 may estimate the presence or absence of microorganisms in each division cell 110 in real-time based on the obtained time correlation. In this specification, real-time refers to estimating the presence of microorganisms M within 3 seconds, preferably estimating the presence of microorganisms M within 1 second.

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

Here, the first image information to the third image information may be at least one of pattern information of a laser speckle and intensity information of a wave. On the other hand, an embodiment of the present invention is to use at least three differences in the image information of the laser speckle detected at different viewpoints, but by extending this, it is possible to use the image information of the plurality of laser speckle at multiple viewpoints. The control unit 400 may calculate the time correlation coefficient between the images using the image information of the laser speckle generated for each of a plurality of preset viewpoints, and based on the time correlation coefficient, the microorganism (M) in the divided cell 110 ) Can be estimated. The time 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 standardized light intensity, (x, y) is the camera's pixel coordinates, t is the measured time, T is the total measurement time,

Indicates a time lag.

The time correlation coefficient may be calculated according to Equation 3, and as an embodiment, the presence or absence of microorganisms may be estimated through analysis in which the time correlation coefficient falls below a preset reference value. Specifically, it can be estimated that microorganisms exist as the time correlation coefficient falls below a reference value beyond a preset error range.

Hereinafter, a method of counting the microbial population using the aforementioned microbial population counting system 10 will be described in detail.

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

3 to 5, the method for counting microbial populations according to an embodiment of the present invention may be provided with the sample placement unit 100 described above (S100). The sample arrangement unit 100 may include a plurality of divided cells 110 each containing a culture material.

Then, the sample (S) to be measured is diluted with a predetermined dilution ratio, and distributed to a plurality of divided cells (110) (S200). At this time, the size of the plurality of divided cells 110 is configured the same, the sample (S) can 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, evenly distributed, as shown in FIG. 4, each divided cell 110 has a microorganism ( M) may or may not be present.

Subsequently, waves are sequentially irradiated to the plurality of divided cells 110 by the wave source 200, and individual waves emitted from samples S accommodated in each divided cell 110 in connection with the sequentially irradiated waves. Information may 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 irradiated wave, the detection unit 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 a laser speckle detected at different viewpoints in the corresponding divided cell 110.

The control unit 400 may determine the presence or absence of microorganisms M in each divided cell 110 using individual wave information (S400). For example, as shown in Figure 4, (1, 1) if the microorganism (M) is present in the divided cell, (2,1) when the microorganism (M) is not present in the divided cell, the control unit 400 May define (1,1) division cell as 1, and (2,1) as 0. The determination of the presence or absence of the microorganism (M) may 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 for all the divided cells 110 is completed, the control unit 400 may calculate the number of microorganisms in the entire sample S using the number of divided cells in which the microorganisms M are present. . In other words, by counting the number of divided cells defined as 1 of the entire divided cells 110, it is possible to calculate the number of microorganisms contained in the entire sample (S).

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

Referring to Figure 6, the microbial population counting system 10-1 according to another embodiment of the present invention, in order to sequentially irradiate the waves irradiated from the wave source 200 to a plurality of divided cells 110, the wave path A changer 210 may be further provided. In other words, waves are not irradiated from the wave source 200 directly to the plurality of divided cells 110, but waves may be irradiated to the plurality of divided cells 110 using the wave path changing unit 210. Other embodiments of the present invention, except for the irradiation method of the wave, the rest of the components are the same as the one embodiment, for the sake of convenience of description, redundant description will be omitted.

The wave path changing unit 210 may receive waves 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 include a reflective surface, and reflect the incident wave toward the plurality of split cells 110. The reflective surface is shown as a flat surface without refractive power, but the present invention is not limited thereto. The wave path changing unit 210 may be finely driven by a driving control unit (not shown).

In another embodiment, the wave path changing unit 210 is finely driven by the control unit 400, and accordingly, the wave may be sequentially irradiated to the plurality of divided cells 110. At this time, 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 wave order irradiated by the wave path changing unit 210.

The micro-mirror constituting the wave path changing unit 210 may employ various configurations in which 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, or the like may be employed. The wave path changing unit 210 is shown as one micro mirror, but this is exemplary, and may be a configuration in which a plurality of micro mirrors are arrayed in two dimensions.

Meanwhile, the wave path changing unit 210 may further include a mirror 215 disposed on the wave path, so that the angle of the wave irradiated to the plurality of divided cells 110 is constant. In other words, when the wave path changing unit 210 irradiates waves to the plurality of divided cells 110 using a micro mirror, the incident angle of the wave may vary according to the position of the divided cell 110. Accordingly, since the degree of multi-scattering within the sample S may vary depending on the positions of the plurality of split cells 110, accurate comparison evaluation may be difficult, so that the mirror 215 is further disposed to divide the cell 110. It can be adjusted so that the angle of the wave incident to the field is uniform. In the drawing, only one mirror 215 disposed on the (1,1) divided cell is schematically illustrated for convenience of description, and a mirror is disposed on each of the divided cells 110 to divide cells ( 110) can control the angle of the wave being irradiated.

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

Referring to FIG. 7, the sample placement unit 100-1 according to another embodiment is fine, unlike the sample placement unit 100 of one embodiment in the form of a petri dish such as an agar plate. 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 placement unit 100-1 includes a plurality of division cells 110 in a multi-well form and an input unit 130 through which the sample S can be introduced, and the plurality of division cells 110 And the input unit 130 may be in communication with the microfluidic channel (135). In the sample placement unit 100-1, when the sample S is input to the input unit 130, the sample S may be evenly distributed to each of the divided cells 110 through the microfluidic channel 135.

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

On the other hand, the sample placement unit 100-1 is provided with a blocking area on at least the side of each of the divided cells 110, so that the wave incident to each divided cell 110 does not flow into the other divided cells 110 I can do it. Through the blocking area, the sample placement unit 100-1 can improve the accuracy of the population count by minimizing wave interference between the split cells 110. In addition, the sample placement unit 100-1 described above may be provided as a disposable kit.

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

9 and 10, the microbial entity counting system is the first wave signal scattered from the sample (S) accommodated in a plurality of divided cells 110, the wave of the wave source 200 is scattered in the sample (S) It may further include an optical unit 550 for modulating 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 scattered wave is incident from the measurement object, the optical unit 550 may control the wave surface of the scattered wave to restore the wave (light) before being scattered again, and provide it to the detection unit 300.

The spatial light modulator 551 may receive waves (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 collect light and provide it to the detection unit 300 again. The detection unit 300 may detect a wave collected by the lens 552 and restore and output a wave output from the original wave source before being scattered.

Here, the optical unit 550 may restore a first wave signal scattered from the sample S to a wave before being scattered when a microorganism is not present in a 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, so that the phase-controlled wavefront cannot be sensed. Cannot be tampered with. The microbial population counting system 10 including the above-described optical unit 550 more accurately estimates the presence or absence of microorganisms M in each of the plurality of split 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 as a unit of population using a chaotic wave sensor. Through this, embodiments of the present invention can quickly calculate the number of microorganisms contained in the entire sample by detecting and counting the presence or absence of microorganisms in each of the plurality of divided cells. The microbial population counting system and the microbial population counting method according to embodiments of the present invention can be detected without culturing microorganisms with colonies, and thus the culture time can be significantly reduced to shorten the time for population counting.

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

10: Microbial population counting system
100: sample placement unit
110: split cell
200: wave source
300: detection unit
400: control unit

Claims (10)

Preparing a sample placement 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 irradiating waves to the plurality of divided cells;
Sequentially detecting individual wave information emitted from samples accommodated in each divided cell in association with the sequentially irradiated wave;
Determining the presence or absence of microorganisms in each divided cell using the individual wave information; And
Comprising the step of calculating the number of microorganisms in the sample using the number of the divided cells in which the microorganisms; includes,
The individual wave information,
It is information obtained by detecting a laser speckle generated by multiple scattering from samples accommodated in each of the plurality of divided cells at preset time points,
The sample placement unit comprises a multi-scattering amplification region for amplifying the number of times the wave incident on the plurality of divided cells is multi-scattered in the sample, the counting method of the number of microorganisms.
delete According to claim 1,
The step of determining the presence or absence of the microorganism,
The step of acquiring temporal correlation of the detected laser speckle using the detected laser speckle and determining the presence or absence of microorganisms in the corresponding divided cell based on the obtained time correlation. , Microbial population counting method. According to claim 3,
The individual wave information includes first image information of the laser speckle detected at a first time point among laser speckles emitted from the corresponding split cell, second image information of the laser speckle detected at a second time point, and 3rd image information of the laser speckle detected at 3 time points,
The first time point to the third time point are different time points,
In the determining of the presence or absence of the microorganism, the presence or absence of the microorganism is determined using a difference between the first image information and the third image information, and the method for counting the number of microorganisms. According to claim 1,
The plurality of divided cells are arranged in a matrix (matrix) form,
The number of the plurality of divided cells is greater than the expected number of microorganisms contained in the sample, the method of counting the number of microorganisms. A sample placement unit each containing a culture material and having a plurality of divided cells for distributing and receiving samples to be measured;
A wave source sequentially irradiating waves to the plurality of divided cells;
A detection unit sequentially detecting individual wave information emitted from a sample accommodated in each divided cell in connection with the sequentially irradiated wave; And
Includes a control unit for determining the presence or absence of microorganisms in each division cell using the detected individual wave information, and calculating the number of microorganisms in the sample using the number of division cells in which the microorganisms are present.
The individual wave information,
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 includes a multi-scattering amplification region for amplifying the number of times the wave incident on the plurality of divided cells is multi-scattered in the sample, the counting system for the number of microorganisms.
delete The method of claim 6,
The control unit obtains a time correlation of the detected laser speckle using a fraud-detected laser speckle, and determines the presence or absence of microorganisms in the divided cell based on the obtained time correlation, the number of microorganisms Counting system. The method of claim 8,
The individual wave information includes first image information of the laser speckle detected at a first time point among laser speckles emitted from the corresponding split cell, second image information of the laser speckle detected at a second time point, and 3rd image information of the laser speckle detected at 3 time points,
The first time point to the third time point are different time points,
The control unit determines the presence or absence of the microorganism by using a difference between the first image information and the third image information, the counting system for the number of microorganisms. The method of claim 6,
The plurality of divided cells are arranged in a matrix (matrix) form,
The number of the plurality of division cells is greater than the expected number of microorganisms contained in the sample, the counting system of the number of microorganisms.

Images