Microorganism-collecting chip, microorganism-collecting kit, method of quantifying microorganisms, specimen for confirming normal state of microorganism-quantifying apparatus and microorganism-quantifying apparatus

Abstract

The invention aims to efficiently collect microorganisms from a test sample and accurately detect and quantify the collected microorganisms. A microorganism-collecting chip of the invention basically comprises a filter for removing contaminants and a filter for trapping microorganisms. A microorganism-collecting kit comprises the foregoing microorganism-collecting chip and a suction filtration unit. The suction filtration unit is, for example, a negative pressure tube provided at an opening with a rubber stopper. The microorganism-collecting chip has a liquid specimen injection container for injecting a liquid specimen and a hollow needle capable of penetrating the rubber stopper mounted at the opening of the negative pressure tube. The liquid specimen injected into the liquid specimen injection container is suction-filtered with a pressure of the negative pressure tube. Contaminants are removed with the filter for removing contaminants, and microorganisms are trapped on a filter for collecting microorganisms. The microorganisms trapped on the filter for collecting microorganisms are then detected and quantified by using a unit including the filter for collecting microorganisms.

1 . A microorganism-collecting chip comprising a combination of a filter for removing contaminants mounted at a front stage as a prefilter and a filter for collecting microorganisms mounted at a back stage, in which the microorganisms are filtered from a liquid specimen probably containing the microorganisms to trap the microorganisms on the filter for collecting microorganisms.  
     
     
         2 . The microorganism-collecting chip according to  claim 1 , wherein a pore diameter of the filter for removing contaminants is from 5 to 20 μm, and a pore diameter of the filter for collecting microorganisms is from 0.2 to 0.8 μm.  
     
     
         3 . The microorganism-collecting chip according to  claim 1 , wherein the filter for removing contaminants is mounted on a bottom of a liquid specimen injection container.  
     
     
         4 . The microorganism-collecting chip according to  claim 1 , wherein the liquid specimen injection container is mounted ahead of the filter for removing contaminants.  
     
     
         5 . The microorganism-collecting chip according to  claim 3  or  4 , wherein a lid with a swab for covering an opening of the liquid specimen injection container is provided.  
     
     
         6 . The microorganism-collecting chip according to  claim 1 , wherein a site including the filter for collecting microorganisms is adapted to be solely removable.  
     
     
         7 . The microorganism-collecting chip according to  claim 1 , wherein a dark filter is used as the filter for collecting microorganisms.  
     
     
         8 . The microorganism-collecting chip according to  claim 1 , wherein a thin film containing at least one metallic component selected from gold, copper, chromium, platinum and palladium is formed on the filter.  
     
     
         9 . The microorganism-collecting chip according to  claim 8 , wherein a film thickness of the thin film is from 10 to 50 nm.  
     
     
         10 . A microorganism-collecting kit comprising a combination of a filter for removing contaminants mounted at a front stage as a prefilter and a filter for collecting microorganisms mounted at a back stage, in which the microorganisms are filtered from a liquid specimen probably containing the microorganisms to trap the microorganisms on the filter for collecting microorganisms, and a suction filtration unit.  
     
     
         11 . The microorganism-collecting kit according to  claim 10 , wherein a negative pressure tube is used as the suction filtration unit.  
     
     
         12 . The microorganism-collecting kit according to  claim 11 , wherein a rubber stopper is mounted at an opening of the negative pressure tube.  
     
     
         13 . The microorganism-collecting kit according to  claim 12 , wherein a central portion of the rubber stopper is made of a thin layer.  
     
     
         14 . The microorganism-collecting kit according to  claim 11 , wherein a hollow needle that reaches the inside of the negative pressure tube is mounted on a lower portion of the filter for collecting microorganisms in the microorganism-collecting chip.  
     
     
         15 . A method of quantifying microorganisms, which comprises contacting a liquid specimen with one or more of a first compound that colors viable and dead cells, a second compound that colors the dead cells with a wavelength different from that of the foregoing coloration and a third compound that colors the viable cells with a wavelength different from that of the foregoing coloration and at least one fourth compound that allows coloration with a wavelength from that of the foregoing coloration by a reaction with a specific microorganism-derived material, staining microorganisms in case of containing the microorganisms in the liquid specimen, then trapping the microorganisms on the filter for collecting microorganisms using the microorganism-collecting chip according to  claim 1 , and thereafter detecting both or either of the viable cells and the dead cells and the specific species of microorganisms at the same time from the difference in wavelength and the amount of coloration.  
     
     
         16 . A method of quantifying microorganisms, which comprises trapping microorganisms on a filter for collecting microorganisms from a liquid specimen probably containing microorganisms using the microorganism-collecting chip according to  claim 1 , then contacting the trapped microorganisms with one or more of a first compound that colors viable and dead cells, a second compound that colors the dead cells with a wavelength different from that of the foregoing coloration and a third compound that colors the viable cells with a wavelength different from that of the foregoing coloration and at least one fourth compound that allows coloration with a wavelength different from that of the foregoing coloration by a reaction with a specific microorganism-derived material, staining the microorganisms, and thereafter detecting both or either of the viable cells and the dead cells and the specific species of microorganisms at the same time from the difference in wavelength and the amount of coloration.  
     
     
         17 . The method of quantifying microorganisms according to  claim 15  or  16 , wherein India ink is added to the liquid specimen.  
     
     
         18 . The method of quantifying microorganisms according to  claim 15  or  16 , wherein after the microorganisms are trapped on the filter for collecting microorganisms, India ink is added from above the filter for collecting microorganisms to blacken the filter for collecting microorganisms.  
     
     
         19 . The method of quantifying microorganisms according to  claim 15  or  16 , wherein a microorganism-quantifying apparatus is confirmed to be in a normal state by previously detecting luminous bodies that emit light with excitation light of a specific wavelength using a specimen for confirming a normal state of a microorganism-quantifying apparatus, the specimen comprising a base material on which surface the luminous bodies are fixed, and both or either of the viable cells and the dead cells and the specific species of microorganisms are detected at the same time.  
     
     
         20 . The method of quantifying microorganisms according to  claim 19 , wherein the luminous bodies are polymeric fluorescent grains.  
     
     
         21 . The method of quantifying microorganisms according to  claim 19 , wherein the luminous bodies are stained microorganisms.  
     
     
         22 . A specimen for confirming a normal state of a microorganism-quantifying apparatus in which the microorganism-quantifying apparatus is confirmed to be in a normal state before quantifying microorganisms, the specimen comprising a base material on which surface luminous bodies that emit light with excitation light of a specific wavelength are fixed.  
     
     
         23 . The specimen for confirming a normal state of a microorganism-quantifying apparatus according to  claim 22 , wherein the base material is darkened.  
     
     
         24 . The specimen for confirming a normal state of a microorganism-quantifying apparatus according to  claim 22 , wherein a thin film containing at least one metallic component selected from gold, copper, chromium, platinum and palladium is formed on the base material on which surface luminous bodies that emit light with excitation light of a specific wavelength are fixed.  
     
     
         25 . The specimen for confirming a normal state of a microorganism-quantifying apparatus according to  claim 24 , wherein a film thickness of the thin film is from 10 to 1,000 nm.  
     
     
         26 . A microorganism-quantifying apparatus comprising a light source for emitting excitation light with a predetermined wavelength region to a fixed micro-area of the filter for collecting microorganisms in the microorganism-collecting chip according to  claim 1 , a light receiving unit for receiving the light of the predetermined wavelength region emitted with the excitation light, a microorganism-identifying unit for receiving the light emitted by the light source in a determined time and identifying the light as the microorganism when the amount of light received is within a determined threshold, a moving unit for moving the fixed micro-area continuously or intermittently and an addition unit for adding up the number of the microorganisms from signals identified as microorganisms by the microorganism-identifying unit, wherein a base material on which surface luminous bodies that emit light with excitation light of a specific wavelength are fixed is provided so as to be able to confirm that the microorganism-quantifying apparatus is in a normal state, before quantifying the microorganisms.