JP6071580B2 - Microorganism detection system and microorganism detection method - Google Patents

Description

  The present invention relates to an environmental evaluation technique, and more particularly to a microorganism detection system and a microorganism detection method.

  In a clean room such as a bio clean room, scattered microorganisms are detected and recorded using a microorganism detection apparatus (see, for example, Patent Document 1 and Non-Patent Document 1). From the detection result of microorganisms, it is possible to grasp the deterioration of the air conditioner in the clean room. In addition, detection records of microorganisms in the clean room may be attached to products manufactured in the clean room as reference materials. For example, the optical microorganism detection apparatus sucks a gas in a clean room and irradiates the sucked gas with light. When microorganisms are contained in the gas, the microorganisms irradiated with light emit fluorescence, so that the number and size of microorganisms contained in the gas can be detected.

JP 2011-83214 A

Hasegawa, M. et al., “Real-time microorganism detection technology in the air and its application”, Yamatake Corporation, azbil Technical Review December 2009, p.2-7, 2009

  However, the present inventor has found that when the gas contains non-microbe particles that emit fluorescence, the microbe detection device may erroneously detect the non-microbe particles as microorganisms. Accordingly, an object of the present invention is to provide a microorganism detection system and a microorganism detection method with few false detections.

  According to the aspect of the present invention, (a) an intake device that takes in microorganisms and soluble particles contained in a gas into a liquid and dissolves soluble particles in the liquid; and (b) lightes the microorganisms taken into the liquid. A microorganism detection system comprising: an in-liquid microorganism detection device that detects at least one of fluorescence emitted by microorganisms and scattered light generated by microorganisms.

  Further, according to the aspect of the present invention, (a) the microorganisms contained in the gas and the soluble particles are taken into the liquid, the soluble particles are dissolved in the liquid, and (b) the microorganisms taken into the liquid A method of detecting a microorganism comprising: irradiating light and detecting at least one of fluorescence emitted by the microorganism and scattered light generated by the microorganism.

  According to the present invention, it is possible to provide a microorganism detection system and a microorganism detection method with few false detections.

It is a schematic diagram of the clean room where the microorganisms detection system which concerns on the 1st Embodiment of this invention is arrange | positioned. It is a mimetic diagram of an impinger concerning the 1st embodiment of the present invention. It is a graph which shows the fluorescence intensity for every kind of microorganisms concerning the 1st Embodiment of this invention. It is a graph which shows typically the relation between the particle size of microorganisms in the liquid concerning a 1st embodiment of the present invention, and fluorescence intensity. It is a schematic diagram of the scrubber which concerns on the 2nd Embodiment of this invention. It is a schematic diagram of the powder separator which concerns on the 3rd Embodiment of this invention. It is a schematic diagram of the impactor which concerns on the 4th Embodiment of this invention. It is a schematic diagram of the acquisition apparatus which concerns on the 5th Embodiment of this invention. It is a schematic diagram of the filter which concerns on the 5th Embodiment of this invention. It is a schematic diagram of the acquisition apparatus which concerns on the 5th Embodiment of this invention.

  Embodiments of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
As shown in FIG. 1, the microorganism detection system according to the first embodiment takes in microorganisms contained in a gas and soluble particles into a liquid, and takes in the soluble particles in the liquid. And an in-liquid microorganism detection device 1 for detecting at least one of fluorescence emitted from the microorganism and scattered light generated by the microorganism by irradiating light to the microorganism taken into the microorganism. The intake device 50 and the in-liquid microorganism detection device 1 are arranged in a clean room 70, for example. A clean room 70 is supplied with a gas such as clean air through a duct 71 and an outlet 72 having an ultra-high performance air filter such as HEPA (High Efficiency Particulate Air Filter) and ULPA (Ultra Low Penetration Air Filter). It is.

  Production lines 81 and 82 are arranged in the clean room 70. The production lines 81 and 82 are, for example, production lines for precision equipment, electronic components, or semiconductor devices. Alternatively, the production lines 81 and 82 are food, beverage, or pharmaceutical production lines. For example, in the production lines 81 and 82, the infusion solution is filled into an infusion or a syringe. Or in the production lines 81 and 82, an oral preparation and a Chinese medicine are manufactured. Alternatively, in the production lines 81 and 82, containers for energy drinks and beer are filled.

  The production lines 81 and 82 are usually managed so that microorganisms and non-microbial particles are not scattered in the gas in the clean room 70. However, the production lines 81 and 82 become sources of microorganisms and non-microbial particles scattered in the gas in the clean room 70 for some reason. In addition, microorganisms and non-microbial particles may be scattered in the gas in the clean room 70 due to factors other than the production lines 81 and 82.

  Bacteria are included as examples of microorganisms that can be scattered in the gas in the clean room 70. Examples of bacteria include gram negative bacteria, gram positive bacteria, and fungi including mold spores. Examples of gram-negative bacteria include E. coli. Examples of gram positive bacteria include Staphylococcus epidermidis, Bacillus subtilis spores, Micrococcus, and Corynebacterium. Examples of fungi containing mold spores include Aspergillus. However, the microorganisms that can be scattered in the gas in the clean room 70 are not limited to these. Examples of non-microbial particles that can be scattered in the gas in the clean room 70 include splashes of chemical substances, chemicals, and foods, dust, dust, and dust such as dust.

  Here, when a microorganism is irradiated with light, nicotinamide adenine dinucleotide, riboflavin and the like contained in the microorganism emit fluorescence. Conventionally, an apparatus for detecting microorganisms in a gas regards particles in the gas that emits fluorescence when irradiated with light as microorganisms. However, even in the case of non-microbial particles, for example, fluorescent particles scattered from cleaned gowns, and infusions, oral preparations, herbal medicines, nutritional drinks, and beer droplets and crystal particles containing vitamins scattered from production lines 81 and 82 Fluorescence may occur when irradiated with light. For this reason, a conventional apparatus for detecting microorganisms in a gas may erroneously detect non-microbial particles that emit fluorescence and emit fluorescence.

  In contrast, the capturing device 50 according to the first embodiment includes, for example, an impinger 51 shown in FIG. 2 that captures microorganisms and soluble particles contained in a gas into a liquid. The impinger 51 includes a container 511 that contains a liquid 516 and a suction pipe 512 that sucks the gas in the clean room 70 and diffuses it into the liquid 516. When the gas is diffused into the liquid 516, the microorganisms 517 contained in the gas are insoluble and thus do not dissolve in the liquid 516. However, soluble fluorescent particles contained in the gas dissolve in the liquid 516. Since the volume of the liquid 516 is sufficiently larger than the volume of the soluble fluorescent particles contained in the gas, the fluorescent particles dissolved in the liquid 516 are diluted and do not emit fluorescence to a detectable level.

  The liquid 516 is pure water or a solution. The container 511 is provided with a supply pipe 514 for supplying the liquid 516 into the container 511. Further, the container 511 is provided with a connecting pipe 515 for passing the liquid 516 in the container 511 shown in FIG. 2 through the submerged microorganism detecting device 1 shown in FIG. However, the mechanism for supplying the liquid 516 in the container 511 to the submerged microorganism detection apparatus 1 is not limited to this. Excess gas in the container 511 is discharged through the exhaust pipe 513.

  As the microorganism detection apparatus 1 in liquid shown in FIG. 1, the apparatus disclosed in US Pat. No. 7430046 or US Pat. No. 8,218,144 can be used, but is not limited thereto. For example, the in-liquid microorganism detection device 1 includes a light source such as a laser. The light source irradiates light such as laser light toward the liquid coming from the capturing device 50. The light may be visible light or ultraviolet light. When the light is visible light, the wavelength of the light is in the range of 400 to 550 nm, for example, 405 nm. When the light is ultraviolet light, the wavelength of the light is in the range of 310 to 380 nm, for example, 340 nm. The submerged microorganism detection apparatus 1 includes an optical system that focuses irradiation light on the focal point, a pipe that allows liquid to pass through the focal point, and the like.

  The in-liquid microorganism detection device 1 further includes a fluorescence detector. The fluorescence detector detects fluorescence emitted by microorganisms and measures fluorescence intensity. The number of microorganisms contained in the liquid can be measured from the number of times the fluorescence detector detects fluorescence. Further, as shown in FIG. 3, the intensity of the fluorescence emitted by the microorganism varies depending on the type of the microorganism. Therefore, it is possible to identify the type of microorganisms contained in the liquid from the intensity of fluorescence detected by the fluorescence detector of the in-liquid microorganism detection device 1 shown in FIG. When Raman scattering occurs in the liquid irradiated with light, a filter for cutting the Raman scattered light may be provided in the fluorescence detector.

  The submerged microorganism detection apparatus 1 may further include a scattered light detector. The scattered light detector detects light scattered by the particles contained in the liquid. The number of particles can be measured from the number of times the scattered light detector detects the scattered light. Further, the intensity of the scattered light by the particles correlates with the particle size of the particles. Therefore, the particle size of the particles contained in the liquid can be obtained by detecting the intensity of the scattered light with the scattered light detector. In addition, the particle size of the microorganism varies depending on the type of microorganism. Therefore, it is possible to identify the type of microorganisms contained in the liquid from the intensity of the scattered light detected by the scattered light detector.

  In addition, when a scattered light detector detects scattered light and a fluorescence detector does not detect fluorescence, it turns out that the particle | grains contained in a liquid are non-living particles. When the scattered light detector detects the scattered light and the fluorescence detector detects the fluorescence, it can be seen that the liquid contains microorganisms. Also, as described above, the soluble fluorescent particles are dissolved and diluted in the liquid 516 in the container 511 of the impinger 51 shown in FIG. Disappear. Therefore, the soluble fluorescent particles scattered in the clean room 70 shown in FIG. 1 are not erroneously detected as microorganisms by the in-liquid microorganism detection device 1.

  The microorganism detection apparatus 1 in a liquid specifies the kind of microorganisms contained in a liquid using at least one of the particle diameter based on the detected fluorescence intensity and scattered light intensity. Methods for identifying the type of microorganism based on both fluorescence intensity and scattered light intensity are disclosed in, but not limited to, US Pat. No. 6,885,440 and US Pat. No. 7,106,442. For example, as shown in FIG. 4, there is a correlation between the particle diameter and the fluorescence intensity depending on the type of microorganism. Therefore, by acquiring a graph as shown in FIG. 4 in advance, it is possible to specify the type of microorganism from the fluorescence intensity and the particle size.

  The in-liquid microorganism detection device 1 may detect a microorganism only by scattered light without detecting fluorescence, and may specify the type of microorganism. Using a concentric scattered light detector, it is possible to detect the scattered light intensity for each angle, and to identify the type of microorganism by a statistical method such as a support vector machine (SVM) (for example, Murugesan Venkatapathi et al., "High speed classification of individual bacterial cells using a model-based light scatter system and multivariate statistics", APPLIED OPTICS, the United States, Optical Society of America, 2 January 10, 2008, Vol.47, No.5 Pp. 678-686).

  Alternatively, the in-liquid microorganisms detection device 1 may detect the fluorescence spectrum by irradiating the liquid coming from the intake device 50 with excitation light having a plurality of wavelengths, and specify the microorganisms contained in the liquid. For example, microorganisms can be identified by irradiating microorganisms with excitation light having wavelengths of 266 nm and 355 nm and detecting fluorescence spectra having wavelengths of 350 nm, 450 nm, and 550 nm (for example, by Vasanthi Sivaprasam et al., “Multiple UV wave excursion and fluorescence of bioarosols”, OPTICS EXPRESS, USA, Optical Society of America, September 20, 2004, Vol.

(Second Embodiment)
In the first embodiment, an example in which the capturing device 50 illustrated in FIG. 1 includes the impinger 51 illustrated in FIG. 2 has been described. In contrast, the capturing device 50 shown in FIG. 1 collects microorganisms and soluble particles contained in a gas in a droplet or liquid film, and liquids the droplets or liquid film in which the microorganisms and soluble particles are collected. You may provide the scrubber 52 shown in FIG.

  The scrubber 52 includes a pipe 521 in which a gas sucked from the clean room 70 flows and a liquid 526 is accumulated at the bottom, and a droplet supply device 528 provided in the pipe 521. The droplet supply device 528 sprays mist containing droplets in the pipe 521. At this time, the microorganisms and soluble particles contained in the gas are collected as droplets, and the droplets collecting the microorganisms and the soluble particles are dropped onto the liquid 526 accumulated at the bottom of the pipe 521.

  The microorganism 527 contained in the gas is insoluble and therefore does not dissolve in the liquid 526. However, soluble fluorescent particles contained in the gas are dissolved in the liquid 526. Since the volume of the liquid 526 is sufficiently larger than the volume of the soluble fluorescent particles contained in the gas, the fluorescent particles dissolved in the liquid 526 are diluted and do not emit fluorescence to a detectable level.

  The liquid 526 is pure water or a solution. The pipe 521 is provided with a supply pipe 524 for supplying the liquid 526 into the pipe 521. Further, the pipe 521 is provided with a connecting pipe 525 for allowing the liquid 526 in the pipe 521 shown in FIG. 5 to pass through the submerged microorganism detecting device 1 shown in FIG. However, the mechanism for supplying the liquid 526 in the pipe 521 to the submerged microorganism detection apparatus 1 is not limited to this.

  The pipe 521 may be provided with a liquid film supply device. In this case, the liquid film supply device supplies a liquid film to the inner wall of the pipe 521, the microorganisms and soluble particles contained in the gas are collected by the liquid film, and the liquid film from which the microorganisms and soluble particles are collected is It flows down to the liquid 526 accumulated at the bottom of the pipe 521. Since the other components of the microorganism detection system according to the second embodiment are the same as those in the first embodiment, description thereof is omitted.

(Third embodiment)
As shown in FIG. 6, the intake device 50 shown in FIG. 1 includes a powder separator 53 that separates microorganisms and soluble particles contained in the gas sucked from the clean room 70, and the separated microorganisms and soluble particles. And a liquid container 54 disposed at the place of dropping.

  In the powder separator 53, a gas containing microorganisms and soluble particles is poured in a vortex from the circumferential direction of a funnel-shaped or cylindrical cyclone. Here, the microorganisms and soluble particles are centrifuged and fall toward the liquid 531 contained in the container 54 by gravity. The gas from which the microorganisms and soluble particles have been removed is discharged upward from the center of the cyclone circle.

  The microorganism 532 contained in the gas is insoluble and therefore does not dissolve in the liquid 531. However, soluble fluorescent particles contained in the gas are dissolved in the liquid 531. Since the volume of the liquid 531 is sufficiently larger than the volume of the soluble fluorescent particles contained in the gas, the fluorescent particles dissolved in the liquid 531 are diluted and do not emit fluorescence to a detectable level. The liquid 531 is sucked and supplied to the submerged microorganism detection apparatus 1 shown in FIG. Since the other components of the microorganism detection system according to the third embodiment are the same as those in the first embodiment, description thereof will be omitted.

(Fourth embodiment)
The intake device 50 shown in FIG. 1 may include an impactor 55 that collects microorganisms and soluble particles contained in the gas sucked from the clean room 70 in the liquid, as shown in FIG. The impactor 55 includes a nozzle 551 through which gas sucked from the clean room 70 flows. A collection container 552 containing a liquid 553 is disposed below the nozzle 551.

  The gas sucked from the clean room 70 is jetted from the orifice of the nozzle 551 toward the collection container 552. At this time, microorganisms having a predetermined size of inertia and soluble particles cannot follow the airflow deflected on the collection container 552 and collide with the liquid 553 contained in the collection container 552. As a result, microorganisms having a predetermined size of inertia and soluble particles are collected by the liquid 553.

  The microorganism 554 contained in the gas is insoluble and therefore does not dissolve in the liquid 553. However, the soluble fluorescent particles contained in the gas are dissolved in the liquid 553. Since the volume of the liquid 553 is sufficiently larger than the volume of the soluble fluorescent particles contained in the gas, the fluorescent particles dissolved in the liquid 553 are diluted and do not emit fluorescence to a detectable level. The liquid 553 is sucked and supplied to the submerged microorganism detection apparatus 1 shown in FIG. Since the other components of the microorganism detection system according to the fourth embodiment are the same as those of the first embodiment, description thereof will be omitted.

(Fifth embodiment)
The uptake device 50 shown in FIG. 1 may take up microorganisms and soluble particles collected by a filter into a liquid. For example, the intake device 50 includes a container 500 containing a liquid 501 as shown in FIG. In addition, as shown in FIG. 9, the gas sucked from the clean room 70 is sucked by a pipe 503, and microorganisms and soluble particles are collected by a filter 504. Furthermore, as shown in FIG. 10, a filter 504 in which microorganisms and soluble particles are collected is conveyed into a container 500 containing a liquid 501.

  At this time, the microorganisms 554 collected by the filter 504 are insoluble, and thus do not dissolve in the liquid 501. However, the soluble fluorescent particles collected by the filter 504 are dissolved in the liquid 501. Since the volume of the liquid 501 is sufficiently larger than the volume of the soluble fluorescent particles contained in the gas, the fluorescent particles dissolved in the liquid 501 are diluted and do not emit fluorescence to a detectable level. The liquid 501 is sucked and supplied to the submerged microorganism detection apparatus 1 shown in FIG. Since the other components of the microorganism detection system according to the fifth embodiment are the same as those in the first embodiment, description thereof will be omitted.

(Other embodiments)
Although the present invention has been described by the embodiments as described above, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques should be apparent to those skilled in the art. For example, as an apparatus for detecting microorganisms in liquid, it has been described in the embodiment that the apparatus disclosed in US Pat. No. 7430046 or US Pat. No. 8218144 can be used. The flow cytometer disclosed in Japanese Patent No. 140148, the microorganism detection means in liquid using the Raman spectrum disclosed in Japanese Translation of PCT International Publication No. 2012-507283, and the mass spectrometer disclosed in Japanese Unexamined Patent Publication No. 2012-507711 Means for collecting microorganisms with a membrane filter disclosed in JP-A-63-053447 and irradiating with a laser, and a luminescence measuring apparatus disclosed in JP-A-2008-249628, etc. It can be used as a microorganism detection device. Thus, it should be understood that the present invention includes various embodiments and the like not described herein.

DESCRIPTION OF SYMBOLS 1 Microbe detection apparatus 50 Intake apparatus 51 Impinger 52 Scrubber 53 Powder separator 54 Container 55 Impactor 70 Clean room 71 Duct 72 Ejection port 81 Production line 500 Container 501 Liquid 503 Pipe 504 Filter 511 Container 512 Suction pipe 513 Exhaust pipe 514 Supply pipe 515 Connection pipe 516, 526, 531, 553 Liquid 517, 527, 532, 554 Microorganism 521 Pipe 524 Supply pipe 525 Connection pipe 528 Droplet supply device 551 Nozzle 552 Collection container

Claims (10)

An uptake device for taking up microorganisms and soluble particles contained in the gas into the liquid, and dissolving the soluble particles into the liquid;
A light source that emits light,
A pipe through which the liquid that has taken in the microorganisms passes through the focal point of the irradiation light; and
Detector for detecting at least one of scattered light generated by the fluorescence and the microorganism microorganism irradiated with the light emitted
An in- liquid microorganism detecting device (excluding an apparatus for concentrating the microorganism in the liquid) ;
A microorganism detection system comprising: Detecting both scattered light generated by the fluorescence and the microorganism said solution microorganism detection apparatus wherein the microorganism emitted, microorganism detection system according to claim 1. Detecting fluorescence the liquid microorganism detection apparatus wherein the microorganism emitted, based on the fluorescence intensity of the fluorescent, identifies the type of the microorganism, the microorganism detection system according to claim 1. Detecting fluorescence the liquid microorganism detection apparatus wherein the microorganism emitted, based on the spectrum of the fluorescence, identifying the type of the microorganism, the microorganism detection system according to claim 1.   The microorganism detection system according to claim 1, wherein the microorganism detection device detects scattered light generated in the microorganism and identifies the type of the microorganism based on the scattered light.   The microorganism detection system according to claim 1, wherein the capturing device includes an impinger that captures microorganisms and soluble particles contained in the gas into the liquid.   The said taking-in apparatus is equipped with the scrubber which collects the microbe and soluble particle | grains which are contained in the said gas in a droplet or a liquid film, and takes in the said droplet or a liquid film in the said liquid. The microorganism detection system according to claim 1.   The intake device includes a powder separator that separates microorganisms and soluble particles contained in the gas, and the liquid container disposed where the separated microorganisms and soluble particles fall. The microorganism detection system according to any one of claims 1 to 5.   The microorganism detection system according to any one of claims 1 to 5, wherein the intake device includes an impactor that collects microorganisms and soluble particles contained in the gas in the liquid.   The microorganism detection system according to claim 1, wherein the capturing device captures the microorganisms and soluble particles collected by a filter into the liquid.