WO1997038128A1 - Method of immediately discriminating bacteria and apparatus therefor - Google Patents

Method of immediately discriminating bacteria and apparatus therefor Download PDF

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Publication number
WO1997038128A1
WO1997038128A1 PCT/JP1996/002370 JP9602370W WO9738128A1 WO 1997038128 A1 WO1997038128 A1 WO 1997038128A1 JP 9602370 W JP9602370 W JP 9602370W WO 9738128 A1 WO9738128 A1 WO 9738128A1
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Prior art keywords
light
fluorescent
bacteria
fungi
cells
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PCT/JP1996/002370
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French (fr)
Japanese (ja)
Inventor
Yoshiyuki Tokuda
Jongchul Park
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Nippon Mizushorigiken Co., Ltd.
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Priority to AU67547/96A priority Critical patent/AU6754796A/en
Publication of WO1997038128A1 publication Critical patent/WO1997038128A1/en

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/10—DNA staining
    • C12Q2304/13—Propidium

Definitions

  • fungi such as bacteria are adhered or collected, and the fungi are appropriately adhered and collected from a specimen to which the fungi adhere or are mixed, and the bacterial species, the amount of attached fungi, the amount of viable bacteria, and the death Bacteria can be distinguished, and especially for food producers and distributors, it is extremely useful for hygiene management, and the method for immediately distinguishing fungi and its discriminating apparatus are used.
  • Background art fungi such as bacteria are adhered or collected, and the fungi are appropriately adhered and collected from a specimen to which the fungi adhere or are mixed, and the bacterial species, the amount of attached fungi, the amount of viable bacteria, and the death Bacteria can be distinguished, and especially for food producers and distributors, it is extremely useful for hygiene management, and the method for immediately distinguishing fungi and its discriminating apparatus are used.
  • Foods especially fresh foods and processed foods with a high moisture content, have conditions that facilitate the propagation of fungi such as bacteria and bacteria, and these foods contain fungi from the beginning or In the process leading to its distribution and consumption, it adheres or falls into the water, which not only propagates in a short time and becomes remarkably unhealthy, but also results in food poisoning along with deterioration and spoilage of foods.
  • preservatives have been added to the extent permitted to prevent the growth of fungi.
  • the use of preservatives is considered dangerous, and consumers are forced to select non-additive processed foods.There is a growing demand for these products, so they must deal with them without additives.Product liability to strengthen consumer protection In conjunction with the enforcement of the liability law, food producers and distributors are in high demand for sanitary D control at the time of production or distribution.
  • fungi collected from specimens are inoculated in a coloring medium in which a coloring enzyme is mixed with an appropriate medium, and the incubator is kept at a required temperature for about 24 hours to 48 hours. Incubate for a period of time to form colonies, which are then retained by Escherichia coli, etc .; S-galactosidase enzyme
  • a fluorescent dye is mixed with physiological saline to an appropriate degree.
  • the luminescence energy is also weak, so a high-precision high-power microscope must be used, and the device for discrimination must use an extremely expensive and large-sized device.
  • the present invention provides a method of appropriately depositing and collecting bacteria from a specimen to which bacteria such as bacteria and the like are contaminated, and using a simple method and an inexpensive device to immediately kill live bacteria.
  • the dye can be penetrated into fungal cells, and in the viable cell ⁇ , the dye is dispersed and emits fluorescence with the absorption of the excitation light, and in the dead cell ⁇ , the dye is taken into the cell tissue to absorb the excitation light.
  • a fluorescent dye consisting of fluorescein or a derivative thereof, which cannot emit fluorescence, and an aropidum iodide which cannot penetrate living cells but penetrates only dead cells and emits fluorescence by absorbing excitation light.
  • Fluorescent dye is mixed with physiological saline at a concentration of 3 to 1 o1 / m1 and from salts, chitinus or cellulase so as to increase the penetration into the cells and to effectively stain the cells with the fluorescent dye.
  • a fungus collected from a specimen is mixed into a staining solution obtained by mixing 1 to 1 Ojmo IZm1 with a staining accelerator, and the mixture is stained while being heated to a required temperature.
  • Fluorescent dye consisting of fluorescein or its derivative penetrated into fungal cells by irradiating it with excitation light having a central wavelength of 488 nm from the lower side, It emits green fluorescent light with a slightly yellowish color of 520 nm, and the fluorescent dye consisting of propidium iodide gives a strong reddish orange fluorescent light with a central wavelength of 625 nm. It emits light.
  • FIG. 1 is an explanatory diagram of a method for instantaneously identifying fungi according to the present invention.
  • FIG. 2 is an explanatory diagram of an apparatus for instantly identifying fungi according to the present invention.
  • FIG. 3 is a diagram for immediately identifying fungi by computer-graphics processing.
  • FIG. 3 is an explanatory view of the apparatus, and shows the best form for implementing the ⁇ invention.
  • FIG. 1 shows a flow chart of a method for immediately discriminating fungi.
  • Bacterial susceptible bacteria collected by the method described in (1) include, for example, adding a sterilized halo stick to physiological saline and attaching it to the surface of the sample.
  • the method includes contacting a water-soluble medium with the surface of a sample and collecting the sample.
  • the fungus 1 thus collected can penetrate the physiological saline 2 into the cells of live and dead bacteria, and is dispersed in the viable cells, and is taken up and dispersed in the dead cells in the dead cells.
  • a fluorescent dye 3 consisting of fluorophore or its derivative, which emits strong fluorescence with a center wavelength of 520 nm due to absorption of excitation light having a center wavelength of 488 nm; It can not penetrate into cells but can die and can penetrate into cells, and can absorb and excite excitation light with a center wavelength of 488 nm, and emit strong fluorescence with a center wavelength of 625 nm
  • Uruvide Fluorescent dyes 4 consisting of umodide, 3 mo 1 / m 1 each The mixture is mixed within the range of 15 mo 1 ⁇ ⁇ 1, and the physiological saline solution 2 is made flexible so that the fluorescent dyes 3 and 4 can sufficiently penetrate and disperse in the fungal cells.
  • Staining solution 20 is prepared by mixing 3 mo 1 / m 1 to 15 mo 1 Nom 1 of dyeing accelerator 5 consisting of salt, chiness or cellulase.
  • Specific examples of the salts used for the dye accelerator 5 include sodium chloride and magnesium chloride.
  • the staining solution 20 formed as described above is mixed with the fungus 1 collected from the specimen to perform staining. In this case, the staining solution 20 is heated to an appropriate temperature in order to enhance the staining property. It is preferable to use a temperature of 25 ° C. to 35 ° C. in general, and the time required for dyeing at such a temperature is about 5 to 15 minutes.
  • the fungi 1 mixed into the staining solution 20 are not affected by the stain-promoting agent 5, but the softening of the membrane promotes the penetration of the fluorescent dyes 3 and 4 into the cells. In this case, the fluorescent dyes 3 and 4 that have permeated into the cells are washed away again, resulting in indistinguishability due to a decrease in fluorescence emission.
  • Fig. 2 is an explanatory diagram of a discrimination device that discriminates the species, attached amount, viable bacteria, and dead bacteria of fungus 1 collected from a sample using the stained bacterial solution 21 in which the staining is stabilized. Since the cells of the fungus 1 stained with the fluorescent dyes 3 and 4 are microscopic, it is necessary to create fluorescent light as highly visible as possible from the fluorescent dyes 3 and 4 in the cells. By the way, the fluorescent dye 3 consisting of fluorescein or its derivative, which penetrates and disperses into viable fungal cells of fungi 1, absorbs the excitation light having a center wavelength of 488 nm and absorbs the center of the fluorescent dye according to the S1 ⁇ 1x rule.
  • the fluorescent dye 4 composed of propidum iodide, which emits strong fluorescent light with a wavelength of 520 nm and which can penetrate and disperse into dead cells but cannot penetrate into viable cells, has a central wave It emits strong fluorescent light with a center wavelength of 625 nm as it absorbs excitation light having a length of 488 nm.
  • an excitation light source 31 capable of irradiating excitation light having a center wavelength of 488 nm is provided below the casing 30 so that the inside thereof is held by a dark room streak.
  • a magnifying lens 32 of a required magnification, preferably about 100 to 500 times, and a light scattering process is provided at an intermediate position of the irradiation optical axis search.
  • the light-transmitting plate 33 is provided with a light-transmitting plate 33, and the light-scattering of the light-transmitting plate 33 preferably has a light scattering rate of about 5 to 50 times.
  • a specific example is frosted glass.
  • the stained bacterial solution 21 is dropped on the light-transmitting plate 33, and the excitation light source 31 provided therebelow is irradiated with excitation light having a center wavelength of 488 nm to thereby perform the staining.
  • Fluorescein or fluorescein which is permeated and stained into cells of fungus 1 mixed in bacterial liquid 21 and penetrates and disperses from viable bacteria as it absorbs excitation light, From the fluorescent dye 3 composed of the derivative, the fluorescence emission of the bacterial species and the fluorescence emission number related to the number of bacteria having a fluorescence emission color having a center wavelength of 50 nm and having different emission shapes and emission sizes are different.
  • a fluorescent dye composed of aropidum iodide is used to generate a fluorescent color having a central wavelength of 625 nm and a fluorescent color related to a bacterial species having a different luminescent shape and luminescent intensity.
  • the number of luminescence and the number of fluorescent light related to the number of bacteria are expanded by light scattering.
  • the large lens 3 2 It will be recognized and can be determined.
  • FIG. 3 is an explanatory diagram of a discrimination device for processing and displaying and storing the fluorescence emission light ⁇ from the fungus 1 visually discriminated by the magnifying lens 3 2 as an image or data in the instant fungus discrimination device of the present invention.
  • a band-pass filter 40 that transmits fluorescent light with a wavelength of 52 nm and a band-pass filter 41 that transmits fluorescent light with a wavelength of 65 nm can be freely used.
  • the filter is provided with a band-pass switching filter 4 that can be switched between the fluorescent light-emitting lights, and the fluorescent light that has passed through the magnifying lens 32 and passed through the band-pass filter 40 or 41 has a focal position at which the fluorescent light is emitted.
  • a video camera 5 is provided for converting the shape and size of the light beam, the number of fluorescent light emission or the number of fluorescent light emission into an electric signal, and the video camera 5 converts the converted electric signal.
  • Wakashi stored ⁇ required surface image processing Ku is connected to a computer 6 which is programmed to data processing is performed.
  • the fluorescent light having passed through the magnifying lens 32 has an image formed by the fluorescent light having a wavelength of 520 nm and an image formed by the fluorescent light having a wavelength of 625 ⁇ by switching the band-pass switching filter 4.
  • the fluorescent light having passed through the magnifying lens 32 has an image formed by the fluorescent light having a wavelength of 520 nm and an image formed by the fluorescent light having a wavelength of 625 ⁇ by switching the band-pass switching filter 4.
  • an image visually recognized by the magnifying lens 32 can be displayed and stored as a photograph or data.
  • a video camera 5 equipped with a band-pass filter 40 with a wavelength of 52 nm and a band-pass filter 141 with a wavelength of 6 25 ⁇ ⁇ were installed.
  • the same result can be obtained by using two video cameras 5 and processing the electric signal from each video camera 5 with the computer 6.
  • a fluorescent dye that emits a different fluorescent color in live and dead cells can be permeated and dispersed in a short time.
  • Stained bacteria solution that can prevent staining of the permeated and dispersed fluorescent dye is dropped onto a light-transmissive light-transmissive plate, and excitation is performed with a center wavelength of 488 nm from below.
  • fluorescence is emitted with a center wavelength of 250 nm, which is clear and highly visible from live bacteria, and a center wavelength of 625 nm, which is clear and highly visible from dead bacteria.
  • Fluorescence is emitted, and the emitted light ⁇ ⁇ is scattered by the light-transmitting plate and is enlarged in advance, so that even a relatively low-magnification magnifying lens can determine not only viable cells and dead cells, but also the shape and shape of the fluorescent light.
  • Bacterial species can be distinguished by the size, and the number of bacteria can be Since the distinction is clearly visible, hygiene management can be performed continuously without stopping the product even in the production and distribution processes of foods, and in the present invention, the determination results can be recorded and stored in the form of photographs and data. As a result, it is possible to appropriately deal with the occurrence of hygiene issues with consumers.

Abstract

A method of immediately discriminating bacteria which comprises mixing bacteria sampled from a specimen into a staining solution prepared by mixing physiological saline with a fluorescent dye comprising fluoresceine or a derivative thereof and another fluorescent dye comprising propidium iodide each in a required concentration and further with a staining accelerator in a given concentration, heating the resultant mixture to a required temperature to stain live and dead bacteria by causing the fluorescent dyes to penetrate into and disperse in the cells of the bacteria selectively, mixing the resultant mixture with an efflux inhibitor in a required concentration to prepare a stained bacteria solution, dropping this solution onto a light-transmitting plate which has been rendered light-scattering, irradiating the solution from under the plate with an exciting light having a central wavelength of 488 nm, and observing the fluorescent light emitted by the absorption of the exciting light, the configuration and size of the fluorescent emission, and the number of fluorescent spots through a magnifier from above, thereby determining the difference between live and dead bacteria, the type of bacteria, and the bacterial count; an apparatus for immediately discriminating bacteria which is provided with a source of an exciting light having a central wavelength of 488 nm in the lower part of a casing whose inside is kept in a state of darkroom, a magnifier above on the optical axis of the exciting light, and a light-transmitting plate which has been rendered light-scattering in the intermediate part; and another apparatus for immediately discriminating bacteria which is further provided with a band-pass filter for selecting the wavelengths of 520 nm and 625 nm above the magnifier and a video camera on the upper end of the filter, thereby converting the images which have passed the magnifier into electric signals and synthesizing the signals with a computer for image processing.

Description

明 細 書 菌類の即時判別方法及びその判別装置 技術分野  Description Immediate identification method for fungi and its identification device
この発明は細菌ゃ敝菌等の菌類が付着或 t、は混入する検体から、適宜 に菌類を被着採取して且即時にその付着や混在する菌類の菌種、 付着量 及び生菌、 死菌の判別ができ、 特に食品類の生産者や流通業者には衛生 管理上極めて利用価值の高い、 菌類の即時判別方法及びその判別装置に 鬨するものである。 背景技術  According to the present invention, fungi such as bacteria are adhered or collected, and the fungi are appropriately adhered and collected from a specimen to which the fungi adhere or are mixed, and the bacterial species, the amount of attached fungi, the amount of viable bacteria, and the death Bacteria can be distinguished, and especially for food producers and distributors, it is extremely useful for hygiene management, and the method for immediately distinguishing fungi and its discriminating apparatus are used. Background art
食品類特には水分率の高い生鮮食品や加工食品は、 細菌ゃ撖菌等の菌 類の繁殖しやすい条件を具備するものであって、 しかもこれら食品類に は菌類が原初から混在したり或いはその流通や消費に至る過程において 付着若しくは落下混入するため、 これが短時間に繁殖して著るしく不衛 生となるばかりか、 食品類の変質や腐敗とともに食中毒を招来する結果 ともなつている。  Foods, especially fresh foods and processed foods with a high moisture content, have conditions that facilitate the propagation of fungi such as bacteria and bacteria, and these foods contain fungi from the beginning or In the process leading to its distribution and consumption, it adheres or falls into the water, which not only propagates in a short time and becomes remarkably unhealthy, but also results in food poisoning along with deterioration and spoilage of foods.
このため生鮮食品においては、 合成樹脂製の容器や紙器等で包装し、 更には保冷しながら流通や消費に供しているものの、 これら容器や紙器 には菌類に対する抗菌性を有せぬため、 原初から混在する菌類は時間経 過とと に繁殖し、 しかも容器や紙器の外表面には流通や消費に至る間 に多量の菌類が付着し或いは落下付着して繁殖するため、 著るしく不衛 生な状態におかれる。  For this reason, fresh foods are packaged in containers and paper containers made of synthetic resin, and distributed and consumed while keeping them cool.However, these containers and paper containers have no antibacterial properties against fungi, Fungi that are mixed in from the ground grow over time, and on the outer surface of containers and paper containers, large amounts of fungi adhere or fall down during distribution or consumption, and grow remarkably unsafe. Put in a raw state.
更に加工食品においては、 許容される範囲で合成保存料を添加し菌類 の繁殖防止を図っていたが、 近年に至って健康指向の高まりとともに合 成保存料の使用が危険視され、 消費者においても無添加加工食品の選択 購入が富みに高まつていることから無添加で対処せねばならず、 しかも 消費者保護強化のための生産物賠責責任法の施行とも相俟って、 食品類 の生産者や流通業者においては生産時点若しくは流通時点における衛生 D 管理が強く求められている実状にある。 Furthermore, in processed foods, synthetic preservatives have been added to the extent permitted to prevent the growth of fungi. The use of preservatives is considered dangerous, and consumers are forced to select non-additive processed foods.There is a growing demand for these products, so they must deal with them without additives.Product liability to strengthen consumer protection In conjunction with the enforcement of the liability law, food producers and distributors are in high demand for sanitary D control at the time of production or distribution.
しかしながら現状においては、 発色酵素を適宜の培地に混合させてな る発色培地に検体より採取した菌類を植菌したるうえ、 これを所用温度 に保持させたィンキュベータ一で略 2 4時間乃至 4 8時間培養してコロ ニーを形成させ、 しかして大腸菌等が保持する; S—ガラクトシターゼ酵 However, at present, fungi collected from specimens are inoculated in a coloring medium in which a coloring enzyme is mixed with an appropriate medium, and the incubator is kept at a required temperature for about 24 hours to 48 hours. Incubate for a period of time to form colonies, which are then retained by Escherichia coli, etc .; S-galactosidase enzyme
10 素で発色酵素を分解し発色させて判別する手段があるが、 判別のための 培養に極めて長時間を要するため実用的でなく、 しかも判別しえる菌類 も ;3—ガラクトシターゼ酵素を有する大腸菌等の一部の菌類に限定され 、 且生菌や死菌の判別もできぬ問題を抱えている。 There is a means to determine by decomposing the chromogenic enzyme with 10 elements to form a color, but it is not practical because the culture for the determination takes an extremely long time, and even fungi that can be distinguished also have a 3-galactosidase enzyme It is limited to some fungi such as Escherichia coli and has a problem that it is not possible to discriminate between live bacteria and dead bacteria.
更に他の手段として、 蛍光染料を生理的食塩水に適宜漉度に混合させ As another method, a fluorescent dye is mixed with physiological saline to an appropriate degree.
ID た染色溶液中に検体より採取した菌類を混入し、 これを 2 4時間乃至 4 8時間培養しコロニーの形成を図ったうえ光学顕微鈸で判別することも 試みられているが、 かかる手段も培養のために長時間を要するため実用 にそぐわず、 而も蛍光染料は菌類の細胞内に入りにくく且仮りに細胞内 に浸透した染料も容易に細胞外に流失するため細胞が十分に染色されずAttempts have been made to mix fungi collected from specimens into the stained staining solution, culture them for 24 to 48 hours, form colonies, and discriminate them by optical microscopy. It takes a long time for cultivation, which is unsuitable for practical use.Furthermore, fluorescent dyes are difficult to enter into fungal cells, and dyes that have penetrated into cells easily flow out of cells easily, so that cells are sufficiently stained. Without
20 、 従って発光エネルギーも微弱となるため精密高倍率の顕微鏡を用いね ばならず、 判別のための装置も極めて高価で大型なものを用いねばなら ぬ等の問題を内在している。 20 Therefore, the luminescence energy is also weak, so a high-precision high-power microscope must be used, and the device for discrimination must use an extremely expensive and large-sized device.
そこで本発明は、 細菌ゃ擻菌等の菌類が混入付着する検体から適宜に 菌類を被着採取して、 且簡便な方法と安価な装置により即時に生菌と死 Therefore, the present invention provides a method of appropriately depositing and collecting bacteria from a specimen to which bacteria such as bacteria and the like are contaminated, and using a simple method and an inexpensive device to immediately kill live bacteria.
25 菌、 菌の種類、 及び菌数を判別することの可能な菌類の即時判別方法と その判別装置の提供を目的とする。 発明の開示 It is an object of the present invention to provide a method for instantly identifying fungi capable of distinguishing bacteria, the type of bacteria, and the number of bacteria, and an apparatus for distinguishing the fungi. Disclosure of the invention
本発明は、 菌類の細胞内に浸透でき且生菌細胞內では染料が分散され て励起光の吸収に伴い蛍光発光し、 死菌細胞內では染料が細胞組織に取 込まれ励起光の吸収に際しても蛍光発光しえないフルォレセイン若し はその誘導体からなる蛍光染料と、 生菌細胞内には浸透できず死菌細胞 内のみに浸透しぇ且励起光の吸収により蛍光発光しえるァロピデューム ィオダイドからなる蛍光染料とを、 生理的食塩水に対し 3乃至 1 o 1 / m 1混合し、 且細胞内への浸透性を高めて細胞が蛍光染料で効果 的に染色されるよう塩類、 キチナス或いはセルラーゼからなる染色促進 剤を 1乃至 1 O j m o I Zm 1混合してなる染色溶液中に検体から採取 した菌類を混入し、 所要温度に加温させた状態で染色する。  According to the present invention, the dye can be penetrated into fungal cells, and in the viable cell 內, the dye is dispersed and emits fluorescence with the absorption of the excitation light, and in the dead cell 內, the dye is taken into the cell tissue to absorb the excitation light. A fluorescent dye consisting of fluorescein or a derivative thereof, which cannot emit fluorescence, and an aropidum iodide which cannot penetrate living cells but penetrates only dead cells and emits fluorescence by absorbing excitation light. Fluorescent dye is mixed with physiological saline at a concentration of 3 to 1 o1 / m1 and from salts, chitinus or cellulase so as to increase the penetration into the cells and to effectively stain the cells with the fluorescent dye. A fungus collected from a specimen is mixed into a staining solution obtained by mixing 1 to 1 Ojmo IZm1 with a staining accelerator, and the mixture is stained while being heated to a required temperature.
そして細胞内に浸透し染色された蛍光染料が再び細胞外に流失せぬよ う、 該染色に引続いてジェチルスチルベストロール或いは Ν， Ν · —ジ シクロへキシルカーボジィミドからなる流失防止剤を、 該染色溶液に 5 乃至 2 1 / m 1の割合で混合し蛍光染料の流失防止を図る。 かかる如き蛍光染料で染色し且流失防止がなされた染色菌液を、 その 菌類細胞内に浸透染色された蛍光染料の蛍光発光を光散乱させて発光拡 大化のなしえる光散乱加工が施された透光板上に滴下させ、 その下側よ り中心波長が 4 8 8 n mの励起光を照射し菌類細胞内に浸透染色された フルォレセイン若しくはその誘導体からなる蛍光染料からは、 その中心 波長が 5 2 0 n mの緑色にやや黄味色の強い蛍光発光をなさしめ、 更に プロピデュームィォダイドからなる蛍光染料からは、 その中心波長が 6 2 5 n mの赤味を帯びた橙色の強い蛍光発光をなさしめる。  Then, in order to prevent the fluorescent dye that has penetrated into the cells and stained from flowing out of the cells again, it is prevented from being washed away with getyl stillbestrol or Ν, Ν · -dicyclohexylcarbodiimide following the staining. An agent is mixed with the staining solution at a ratio of 5 to 21 / m1 to prevent the fluorescent dye from flowing out. The stained bacterial solution stained with such a fluorescent dye and prevented from flowing out is subjected to a light scattering process in which the fluorescent emission of the fluorescent dye penetrated and stained into the fungal cells is light-scattered to increase the light emission. Fluorescent dye consisting of fluorescein or its derivative penetrated into fungal cells by irradiating it with excitation light having a central wavelength of 488 nm from the lower side, It emits green fluorescent light with a slightly yellowish color of 520 nm, and the fluorescent dye consisting of propidium iodide gives a strong reddish orange fluorescent light with a central wavelength of 625 nm. It emits light.
かくして発光拡大化されたこれら蛍光発光をその上側より拡大レンズ で視認することにより、 検体から採取した菌類についてその蛍光発光の 色光の相違により生菌及び死菌を、 蛍光発光の形状及び大小により菌類 の種類を、 更に蛍光発光数により菌数を即時に判別する菌類の即時判別 方法及びその判別装置であり、 更には拡大レンズで拡大させた蛍光発光 を波長 5 2 0 n m及び 6 2 5 n mの切替バンドパスフィルターを介して ビデオカメラにより電気信号に変換させたうえ、 コンピューターグラフ ィックス処理する菌類の即時判別装置に存する。 図面の簡単な説明 By visually observing the fluorescent light thus expanded in emission from above, using a magnifying lens, viable and dead bacteria can be identified for the fungi collected from the specimen due to differences in the color of the fluorescent light, and fungi depending on the shape and size of the fluorescent light. And an apparatus for immediately discriminating the number of bacteria based on the number of fluorescent light emission, and a device for discriminating the fungus. It is converted into an electric signal by a video camera via a switchable bandpass filter, and is present in a device for immediate identification of fungi to be processed by computer graphics. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 この発明にかかる菌類の即時判別方法の説明図であり、 第 2図はこの発明の菌類の即時判別装置の説明図、 第 3図はコンピュータ 一グラフィックス処理による菌類の即時判別装置の説明図である β 発明を実施するための最良の形筋 FIG. 1 is an explanatory diagram of a method for instantaneously identifying fungi according to the present invention. FIG. 2 is an explanatory diagram of an apparatus for instantly identifying fungi according to the present invention. FIG. 3 is a diagram for immediately identifying fungi by computer-graphics processing. FIG. 3 is an explanatory view of the apparatus, and shows the best form for implementing the β invention.
本発明をより詳細に説述するために、 添付された図面に従ってこれを 説明すれば、 第 1図は菌類の即時判別する方法を流れ図として示したも ので、 判別に供する菌類 1は検体より適宜の手段で採取される細菌ゃ敏 菌であって、 検体から菌類 1を採取する具体的手段としては滅菌睇棒に 生理的食塩水を含ませたうえ検体面に被着させて採取することや、 水溶 性培地を検体面と接触させて被着採取することが挙げられる。  In order to explain the present invention in more detail, the method will be described with reference to the attached drawings.Fig. 1 shows a flow chart of a method for immediately discriminating fungi. Bacterial susceptible bacteria collected by the method described in (1) .Specific means for collecting fungus 1 from a sample include, for example, adding a sterilized halo stick to physiological saline and attaching it to the surface of the sample. The method includes contacting a water-soluble medium with the surface of a sample and collecting the sample.
かくして採取された菌類 1は、 生理的食塩水 2に生菌及び死菌の細胞 内に浸透しえ且生菌細胞内には分散され死菌細胞内では該死菌細胞に取 込まれて分散されず、 しかも中心波長が 4 8 8 n mの励起光の吸収によ りその中心波長が 5 2 0 n mの強い蛍光発光をするフ /レオレセィン若し くはその誘導体からなる蛍光染料 3と、 生菌細胞内には浸透できず死菌 細胞内には浸透し分散でき、 しかも中心波長が 4 8 8 n mの励起光の吸 収に ί半いその中心波長が 6 2 5 n mの強い蛍光発光をなしうるプロビデ ユームィォダイドからなる蛍光染料 4が、 それぞれ 3 m o 1 / m 1乃 至 1 5 m o 1 Ζ π 1の範囲で混合され、 更に該生理的食塩水 2にはこ れら蛍光染料 3及び 4を菌類細胞内に十分に浸透させ且分散されるよう 細胞膜の柔軟化を図る塩類、 キチネス或いはセルラーゼからなる染色促 進剤 5が 3 m o 1 / m 1乃至 1 5 m o 1ノ m 1混合されて染色溶液 2 0が作成される。 かかる染色促進剤 5に用いられる塩類の具体的なも のとしては、 塩化ナトリウムや塩化マグネシウム等が挙げられる。 そしてかかる如く形成された染色溶液 2 0に、 検体より採取した菌類 1を混入し染色を図るものであるが、 この場合染色性を高めるうえから は該染色溶液 2 0を適宜温度に加温 6することが望ましく、 通常におい ては摂氏 2 5 °C乃至 3 5 °Cの温度が用いられるもので、 かかる溫度にお ける染色に要する時間は概ね 5乃至 1 5分程度である。 The fungus 1 thus collected can penetrate the physiological saline 2 into the cells of live and dead bacteria, and is dispersed in the viable cells, and is taken up and dispersed in the dead cells in the dead cells. And a fluorescent dye 3 consisting of fluorophore or its derivative, which emits strong fluorescence with a center wavelength of 520 nm due to absorption of excitation light having a center wavelength of 488 nm; It can not penetrate into cells but can die and can penetrate into cells, and can absorb and excite excitation light with a center wavelength of 488 nm, and emit strong fluorescence with a center wavelength of 625 nm Uruvide Fluorescent dyes 4 consisting of umodide, 3 mo 1 / m 1 each The mixture is mixed within the range of 15 mo 1 π π 1, and the physiological saline solution 2 is made flexible so that the fluorescent dyes 3 and 4 can sufficiently penetrate and disperse in the fungal cells. Staining solution 20 is prepared by mixing 3 mo 1 / m 1 to 15 mo 1 Nom 1 of dyeing accelerator 5 consisting of salt, chiness or cellulase. Specific examples of the salts used for the dye accelerator 5 include sodium chloride and magnesium chloride. The staining solution 20 formed as described above is mixed with the fungus 1 collected from the specimen to perform staining. In this case, the staining solution 20 is heated to an appropriate temperature in order to enhance the staining property. It is preferable to use a temperature of 25 ° C. to 35 ° C. in general, and the time required for dyeing at such a temperature is about 5 to 15 minutes.
しかしながら該染色溶液 2 0に混入された菌類 1は、染色促進剤 5に よる細 !¾膜の柔軟化で蛍光染料 3及び 4の細胞内への浸透が促進される ものの、 かかる状筋のままでは再び細胞内に浸透した蛍光染料 3及び 4 が流失し蛍光発光の低下に伴う判別不能が招来される。 そこで該染色溶 液 2 0の中で細胞内に十分に蛍光染料 3及び 4を浸透させた後、 再び細 胞膜の強靭化を図り流失を防止させるため、 ジェチルスチルベストロー /レ若しくは N， N ' ージシクロへキシルカーボジイミドからなる流失防 止剤 7を、 染色溶液 2 0を構成する生理的食塩水 2に対し 5 m o 1 m 1乃至 2 O rn o 1 / m 1の割合で混合させることにより、 菌類 1に 対して染色の安定化が図られた染色菌液 2 1が作成される。  However, the fungi 1 mixed into the staining solution 20 are not affected by the stain-promoting agent 5, but the softening of the membrane promotes the penetration of the fluorescent dyes 3 and 4 into the cells. In this case, the fluorescent dyes 3 and 4 that have permeated into the cells are washed away again, resulting in indistinguishability due to a decrease in fluorescence emission. Therefore, after sufficiently penetrating the fluorescent dyes 3 and 4 into the cells in the staining solution 20, in order to strengthen the cell membrane again and to prevent the cells from flowing out, getyl stillbestrow / N or N , N 'dicyclohexylcarbodiimide, and a washout inhibitor 7 is mixed with physiological saline 2 constituting the staining solution 20 at a ratio of 5 mo 1 m 1 to 2 Orn o 1 / m 1. As a result, a stained bacterial solution 21 in which staining of fungus 1 is stabilized is prepared.
図 2は、 染色の安定化が図られた染色菌液 2 1を用いて検体から採取 した菌類 1の菌種、 付着量及び生菌、 死菌の判別をする判別装置の説明 図であつて、 蛍光染料 3及び 4で染色された菌類 1の細胞は微細なもの であるから該細胞内の蛍光染料 3及び 4から可能な限り視認性の高い蛍 光発光を創出させる必要がある。 ところで菌類 1の生菌細胞内に浸透し分散されるフルォレセイン若し くはその誘導体からなる蛍光染料 3は、 中心波長が 4 8 8 n mの励起光 の吸収によりス 1 ^一クス則に従ってその中心波長が 5 2 0 n mの強い蛍 光発光をなすこと、 及び生菌細胞内には浸透できず死菌細胞内に浸透し 且分散しえるプロピデュームィォダイドからなる蛍光染料 4は、 中心波 長が 4 8 8 n mの励起光の吸収に伴いその中心波長が 6 2 5 n mの強い 蛍光発光をなすものである。 Fig. 2 is an explanatory diagram of a discrimination device that discriminates the species, attached amount, viable bacteria, and dead bacteria of fungus 1 collected from a sample using the stained bacterial solution 21 in which the staining is stabilized. Since the cells of the fungus 1 stained with the fluorescent dyes 3 and 4 are microscopic, it is necessary to create fluorescent light as highly visible as possible from the fluorescent dyes 3 and 4 in the cells. By the way, the fluorescent dye 3 consisting of fluorescein or its derivative, which penetrates and disperses into viable fungal cells of fungi 1, absorbs the excitation light having a center wavelength of 488 nm and absorbs the center of the fluorescent dye according to the S1 ^ 1x rule. The fluorescent dye 4 composed of propidum iodide, which emits strong fluorescent light with a wavelength of 520 nm and which can penetrate and disperse into dead cells but cannot penetrate into viable cells, has a central wave It emits strong fluorescent light with a center wavelength of 625 nm as it absorbs excitation light having a length of 488 nm.
そこでその内部が暗室状筋に保持されるようなケーシング 3 0の下部 に、 その中心波長が 4 8 8 n mの励起光を照射できる励起光源 3 1が設 けられてなり、 この励起光の照射光軸樣上の上端には所要の倍率好まし くは 1 0 0乃至 5 0 0倍程度の拡大レンズ 3 2が設けられている， そし て該照射光軸探の中間位置には光散乱加工が施され且透光性を有する透 光扳 3 3が設けてなるもので、 この透光板 3 3の光散乱は望ましくは 5 乃至 5 0倍程度の光散乱率を有するものが用いられ、 具体的なものとし ては摺りガラスが挙げられる。  Therefore, an excitation light source 31 capable of irradiating excitation light having a center wavelength of 488 nm is provided below the casing 30 so that the inside thereof is held by a dark room streak. At the upper end of the optical axis, there is provided a magnifying lens 32 of a required magnification, preferably about 100 to 500 times, and a light scattering process is provided at an intermediate position of the irradiation optical axis search. The light-transmitting plate 33 is provided with a light-transmitting plate 33, and the light-scattering of the light-transmitting plate 33 preferably has a light scattering rate of about 5 to 50 times. A specific example is frosted glass.
従って染色菌液 2 1を該透光板 3 3の上に滴下し、 その下方に設けて なる励起光源 3 1よりその中心波長が 4 8 8 n mの励起光を照射するこ とにより、 該染色菌液 2 1に混在する菌類 1の細胞内に浸透染色されて なる蛍光染料 3及び 4が、 励起光を吸収することに伴い生菌からはその 細胞内に浸透し且分散されてなるフルォレセイン若しくはその誘導体か らなる蛍光染料 3より、 その中心波長が 5 2 0 n mの蛍光発光色で且発 光形状及び発光の大小を異にした菌種に係る蛍光発光、 菌数に係る蛍光 発光数が、 更には死菌からはァロピデュームィォダイドからなる蛍光染 料によって、 その中心波長が 6 2 5 n mの蛍光発光色で且その発光形状 及び発光の大小を異にした菌種に係る蛍光発光、 菌数に係る蛍光発光数 が光散乱により拡大され、 しか 上端の拡大レンズ 3 2により明確に視 認され判別できることとなる。 Therefore, the stained bacterial solution 21 is dropped on the light-transmitting plate 33, and the excitation light source 31 provided therebelow is irradiated with excitation light having a center wavelength of 488 nm to thereby perform the staining. Fluorescein or fluorescein, which is permeated and stained into cells of fungus 1 mixed in bacterial liquid 21 and penetrates and disperses from viable bacteria as it absorbs excitation light, From the fluorescent dye 3 composed of the derivative, the fluorescence emission of the bacterial species and the fluorescence emission number related to the number of bacteria having a fluorescence emission color having a center wavelength of 50 nm and having different emission shapes and emission sizes are different. Furthermore, from the dead bacteria, a fluorescent dye composed of aropidum iodide is used to generate a fluorescent color having a central wavelength of 625 nm and a fluorescent color related to a bacterial species having a different luminescent shape and luminescent intensity. The number of luminescence and the number of fluorescent light related to the number of bacteria are expanded by light scattering. Clearly seen by the large lens 3 2 It will be recognized and can be determined.
図 3は、 この発明における菌類の即時判別装置において、 拡大レンズ 3 2により視認判別される菌類 1からの蛍光発光光緣を画像やデーター として処理し表示保存する判別装置の説明図であって、 拡大レンズ 3 2 の上方に 5 2 0 n mの波長の蛍光発光光線を透過させるバンドパスフィ ルター 4 0、 及び 6 2 5 n mの波長の蛍光発光光緣を透過させるバンド パスフィルター 4 1とを自在に切替え可能なバンドパス切替フィルター 4が設けられてなるとともに、 拡大レンズ 3 2を通過し且バンドパスフ ィルター 4 0若しくは 4 1を透過したそれぞれの蛍光発光光緣の焦点位 置には、 該蛍光発光光綠の形状や大小、 蛍光発光数或いは蛍光発光色を 電気信号に変換させるビデオカメラ 5が設けられており、 しかもこのビ デォカメラ 5からは変換された電気信号を記憶し且所要の面像処理若し くはデーター処理がなされるようプログラミングされたコンピューター 6に接続されている。  FIG. 3 is an explanatory diagram of a discrimination device for processing and displaying and storing the fluorescence emission light の from the fungus 1 visually discriminated by the magnifying lens 3 2 as an image or data in the instant fungus discrimination device of the present invention. Above the magnifying lens 3 2, a band-pass filter 40 that transmits fluorescent light with a wavelength of 52 nm and a band-pass filter 41 that transmits fluorescent light with a wavelength of 65 nm can be freely used. The filter is provided with a band-pass switching filter 4 that can be switched between the fluorescent light-emitting lights, and the fluorescent light that has passed through the magnifying lens 32 and passed through the band-pass filter 40 or 41 has a focal position at which the fluorescent light is emitted. A video camera 5 is provided for converting the shape and size of the light beam, the number of fluorescent light emission or the number of fluorescent light emission into an electric signal, and the video camera 5 converts the converted electric signal. Wakashi stored 且 required surface image processing Ku is connected to a computer 6 which is programmed to data processing is performed.
即ち拡大レンズ 3 2を通過した蛍光発光光樣は、 バンドパス切替フィ ルター 4の切替えにより波長 5 2 0 n mの蛍光発光光緣による映像と、 波長 6 2 5 η πιの蛍光発光光樣による映像とが同一視野において別々に ビデオカメラ 5に結像され、 それぞれ電気信号に変換される。  That is, the fluorescent light having passed through the magnifying lens 32 has an image formed by the fluorescent light having a wavelength of 520 nm and an image formed by the fluorescent light having a wavelength of 625 ηπι by switching the band-pass switching filter 4. Are separately imaged on the video camera 5 in the same field of view, and are converted into electric signals.
従って、 かかる電気信号を合成させて画像処理を施すことにより拡大 レンズ 3 2にて視認判別される映像が、 写真或いはデーターとして表示 され保存できる。 かかる場合にバンドバス切替フィルター 4の使用に代 えて、 波長 5 2 0 n mのバンドパスフィルター 4 0を装備したビデオ力 メラ 5、 及び波長 6 2 5 η τηのバンドパスフィルタ一 4 1を装備したビ デォカメラ 5の 2台を用いて、 それぞれのビデオカメラ 5からの電気信 号をコンピューター 6で ^理しても同様の結果が得られる。 産業上の利用可能性 Therefore, by combining such electric signals and performing image processing, an image visually recognized by the magnifying lens 32 can be displayed and stored as a photograph or data. In such a case, instead of using the band-pass switching filter 4, a video camera 5 equipped with a band-pass filter 40 with a wavelength of 52 nm and a band-pass filter 141 with a wavelength of 6 25 η τη were installed. The same result can be obtained by using two video cameras 5 and processing the electric signal from each video camera 5 with the computer 6. Industrial applicability
本発明は上述したように、 検体から採取した菌類を直接染色溶液中に 混入することにより、 生菌細胞内と死菌細胞内に異る蛍光発光色で発光 する蛍光染料を短時間に浸透分散せさて染色でき、 更に該浸透分散され た蛍光染料の流失防止をなした染色菌液を、 光散乱加工の施された透光 板上に滴下しその下方より中心波長が 4 8 8 n mの励起光を照射させる ことで、 生菌からは鮮明で且視認性の高い中心波長が 5 2 0 n mの蛍光 発光をなさしめ更に死菌から 鮮明で且視認性の高い中心波長が 6 2 5 n mの蛍光発光がなされ、 しかもこれら発光光綠が透光板で光散乱され て予め拡大化されるため比較的低倍率の拡大レンズによっても生菌並び に死菌の判別はもとより、 蛍光発光の形状や大小によって菌種の判別が、 更に蛍光発光数により菌数の判別が明確に視認できるため、 食品類の生 産工程や流通過程においても製品を留めることなく連統的に衛生管理が なしえ、 しかも本発明では判別結果を写真やデータ一で記録保存ができ ることから、 消費者との間の衛生管理上の問題の発生にも適確な対処が できる。  As described above, according to the present invention, by mixing fungi collected from a specimen directly into a staining solution, a fluorescent dye that emits a different fluorescent color in live and dead cells can be permeated and dispersed in a short time. Stained bacteria solution that can prevent staining of the permeated and dispersed fluorescent dye is dropped onto a light-transmissive light-transmissive plate, and excitation is performed with a center wavelength of 488 nm from below. By irradiating the light, fluorescence is emitted with a center wavelength of 250 nm, which is clear and highly visible from live bacteria, and a center wavelength of 625 nm, which is clear and highly visible from dead bacteria. Fluorescence is emitted, and the emitted light さ れ is scattered by the light-transmitting plate and is enlarged in advance, so that even a relatively low-magnification magnifying lens can determine not only viable cells and dead cells, but also the shape and shape of the fluorescent light. Bacterial species can be distinguished by the size, and the number of bacteria can be Since the distinction is clearly visible, hygiene management can be performed continuously without stopping the product even in the production and distribution processes of foods, and in the present invention, the determination results can be recorded and stored in the form of photographs and data. As a result, it is possible to appropriately deal with the occurrence of hygiene issues with consumers.

Claims

請 求 の .範 囲 The scope of the claims
1 . 菌類を適宜手段で被着採取したうえ、 フルォレセイン若しくはその 誘導体からなる蛍光染料と、 プロピデュームィォダイドからなる蛍光染 料とが生理的食塩水に対しそれぞれ 3乃至 1 5 m o 1 Z m 1の漉度で、 及び塩類、 キチネス若しくはセルラーゼからなる染色促進剤が 1乃至 I O ju m o 1 / m 1の濃度で混合された染色溶液中に混入して且所用溫度 に加温させた状態で菌類の細胞を染色し、 更に染色された染料の流失防 止を図るためジェチルスチルベストロール或いは N， N ' —ジシクロへ キシルカーボジィミドからなる流失防止剤を 5乃至 2 O ITI O I / m 1 の濃度で混合して染色菌液となしたうえ、 光散乱加工の施された透光板 上に該染色菌液を滴下し、 その下方より中心波長が 4 8 8 n mの励起光 を照射させて、蛍光発光する蛍光色光、 蛍光発光の形状や大小或いは蛍 光発光数を上方より拡大視認し、 生菌及び死菌、菌の種類並びに菌数を 判別する菌類の即時判別方法。 1. After collecting and collecting fungi by appropriate means, the fluorescent dye consisting of fluorescein or a derivative thereof and the fluorescent dye consisting of propidum iodide are each 3 to 15 mo 1 Zm in physiological saline. With a strainer of 1 and a dyeing accelerator consisting of salts, chitines or cellulase mixed in a dyeing solution mixed at a concentration of 1 to IO jumo 1 / m1 and heated to the required temperature. 5 to 2 O ITI OI / m to stain fungal cells and to prevent run-off of the stained dye, using effluent preventive agent consisting of getylstilbestrol or N, N'-dicyclohexylcarbodiimide After mixing at a concentration of 1 to form a stained bacterial solution, the stained bacterial solution is dropped onto a light-transmissive translucent plate, and irradiated with excitation light having a center wavelength of 488 nm from below. Fluorescent color light that emits fluorescent light, fluorescent light The shape and size or fluorescent emission of the light enlarged viewing from above, live cells and dead cells, an immediate determination method fungi determining the type and number of bacteria of fungi.
2 . 内部が暗室状態に保持するケーシングの下部には、 その中心波長が 4 8 8 n mの励起光を照射する励起光源が設けられ、 且この励起光の照 射光軸緣上の上部には、 所用の拡大倍率に拡大できる拡大レンズが設け られ、 しかもその中間位置には滴下される染色菌液が励起光の吸収に伴 い発光する蛍光発光光緣を散乱拡大する透光板が設けられてなる菌類の 即時判別装置。  2. An excitation light source for irradiating excitation light having a center wavelength of 488 nm is provided at a lower portion of the casing which holds the interior in a dark room state, and an upper portion on the illumination light axis 照 of the excitation light is provided at an upper portion thereof. A magnifying lens that can be enlarged to the required magnification is provided, and a translucent plate that scatters and expands fluorescent emission light す る, which is emitted by the stained bacterial solution dropped upon absorption of the excitation light, is provided at an intermediate position. An instantaneous identification device for fungi.
3 . 拡大レンズの上部に波長 5 2 0 n mの蛍光発光光線及び波長 6 2 5 n mの蛍光発光光緣を透過させるバンドバス切替フィルターを配し、 且 その上端にビデオカメラを設けて拡大レンズを透過させたそれぞれの波 長の映像を電気信号に変換させ、 しかしてこの電気信号をコンピュータ 一で合成し画像処理をおこなう前記菌類の即時判別装置。  3. At the top of the magnifying lens, a band-bass switching filter that transmits fluorescent light with a wavelength of 520 nm and fluorescent light with a wavelength of 625 nm is disposed, and a video camera is provided at the upper end of the filter to mount the magnifying lens. The fungus real-time discrimination device which converts the transmitted image of each wavelength into an electric signal, and synthesizes the electric signal with a computer to perform image processing.
PCT/JP1996/002370 1996-04-11 1996-08-26 Method of immediately discriminating bacteria and apparatus therefor WO1997038128A1 (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
US6309835B1 (en) 1999-05-27 2001-10-30 Koninkiijke Philips Electronics N.V. Methods for quantitating the efficacy of oral care products
US6979828B2 (en) * 2001-02-15 2005-12-27 Nippon Mizushori Giken Co. Ltd. Method and apparatus for immediately determining microorganism
GB2510365A (en) * 2013-01-31 2014-08-06 Blood Analysis Ltd Quantification of bacteria
CN108350409A (en) * 2015-12-28 2018-07-31 日本理化学开发公司 Viable bacteria/dead bacterium state determining apparatus and the viable bacteria/dead bacterium condition judgement method for using the device
CN111006991A (en) * 2018-10-30 2020-04-14 江南大学 Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003102496A (en) * 2001-09-27 2003-04-08 Matsushita Ecology Systems Co Ltd Microorganism testing system
JP4876251B2 (en) * 2006-08-29 2012-02-15 国立大学法人山口大学 Method for determining the presence ratio of live bacteria, dead bacteria and pseudo-viable bacteria
CN101225428B (en) * 2007-01-19 2012-01-25 华南农业大学 Dyeing liquid as well as dyeing method and uses thereof
CN113533004A (en) * 2021-07-30 2021-10-22 深圳联合医学科技有限公司 Multiple fluorescent staining solution and preparation method and use method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BY THE FOUNDATION JAPAN CHEMICAL SOCIETY, "New Biochemical Experimental Lecture 17 Microbial Experimental Method", (23-03-92); & TOKYO KAGAKU DOJIN CO. LTD., p. 66-69. *
CYTOMETRY, Vol. 15, (1994), M.J. HUMPHREYS et al., "Determination of the Viability of Trichomonas Vaginalis Using Flow Cytometry", p. 343-348. *

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US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
US6416969B2 (en) 1999-03-12 2002-07-09 Akzo Nobel N.V. Susceptibility plates for microbial antibiotic susceptibility testing
US6309835B1 (en) 1999-05-27 2001-10-30 Koninkiijke Philips Electronics N.V. Methods for quantitating the efficacy of oral care products
US6979828B2 (en) * 2001-02-15 2005-12-27 Nippon Mizushori Giken Co. Ltd. Method and apparatus for immediately determining microorganism
GB2510365A (en) * 2013-01-31 2014-08-06 Blood Analysis Ltd Quantification of bacteria
WO2014118544A1 (en) * 2013-01-31 2014-08-07 Blood Analysis Ltd Quantification of bacteria
CN108350409A (en) * 2015-12-28 2018-07-31 日本理化学开发公司 Viable bacteria/dead bacterium state determining apparatus and the viable bacteria/dead bacterium condition judgement method for using the device
CN108350409B (en) * 2015-12-28 2022-06-14 日本理化学开发公司 Viable/dead bacteria state determination device and viable/dead bacteria state determination method using same
CN111006991A (en) * 2018-10-30 2020-04-14 江南大学 Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment
CN111006991B (en) * 2018-10-30 2021-06-25 江南大学 Method for determining optimal storage temperature of aerobic denitrifying bacteria for sewage treatment

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