JP2002501363A - Microbial monitoring method - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for detecting the presence of respiring microorganisms in a solution. The method comprises the following steps. It involves (i) placing the solution in a container that can substantially isolate the solution from atmospheric oxygen, and exposing it to light when irradiated with light containing a wavelength that causes the fluorescent compound to fluoresce Placing a sensor composition containing a fluorescent compound capable of exhibiting a decrease in fluorescence intensity into the container without directly touching the solution so that microorganisms are not destroyed by the sensor composition; and (ii) the sensor Irradiating the composition with light containing a wavelength that causes the fluorescent compound to emit fluorescence; and (iii) measuring or visually observing the fluorescence from the fluorescent compound while irradiating the sensor composition with the light. (Iv) a respiratory microorganism-free control selected from the group consisting of: (iv) a calculated threshold and a reagent control not in contact with the respiring microbe. Comparing the results of the measurements of the control with the results of the above measurements, wherein the change in fluorescence intensity relative to the fluorescence intensity for the control indicates the presence of respiring microorganisms; When the increase is not measured or observed, the above steps (ii), (iii) and (iv) are repeated as necessary to detect the presence of respiring microorganisms in the solution. .

Description

DETAILED DESCRIPTION OF THE INVENTION                            Microbial monitoring method                                Background of the Invention                                Field of the invention   This application is a continuation of the patent application Ser. No. 07 / 687,359 filed Apr. 18, 1991. No. 08 / 025,899 filed on March 3, 1993 is there.                              Description of related technology   In our living environment, there are countless microorganisms that we always use. this These microorganisms benefit from fermenting to produce wine, vinegar, and antibiotics. And harmful substances that may cause infectious diseases. Because these microorganisms exist universally, their presence and metabolic activity Motion needs to be identified, studied and detected.   Microbiology has changed a lot in the last 25 years, but detecting microbial habits Many methods of identification and analysis are still time consuming. For example, antibiotics In the field of susceptibility testing, more than half of all U.S. hospital tests remain Bauer- Kirby Dics method is used. This method visually confirms the presence or absence of microbial growth This is a method to show the effect of the antibiotic compound. In general, you can get results with this method Requires 18 to 24 hours of incubation to grow the microorganisms. You. It is necessary to reduce the time required to obtain information on antimicrobial susceptibility. You.   Another popular antimicrobial susceptibility test method is microbial identification and antimicrobial susceptibility. dilution method (Becton Dickinson Diagnostic Instrumentation Systems, Sparks , MD). The system consists of several small cupulas (each cupula). Different tests using disposable plastic panels with Dry compound or different concentrations of test compound on each cupra surface To use. The microorganism to be tested is suspended in the desired test medium and the test The individual cupra of the flannel is filled with the aliquot. This system After dissolving the dried reagent on the panel and dissolving it in the sample, To allow enough time for interaction and growth to be visually confirmed, overnight ( (18 to 24 hours). This panel is It is used for a test to visually confirm the growth of microorganisms continuously. Obtain information about the susceptibility of the tested microorganism. In this case, the well The greater the number, the easier the identification of the microorganism. However, this test method is It has the disadvantage of requiring a long incubation period.   One way to shorten the incubation period is to grow microbial colonies. Rather, it is a way to monitor its metabolic activity. Such quick and accurate Many proposals have been reported for various monitoring attempts.   For example, a device using optical means called light scattering can be used in the presence of various antibiotic compounds. Used to determine susceptibility by examining changes in the number and size of microorganisms Have been. A commercially available device using this principle is a Vitec System (BioMerieux Crop.) Has been realized. This system is compatible with many microbial and drug combinations. And antimicrobial susceptibility It is reported that information can be obtained within six hours. Other combinations In some cases, this device can take up to 18 hours to determine the antimicrobial susceptibility of a microorganism.   Bauer-Ki, so that several samples can be observed within 4 to 6 hours Further improvements to the rby method are being developed. However, in such a system, It is necessary to spray the developing solution of the color stain on the test plate. It is "destructive" for the condition. Reincubate and measure later Becomes impossible, and if the fast test method fails, then the standard The law cannot be used subsequently.   Bioluminescent methods based on the amount of ATP present in growing microorganisms require specific compositions About 4 and a half hours can produce antimicrobial susceptibility test results. (Wheat et al.). However, this method is cumbersome and widely used. Not used.   Other approaches include measuring changes in pH and / or hemoglobin color, or salt Liquid test media such as triphenyltetrazolium bromide and resazurin The use of dyes that change color in response to the total redox potential of the nutrient solution allows for Includes those that monitor the oxygen consumption of organisms.   How to monitor the consumption of dissolved oxygen as a marker of the metabolism of this microorganism Has been studied for many years. For example, C.E.Clifton was Microbial oxygen consumption was monitored for several days using a Warburg flask. This method measures changes in oxygen concentration and is slow and cumbersome. there were.   A newer electrochemical device, the “Clark” type electrode, It is commonly used to measure dissolved oxygen. However, Clark-type The poles consume oxygen during use (this reduces the available oxygen for microorganisms). `` Standard '' size to avoid interference with electrode measurements. The electrode of the size is usually used only for measuring the volume of 100 mL or more.   A "miniature" Clark-type electrode is disclosed, which also comes in contact with the solution It is a part that cannot be measured unless it is done and consists of multiple complicated parts. In this case, Oxygen permeable membranes to protect the electrode components of the equipment from interaction with the components of the solution. Oxygen may be used, but oxygen will equilibrate between the test solution and the measurement system. On the other hand, once through the membrane oxygen is consumed (by the electrodes).   Obtaining oxygen concentration data overcomes the shortcomings of Clark-type electrode systems Optical systems have been developed. The main advantage of this optical system is that the oxygen concentration The equipment used for quantitative determination does not require physical contact with the test solution That is. Use either colorimetric or fluorometric optical techniques In addition, any of these methods can perform the analysis for oxygen reproducibly and quickly. And the cost of the analysis is considerably lower. For example, many Due to the fluorescent nature of the compound and the nature of oxygen quenching the emission of phosphorescence, oxygen Some luminescence-based techniques have been disclosed to determine the amount of . However, such methods are not suitable for monitoring microorganisms.   Provide information on the presence, identification, and antimicrobial susceptibility of microorganisms within a period of less than 8 hours Other systems that can be used have already been disclosed. Given to Wilkins and Stone U.S. Pat. A system for determining the presence of microorganisms using a dance potentiometer has been disclosed. Have been. U.S. Pat.No. 3,907,646 to Wilkins et al. For monitoring the excess headspace pressure in the flask associated with microbial growth Analytical methods that utilize change are described. U.S. Patent No. 4,220, granted to Ahnell In issue 715, the headspace on the test sample was determined to determine the presence of microorganisms. A system for passing a gas through an external oxygen detector is described. Given to Ahnell U.S. Pat.No. 4,152,213 discloses a headspace on a closed test sample. Describes an analytical system that monitors the vacuum created by growing microorganisms. I'm done. U.S. Patent No. 4,116,775 to Charles et al. To detect and monitor the turbidity or optical density of the growing microbial culture, Examples are given of the use of amplification-based optical means. European Patent Publication No. 0092 No. 958 (Lowe and Meltzer) uses a combination of birefringence in a test solution containing microorganisms. An aero-optical measuring method is described.   Increased incidence of tuberculosis and the emergence of Multiple Drug Reslitant (MDR) strains Today, it is a threat to the control of this disease. Therefore, if the strain is rifampin And the duration of treatment if resistant to more than one drug such as isoniazid Extends from June to April, and its cure rate drops from 100% to 60%.   Mycobacterium tuberculosis (TB, Mycobacterium tuberculosis) is a slow growing strain. Typically Is it necessary to grow the species to a sufficient cell volume for identification and sensitivity testing It requires a period of 3 to 5 weeks in solid or liquid medium. Most commonly The TB sensitivity test method used is the Modified Proportion Method (NCCLS M24-T). You. In this way, you can use A further growth period of 3 to 4 weeks is required to achieve the desired results. After all, generally It takes more than two or three months to combine the ports.   The BACTEC 460 device (Becton, Dickinson and Company, Franklin Lakes, NJ) The measurement time can be relatively reduced. In this BACTEC 460 method, mycobacterial Radioactive CO<sub>Two</sub>Can be detected to detect its presence. With the BACTEC system Is radioactive CO in the presence of an anti-mycobacterial agent.<sub>Two</sub>Microbial resistance due to continuous generation of Objects can also be detected.   A wide variety of different methods are available for detecting microorganisms and testing their sensitivity to antibiotics. It is clear that it is used for strikes. In many of these ways, this challenge Useful data only when monitored by equipment designed to solve Brought. Therefore, no special equipment is required, and the microbial properties and their existence are determined. There is a real need for a system that can do this. In addition, the nature of the microorganism No antibiotic-like compounds on microbial samples in a short time without effect There is also a real need for a system that can determine the effect of an object.                                Summary of the Invention   Therefore, one of the objects of the present invention is to detect the presence of microorganisms in a liquid or solid medium. Provide an improved means for releasing and examining its metabolic activity . A further object of the present invention is to provide a microorganism which can be easily measured and judged visually. Provide a monitoring device or system. This device or system Usually, results can be obtained within 6 hours, which is shorter than before. Furthermore, the book The invention uses a special instrument to measure the activity of enzymes or enzyme systems that consume oxygen. Without It is intended to provide means for detection and / or monitoring.   The above objects and related objects are realized by the process of the present invention. This process Fluorescence that can quantify and show the degree of quenching when exposed to oxygen A fluorescence detection system containing a sensor is used. In one embodiment, a sensor Compounds (either directly or sequestered by an oxygen permeable membrane) The fluorescence to the test sample and measure or observe the fluorescence with appropriate auxiliary means. Observe visually. In some embodiments, the increase in fluorescence utilizes oxygen in the sample. A sign of the presence of aerobic micro-organisms breathing (thereby reducing oxygen) is there.   This sensor does not require direct contact with the sample. Tessa Samples and sensors are usually housed in containers that are substantially isolated from atmospheric oxygen. Must be installed so that the sensor can detect if there is a change in oxygen in the container. Can be shown.   This system can be used to detect many respiring microorganisms as described above. Wear. In addition, the system detects the presence of oxygen-dependent compositions such as enzymes. Can be used to                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows the fluorescence of the labeling agent in contact with the liquid medium containing microorganisms and the liquid medium without microorganisms. 5 is a graph depicting intensity as a function of time.   Figure 2 shows the fluorescence intensity of the labeling agent in contact with the liquid medium inoculated at different microbial concentrations. Is a graph depicting as a function of time.   FIG. 3 shows contact with a liquid medium of the same number of microorganisms containing different phenol concentrations. 4 is a graph depicting the fluorescence intensity of a labeled agent as a function of time.   FIG. 4 shows the labeling of the same number of microorganisms in contact with liquid medium containing different amounts of copper sulfate. 5 is a graph depicting the fluorescence intensity of an agent as a function of time.   FIG. 5A shows a liquid medium containing different concentrations of cefuroxime but with the same concentration of microorganisms. 7 is a graph depicting the fluorescence intensity as a function of the labeling agent contacted with. Some way The oil was sealed with mineral oil to prevent oxygen from diffusing into the wells. fluorescence Luminosity is expressed as a percentage of the presence of the growth control.   FIG. 5B shows wells or oil-sealed wells measured at several different times. Is the percentage of growth control in wells left in air It is a graph depicting the fluorescence intensity.   FIG. 6 shows the fluorescence of the labeling agent in the blood culture bottle when the measurement was continued for 16 hours or more. 4 is a graph depicting light intensity. The arrow points to the sample to quantify the concentration of microbial presence. Indicates when the pull was moved. 5 is a graph depicting data collected by the device. 5 is a graph depicting data collected by the device. 6 is a graph depicting the data collected in FIG.                             Detailed description of the invention   The process of the present invention is aerobic breathing by visual observation or measurement of fluorescence. It is a fast, simple and clear way to measure and / or detect microorganisms. this The fluorescent sensor composition is illuminated with light containing a wavelength that causes fluorescence, and Is measured by any standard means or evaluated visually. This fluorescent compound has the concentration normally observed in test liquids (typically 0.4%). Exposure to oxygen) can show significant quenching No. In practice, any fluorescent compound can be used. Preferred of the present invention Fluorescent compounds are tris-2,2'-bipyridylruthenium (II) salts (tris-2,2'-bi pyridyl ruthenium (II) salt), of which the most preferred is chloride hexahydrate ( Ru (BiPy)<sub>Three</sub>Cl<sub>Two</sub>) And also tris-4,7-diphenyl-1,10-phenanthroline Ruthenium (II) salts (tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II ) salt), among which the chloride (Ru (DPP)<sub>Three</sub>Cl<sub>Two</sub>) And 9,10-di Phenylanthracene (DPA).   The fluorescent compound chemically reacts with oxygen in the test sample to indicate quenching. Must be in a place where communication is possible. This directly samples fluorescent compounds Can be achieved by contacting However, in a preferred embodiment, Materials that can transmit oxygen but are relatively impermeable to other compounds in the sample The fluorescent compound and the sample can mutually Sequestered, thus preventing interaction between the sample and the fluorescent compound. Fluorescence Compounds, membranes with embedded fluorescent compounds, samples, liquid media, or solutions There is no need for direct contact with oxygen, just the presence or absence of oxygen as a result of microbial respiration. As long as the compound and the sample are substantially isolated from atmospheric oxygen. Must be released, thus preventing any false positives due to the presence of oxygen in the atmosphere Do).   This system allows the internal presence of fluorescence to be observed for a predetermined Can be left unmeasured while the interaction takes place, and Often, compared to troll samples, The result can be obtained by two comparison observations. One of the advantages of this system is that That the measurement is non-destructive and that it has been Reincubation and measurement, even if results obtained are indeterminate Is what you can do. In addition, it is assumed to be compared with the reagent control. However, such a comparison is not always necessary and the appropriate selection of the fluorescent compound This allows those skilled in the art to conclude whether microbial activity is present or not. It is believed that the fruit can be determined independently.   Detection of fluorescence intensity can be performed using any commonly used measurement technique, such as a fluorometer. It can be performed by steps. In particular, the intensity of the fluorescence is visually observable and may Reagent control (e.g., for systems containing no viable microorganisms or for testing) (System without added chemicals). Thus, this method Providing a quantitative measurement of relative activity using a fluorimeter, or Can provide any qualitative assessment of such activity.   In a preferred embodiment of the present invention, when oxygen is present, There is an embodiment in which a fluorescent compound is selected from those which do not show fluorescence at all. Something The presence of fluorescence (in other words, fluorescence above negligible background fluorescence) was observed. The tester can determine that the tester has microbial activity. There is no need to use rolls. Such results can be obtained using a fluorimeter or other measurement. Obtained by visual inspection, or more preferably by A qualitative expectation of activity such as is quickly obtained. In this aspect, preferred Fluorescent compounds include Ru (BiPy)<sub>Three</sub>Cl<sub>Two</sub>And Ru (DPP)<sub>Three</sub>Cl<sub>Two</sub>There is.   Fluorescent compound or fluorescent compound embedded in membrane is used for liquid culture, test Isolate from atmospheric oxygen to use the sample or system that contacts the medium Tests are performed on the system, as well as systems exposed to atmospheric oxygen. It has also been shown that accurate results can be obtained. In fact, this is It is particularly preferred when the object is incubated for more than 2 hours. If exposed to the atmosphere Otherwise, all dissolved oxygen in the system will be consumed, resulting in erroneous This is because a measured value can occur. Thus, the use of the system of the present invention is It is quite broad and can be used under a wide range of conditions.   Furthermore, it is an advantage of the present invention that this integrated working device is assembled. You. In short, this device is a room for storing samples, or a test sample or Contains other liquids, soluble components for special applications (eg, nutrient solutions, etc.) A device that normally contains a suitable similar chamber. This chamber contains the oxygen level of the solution Can be added. As the labeling element of the present invention, Well known to have a large quenching effect on its fluorescence emission when exposed to oxygen Can be used.   As a further embodiment of the present invention, it is permeable to oxygen gas, Fluorescent compounds on plastics and rubbers that are relatively impermeable to non-oxygen solutes There is an embodiment in which objects are mixed or dispersed. Silicone rubber is particularly useful in such an embodiment Material. The test solution containing the microorganism may be slightly The metabolic activity of the organism causes a decrease in the dissolved oxygen level in the sample, The sample produces a high fluorescence signal upon excitation. Microbial-free sample liquid Shows no decrease in oxygen level and high quenchin against the fluorescence of oxygen. Only low levels of fluorescence due to the   In particular, an oxygen-sensitive fluorescent substance is a microcapsule made of a material having oxygen permeability. It is preferably contained in a preparation or granules. In addition, individually introduced into the test solution The fluorescent material is added to a separately manufactured structure such as a ball, a plate, or a protrusion. It could be included. The use of a protruding structure allows it to be used in lids and other devices. The advantage is particularly large because it can be easily handled by pasting. As a particularly preferred embodiment, Multiple protrusions to ensure proper orientation and allow simultaneous entry into multiple sample chambers In this embodiment, the origin is attached to a single film or another cover. In this case, Depending on the choice of substance, this protrusion can be made permeable to oxygen without penetrating the labeled molecules and microorganisms. Can be made to have.   Is the solution analyzed by a membrane permeable to oxygen but impermeable to labeled molecules and microorganisms? In the separated liquid phase, a fluorescent substance (fluorophore) can be added. Especially A low sensitivity sensor may be a less oxygen permeable polymer or excited state time It can be prepared by using shorter compounds.   In addition, the method of the present invention can be used to determine, for example, the activity of growth and / or metabolism of microorganisms. To test the susceptibility of microorganisms to compounds such as antibiotics Can also be used. In general, the increase in fluorescence signal normally caused by microorganisms Is suppressed by the presence of such a compound. Changes in the fluorescence signal from the container Strike components have a negative effect on the normal oxygen consumption of microorganisms added to the container Indicates that   Furthermore, in any of the above embodiments, the test sample in which the microorganism is present, Without the need for direct contact with liquid media, sensors, fluorescent compounds, or Embedded membranes can be utilized. In such cases, sensors, Compound or compound embedded The membrane is simply placed in the same container as the test sample, solution or medium. This system is required to function as an indicator of the presence or absence of respiratory microorganisms. The system must be substantially isolated from atmospheric oxygen.                                  Example   The following examples describe some preferred embodiments of the present invention, It is not intended to describe every embodiment. Embodiment 1 FIG. Preparation of microtitration tray for oxygen-sensitive labeling agent   Tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) Ru (DPP)<sub>Three</sub>C l<sub>Two</sub>) Using the procedure of Watts and Crosby (J. Am. Chem. Soc. 93, 3184 (1971)) Synthesized by 3.6 mg of the obtained compound was 2.0 mL of dimethyl sulfoxide (D-5879, Sigma Chemcal St. Louis MO) and slowly agitate the resulting solution. , 1300mL silicone rubber solution (Water Based Emulsion # 3-5024, Dow Cornin g Midland MI). Subsequently, the bottom is flat and the white microtiter tray (# 0 11-010-7901 Dynatech Chantilly VA) with 35 μL aliquots of the mixture in 96 wells Add each, then allow the system to run overnight in a low humidity (<25% RH) environment. At the boundary, curing was performed in a 60 ° C. incubator. After curing, soak the tray Washing by filling or filling method, and a) absolute ethanol, b) 0.1 of pH 7.2 M phosphate buffer, c) hot distilled water (about 45 ° C), d) ambient temperature distilled water Each well is emptied with these reagents over several hours.   Subsequently, 35% of Muller Hinton II (BBL 322124322 BD Microbiology System, Coc keysville MD), 15% Brucalla (BBL # 11088) and 50% steam Separate wells of 150 μL of liquid medium A containing distilled water into each of the trays. In a box containing the desired oxygen concentration and the appropriate nitrogen so that the total volume is 1 atm. Oita. Leave the tray in the box for at least 24 hours, then Sealed with a wearing Meyer sheet and moved.   The fluorescence emission of the fluorescent compound from the bottom of each tray was performed at an excitation wavelength of 485 nm. The emission window has a 550 nm cut-on filter and a 10 nm excitation slit. Equipped with microtiter tray reader with emission slit set at 5nm And Perkin-Elmer LS-5B. The results are shown in the table.                                   Table 1              Fluorescence of trays balanced at various oxygen gas levels   As shown in Table 1, in a tray (tray 7), which is in equilibrium with air in the atmosphere Labels in wells indicate trays that are in equilibrium with a gas mixture containing a low oxygen concentration. -1 to 6) showed a very small fluorescence signal. This means that silicone The fluorescence emission of the fluorescent display compound embedded in the rubber is related to the concentration of oxygen, This system makes it easy to balance even when oxygen levels change. Is shown. The system includes 96 sample wells (containing 0.1-0.3 mL samples) The operations can be made easier by grouping the parentheses into one unit. Embodiment 2. FIG. Use of a labeling system to measure the relative oxygen concentration produced by reducing agents   Oxygen concentration in wells of labeled microtiter tray prepared as in Example 1 The addition of a strong reducing agent, sodium sulfite (which reduces the oxygen content) So changed. 150 μL aliquots of reducing agent (concentration 0 to 1083 ppm (parts per million) )) Was added to the wells of the tray. 30 minutes in well After each reaction of 460 nm, it is exposed to the atmosphere, Fluoroskan II Fluorometer with filter and 570 nm cut-on filter (Flo w Fluorescence display was measured in Laboratories, McLean VA). The results are shown in Table 2. did.                                   Table 2                   Effect of sodium sulfite on fluorescence *: Average value of 4 wells   As shown in Table 2, the wells containing the highest concentration of reducing agent (so that the oxygen concentration Has the highest fluorescence intensity, which is the relationship between oxygen concentration and fluorescence. It shows the person in charge. Embodiment 3 FIG. Use of labeling systems to determine the presence of microorganisms   1.5 × 10<sup>8</sup>A suspension of 0.5 McFarland E. coli (ATCC # 25922) containing CFU / mL was A-Just Prepared using a nephelometer (Abbott Labs, Chicago IL). This suspension in liquid medium A (See Example 1) 1.0 × 10<sup>7</sup>Diluted to CFU / mL I shouted. A 150 μL aliquot of this suspension was placed in the wells of the labeling tray and And incubated at 37 ° C. 1 to 3 and a half hours At intervals, fluorescence was measured with a Fluoroskan II fluorimeter. Increased fluorescence The signal was observed over time as shown in FIG. From microbial-free wells Showed only very small changes in the fluorescence signal. Wells containing microorganisms are ultraviolet It was much brighter when viewed with a line. Thus, the fine The fluorescent signal appears to increase due to the metabolic activity of the organism (perhaps a decrease in oxygen According to). Embodiment 4. FIG. Depends on fluorescence change with microbial concentration   0.5 McF in sterile trypticase soy broth (TSB BBL # 11768) arland E. coli (ATCC # 25922) suspension was applied to an A-Just nephelometer (Abbott Labs, Chicago IL). ). And 1.0 × 10<sup>7</sup>E. coli suspension from CFU / mL to about 10 CFU / mL A series of liquors was prepared by dilution. A 200 μL aliquot of each suspension was prepared in the Example Placed in 8-well labeling trays such as 1. Incubate at 37 ° C Fluorescence was measured every 30 minutes with a Fluoroskan II fluorimeter. Fluorescence value of 8 wells Were averaged and corrected by subtracting the fluorescence of the sterile TSB well background. Of that time series The changes are shown in FIG.   As shown in FIG. 2, the change in the starting concentration of the microorganism by a factor of 10 (1 log unit) resulted in 2,0 There was a delay of about one hour or more to exceed the fluorescence unit. That this open to the atmosphere It is presumed to be one cause of the delay. Oxygen in the air diffuses freely into the microorganisms Can be added. In addition, the presence or presence of microorganisms Due to their large breeding, the rate of oxygen consumption due to metabolic activity increases the spread of oxygen from air. Equal to scattering speed Or only faster, the fluorescence signal generated by the labeling element at the bottom of the well increases. It is thought to add. Embodiment 5 FIG. Labeling microtitration trays with alternative fluorescent display molecules Preparation   In the silicone mixture (Ru (DPP)<sub>Three</sub>Cl<sub>Two</sub>) Is replaced by tris chloride (2,2 ' Ru) Ruthenium (II) hexahydrate (Aldrich Chemical Commany, Milwaukee, WI) (Ru (BiPy )<sub>Three</sub>Cl<sub>Two</sub>), Except that a 96-well microtiter tray was used. Was prepared. Similarly, a second microtaper containing 9,10-diphenylanthracene (DPA) I prepared an tray. For all wells, 150 μL of 1.0 × 10<sup>7</sup>CFU / mL E. coli (A TCC # 25922) was added to the liquid medium. 0, 1, 2, 3 and 4 hours after adding the microorganism Table 3 shows the results.                                   Table 3                   Fluorescence counting on instruments with different fluorescent materials A = Dow-Corning 3-5024 water-based silicone B = Wacker white SWS-960 + silicone   As shown in Table 3, each of the fluorescent sensor compounds showed a large Representing the increase in light suggests that they can be fully used in the system did. Embodiment 6 FIG. Preparation of Labeled Microtiter Tray Using Alternative Silicone   A 96-well microtiter tray was prepared as in Example 1 and That the fluorophore can function even when filled with Testify. In this example, 10 mg of Ru (DPP) per liter<sub>Three</sub>Cl<sub>Two</sub>10 μL white containing Add SWS-960 RTV silicone (Wacker Silicones, Adrian MI) to each well of the tray. And cured. The resulting tray was not washed. The results are shown in Table 3. You. As in Example 1, 1.0 × 10<sup>7</sup>150μL containing CFU / mL E. coli (ATCC # 25922) The liquid medium in the wells is very poor after several hours of incubation at 37 degrees Celsius. Had a high fluorescence intensity. Embodiment 7 FIG. Effects of toxic substances on oxygen consumption of microorganisms   3.0 × 10 in liquid medium A<sup>8</sup>Suspension containing CFU / mL Pseudomonas aeruginosa (ATCC # 10145) The suspension was prepared using an A-Just nephelometer. A total of 150 μL of suspension was applied as in Example 1. Put in each well of the display tray at 1.5 × 10<sup>8</sup>Bring the suspension to phenol to CFU / mL. Alternatively, it was diluted with a solution of copper sulfate (a substance that adversely affects the growth of microorganisms). This Incubate the cells at 37 ° C and measure fluorescence with a Fluoroskan II fluorimeter. Measured at minute intervals. Figures 3 and 4 show the phenols obtained with this system. And the effect of copper sulfate.   As shown in the figure, high levels of addition inhibit growth and increase fluorescence values. Did not increase over time. Contains 1 g / L or more of phenol and 500 mg / L or more of copper sulfate In the wells, no increase in the fluorescence signal was observed for 2 hours, and No metabolic activity was indicated. In this way, the measurement of oxygen consumption Can be closely examined for metabolism. Embodiment 8 FIG. Effects of antibiotics on Escherichia coli   E. coli (ATCC # 25922) of 0.5 McFarland (see Example 1) was added to the liquid medium A by A-Jus. Prepared using a t-turbidimeter. As in Example 1, the final Ciprofloxacin, Cefoxitin (Ce) at concentrations of 0.5 to 8 μg / mL foxitin) and Cefurxime antibiotics. × 10<sup>7</sup>Labeled trays diluted to CFU / mL were prepared. The tray was incubated at 37 ° C for 4 hours, and the fluorescence was changed to Fluoroskan II. It was measured with a fluorimeter. Table 4 shows the results.                                   Table 4                Fluorescence from labeled trays containing E. coli and antibiotics                           4 hours relative fluorescence   As shown in Table 4, at all concentrations, E. coli was sensitive to ciprofloxacin. And low fluorescence values were observed. On the other hand, resistance to cefuroxime concentration And a high fluorescence value was observed. In addition, this E. coli has a 0.5, 1, 2 μg / mL concentration Resistant to cefoxitin and high fluorescence values were observed, but 4 and 8 μg / mL Sensitive to high concentrations of cefoxitin and low fluorescence values were observed . Thus, the correlation between the fluorescence value and the concentration of the antibiotic can be seen by the system of the present invention. It is possible to evaluate the effects of raw materials and to determine the minimum effective concentration composition. Prove that it can be. Embodiment 9 FIG. Fluorescent indicator Ru (BiPy)<sub>Three</sub>Cl<sub>Two</sub>Against oxygen consumption of Escherichia coli when using The effect of raw materials   A-Just 0.5 McFarland E. coli (ATCC # 25922) (see Example 1) in liquid medium A Prepared using a nephelometer. Final concentration 0.5 to 8 μg / mL Containing ciprofloxacin, cefoxitin and cefuroxime antibiotics 1.0 × 10<sup>7</sup>Labeled trays diluted to CFU / mL were prepared as in Example 5 (Ru (BiPy )<sub>Three</sub>Cl<sub>Two</sub>Indicator) was prepared. Incubate the tray at 37 ° C for 4 hours. Fluorescence was measured with a Fluoroskan II fluorimeter. Table 5 shows the results.                                   Table 5                Fluorescence from labeled trays containing E. coli and antibiotics                           4 hours relative fluorescence   As shown in Table 5, as in Example 8, these E. coli cells were ciprofloxacin. And low fluorescence values were observed. On the other hand, the concentration of cefuroxime In this case, the cells were resistant and high fluorescence values were observed. In addition, this E. coli is 0.5, 1, Although resistant to cefoxitin at a concentration of 2 μg / mL, high fluorescence values were observed. , Sensitive to cefoxitin at high concentrations of 4 and 8 μg / mL and low fluorescence values Was observed. Thus, the result of the example is Ru (BiPy)<sub>Three</sub>Cl<sub>Two</sub>Used as a fluorescent labeling agent Suggested that it can be used. Embodiment 10 FIG. Antibiotics against oxygen consumption of Escherichia coli when using the fluorescent labeling agent DPA Effect   A-Just 0.5 McFarland E. coli (ATCC # 25922) (see Example 1) in liquid medium A Prepared using a nephelometer. Ciprofloxacin at a final concentration of 0.5 to 8 μg / mL, Contains cefoxitin and cefuroxime antibiotics And add 1.0 × 10<sup>7</sup>Labeled tray diluted to CFU / mL as in Example 5 (DPA labeling agent) was prepared. Incubate the tray at 37 ° C for 4 hours. Fluorescence was measured with a Fluoroskan II fluorimeter. Table 6 shows the results.                                   Table 6                Fluorescence from a label tray containing E. coli and antibiotics                           4 hours relative fluorescence   As shown in Table 6, as in Examples 8 and 9, these E. coli were It was sensitive to sacin and low fluorescence values were observed. On the other hand, cefuroxime It was resistant to the concentration and a high fluorescence value was observed. In addition, this E. coli contains 0. It is resistant to cefoxitin at concentrations of 5, 1, and 2 μg / mL, and high fluorescence values are observed. But sensitive to low concentrations of cefoxitin as high as 4 and 8 μg / mL Fluorescence values were observed. Thus, the results of the examples show that DPA is used as a fluorescent labeling agent. Suggested that it can be used. Embodiment 11 FIG. Effect of open and closed systems on oximetry   A 96-well labeled microtiter tray was prepared as in Example 1. And Prepare another copy in the same way and give them a concentration of 0.25 to 32 μg / mL. The antibiotic cefuroxime was prepared. There, 150μL of E. coli (ATCC # 11775) Add the suspension to the wells and bring its concentration to about 3.0 × 10<sup>7</sup>It was adjusted to CFU / mL. Two trays In one of the wells Apply mineral oil over the wells to prevent oxygen from diffusing into the wells. One was exposed to air. Incubate these trays at 37 ° C for 5 hours The fluorescence is measured with a Fluoroskan II fluorimeter and the resulting fluorescence value is At each antibiotic concentration compared to the average of several wells without The percentage of long control was determined. FIG. 5A shows an open system exposed to air. 5 hours of cefuroxime concentration in well-covered closed systems showed that. FIG. 5B shows the fluorescence of the growth control well in the open system and the closed system. Degrees of change were indicated.   In wells that were not covered with mineral oil, microorganisms at this concentration It has been shown that it is susceptible to shims, but is covered with mineral oil. In a `` closed system '', the concentration of antibiotics at 4 and 8 μg / mL can affect oxygen consumption. No sound appeared. This difference is probably due to the fact that the effects of antibiotics on This is because there was a shift. During this time, oxygen is Probably because it was led to a low level by engineering.   In this way, optimal sensitivity for the detection of the effect of the toxin on the microorganism is obtained. In order to utilize the present invention, the influx of oxygen into the sample solution should be tolerated. is there. Embodiment 12 FIG. Effect of sample volume on display tray   A suspension of E. coli (ATCC # 7133) of 0.5 McFarland was added to liquid medium A at 1.0 × 10⁷CFU / mL Diluted. Different volumes (10 μL to 300 μL) of the diluted suspension were prepared in Example 1 As in the wells of the display tray. Incubate the tray at 37 ° C for 4 hours. And measure the fluorescence with a Fluoroskan II fluorimeter at 30 minute intervals. Specified. the same By subtracting the fluorescence from a volume of sterile liquid medium, the fluorescence The change was measured. Table 7 shows the results.                                   Table 7                   Effect of sample amount in fluorescent labeling tray                              Relative fluorescence   Briefly, if the time is longer than 2 hours, the sample is The increase in the relative signal observed in each well is half that of wells with more than 80 μL of sample. Less than a minute was indicated. Microorganisms are in wells containing up to 40 μL of sample. The rate at which oxygen diffuses into a small sample is faster than the rate at which it is effectively consumed. It is likely that too little sample was present. Embodiment 13 FIG. Use of a labeling system without a fluorescence meter   A labeling tray was prepared using the same fluorescent compound and silicone as in Example 1. However The tray (# 4-3918, BD Labware Lincoln Park NJ) is made of clear plastic Made and the bottom of the well is circular. For every 10 μL of silicone, 2 nanograms of Ru ( DPP)_ThreeCl_TwoWas placed in the wells of each tray and performed without the washing step. Ps. ae ruginosa (BDMS Culture collection # N111) and E. coli (ATCC # 25922) 1.0 × 10⁷CFU / mL liquid culture To the medium A (see Example 1), and add 0 to 32 μg / mL Roxime, ciprofloxacin at 0.12 to 8 μg / mL, cefoxy at 0 to 32 μg / mL One of the tins was added to the tray. Incubate the tray at 37 ° C for 4 hours. Followed by an ultraviolet light box (# TX-365 A, Spectronics Corp., Westbury, NY). Away from the tray 550 cut-on filter 1 foot above (# LL-550-S-K962, Corion, Holliston, MA ) Could directly observe the resulting fluorescence. Contains no antibiotics Wells or wells that contain only antibiotic concentrations that do not affect microorganisms It is immediately apparent that all of the gels showed high fluorescence levels . Wells without microorganisms or wells with higher levels of antibiotics are very Showed low levels of fluorescence. Using an overnight microdilution antimicrobial susceptibility test Together with the determined MIC concentration, can effectively reduce fluorescence emission for each microorganism. Table 8 shows the lowest antibiotic concentrations that can be achieved.                                   Table 8                   Fluorescence results obtained without using MIC equipment Embodiment 14 FIG. To detect the presence of low-level bacteria in media containing blood Use of labeling agents   68ng Ru (DPP)_ThreeCl_TwoOne side coded with 3 mL of Dow Corning water-based emulsion containing Tissue culture flask (Falcon # 3804, BD Labware, Lincoln Park NJ) was prepared. The flask was sterilized with ethylene oxide. About 0.05 135 mL TSB (BBL # 11768) containing CFU / mL E. coli (ATCC # 25922) and 15 mL defibrinated blood The liquid was added to one flask. One flask contains 135mL of TSB without microorganisms Controls contain 15 mL of blood. Put a loose cap on the cap to allow air to flow. After scoring, incubation was performed at 37 ° C. in the light. fluorescence A few feet from the incubator, with a fiber optic probe to measure At this time the fluorescence from the flask was directed to a Perkin Elmer LS-5B fluorescence spectrophotometer. 10nm Flask at 485 nm excitation wavelength with slit width of 550 nm and cut-on filter of 550 nm Was measured with a fluorimeter. To monitor fluorescence for 16 hours continuously, print a band on the fluorometer. I attached the recording paper. Stand for 7.5, 10.5, and 16 hours during incubation Each time, remove 100 μL aliquots from test flasks and make 1: 100 with sterile TSB Diluted, spread 100 μL of the diluted solution on each plate of 3 TSA, The CFU / mL amount of the microorganism in the sample was determined. The results are shown as a graph in FIG.   As shown in FIG. 6, the technology that does not invade healthy tissue of the present invention is very important and demanding. It can be used to detect important microorganisms in blood. Cloudy and cloudy solution The containing flask is monitored continuously for an additional 16 hours, and the measurement of fluorescence is Direct association was shown. Microorganism concentration of 10⁶When CFU / mL is exceeded, grow for 11 hours Could be detected immediately. Embodiment 15 FIG. Labeling agent coated on the circular bottom of the protrusion of the lid of the FAST tray   This example coats the circular edge of the protrusion on the lid of a FAST tray (Becton Dickinson). Bacterial respiration was monitored with the removed oxygen label. Three Different labeling agents were selected for evaluation.   1 mL Ru (DPP)_ThreeCl_Two2 mg / mL dichloromethane solution and 10 mL Dow-Corning 3-5024 A mixture with a water-based emulsion was provided as the first labeling agent. FAST tray lid protrusion Immerse the circular end of the raised part in a shallow marker solution, move it, and tap the protrusion downward. And cured by evaporation. 3mL Wacker SWS-960 clear silicone Dispersion, 6mL petroleum ether, 0.5mL Ru (DPP)_ThreeCl_Two2 mg / mL dichloromethane A mixture with the solution was prepared as a second labeling agent. FAST tray lid protrusion The edge is coated with this label in the same manner as the first label, and the solution is allowed to evaporate. Cured by reaction with atmospheric moisture. Wacker SWS-960 using white silicone Other than that, a third labeling agent was prepared in exactly the same manner as the second labeling agent.   1.0 × 10 in Mueller Hinton liquid medium⁷CFU / mL suspension of E. coli (ATCC # 25922) Prepare a 150 μL aliquot of the microtiter tray in an odd-numbered row Aliquot a 150 μL aliquot of uninoculated MuellerHinton liquid medium added to The coat was added to the even-numbered rows of the microtiter tray. Labeling agent Place the lid with the coated protrusions on the tray and place the lid on the tray at 37 ° C 3 hours in the incubator.   After a 3 hour incubation, place the tray on a clear glass plate. Was. Place the mirror under the glass plate so that the bottom of the tray is visible through the mirror installed. A 365nm UV light source that evenly illuminates the entire tray 1 inch above the tray It was installed in. Place a box with a small window through which a mirror can be seen over the Assemble to block the light, then put a 550nm cut-on filter in the box window installed. Labeling agent The fluorescence from the circular bottom of the protrusion of the lid of the FAST tray coated with Can be visualized through the bottom of the tray. Visual inspection of the tray evaluated by this means The results of typical observations are included in Table 9.                                   Table 9 Visual observation of the label coated on the protrusion of the lid looking through the bottom of the tray   Thus, in Dow Corning and Wacker White, the The differences between the wells that are not All of the systems obtained the desired results. Embodiment 16 FIG. Adsorbs on silica gel particles embedded in UV-cured silicone rubber Ru (DPP)_ThreeCl_TwoLabeling agent consisting of   Ru (DPP) on silica gel particles_ThreeCl_TwoAdsorb to Loctite Nuva-Sli silicone rubber A labeling agent having the particles embedded therein was prepared. Silica gel with different mesh sizes Particles, different adsorbed fluorophore amounts, different ratios of silicone to silicone Two types of licagel and Loctite Nuva-Sli (Nuva-Sli 5091 and Nuva-Sli 5147) Various labeling agents were prepared using silicone. Characteristics of the prepared labeling agent and Table 10 summarizes the visual results obtained from the labeling agent in contact with the microbial suspension. The experimental procedure used for preparing the labeling agent is shown below.   10-g Davisil silica gel with 100-200 mesh (Aldrich, Milwaukee, WI) was measured in a 500 mL round bottom flask. 0.14mg / mL Ru (DPP)_ThreeCl_TwoEthanol 43 mL of the solution was placed in the flask. The ethanol is removed by rotary vacuum evaporation and Kagel concentration 0.6mg Ru (DPP)_ThreeCl_TwoRu (DPP) of silica gel which is / gm_ThreeCl_TwoSorbent Obtained. Mix 4 g of this silica gel with 16 g of Nuva-Sli 5091 (Loctite, Newington, CT) As a result, a 20% (w / w) ratio of silica / silicone was obtained. 25 mL aliquots Was placed in the wells of a microtiter tray. Loctite Zeta 7200UV Cure by exposure to high intensity UV radiation for 15 seconds in the curing room. table Other labeling agents in No. 10 were prepared similarly.   To evaluate the labeling agent, 150 μL of 1 × 1 in Mueller Hinton II broth (BBL) 0⁷CFU / mL suspension of E. coli (ATCC # 25922) was selected in microtiter trays. In the wells, uninoculated liquid medium was placed in the other wells. 35 ℃ For 3 hours. To visualize fluorescence from labeling agents And place the tray on a benchtop with a 365nm UV light box, Can be observed from above through a 550 nm cut-on filter. In the response column of Table 10 One "+" sign is a clear increase from wells containing microorganisms. This indicates that great fluorescence was observed.                                  Table 10                         Official name of labeling agent and its response *: Results were obtained from all 18 cases (9 cases each for silicone 5091 and 5147)   When repeated experiments were performed in wells containing no microorganism, the labeling agent showed little light. Or little (except at higher label concentrations (0.6 mg / mL) Dark light was observed).   Many modifications and variations of the present invention described above are within the scope and spirit of the present invention. Real The embodiments provided by the examples are only special embodiments of the present invention, and the scope of the present invention is not limited to the embodiments. It is specified by the appended claims. Embodiment 17 FIG. Oxygen sensor without direct contact with sample solution   Multiple test vials (60 mL capacity) containing 60 mL media and oxygen sensor (OS) The microorganism, Pseudomonas aeruginosa, Mycobacterium fortuitum and Escherichia c oli inoculated. These vials were combined with 80 mL vials without liquid medium. Connect with a rubber tube through which oxygen cannot pass Was collected over 50 hours. The results are shown in FIGS. Figure The collected data was drawn. In FIG. 8, fluorescence showing growth of M. fortuitum Painted. In each figure, the thick line was collected from the vial containing the liquid medium. Represents the data, and the thin line represents the data collected by the sensor not directly contained in the liquid medium. Data. In these three cases, the consumption of oxygen in vials without liquid medium Was observed. The pattern of oxygen consumption that appears in these vials is It represents enzyme consumption as a logarithm indicating the growth of an object.   This data is useful when no sensor is in direct contact with the liquid medium. Shows that OS can be used to detect microbial growth ing. The delay in detection seen in vials containing no medium was due to this particular test. Related to the configuration environment. One of ordinary skill in the art will recognize the parameters of this system. Can be optimized, and by way of example, but not limited to, Reduce headspace volume and oxygen concentration to increase sensitivity and improve liquid culture Compared to measuring by touching (liquid phase), measuring without contacting the liquid medium (Ga Phase) can be made more similar.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, HU, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, N O, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Barrel, Gregory Jay             United States Pennsylvania             17356, Red Lion, Acorn Les             Inn 3055 (72) Inventor Beatty, Sean             United States Pennsylvania             17322, Felton, Water Street             Port 50 (72) Inventor Fu, Joanna Kwok You             45069, Ohio, Ohio, USA             Stochester, Quail Meadow             Rain 8065 (72) Inventors Manson, James F             United States Maryland 21074,             Hampstead, Linkrest Drive             4618 (72) Inventor Sapuitwitz, Robert             United States Maryland 21014,             Bel Air, East Ring Fact             Lee Lord 967 (72) Inventor Foley, Timothy G             United States Maryland 21050,             Forest Hill, Pleasantville Lo             Mode 1722

Claims (1)

[Claims] 1. (I) in a container that can substantially isolate the solution from atmospheric oxygen; The solution was added, and the fluorescent compound was irradiated with light containing a wavelength causing fluorescence. At the same time, a sensor containing a fluorescent compound that can show a decrease in fluorescence intensity when exposed to oxygen. The sir composition to the solution so that microorganisms are not destroyed by the sensor composition. Placing in the container without direct contact; (Ii) The sensor composition contains a wavelength that causes the fluorescent compound to emit fluorescence. Irradiating light; (Iii) while irradiating the light to the sensor composition, Measuring or visually observing (Iv) The result of the measurement of the control containing no respiring microorganisms and the above Comparing the result of the measurement with the calculated threshold value. And a reagent control that is not in contact with breathing microorganisms Selected, the change in fluorescence intensity relative to the control fluorescence intensity Indicating the presence of a microorganism (V) If no such increase is measured or observed, the above (ii), (i) Repeat steps ii) and (iv) as necessary to obtain the fine breathing solution in the above solution. Detecting the presence of an organism; A method for detecting the presence or absence of microorganisms in a solution, comprising: 2. Fluorescent compounds have high permeability to oxygen, but water and non-gaseous solutes 2. The method according to claim 1, wherein the matrix is included in a matrix having non-permeability. The method described in. 3. The above matrix is a rubber or plastic matrix The method according to claim 2, characterized in that: 4. Wherein the matrix is a silicone rubber matrix; The method of claim 2. 5. Wherein the fluorescent compound is adsorbed on solid silica fine particles, The method according to claim 2. 6. The fluorescent compound is tris-4,7-phenyl-1,10 phenanthroline ruthenium (II) salt (tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) salt) The method of claim 1, wherein: 7. The fluorescent compound is tris-4,7-phenyl-1,10 phenanthroline The method according to claim 6, characterized in that it is ruthenium (II). 8. The above fluorescent compound is a tris-2,2-bipyridyl ruthenium (II) salt (tris-2,2 ′ -bipyridyl ruthenium (II) salt), characterized in that: Law. 9. The fluorescent compound is tris-2,2'-bipyridyl ruthenium (II) chloride The method according to claim 8, characterized in that: 10. The fluorescent compound is 9,10-diphenylanthracene (9,10-diphenylanth racene). 11. (I) providing a culture medium for respiring microorganisms; (Ii) The above medium is placed in a container capable of substantially isolating the medium from atmospheric oxygen. And then irradiate the fluorescent compound with light containing a wavelength that causes fluorescence to occur. , A sensor containing a fluorescent compound that can show a decrease in fluorescence intensity when exposed to oxygen The composition is added directly to the medium so that microorganisms are not destroyed by the sensor composition. Placing in the container without touching; (Iii) mixing an antibiotic or antibacterial composition into the medium When, (Iv) irradiating the sensor composition with light containing a wavelength that causes the fluorescent compound to emit fluorescence. Steps (V) irradiating the fluorescent light from the fluorescent compound while irradiating the light to the sensor composition. Measuring or visually observing; (Vi) Calculated threshold and respiring microorganisms or antibiotic or antimicrobial composition Reagent controls that do not come in contact with substances and antibiotics or other substances that come into contact with respiring microorganisms Is selected from the group consisting of a reagent control that does not contact the antimicrobial composition. Steps to compare the measurement results of the control with the results of the above measurement or observation Change in fluorescence intensity relative to the fluorescence intensity of the control. Exhibit the sensitivity or resistance of the microorganism to the amount of the antibiotic or antimicrobial composition Steps and (Vii) when no such change is measured or observed, the above (iv), Repeat steps (v) and (vi) as necessary to reduce Determining the effect of the antibiotic or antimicrobial composition; Antibiotics or antibacterial properties against respiring microorganisms, characterized by comprising A method for determining the effect of a composition. 12. The fluorescent compound is not permeable to water and non-gaseous solutes but is Is characterized by being contained in a highly permeable matrix. 12. The method according to claim 11. 13. A contractor characterized in that the matrix is rubber or plastic. 13. The method according to claim 12. 14． The above rubber or plastic matrix is silicon rubber mat 13. The method according to claim 12, wherein the method is a risk. 15. Wherein the fluorescent compound is adsorbed on solid silica fine particles. The method of claim 12, wherein: 16. The fluorescent compound is tris-4,7-phenyl-1,10 phenanthroline 13. The method according to claim 12, characterized in that the salt is a (II) salt. 17． The fluorescent compound is tris-4,7-phenyl-1,10 phenanthroline 17. The method according to claim 16, wherein the method is ruthenium (II). 18. The fluorescent compound is a tris-2,2'-bipyridyl ruthenium (II) salt. The method according to claim 11, characterized in that: 19. The fluorescent compound is tris-2,2'-bipyridyl ruthenium (II) chloride The method according to claim 18, characterized in that: 20. The fluorescent compound is 9,10-diphenylanthracene, The method of claim 11, wherein 21. (I) in a container capable of substantially isolating the solution from atmospheric oxygen Solution and add tris-4,7-phenyl-1,10 phenanthroline (II) and tris-2,2'-bipyridylruthenium (II) hexahydrate A microorganism comprising the sensor composition comprising a fluorescent compound selected from the group consisting of Into the container without direct contact with the solution so that it is not destroyed by the sensor composition Steps and (Ii) The sensor composition contains a wavelength that causes the fluorescent compound to emit fluorescence. Irradiating light; (Iii) while irradiating the light to the sensor composition, Measuring or visually observing (Iv) Calculated thresholds and reagent controls not in contact with respiring microorganisms Measurement results for controls selected from the group consisting of Comparing the result of the measurement or observation with the fluorescence intensity of the control. Changes in fluorescence intensity relative to indicate the presence of respiring microorganisms; (V) If no such increase is measured or observed, the above (ii), (i) Repeat steps ii) and (iv) as necessary to obtain the fine breathing solution in the above solution. Detecting the presence of an organism; A method for detecting the presence of respiring microorganisms in a solution, comprising: . 22. The fluorescent compound is not permeable to water and non-gaseous solutes but is Is characterized by being contained in a highly permeable matrix. 22. The method according to claim 21. 23. A contractor characterized in that the matrix is rubber or plastic. 23. The method according to claim 22. 24. A contractor characterized in that the matrix is a silicone rubber matrix. 23. The method according to claim 22. 25. Wherein the fluorescent compound is adsorbed on solid silica fine particles. 23. The method of claim 22. 26. (I) providing a culture medium for respiring microorganisms; (Ii) The above medium is placed in a container capable of substantially isolating the medium from atmospheric oxygen. And tris-4,7-phenyl-1,10 phenanthroline ruthenium (II) and tris-2,2'-bipyridyl ruthenium (II) hexahydrate A microorganism comprising the sensor composition comprising a fluorescent compound selected from the loop. So that it is not destroyed by the composition Placing the medium in the container without directly touching the medium; (Iii) mixing an antibiotic or antibacterial composition into the medium; (Iv) irradiating the sensor composition with light containing a wavelength that causes the fluorescent compound to emit fluorescence. Steps (V) irradiating the fluorescent light from the fluorescent compound while irradiating the light to the sensor composition. Measuring or visually observing; (Vi) Calculated threshold and respiring microorganisms or antibiotic or antimicrobial composition Reagent controls that do not come in contact with substances and antibiotics or other substances that come into contact with respiring microorganisms Is selected from the group consisting of a reagent control that does not contact the antimicrobial composition. Step of comparing the measurement results of the The change in fluorescence intensity relative to the control fluorescence intensity A sample showing the susceptibility or resistance of a microorganism to the amount of an antibiotic or antibacterial composition. Tep, (Vii) when no such change is measured or observed, the above (iv), Repeat steps (v) and (vi) as necessary to reduce Determining the effect of the antibiotic or antimicrobial composition; Antibiotics or antibacterial properties against respiring microorganisms, characterized by comprising A method for determining the effect of a composition. 27. The fluorescent compound is not permeable to water and non-gaseous solutes but is Is characterized by being contained in a highly permeable matrix. The method of claim 26. 28. The matrix is made of rubber or plastic 28. The method of claim 27, wherein 29. A contractor characterized in that the matrix is a silicone rubber matrix. 28. The method of claim 27. 30. Wherein the fluorescent compound is adsorbed on solid silica fine particles. 28. The method of claim 27.