KR20170129308A - optical technique for detecting microbes or bacteria in bacterial culture dishes using laser speckle patterns - Google Patents

Abstract

The present invention proposes a technology capable of quickly and precisely measure a presence or absence of microorganisms in a culture dish using a laser speckle. That is, the present invention is configured to measure a laser speckle shape reflected or transmitted after a light source with good coherency is irradiated onto a bacterial culture dish, analyze a change in time of the measured speckle signal, and quickly and precisely measures the presence of microorganisms in the culture dish, activities of the microorganisms, resistance to antibiotics, etc., in a non-contact manner.

Description

[TECHNICAL FIELD] The present invention relates to a method for detecting bacteria in a culture dish using a laser speckle,

The following description relates to a technique for measuring bacteria in a culture dish using laser speckle.

Currently, a commonly used method for determining the presence or absence of microorganisms in a culture dish is a culture observation (Fig. 1A). When a specimen (mainly a microorganism such as bacteria) is injected into a culture dish in which the culture liquid is applied in a semi-solid state or a culture liquid is mixed in a liquid state, and the microorganism is cultured for about 1-2 days after application to a large area, It grows throughout the dish. At this time, the presence or absence of the microorganisms generally determines whether the microorganisms cultured through the ducts are forming colony. After the microorganism specimens thus cultured were injected with various antibiotics, the microorganisms were examined for resistance to antibiotics by observing whether or not the microorganisms were growing around the antibiotics (Fig. 1B).

This conventional method has a disadvantage in that it takes a very long time (one to four days) to cultivate the bacteria. It takes time to grow to such an extent that it can be observed in the duct. In addition, some bacteria are generally easier to culture, but certain bacterial species have been found to be very difficult or difficult to cultivate because of certain bacterial species. In addition, there is a limitation that quantification can not be performed because observation is made with oil pipes.

Understanding the presence and activity of microorganisms in culture dishes is a critical issue in life sciences and medicine. For example, in infectious diseases, antibiotic resistance of bacteria extracted from patient's specimens is measured and appropriate treatment methods are determined.

Through the present invention, it is possible to quickly and accurately measure the presence of microorganisms in a culture dish. It is possible to shorten the time of life science and medicine dealing with microorganisms by omitting the culturing process from the first 1 to 4 days and preparing the specimens and immediately measuring the presence of the microorganisms and the reactivity of the microorganisms to the antibiotics . Also, since it is a simple principle that can be realized by only a laser light source and an image sensor, it has high price competitiveness in commercialization. Also, since it can be manufactured in a small size, it can be widely applied in various experiment environments.

And the proposed invention uses optical measurement. Therefore, there is no need to use target substances such as antigen-antibody and there is no need to collect specimens such as gene amplification technology. Therefore, the cost of microbial measurement can be greatly reduced.

FIG. 1A is a schematic view of a method for generally observing the presence or absence of microorganisms in a culture dish after cultivation.
FIG. 1B is a graph showing the resistance of microorganisms to antibiotics by observing whether or not microorganisms grow around the antibiotics after injecting various antibiotics into the cultured microorganism specimens.
2 is a view for explaining the form of speckle.
3 is a diagram for explaining the application of the entire invention of the present invention.
FIG. 4A is a view for explaining the measurement of the presence or absence of microorganisms by comparing differences between two speckle images measured at predetermined time intervals. FIG.
FIG. 4B is a graph showing the results of an experiment in which microorganisms are detected on a culture dish by applying a time correlation analysis method. FIG.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings.

It is an object of the present invention to provide an optical method capable of quickly and accurately measuring the presence and activity of microorganisms and the reactivity (resistance) to antibiotics.

Although the culture dish appears to be slightly transparent, due to the heterogeneous distribution of the internal refractive index, multiple scattering occurs when irradiated with a coherent light source such as a laser. The spatial distribution of intensity of multiple scattered light is very complex and is called a laser speckle pattern. The physical reason for the laser speckle is that the light has different degrees of constructive or destructive interference at each point of the measurement plane due to complex multiple scattering. Although the form of this speckle appears to be very complex, the form of speckle does not change if it does not move the spawning material, the culture dish, in this invention. This is because the scattering phenomenon is determined by the wave equation (Fig. 2).

However, when microorganisms are present in the culture dish, microscopic biological activities (microbial movement, microbial movement, etc.) of the microorganisms cause microscopic changes in the optical path over time. Such a fine change in the light path causes a large change in the speckle pattern because the speckle pattern is a phenomenon caused by light interference. Thus, by measuring the temporal changes in the speckle pattern, the life activity of the microorganism can be measured quickly.

Using this, it is possible to precisely measure the presence or absence of microorganisms and the reactivity to antibiotics in a few seconds, without incubating the microorganism for several days.

As a result of intensive studies in view of the above object, the inventor of the present invention has found that the activity and bacterial resistance of a microorganism in a culture dish can be simply and accurately measured.

Hereinafter, the present invention will be described in detail. The application of the whole invention is represented in FIG.

[1] Generation of speckle pattern on petri dish

In order to implement the present invention, speckle should be formed in a culture dish, and the time change of the speckle pattern should be measured.

In order to form the speckle, a laser light source having good coherence can be used. In this case, the shorter the spectral bandwidth of the light source that determines the coherence of the laser light source (ie, the longer the coherence length and the interference length), the greater the measurement accuracy. To realize the invention under realistic conditions, the following conditions must be maintained.

[2] Measurement of speckle pattern

The speckle pattern formed in this way can consist of a shape (Figure 3, left, middle) in which the laser is reflected on the specimen or a form in which the laser penetrates the specimen (Figure 3 right).

In order to measure the speckle pattern, a two-dimensional image sensor or a one-dimensional optical sensor can be used. In order to realize the invention, the size (d) of one pixel of the image sensor should be equal to or smaller than the grain size of the speckle pattern.

Also, if the pixel size becomes too small, the undersample becomes severe and the pixel resolution of the sensor is not utilized well. Therefore, to achieve an effective signal-to-noise ratio, it is advantageous to arrange the sensor so that no more than five pixels are located in the speckle grain size.

[3] Analysis of speckle signals

In order to compare the dynamic changes of the speckle signal, at least two images measured at different times are needed. The following speckle signal analysis example can be used.

As a first example, it is possible to measure the presence or absence of microorganisms by comparing differences between two speckle images measured at regular time intervals (FIG. 4A). In the absence of microorganisms (agar plate), the difference between the speckle image measured at 0 second and the speckle image measured at 10 seconds is negligible. The small signal difference can be seen as a contribution to all the noise existing in the experiment such as moisture evaporation and vibration. However, in the case of microorganisms (B. cereus, E. coli bacteria), the presence of bacteria can be determined by a large change in the signal measured between 0 and 10 seconds.

에 대해 측정한 스페클 영상을 평준화 한 데이터를

라고 하면, 특정 지연 시간

에 대해, 각 지점에서 시간 상관 계수를 다음 수식으로 계산할 수 있다.As another example, time correlation analysis can be performed on a plurality of speckle images measured at regular time intervals. Each hour

Data obtained by normalizing the speckle image

, A specific delay time

, The time correlation coefficient at each point can be calculated by the following equation.

The result of the experiment in which microorganisms are detected on the culture dish by applying the time correlation analysis method is shown in FIG.

The apparatus described above may be implemented as a hardware component, a software component, and / or a combination of hardware components and software components. For example, the apparatus and components described in the embodiments may be implemented within a computer system, such as, for example, a processor, a controller, an arithmetic logic unit (ALU), a digital signal processor, a microcomputer, a field programmable gate array (FPGA) , A programmable logic unit (PLU), a microprocessor, or any other device capable of executing and responding to instructions. The processing device may execute an operating system (OS) and one or more software applications running on the operating system. The processing device may also access, store, manipulate, process, and generate data in response to execution of the software. For ease of understanding, the processing apparatus may be described as being used singly, but those skilled in the art will recognize that the processing apparatus may have a plurality of processing elements and / As shown in FIG. For example, the processing unit may comprise a plurality of processors or one processor and one controller. Other processing configurations are also possible, such as a parallel processor.

The software may include a computer program, code, instructions, or a combination of one or more of the foregoing, and may be configured to configure the processing device to operate as desired or to process it collectively or collectively Device can be commanded. The software and / or data may be in the form of any type of machine, component, physical device, virtual equipment, computer storage media, or device , Or may be permanently or temporarily embodied in a transmitted signal wave. The software may be distributed over a networked computer system and stored or executed in a distributed manner. The software and data may be stored on one or more computer readable recording media.

The method according to an embodiment may be implemented in the form of a program command that can be executed through various computer means and recorded in a computer-readable medium. The computer-readable medium may include program instructions, data files, data structures, and the like, alone or in combination. The program instructions to be recorded on the medium may be those specially designed and configured for the embodiments or may be available to those skilled in the art of computer software. Examples of computer-readable media include magnetic media such as hard disks, floppy disks and magnetic tape; optical media such as CD-ROMs and DVDs; magnetic media such as floppy disks; Magneto-optical media, and hardware devices specifically configured to store and execute program instructions such as ROM, RAM, flash memory, and the like. Examples of program instructions include machine language code such as those produced by a compiler, as well as high-level language code that can be executed by a computer using an interpreter or the like. The hardware devices described above may be configured to operate as one or more software modules to perform the operations of the embodiments, and vice versa.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. For example, it is to be understood that the techniques described may be performed in a different order than the described methods, and / or that components of the described systems, structures, devices, circuits, Lt; / RTI > or equivalents, even if it is replaced or replaced.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (1)

Irradiating a bacterial culture dish with a coherent light source and measuring the reflected or transmitted laser speckle pattern; And
Analyzing the time change of the measured speckle signal to quickly and accurately measure the presence or absence of microorganisms in the culture dish, the activity of the microorganism,
Lt; RTI ID = 0.0 > luciferase < / RTI >

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