JP3055017B2 - Fungi instant discrimination device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for easily and accurately determining the number of viable or dead bacteria or species of bacteria such as bacteria and fungi that are mixed or adhered to foods that are particularly required to be hygienically controlled. The present invention relates to a discriminating device that can be immediately discriminated.

[0002]

2. Description of the Related Art In recent years, there has been an increasing trend toward health with the social background of aging and a declining birthrate. Purchase refusals of fresh foods and processed foods containing preservatives or coloring agents have been further strengthened. On the other hand, bacteria and fungi are cultivated under nutrient sources, moisture, temperature and oxygen, so foods have favorable breeding conditions. Or, combined with the non-use of preservatives related to antibacterial, mixed with the raw materials of food from the beginning, or propagated in a short time if attached or mixed at the processing stage or distribution stage, not only deterioration of food and spoilage Not only will this result in a serious accident such as food poisoning, but the occurrence of such a result will also result in the inability to commerce and the demand for enormous damages in accordance with the PL Act. For this reason, producers of foods have been required to further improve sanitary control at the stage of raw materials and processing and shipping, and also at distributors at the stage of receiving or selling them.

[0003] Conventionally, regarding the hygiene management of foods, which is a so-called fungus test, fungi collected from a desired specimen are diluted with physiological saline or the like, inoculated in an appropriate medium, cultured for about 24 to 48 hours, and then visually enlarged. Because it is a judgment, not only takes a lot of time for the judgment, but also the judgment result is low in reliability,
In addition, production and shipping must be interrupted until the discrimination result is found. Furthermore, recently, means for increasing the reliability of visual discrimination by emitting light from fungal cells using a luminescent enzyme has been proposed. In the case of (1), meat chips and meat juice mixed in with the collection of fungi also emit light, so that there is an inherent problem that it is difficult to distinguish them from fungi.

In view of such circumstances, the present inventors have developed a fluorescent-stained bacterium in which viable cells and dead cells of fungi collected by appropriate means are selectively penetrated with fluorescent dyes having different fluorescent wavelengths. The liquid is irradiated with excitation light having a center wavelength of 488 nm, and the fluorescent light emitted according to the Stokes law from the fluorescent dye in the viable cell and the fluorescent dye in the dead cell is enlarged and discriminated visually. Accordingly, a method and apparatus for immediately discriminating fungi have been developed, and the contents thereof have already been disclosed in Japanese Patent Application No. 8-125218 as a prior application.

[0005] However, as a result of many practical uses over a wide range of specimens using the prior invention, the adoption of further new technologies has been demanded. That is, the fungi collected by appropriate means at the time of preparing the fluorescent stained bacterial solution include various oils and fats in addition to meat juice, fruit juice, water, etc.
Dust, etc. are also mixed, and the concentration of fungi collected by the specimen is also diverse, so that the turbidity of the fluorescent stained bacterial solution itself that has been subjected to fluorescent staining is also wide, while the excitation light of a specific wavelength is previously determined. The excitation light from the excitation light source that emits light has low energy, and the emission intensity of the fluorescent dye by the excitation light is the strongest immediately after irradiation and decreases very quickly. Excitation light irradiation makes it difficult to obtain sufficient fluorescence emission due to the turbidity of the fluorescent stained bacterial solution, and the fluorescent light emitted in the fluorescent stained bacterial solution having a relatively high turbidity is not affected. However, since the so-called blurring occurs in the fluorescent light beam due to the diffraction phenomenon when passing through the fluorescent stained bacterial solution, even if the fluorescent light beam is temporarily expanded, it is still not possible to accurately determine the number of viable and dead bacteria. There are difficulties, and it is especially difficult to distinguish bacterial species from fluorescent light images. In addition, it is difficult to make an accurate determination because live bacteria move during the visual recognition, so that it is strongly desired to make a clear fluorescent light beam and convert it into an electric signal and perform numerical processing or image processing. .

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve such a problem, and the present invention has been made to solve the above-mentioned problems by using fluorescent light in a cell with strong light energy and pulsed excitation light having a required wavelength and pulse interval. By irradiating the stained bacterial solution, it emits fluorescent light with strong luminescence energy, transmits only a specific wavelength from the fluorescent light with high precision, and further enters a doubled electron tube as a clear fluorescent light by adjusting for blur. Accordingly, it is an object of the present invention to provide a device which can easily and accurately determine the number of viable bacteria, the number of dead bacteria, and the type of bacteria in a simple and immediate manner.

[0007]

The technical means used by the present invention to solve the above-mentioned problems is to selectively penetrate viable fungal cells and dead bacterial cells collected by appropriate means, respectively. The cell is filled with an appropriate amount of a fluorescent stained bacterium solution that has been fluorescently stained with the fluorescent dye to be prepared, and the pulse interval is strong within the fluorescence emission and decay time in an electromagnetic shield tube having one side opened. Fluorescence is emitted every time the pulsed excitation light is irradiated, and it is captured as an electrical signal. In addition, by capturing the time-dependent behavior of the live bacteria moving in the fluorescence stained bacterial solution of the cell, an accurate viable count can be obtained. 0.2 to 5 pulses /
A white pulse light source having a strong luminous energy is provided at intervals of seconds, that is, 5 pulses per second to 1 pulse every 5 seconds, and viable cells and dead cells in a cell are obtained from the white pulse light from the white light pulse light source. Energy is generated by an excitation light generating tube equipped with a band-pass filter that transmits a specific wavelength for exciting and emitting fluorescent dyes that have been dyed by penetration into cells, and a conductive filter that removes electric noise generated by pulse emission. While irradiating the pulse excitation light with high intensity, the fluorescent light emitted at a high luminous intensity by the pulse excitation light in two directions orthogonal to the irradiation optical axis of the pulse excitation light. On the other side, only the specific wavelength related to the fluorescent emission from the killed cells is used for the high wavelength region cut filter and the low wavelength region cut filter on the other side. A transparent gap is provided by a light source to transmit light with high precision, and a diffraction grating is interposed to compensate for the blur caused by diffraction in the fluorescent light and input to the doubled electron tube as a clear fluorescent light to convert it into an electric signal. In addition, the present invention resides in a fungus real-time discriminating device for performing numerical processing and image processing.

[0008]

The present invention has the following functions since it uses such technical means. That is, a white pulse light source which emits pulses at a pulse interval of 0.2 to 5 pulses / sec is provided in an electromagnetic wave shield tube having one side opened, so that a white pulse light having a strong light energy is provided. The pulsed excitation light is emitted by the excitation light generating tube provided with the band-pass filter and the conductive glass filter provided with a band-pass filter and a conductive glass filter through which a specific wavelength is transmitted from the white pulsed light. Electric noise mixed with the generation of the pulse is also removed by the conductive glass filter and the electromagnetic wave shielding tube, and the pulse excitation light having a required pulse interval and a specific wavelength is irradiated. In addition, fluorescent dyes selectively penetrated into living cells and dead cells by irradiation with pulse excitation light having strong light energy have a specific wavelength in accordance with Stokes' rule and a pulse interval. Correspondingly strong fluorescence, the viable and dead cells of each microbial microscopically differ in shape and size depending on the microbial species. Even in the case of living cells such as live bacterial cells, accurate grasp can be achieved by capturing an arbitrary number of fluorescent light beams for each pulse. The strong fluorescent light emitted by the irradiation of the pulsed excitation light has two sides orthogonal to the irradiation optical axis of the pulsed excitation light.
Further, on the other side, the fluorescent light from the killed cells is transmitted through the transmission gap between the high wavelength region cut filter and the low wavelength region cut filter having a sharp wavelength region cut gradient, so that the fluorescent light is further narrowed down to only a specific wavelength, Since the diffraction grating is interposed, the blur caused by the diffraction lurking in the narrowed fluorescent light of a specific wavelength is corrected, and the fluorescent light is input to the doubled electron tube as a clear and highly reliable fluorescent light, converted into an electric signal, and processed. Therefore, the number of viable bacteria, the number of dead bacteria, and the type of bacteria can be accurately determined.

[0009]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic explanatory view of the present invention, and FIG. 2 is a cross-sectional explanatory view of an excitation light generating tube. A fluorescently stained bacterial solution obtained by selectively permeating the fungi collected by more appropriate means into the viable cells and the dead cells, and performing fluorescent staining with a fluorescent dye having a specific fluorescent emission. A cell 1 into which 1A is appropriately injected is used. Many fluorescent dyes have been proposed for selectively penetrating viable cells and dead cells when preparing the fluorescent stained bacterial solution 1A, but it is important to note that the wavelength of the excitation light to be irradiated is important. Since the Stokes law is established between the fluorescent dye and the fluorescent wavelength of the fluorescent dye, the wavelength of the excitation light is also determined by the fluorescent dye used. As a fluorescent dye suitable for preparing the fluorescent stained bacterial solution 1A in the present invention, a fluorescent dye composed of fluorescein or a derivative thereof is exemplified as a fluorescent dye excellent in permeation staining into living cells. Fluorescent dyes from propidium iodide are ones that are excellent in infiltration dyeing into the inside. In the case of such fluorescent dyes, by using 488 nm as the wavelength of excitation light, a fluorescent dye composed of fluorescein or a derivative thereof is used. A fluorescent dye consisting of 520 nm as a fluorescent wavelength and 625 nm as a fluorescent dye emits fluorescent light at 625 nm as a fluorescent wavelength.

In the cell 1 into which the fluorescent stained bacterial solution 1A is injected in an appropriate amount, the excitation light irradiated to excite the injected fluorescent stained bacterial solution 1A is sufficiently transmitted without being absorbed or blocked. It is made of a material having excellent translucency because it is necessary to sufficiently transmit fluorescent light emitted from live cells or dead cells excited by the excitation light, and is usually made of glass or acrylic resin. Material is used. Further, in order to determine the number of viable cells, the number of dead cells, or the type of bacteria by fluorescence emission from viable cells or dead cells, at least about 1 ml to about 3 ml of the fluorescent stained bacterial solution 1A is injected into the cell 1. The capacity of the cell 1 is formed so that the fluorescent light emitted by the irradiation of the excitation light is received in two directions orthogonal to the irradiation optical axis of the excitation light and converted into an electric signal.
The shape of the cell 1 is formed in a cylindrical shape having a bottom, or preferably in a prismatic shape.

According to the present invention, the fluorescent dye which has been permeated and stained into the viable cells and the dead cells of the fluorescent-stained bacterial solution 1A emits fluorescent light at a high luminous intensity, and then the excitation light itself to be irradiated is irradiated. When increasing the intensity of the excitation light and irradiating the excitation light consisting of continuous light, the fluorescence emission intensity decreases within a short period of time. It is desirable from the viewpoint of accurate determination of the number of viable cells, the number of dead cells, or the type of bacteria, and that it can be more accurately determined even when the cells are always moving like live cells. Become.

The present invention solves such a problem by using a pulsed excitation light 20. The pulsed excitation light 20 is formed and irradiated by an excitation light generating tube 2 as shown in FIG. The excitation light generating tube 2 has a pulse interval of 0.1 mm in an electromagnetic wave shielding tube 2A having one side open.
A white pulse light source 2B having a strong light emission intensity of 2 to 5 pulses / second is provided, and live and dead cells of the fluorescent stained bacterial solution 1A are obtained from white pulse light from the white pulse light source 2B. A band-pass filter 2C that transmits a specific wavelength that excites and emits a fluorescent dye that has been dyed by permeation dyeing is provided, and a conductive glass filter 2D for removing electric noise that is mixed when white pulse light is emitted is provided. It has a configuration provided. In such a case, a xenon lamp is generally used as the white pulse light source 2B because the emission wavelength has a wide wavelength range of 250 nm to 2 μm and the emission intensity is extremely high. Then, the pulse interval of the white pulse light source 2B is irradiated with the pulse excitation light 20 formed of the white pulse light, and the fluorescent dye solution 1A
The fluorescent dye, which has been penetrated and stained into live and dead cells, fluoresces and attenuates for about 200 to 250 μsec. Since there is a latent time lag of approximately 30 μs before the light emission, the fluorescent light emission at the time of the maximum light emission may be captured for discrimination. Therefore, the pulse interval is limited to a maximum of 5 pulses / second or less. On the other hand, the fluorescence emission from the viable bacterial cells fluctuates every moment due to the movement of the viable bacterial cells. Although it is necessary to determine the number of bacteria, the surplus time is at least 0.2 pulses / sec or more and the number of fluorescent light emission is 5 to 10 pulses since the extra time may adversely affect the propagation dynamics of live bacteria. Can be sufficiently determined by the average value of In the practical use of the present invention, the pulse interval of the pulse excitation light 20 is used at 0.5 or 1.0 pulse / second.

Thus, the pulse excitation light 2 is converted from the white pulse light emitted from the white light pulse light source 2B at a required pulse interval.
The band-pass filter 2C for forming the fluorescent light of the fluorescent dye according to the Stokes law for the excitation light of the fluorescent dye which is selectively penetrantly stained into the viable cells and the dead cells of the fluorescent stained bacterial solution 1A. And a fluorescent dye composed of fluorescein or a derivative thereof that selectively penetrates and stains live cells, and is used for dead cells. When a fluorescent dye consisting of propidium iodide which is to be dyed by permeation is used, the band-pass filter 2C which transmits a specific wavelength of 488 nm according to the Stokes law is used. Further, a conductive glass filter 2D is provided on the opening side of the electromagnetic wave shield tube 2A.
By D, the electric noise mixed in the emission of the white pulse light is conductively removed, and the pulse excitation light 20 having the required emission intensity, wavelength and pulse interval is emitted from the excitation light emission tube 2. In such a case, a specific example of the conductive glass filter 2D is one in which indium oxide or tin oxide is applied to one side surface of a light-transmitting glass and has an electric resistance of about 10 Ω / cm ² . In addition, the conductive glass filter 2D is naturally fixed to the electromagnetic wave shielding tube 2A with a conductive adhesive or the like and grounded.

By irradiating the cell with the pulsed excitation light 20 having a high emission intensity and a required wavelength and pulse interval, the viable cells and the dead cells of the fluorescently stained bacterium solution 1A in the cell 1 are scattered. The fluorescent dye, which has been subjected to penetration dyeing, emits fluorescent light with a high fluorescent intensity at each pulse interval according to the Stokes' law, and such fluorescent light is emitted by the pulse excitation light 20.
In each of the two directions perpendicular to the irradiation optical axis, the fluorescent light is corrected with high precision and clear, and then converted into an electric signal by a doubling electron tube and then subjected to necessary processing.

That is, in the fluorescent light of high fluorescence intensity emitted from the viable cells and the dead cells by irradiation with the pulse excitation light 20, approximately 15 to 15 wavelengths before and after a specific wavelength according to the Stokes law are centered. Within the wavelength range of 25 nm,
A peak having a fluorescence intensity of about 1/2 to 1/3 of the maximum fluorescence intensity, a so-called half width,
When the fluorescent light in such a state is converted into an electric signal and subjected to a required process, not only a significant error occurs in the number of viable bacteria and the number of dead bacteria, but also it is difficult to capture even fine shapes and sizes. Species identification is also difficult. Therefore, in order to transmit only a specific wavelength of the fluorescent light from the viable cell and the dead cell with high precision and to obtain a highly reliable fluorescent light, a high wavelength region cut with a sharp wavelength region cut gradient as shown in FIG. The filter 3 and the low-wavelength region cut filter 3A are provided with a transmission gap 30 with high precision to limit transmission of only a specific wavelength. For example, when the wavelength of fluorescent light from live bacterial cells is 520 nm, Is formed by combining a high wavelength region cut filter 3 having a sharp cut gradient in a high wavelength region of 521 nm or more and a low wavelength region cut filter 3A having a sharp cut gradient in a low wavelength region of 519 nm or less. 51 in the gap 30
Only the fluorescent light in the range of 9 to 521 nm can be transmitted, and it is desired that the allowable range of the fluorescent light transmitted through the transmission gap 30 be as small as possible.

In addition, it is important that the high wavelength region cut filter 3 and the low wavelength region cut filter 3A
Fluorescent light from live cells and dead cells can be transmitted through the fluorescent stained bacterial solution 1A
When the light passes through various turbid substances in the inside or through the narrow transmission gap 30, a diffraction phenomenon acts, and blurs are mixed in the fluorescent light. For this reason, the fluorescent light of a specific wavelength transmitted through the transmission gap 30 is further transmitted through a diffraction grating, thereby compensating for diffraction in the fluorescent light, thereby providing an accurate and clear fluorescent light. In addition, it is necessary to make the light enter the doubled electron tube 5 and convert it into an electric signal to perform numerical processing and image processing. The diffraction grating 4 is formed by forming an extremely fine groove grating on the surface of a light-transmitting glass plate. The formation density of the groove grating varies depending on the specific wavelength of a specific wavelength to be transmitted, but the transmission wavelength is approximately 520 to 620 nm. In this case, a groove grating having a formation density of about 920 lines / mm is suitable for diffraction compensation. Since the fluorescent light whose diffraction has been corrected by the diffraction grating 4 is polarized at an angle of about 30 to 36 with respect to the light incident axis before transmission, the diffraction-corrected fluorescent light is incident on the fluorescent light. A doubling electron tube 5 for converting into a signal is provided so as to correspond to the axial light of the polarized fluorescent light beam.

The doubling electron tube 5 is not particularly limited, and has a doubling ability capable of accurately detecting fluorescent light rays from viable cells and dead cells and discriminating the number of viable cells, dead cells, and species. Anything is fine. There is no particular limitation on the processing unit 6 for performing desired numerical processing or image processing after converting the fluorescent light beam into an electric signal by the doubling electron tube 5, but of course the pulse excitation light irradiation trigger and the fluorescent light emission Algorithm to remove the time lag until and hold the maximum fluorescence intensity
In addition, an algorithm for obtaining an average value from an arbitrary number of hold values of the maximum fluorescence emission intensity for each pulse of the pulse excitation light is incorporated.

FIG. 4 is an explanatory view showing the inside of the notch according to the present invention.
The method of use of the present invention will be described with reference to FIG. 4. In this method, fungi collected from a specimen by appropriate means are selectively penetrated into a physiological saline solution into viable cells and dead cells, respectively, and fluorescently stained. A cell 1 which is mixed with a dye solution in which a light dye is dissolved to form a fluorescent stained bacterial solution 1A and into which a suitable amount of the fluorescent stained bacterial solution 1A is injected is placed above the casing 7 of the present invention. The lid 7A, which is provided to be openable and closable, is opened to be inserted and held, and then the lid 7A is closed to be in a dark room state. By irradiating the cell 1 with the required pulsed excitation light 20 from the excitation light generating tube 2, fluorescent cells of different specific wavelengths are emitted from live cells and dead cells, and this fluorescent light is emitted. High wavelength region cut filter 3 and low wavelength region cut filter 3A provided in two directions orthogonal to the irradiation optical axis of the pulse excitation light, respectively.
Through the transmission gap 30 and the diffraction grating 4 and the doubled electron tube 5 transmits a fluorescent light beam relating to the number of viable bacteria and the viable bacterial species, and a fluorescent light beam relating to the number of dead bacteria and the viable bacterial species to an electric signal. Then, numerical processing or image processing is performed in the processing device 6 and displayed on the display unit 7B.

[0019]

As described above, the present invention allows a band-pass filter, which has a strong luminous intensity and transmits a specific wavelength from white pulse light having a pulse interval of 0.2 to 5 pulses / second, to pass therethrough. Because pulse excitation light is formed, pulse excitation light with high light energy is applied, and even in turbid fluorescent stained bacterial solutions, fluorescent dyes that have been penetrated into live and dead cells can be used. High fluorescence emission can be achieved by sufficient excitation. Also, because of the pulse excitation light, the maximum fluorescent light emission can be captured for each pulse, and an arbitrary number of the maximum fluorescent light emission can be captured within a very short time. The number and the number of dead bacteria can be grasped. The fluorescent light emitted by the pulse excitation light receives the fluorescent light from live cells and dead cells in two directions perpendicular to the irradiation optical axis of the pulse excitation light. Fluorescent rays of specific wavelengths from the living and dead cells are not affected by light, and the high-precision and low-wavelength cut filters with sharp cut-off gradients in the wavelength range respectively provide high precision. By transmitting the light through the transmission gap, a fluorescent light having a very limited specific wavelength is obtained. Since the light is transmitted through the diffraction grating, diffraction mixed in the fluorescent light is removed, and the fluorescent light is doubled as an accurate and clear fluorescent light. Since the light is incident on the electron tube, converted into an electric signal, and then processed, the type of bacteria can be reliably determined in addition to the accurate number of viable and dead bacteria. In the present invention, the operation is simple because only the cells are inserted and held, and the number of viable bacteria, the number of dead bacteria, or the type of bacteria that have been quantified or imaged can be determined within at most several minutes. It can be said that it is an instantaneous discrimination device for fungi that has extremely many features, such as immediate hygiene control of foods distributed from factories as well as food products.

[Brief description of the drawings]

FIG. 1 is a schematic explanatory diagram of the present invention.

FIG. 2 is an explanatory sectional view of an excitation light generating tube.

FIG. 3 is an explanatory diagram of a high wavelength region cut filter and a low wavelength region cut filter.

FIG. 4 is an explanatory view of the inside of the notch according to the present invention.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 cell 1A Fluorescent staining bacteria solution 2 Excitation light generating tube 2A Electromagnetic wave shielding tube 2B White pulse light source 2C Bandpass filter 2D Conductive glass filter 20 Pulse excitation light 3 High wavelength region cut filter 3A Low wavelength region cut filter 30 Transmission gap 4 diffraction grating 5 double electron tube 6 processing unit 7 casing 7A lid

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) C12M 1/34 G01N 21/64 G01N 33/48 G01N 21/76-21/78

Claims (1)

(57) [Claims] 1. A fluorescently stained bacterial solution obtained by penetrating a fungus, which has been collected from a specimen by a suitable means, into a cell with a required fluorescently stained solution, is irradiated with excitation light, and the fluorescence emission is converted into an electric signal. In an instant fungus discriminating apparatus for discriminating the number of viable bacteria, the number of dead bacteria and the type of bacteria by conversion, a pulse interval of 0.2 to 5 pulses /
Excitation provided with a white pulse light source that emits light in seconds, and a bandpass filter that transmits a specific wavelength in the white pulse light, and a conductive glass filter that removes electric noise mixed in the white pulse light. The light-generating tube and the fluorescence-stained bacterium solution cell irradiated with the excitation light are irradiated in two directions perpendicular to the excitation light irradiation optical axis, and each of the specific wavelengths related to the fluorescence emission from viable cells and dead cells is used. Is transmitted through the transmission gap of the high-wavelength region cut filter and the low-wavelength region cut filter with high precision, and is passed through a diffraction grating to compensate for a clear fluorescent light, input to a doubling electron tube, and convert it into an electric signal. A fungus instant discrimination device that discriminates the number of viable and dead bacteria and the type of bacteria easily and with high accuracy by performing required processing.