JP5245379B2 - Collection device and analysis system using the same - Google Patents

Description

  The present invention relates to a collection device and an analysis system using the same, for example, a collection device for collecting microorganisms floating in the atmosphere, and an analysis system using the same.

  As a method for inspecting the number of microorganisms such as bacteria floating in the air, a method of culturing microorganisms collected on the surface of a gel-like agar medium using a dust collector and counting the number of colonies generated has been put into practical use.

  However, the culture method has a problem that it takes a long time of several days, and it is difficult to automate and takes time. Microbiological testing is often used for verification of microbial cleanliness (sterility) of environments requiring high cleanliness, such as pharmaceutical factories, cell processing centers, and food factories. When the culture method is applied to on-site microorganism testing in the above environment, the purpose of ensuring the cleanliness of the environment may be destroyed if a large amount of amplified microorganisms, which are products of the inspection process, enter the site. It is difficult to perform inspections, and there is a problem that it is necessary to set up an inspection room, transport samples, and strictly manage products.

  Various methods have been devised for improving the number of microorganisms without culturing, that is, quickly, simply, and without risk of biological contamination. As a first method, there is a method (fluorescence method) in which microorganisms in a sample are stained using two types of fluorescent reagents, and live and dead bacteria are discriminated and counted by fluorescence microscopic image analysis or the like. As a second method, there is a method (ATP method) in which ATP is extracted from microorganisms in a sample and the amount of ATP is quantified by measuring the bioluminescence reaction of ATP using a luminometer and converted to the number of viable bacteria.

  However, in principle, the first method can measure one bacterium, but if a fluorescent contaminant is mixed in the sample, it causes an error. Also, it is not always easy to distinguish between live and dead bacteria. For example, the average fluorescence intensity of killed bacteria of propidium iodide (PI), which is supposed to selectively stain dead bacteria, may be lower than that of live bacteria (Non-Patent Document 1, FIG. 1C, the vertical axis represents the fluorescence of PI. Strength), a simple interpretation is likely to lead to the opposite conclusion.

  On the other hand, since the second method is based on a bioluminescence reaction that selectively responds to ATP, it is not influenced by fluorescent contaminants in principle. In addition, an ATP scavenger that erases ATP contained outside the cells of live cells in advance, an ATP extraction reagent that extracts ATP from live cells, and luminescence for measuring ATP in live cells by emitting light through a bioluminescence reaction A reagent kit (product name “Lucifer HS Set”) including a reagent is also available from Kikkoman Corporation (Non-patent Document 2). If this kit is used in combination with a luminometer, the effects of dead bacteria and the like are eliminated, and the amount of ATP derived only from live bacteria can be selectively measured.

  However, the test target sample of the second method is a microorganism dispersed in an aqueous solution and is not assumed to be applied to the test for airborne bacteria. A product that can be equipped with an impactor-type dust collection mechanism and can contain a collection carrier made of gel-like agar (sugar) medium (for example, M Air samplers applicable to the ATP method are not commercially available. When applying the ATP method to the inspection of airborne bacteria, the bacteria are collected on a collection carrier using an impactor-type air sampler for the culture method, and the bacteria are taken out from the collection carrier to form a bacterial suspension. Lucifer A method of measuring by the ATP method using a reagent kit based on a bioluminescence method such as an HS set is conceivable. Hereinafter, a simple combination of the conventional air sampler and the ATP method will be referred to as a conventional method in this specification.

Product Technical Information "Live / Dead Backlight TM Bacterial Viability and Counting Kit (L34856)" Molecular Probes, Revised February 2, 2004 Murakami Seiji et al. “Applied development of firefly luciferase” Journal of Japanese Society for Agricultural Chemistry, Vol. 78, No. 7, p. 630-635 (2004) Supervised by Kunio Furusawa, “New Science of Dispersion and Emulsification and New Development of Applied Technology”, ISBN 4-924728-50-0, Techno System, p. 370 (2006)

  However, the collection carrier comprising a gel-like agar medium used in an air sampler in the conventional method is intended to culture the collected bacteria on the collection carrier. For this reason, the conventional method does not consider the step of taking out the bacteria from the collection carrier (hereinafter referred to as the taking-out step). The extraction process has the following problems. That is, since the collection carrier is in the form of a gel, it can capture bacteria well, but it is difficult to take it out and the collection efficiency of bacteria is low. If the agar gel is heated, it can be made into a sol that can be handled substantially like a bacterial suspension.

  However, since a high temperature of 80 ° C. or higher is required for solification of agar gel, most of the live bacteria are damaged or killed, and the recovery efficiency of live bacteria is low, and there is a problem that live bacteria and dead bacteria cannot be distinguished. is there. Therefore, in order to increase the recovery efficiency of viable bacteria, do not sol agar gel, repeat the operation of adding a solution at a temperature close to room temperature on the gel and recovering the bacteria transferred to the solution as a bacterial suspension, etc. The mild conditions are necessary. However, this requires time-consuming and delicate manual work by skilled workers, is difficult to automate, has low reproducibility, and has the risk of false positives due to worker-derived contamination.

  On the other hand, a method of simplifying the operation by increasing the number of solutions used for recovery is also conceivable, but if the amount of the bacterial suspension is large, the number of bacteria (concentration) per unit liquid is low, resulting in the measurement process. The utilization efficiency of bacteria is low. For example, according to the protocol (conventional protocol) in the Lucifer HS set of Kikkoman Co., Ltd., which is a representative product of the ATP method, the sample solution volume is 0.1 mL.

  Accordingly, the utilization efficiency of the suspension in the measurement step is 10% if the volume of the bacterial suspension is 1 mL, and 1% if the volume is 10 mL, and the utilization efficiency of the suspension is lower as the amount of the suspension is larger. Therefore, this method has a problem that convenience and utilization efficiency are contradictory.

  Furthermore, according to the conventional protocol, since 1/10 of the sample solution is actually used for luminescence measurement, there is a problem that the overall utilization efficiency is only 10% of the bacteria recovery efficiency. In addition, if the purpose is to examine a few to tens of extremely small amount of airborne bacteria in a clean environment such as a clean room, if the collection efficiency and usage efficiency are low, it will cause a measurement leak of the bacteria. There is a false negative issue. In order to avoid measurement omission, measures such as increasing the sampling volume of the air sample are required, and there is a problem that the sampling time is long and the result cannot be obtained quickly.

  As described above, if the above method is automated as it is, the system configuration is complicated, and it is necessary to use an excessive amount of the solution used for collection in order to secure the tolerance, and there is a problem that the utilization efficiency is further lowered. In addition, when this method is applied to an actual sample, a large amount of contaminants other than microorganisms are mixed in the collected sample. However, in the conventional method, contaminants affect the ATP elimination reaction, ATP extraction reaction, and ATP luminescence reaction. There was a problem that the measurement accuracy was low.

  The present invention has been made in view of such a situation, the problem in the case of applying the ATP method to the inspection of airborne bacteria, that is, the step of taking out the bacteria from the collection carrier is not considered, Problems such as low collection efficiency and low use efficiency, low reliability in the measurement of extremely small numbers of bacteria, difficulty in automation and complicated construction, and low measurement accuracy due to contamination It is an object of the present invention to provide a microorganism collection device and an analysis system including the same, which solve at least one of the points.

  In order to solve the above-described problems, the polymer retained by the microorganism-collecting device according to the present invention performs a phase transition between gel and sol at a temperature in the range of 15 degrees to 37 degrees. Moreover, you may make it contain alcohols which do not have a bactericidal action other than a polymer | macromolecule. Examples of the alcohols include glycerol, ethylene glycol, and propylene glycol. The polymer is preferably either gelatin or NAGAm / MBPDA.

  In addition, when alcohol is glycerol, it is preferable that this glycerol is 30 to 60% of weight ratio with respect to the member (substance) which a collection device hold | maintains. When the polymer is gelatin, the gelatin is preferably 5% or more and 10% or less by weight with respect to the member (substance) held by the collection device. Furthermore, when the polymer is NAGAm / MBPDA, the NAGAm / MBPDA is preferably 1% to 10% by weight with respect to the member (substance).

  Furthermore, you may make it the collection device comprise a bottom face with a filter.

  Further, the automatic measuring apparatus according to the present invention comprises a temperature-sensitive resin that undergoes a sol-gel phase transition in a temperature-dependent manner, preferably a temperature-sensitive resin that undergoes a sol-gel phase transition at a temperature of 40 ° C. or less. A device including a collecting carrier, a temperature control mechanism, preferably a filtration mechanism, and a dilution mechanism as necessary. In the present invention, first, a trapping carrier made of a temperature-sensitive resin is gelled using a temperature control mechanism to collect airborne bacteria, and secondly, this trapping carrier is sol-like using a temperature control mechanism. To give a bacterial suspension.

  Since the phase transition temperature of the temperature-sensitive resin material in the present invention is 40 ° C. or less, the bacteria are collected and collected under mild temperature conditions. Applying the conventional ATP method to the bacterial suspension to measure the bacterial ATP, or preferably filtering the bacterial suspension to capture the bacteria in the bacterial suspension on the filter for improvement ATP measurement is performed using the measurement protocol of the ATP method, and airborne bacteria are inspected.

  Further features of the present invention will become apparent from the best mode for carrying out the present invention and the accompanying drawings.

  According to the collection device of the present invention, it is easy to take out the bacteria from the collection carrier, the collection efficiency and the utilization efficiency are increased, the reliability in the measurement of the extremely small number of bacteria can be improved, and the automation is simple Thus, at least one of the effects can be expected, for example, the measurement accuracy can be increased even if it is affected by contamination.

  The present invention uses a collection carrier made of a temperature-sensitive resin, and the collection carrier is made into a gel during collection by temperature control, and the collection carrier is made into a sol during collection, and a mild temperature condition of 40 ° C. or less. The phase transition of gel and sol is performed. And by doing this, avoid problems such as damage and death due to the heat of the bacteria, increase the recovery efficiency of the bacteria from the collection carrier, increase the utilization efficiency by reducing the volume of the bacterial suspension, A highly sensitive test for airborne bacteria can be achieved easily and quickly.

  In addition, in many cases, humans bring germs that are problematic at various sites to the sites. Therefore, it is necessary to collect and analyze bacteria that survive at a temperature close to human body temperature, and the temperature is set to 40 ° C. or lower.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be noted that this embodiment is merely an example for realizing the present invention, and does not limit the technical scope of the present invention. In each drawing, the same reference numerals are assigned to common components.

<First Embodiment>
(1) About collection device First, the structure outline | summary of the collection device 1 by the 1st Embodiment of this invention is demonstrated. FIG. 1 is a cross-sectional view showing a schematic configuration of a collection device 1 according to a first embodiment of the present invention. The collection device 1 includes a container 3 and a collection carrier 2 accommodated therein.

  In the present embodiment, a resin having the following composition is used as the material of the collection carrier 2. Glycerol is 50% by weight, temperature-sensitive resin material (in this embodiment, gelatin (protein hydrolyzate), 7.5% by weight), ATP scavenger 0.03%, and the rest is balanced with physiological saline. As the container 3, a petri dish made of polystyrene having a cylindrical recess having a diameter of 40 mm and a depth of 5 mm is used.

  Next, a method for forming the collection carrier 2 according to the present invention will be described. A uniform raw material sol is obtained by mixing the materials of the collection carrier 2 with heating as necessary. The subsequent operations are performed in a sterile environment. The raw material sol is sterilized by filtration through a syringe filter having a pore size of 0.22 microns while heating. 1 mL of the obtained sterilized sol is collected and uniformly injected into the bottom of the sterilized container 3 and cooled to 4 ° C. for 1 week or longer to form a uniform gel. Therefore, the shape of the collection carrier 2 is also generally cylindrical, and its diameter is 40 mm, which is the same as that of the recess of the container 3, and its thickness is about 0.8 mm. The collection carrier 2 is in a gel state at room temperature (25 ° C.), but when heated, the phase transition from the gel to the sol occurs at the phase transition temperature, and is in the sol state at 37 ° C. Therefore, the collection carrier 2 functions as a temperature-sensitive resin having an upper critical solution temperature (UCST: less than this temperature is a gel), and the upper critical solution temperature is higher than 25 ° C. and lower than 37 ° C. The temperature-dependent sol-gel phase transition of the collection carrier 2 is reversible. In addition, you may use the support | carrier (after-mentioned) which has a lower critical solution temperature (it is a gel form in the temperature higher than the said temperature) as a collection support | carrier. By adopting), the conditions of sol and gel should be matched.

(2) About collection device The outline of the configuration of the collection device 11 according to the present invention will be described with reference to FIG. FIG. 2 is a schematic configuration diagram of the collection device 11 according to the first embodiment of the present invention. The collection device 11 includes an upper member 11a, a lower member 11b, an intake nozzle 12, a holder 13, a temperature adjustment mechanism 14, a pump 16, an exhaust duct 17, and an exhaust filter 18, and includes an inside of the device. An air gap (space) 15 is formed in. In this collection device 11, the upper member 11a is detachable from the lower member 11b. When the upper member 11a is mounted on the lower member 11b to form the collection device 11, the joint between the two becomes airtight, and the external opening of the collection device 11 is connected to the intake nozzle 12 in the upper member 11a and the lower member 11b. An exhaust filter 18 is provided. A holder 13, a pump 16, and an exhaust filter 18 are fixed inside the lower member 11b. Although not shown, a control system and a power source for the pump 16 and the temperature adjustment mechanism 14 are incorporated, and operation buttons, a handle, and the like are provided on the outer surface. Moreover, the holder 13 accommodates the temperature adjustment mechanism 14, and the collection device 1 is detachably accommodated. When the collection device 1 is accommodated, the upper surface of the temperature adjustment mechanism 14 contacts the lower surface of the collection device 1. Inside the collection device 11, a gap 15 is provided to communicate from the outlet of the intake nozzle 12 to the inlet of the pump 16 via the collection device 1, and the exhaust duct 17 is airtight from the outlet of the pump 16 to the exhaust filter 18. Contact Since the so-called impactor-type collection device such as this embodiment has a high flow rate of sample air, it has the advantage that airborne bacteria can be collected by sucking a predetermined amount of sample in a short time. In FIG. 2, the single-hole configuration is illustrated as the intake nozzle 12. However, the intake nozzle 12 usable in the present invention is not limited to this configuration, and various intake nozzles such as a multi-hole type having many smaller holes may be used. It can be adopted.

(3) Bacteria measurement procedure (i) First, the outline of the bacteria measurement procedure according to the present invention will be described with reference to FIG. The measurement procedure of this bacterium is as follows: microbial collection step (a), microbial recovery step (b), ATP elimination step (c), filtration step (d), ATP extraction step (e), and ATP recovery step (F) and an ATP measurement step (g).

  In the bacteria collection step (a), the airborne bacteria are collected on the collection carrier 2 (here, in a gel form) of the collection device 1 using the collection device 11.

  In the bacteria recovery step (b), the collection carrier 2 is phase-shifted from a gel state to a sol state to obtain a bacterial suspension (sample).

  In the ATP elimination step (c), ATP other than bacteria in the sample, that is, extracellular ATP and ATP in dead bacteria are erased.

  In the filtration step (d), by filtering the sample, contaminants such as a water-soluble component and a solid component having a particle diameter smaller than that of the bacteria are filtered, and the live bacteria in the sample are captured on the filter.

  In the ATP extraction step (e), the cell walls of the bacteria are dissolved, and ATP contained in the cytoplasm of the living bacteria is extracted into the sample solution.

  In the ATP recovery step (f), a sample solution containing ATP extracted from the bacteria is obtained.

  In the ATP measurement step (g), a bioluminescence reaction is performed based on ATP in the sample solution, and the amount of luminescence is measured using a luminometer to determine the ATP content in the sample. As the number of airborne bacteria per unit volume Provide test results.

(Ii) Next, the operation of each component according to the present invention will be described in detail along the measurement procedure. Prior to the fungus collection step (a), the collection device 1 is prepared by the method described above. Clean the upper member 11a of the collection device 11 by sterilization by autoclave and installed in the vicinity of the place to collect the air to be measured (hereinafter referred to as collection place) together with the lower member 11b and the collection device 1 prepared as described above. Bring it to the bench. Hereinafter, the operation in the clean bench is performed aseptically. After sterilizing the holder 13, the gap 15, and the outer surface of the lower member 11 b using disinfecting ethanol or the like, the collection device 1 is placed on the holder 13, and the sterilized upper member 11 a is attached to the collection device 11. Form. A cap (not shown) that protects the intake nozzle 12 of the collection device 11 from contamination by bacteria is attached, and the collection device 11 is unloaded from the clean bench and installed at the collection location to complete the preparation. At this time, the room temperature of the collection place and the clean bench is controlled to about 25 ° C. by air conditioning, and the temperature of each member is also about 25 ° C. Further, the temperature adjusting mechanism 14 is operated, and the temperature is set to 25 ° C. Since the collection carrier 2 of the collection device 1 is a temperature-sensitive resin material having an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C., it is gel-like at this temperature.

  In the bacteria collection step (a), the collection device 11 is operated to inhale airborne bacteria at the collection site and collect them in the collection device 1. Specifically, by rotating the pump 16, the air outside the collection device 11 is sucked into the pump 16 through the intake nozzle 12 and the gap (space) 15, and further through the exhaust duct 17 and the exhaust filter 18. To the outside of the collecting device 11. At this time, airborne bacteria in the air are transported along the airflow that is substantially perpendicular to the collection device 1 through the intake nozzle 12, and collide with the gel-like collection carrier 2 due to inertia and collected. The air and fine particles having a mass smaller than that of the bacteria are collided with the collection carrier 2, then turned in a direction parallel to the surface of the collection carrier 2, and carried to the gap 15, the pump 16, the exhaust duct 17, and the exhaust filter 18. . Fine particles having a mass smaller than that of the bacteria are captured by the exhaust filter 18, and clean air not containing the fine particles is discharged to the outside of the collection device 11 through the exhaust filter 18. In this embodiment, the pump 16 exhibits a constant flow rate of 200 L / min, and sucks a certain amount of air by 3 cubic meters by setting the collection time to 15 minutes. As a result of this step, airborne bacteria in air of 3 cubic meters are collected on the gel-like collection carrier 2.

  In the bacteria recovery step (b), the temperature control mechanism 14 is operated to increase the temperature from 25 ° C. to 37 ° C. Heat is conducted from the upper surface of the temperature adjusting mechanism 14 to the lower surface of the collection device 1, that is, the container 3, and further to the collection carrier 2, and the temperature of the collection carrier 2 rises quickly to 37 ° C. The collection carrier 2 employed in this embodiment undergoes a phase transition from gel to sol by increasing the temperature from 25 ° C. to 37 ° C., that is, has an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C. Functions as a temperature sensitive resin. Fine particles such as viable bacteria collected on the collection carrier 2 are dispersed in this sol and become a suspension liquid. Of the components other than viable bacteria collected by the collection carrier 2, the water-soluble components are dissolved in this sol. As a result of this step, 1 mL of the bacterial suspension liquid sample is collected in the container 3.

  In the ATP erasing step (c), 30 μL of the ATP erasing agent contained in the lucifer HS set is added to and mixed with the bacterial suspension liquid sample, and left at room temperature for 30 minutes. By this step, ATP other than viable bacteria in the sample, that is, extracellular ATP, dead ATP, and a very small amount of ATP remaining on the collection carrier 2 are erased. As a result of this step, 1.03 mL of a reaction solution from which ATP other than viable bacteria has been eliminated is obtained in the container 3.

  Prior to the filtration step (d), a filtration device (not shown) containing a membrane filter (Millipore HAWP02500, diameter 25 mm, membrane material MF-Millipore, nominal pore size 0.45 micron) on the eye plate was autoclaved. Prepare things in advance.

  In the filtration step (d), the reaction solution is dropped onto the upper surface of the filter, and the lower surface (the surface of the countersink) is decompressed and suctioned for filtration. After filtration is complete, return the bottom of the filter to atmospheric pressure. As a result of this step, a filter that traps viable bacteria (greater than 0.45 microns) by filtering out water-soluble components in the reaction solution and solid components other than bacteria whose particle size is smaller than 0.45 microns. A filtration device containing the product can be obtained.

  In the ATP extraction step (e), 100 μL of the ATP extraction reagent contained in the lucifer HS set is added to the upper surface of the filter of the filtration device and mixed at room temperature for 20 seconds. The filter is placed on an O-ring made of, for example, silicon rubber, and the lower surface of the filter is at atmospheric pressure. Therefore, filtration is not performed, and the added reagent solution remains on the filter due to surface tension. As a result of this step, the cell walls of viable bacteria are dissolved, ATP contained in the cytoplasm of the bacteria is extracted, and 100 μL of a sample solution in which this ATP is dissolved is obtained on the filter.

  In the ATP recovery step (f), 30 μL of the sample solution is sucked with a pipette. As a result of this step, 30 μL of 100 μL of the sample solution in which ATP contained in the living bacteria is dissolved is obtained as a recovered liquid in the pipette. Since 30% of the ATP in the living bacteria present in the collected sample is recovered in the recovery liquid, the utilization efficiency of the recovery liquid is 0.3 in this case.

  Prior to the ATP measurement step (g), 0.2 mL of ATP luminescent reagent is injected into a polypropylene test tube for luminometer, and installed in a luminometer (Berthold Detection Systems FB12 type) to measure the amount of luminescence (baseline) . The amount of light emitted from the baseline is approximately 40 to 60 cps (count per second). Next, a 200 amol standard sample (ATP standard solution 10 μL at a concentration of 20 pmol / L) is additionally injected into a test tube containing 0.2 mL of the ATP luminescent reagent, and after mixing, the amount of luminescence is measured (standard sample). The light emission amount of the standard sample is approximately 2000 cps. The effective light emission amount of the standard sample is obtained by subtracting the baseline light emission amount from the light emission amount of the standard sample. By dividing the effective light emission amount of the standard sample by the ATP amount, the sensitivity, that is, the effective light emission amount per unit ATP amount is obtained. The sensitivity is approximately 10 to 20 cps / amol.

  In the ATP measurement step (g), 30 μL of the recovered solution is additionally injected into a test tube containing the ATP luminescent solution, and after mixing, the amount of luminescence is similarly measured (sample). The effective light emission amount of the sample is obtained by subtracting the light emission amount of the baseline from the light emission amount of the sample. The amount of ATP in the collected liquid is determined by dividing the effective light emission amount of the sample by the sensitivity. By dividing this value by the recovery liquid utilization efficiency of 0.3, the amount of ATP contained in viable bacteria in the sample is determined. The average number of viable bacteria in the sample is determined by dividing the amount of ATP in this sample by the average value of ATP content per live cell (about 2 amol). By dividing the average viable cell count in the sample by 3 cubic meters which is the volume of the collected gas sample, the average viable cell count contained in the unit volume gas sample is obtained. As a result of this step, the ATP content in the sample solution is measured, and the test result is obtained as the number of airborne bacteria per unit volume.

(4) Preliminary examination of collection carrier As described above, the collection carrier 2 is mentioned as a characteristic component of the first embodiment. Therefore, a preliminary study on the composition of the collection carrier 2 is as follows.

(I) First, in this embodiment, the volume of air to be sucked is 3 cubic meters, the flow rate of the pump 16 is 200 L / min, and the diameter of the intake nozzle 12 is 0.48 cm. Under these operating conditions, the air linear velocity at the intake nozzle 12 reaches an average of 181 m / s, and the volume of air to be collected is as large as 3 cubic meters. Therefore, when a conventional collection carrier made of agar gel or the like is used, moisture evaporates. Thus, there is a problem that the volume of the gel is drastically reduced, the surface of the gel is dried and changed into a solid state, and the bacteria cannot be efficiently collected. Therefore, in this embodiment, by using the collection carrier 2 containing 50% glycerol, deformation and drying of the collection carrier 2 are prevented, and high collection efficiency in collecting airborne bacteria on the collection carrier 2 Is realized. As a material for preventing deformation and drying of the collection carrier 2, various alcohols (drying prevention) such as ethylene glycol and propylene glycol can be used in addition to glycerol employed in the present embodiment. In addition, the kind of alcohol needs to be alcohol with no bactericidal action. This is to prevent the bacteria captured by the collection carrier 2 from being killed. Thus, for example, ethanol is not suitable.

(Ii) As a second preliminary study on the collection carrier 2, a collection carrier prepared from glycerol, gelatin and physiological saline is examined. In this case, even if filtration is performed, the problem that the measurement result of the blank sample that does not contain bacteria shows an abnormally high value of about 10 to 20 amol is confirmed. Among the materials for the collection carrier, gelatin, in particular, contains a large amount of ATP as compared with a very small amount (several amol) of ATP to be measured, so it is considered that it remains and causes an abnormally high value.

  Therefore, prior to the implementation of the present embodiment, the following model experiment is performed, and a method for reducing the blank measurement value by removing ATP contained in the collection carrier in advance is studied. That is, this is a pretreatment for removing ATP derived from gelatin.

  Specifically, a model collection carrier is prepared by adding 5% by weight of gelatin and 50% by weight of glycerol in physiological saline and adding various amounts of the ATP scavenger contained in the Lucifer HS set. This model collection carrier was made into a sol by heating at 37 ° C., 10 μL of a solution obtained by diluting it directly or with pure water was taken, added to 0.2 mL of ATP luminescence reagent, and the amount of luminescence was measured. An example is shown in FIG. When no ATP eraser is added, a very high blank signal of 7178 cps is generated.

  On the other hand, the blank signals of the model collection carriers to which 0.03 and 0.1% of ATP erasing agent are added by volume ratio are 103 and 57 cps, respectively, which are drastically reduced compared to the system without addition. The effective luminescence amount, which is the difference from the baseline measurement value (56 cps in this experiment) using pure water not containing ATP as a sample, is 7122 (== 1222 for the ATP eraser 0, 0.03, and 0.1% systems, respectively. 7178-56), 47 (= 103-56), 1 (= 57-56) cps, and the respective ATP contents determined using a sensitivity of about 11.2 cps / amol are about 636, 4.2,. 1 amol.

  Therefore, it is possible to prepare a collection carrier that effectively decomposes and removes ATP derived from gelatin and the like by adding 0.03% or more of an ATP erasing agent to the collection carrier. It can be seen that the influence of the above can be suppressed to 1/100 or less, and by combining with filtration, the above-mentioned abnormally high value can be avoided and the level can be suppressed to substantially no influence.

  Based on the above examination results, it has been found that a composition containing an ATP erasing agent is suitable as the collection carrier 2 containing gelatin, and the required composition is 0 when the ATP erasing agent attached to the Lucifer HS set is used. It turns out that it is 0.03% or more. By adopting 0.03% as the lower limit of this necessary composition as the ATP erasing agent composition of this embodiment, ATP contamination mainly derived from gelatin is excluded from the collection carrier 2.

(Iii) As a third preliminary study on the collection carrier 2, the gelatin composition used as the temperature sensitive resin material is examined after fixing the ATP erasing agent content ratio of the collection carrier to 0.03%.

  FIG. 5 shows a case where the gelatin concentration is changed to 5, 7.5, and 10% by weight in a model collection carrier prepared by 0.03% of ATP erasing agent content, 50% by weight of glycerol, and physiological saline balance. Shows the temperature characteristics and filtration characteristics of the model collection carrier. As shown in FIG. 5, in any composition, the model collection carrier becomes a sol form within 5 minutes when stepped from 25 ° C. to 37 ° C., and can be suitably applied to the recovery process. When the gelatin concentration is 7.5% by weight or higher, the gel strength at 25 ° C. is high and can be used as a collection carrier. However, when the gelatin concentration is 5% by weight, the gel strength at 25 ° C. is slightly low.

  Therefore, by setting the gelatin concentration to 7.5% by weight or more, a temperature-sensitive resin material having an upper critical solution temperature that is gel at 25 ° C., sol at 37 ° C., that is, higher than 25 ° C. and lower than 37 ° C. is obtained. .

  On the other hand, as for the filtration characteristics under the conditions of the filtration process described above, when the gelatin concentration is 7.5% by weight or less, it is possible to filter 1 mL of sol in 40 seconds at the longest. Applicable.

  However, when the gelatin concentration is 10% by weight, the viscosity is so high that it is difficult to filter, and some components that are not filtered may remain on the filter.

  From the above, the gelatin concentration of the collection carrier material having an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C. and showing good filtration characteristics is preferably 5 to 10% by weight, and the optimum concentration condition is It turns out that it is 7.5 weight%. The optimum concentration condition of 7.5% by weight is adopted as the gelatin concentration condition of this embodiment.

(Iv) As a fourth preliminary study on the collection carrier 2, the glycerol composition is examined after fixing the ATP quencher content ratio of the collection carrier to 0.03%.

  FIG. 6 shows a model collection carrier prepared with an ATP erasing agent content ratio of 0.03%, a gelatin concentration of 5, 7.5, 10% by weight and a physiological saline balance, and a glycerol concentration of 30, 40, 50, 60. The temperature characteristics and filtration characteristics of the model collection carrier when the weight percentage is changed are shown.

  As shown in FIG. 6, the model collection carrier becomes a sol at 37 ° C. in any composition, and can be suitably applied to the recovery process. When the glycerol concentration is 50% by weight or higher, the gel strength at 25 ° C. is high, and it can be used as a collection carrier.

  However, when the glycerol concentration is 40% by weight or less, the gel strength at 25 ° C. is slightly low. Therefore, by setting the glycerol concentration to 50% by weight or more, a temperature sensitive resin material having an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C. can be obtained.

  On the other hand, as for the filtration characteristics, when the glycerol concentration is 50% by weight or less, 1 mL of sol can be filtered if it is 40 seconds at the longest, and can be favorably applied to the filtration process described above. However, when the glycerol concentration is 60% by weight, the viscosity is so high that it is difficult to filter, and some components that are not filtered may remain on the filter.

  As described above, the glycerol concentration of the collection carrier material having an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C. and showing good filtration characteristics is preferably 30 to 60% by weight, and the optimum concentration condition is 50 It is found to be% by weight. The optimum concentration condition of 50% by weight can be adopted as the glycerol concentration condition of this embodiment.

(5) Evaluation of collection characteristics The collection characteristics of this embodiment are evaluated by the following method. As a model sample of airborne bacteria, a suspension prepared by diluting a standard strain (Easy QA Ball Escherichia coli 10,000 cfu of Nissui Pharmaceutical Co., Ltd.) with pure water is prepared. The step of collecting floating bacteria on the collection device 1 by the collection device 11 from the gas sample in the fungus collection step (a) is substituted by spotting a certain amount of model sample on the collection carrier 2. Subsequent steps are evaluated as in this example. For comparison, an agar gel is used as the collection carrier 2 in the conventional method, that is, the bacteria collection step (a), and 1 mL of physiological saline is divided into two times as a method for collecting the bacteria in the bacteria collection step (b). The method of dispensing on the surface and collecting it again from the surface, and the fungus collection step (a) will be compared with the method of substituting with the model sample spotting as in this example. A commercially available agar medium (Tryptic Soy Agar) is used as the collection carrier 2 of the conventional method.

  FIG. 7 shows an example of the measurement result of two series by both methods when a model sample of 500 CFU is used. As a result of the conventional method, the amount of ATP, the average viable cell count, and the recovery rate are extremely low, and in one of the two series, no bacteria are recovered.

  On the other hand, the results according to this embodiment are an average of about 540 amol as the amount of ATP, an average number of viable bacteria of about 270 CFU, and an average of about 55% (= (44 + 65) / 2) as the recovery rate. This result is an average of about 4 (= 55/13) times closer to the predetermined value than the conventional method (that is, closer to a recovery rate of 100%).

  As described above, this embodiment can obtain a measurement result with higher accuracy than the conventional method. The greatest difference between the present invention and the conventional example is in the collection device 1 and the step of recovering the bacteria therefrom. The reason why the present invention was more accurate than the conventional method is considered to be that bacteria were efficiently recovered from the collection device 1 in the bacteria recovery step (b). In fact, as a reference experiment, the collection carrier 2 used in the conventional method (remaining after collecting bacteria) was cultured at 35 ° C. for 2 days, and each collection carrier exhibited many colonies.

  Therefore, it is considered that the reason why the conventional method showed a result of separation from the predetermined value is that the collection efficiency in the bacteria collection step (b) is low.

  On the other hand, the method of this embodiment, that is, the collection carrier itself can undergo gel-sol phase transition without contamination by a mild and non-contact means of temperature change of 40 ° C. or less, and can be handled as a bacterial suspension. By adopting the novel method, the bacteria recovered on the collection carrier can be efficiently provided to the next step.

(6) Evaluation of overall characteristics The overall characteristics of this embodiment are evaluated by the following method. As a model sample of airborne bacteria, bacteria contained in the atmosphere in the laboratory are used. That is, using this laboratory atmosphere as a sample, the bacteria are examined according to the bacteria measurement procedure based on this embodiment. For comparison, the results are compared with the results of the culture method. The culture method uses Millipore air sampler (M Air T type) and agar medium (Tryptic Soy Agar), and collects 1 cubic meter of air according to the attached instruction manual. Incubate at 0 ° C. for 2 days and count colonies. Result of this embodiment and the culture method are each ^{950CFU / m 3, 1100CFU / m} 3, results of both coincide very well. The time required for measurement is about 1 hour and 50 hours including collection. Therefore, this embodiment according to the present invention has an effect that it can provide a highly accurate test result equivalent to the culture method in an extremely short time of about 1/50.

  As described above, according to the present embodiment, since the bacteria recovery efficiency from the collection carrier is high, the number of bacteria can be measured with high reliability. Furthermore, by applying this embodiment, there is an effect that highly accurate measurement of viable bacteria can be performed in an extremely short time.

<Second Embodiment>
(1) About collection device FIG. 8: is sectional drawing which shows schematic structure of collection device 1 'by the 2nd Embodiment of this invention. The collection device 1 ′ includes a collection carrier 2, a container wall 3 ′, and a filter 4. The collection device 1 ′ has the same filter 4 as that used in the first embodiment adhered to the bottom of the hollow container wall 3 ′, and the collection carrier 2 prepared by the same method as in the first embodiment. The raw material is poured onto the cooled filter 4.

(2) Automatic Measurement Device FIG. 9 is a diagram showing a schematic configuration of the automatic measurement device 21 according to the second embodiment of the present invention. The automatic measuring device 21 includes a collection device holder 22, an eye plate holder 23, an ATP erasing agent dispenser 24, an ATP extraction reagent dispenser 25, an ATP recovery pipettor 26, and a luminescent reagent dispenser. The injection device 27, the test tube holder 28, the control device 29, the temperature adjustment mechanism 30, the sterilization and cleaning mechanism 31, the electromagnetic valve 41, the waste liquid trap 42, the suction pump 43, and the photon counter 44. I have. The collection device holder 22, the eye plate holder 23, and the test tube holder 28 can each be provided with a collection device 1 ′, an eye plate, and a test tube (not shown). Can be transported to a predetermined position.

  The ATP erasing agent dispenser 24, the ATP extraction reagent dispenser 25, the ATP recovery pipettor 26, and the luminescent reagent dispenser 27 are each supplied with various reagents (not shown) by control signals from the control device 29. It has a mechanism that automatically performs dispensing and pipetting operations.

  The eye plate holder 23, the electromagnetic valve 41, the waste liquid trap 42, and the suction pump 43 constitute a suction mechanism for filtration together with a not-shown eye plate, and further, a filtration mechanism in combination with the filter 4 at the bottom of the collection device 1 ′. Configure.

  The electromagnetic valve 41 and the suction pump are controlled by the control device 29. Under the control of the control device 29, the temperature adjustment mechanism 30 controls the temperature inside the device, particularly the temperature of the collection device holder 22, to 37 ° C. or more and 40 ° C. or less. The photon counter 44 measures the intensity of light emission generated in a test tube (not shown) installed in the test tube holder 28 as the number of photons, and outputs the number of photons to the control device 29.

(3) About measurement operation Then, operation | movement at the time of using the collection device 1 'by this invention, the collection apparatus 11 (FIG. 2), and the automatic measurement apparatus 21 is demonstrated. Although the operation of this embodiment is basically the same as that of the first embodiment, the automatic measurement apparatus 21 is used to automatically perform the bacteria collection step (b) to ATP measurement step (g) in FIG. Is mainly different. In addition, since the main body of this embodiment exists in the collection | recovery method of the microbe collected on the collection device, the detailed description of the electric system, the control system, the optical system including light shielding, and the mechanism system is omitted.

  In the present embodiment, prior to the start of use of the automatic measuring device 21, the inside of the automatic measuring device 21 is sterilized using the sterilization cleaning mechanism 31, and ozone gas (when using an ultraviolet sterilization method) and fine particles are removed. Further, during use of the automatic measuring device 21, the sterilization and cleaning mechanism 31 is used while removing ozone gas and fine particles.

  In the bacteria collection step (a) of the present embodiment, as in the first embodiment, fine particles in the air sample on the collection carrier 2 (gel) of the collection device 1 ′ at room temperature (25 ° C.). Is collected using the collection device 11.

  Prior to the bacteria collection step (b), the temperature adjustment mechanism 30 is operated to maintain the temperature of the collection device holder 22 at 37 ° C. or higher and 40 ° C. or lower. In the bacteria collection step (b), the collection device 1 ′ is aseptically installed in the collection device holder 22 of the automatic measuring device 21. Subsequent bacteria collection step (b) to ATP measurement step (g) are performed automatically and aseptically under the control of the control device 29. Heat is conducted from the collection device holder 22 to the collection device 1 ′ and further to the collection carrier 2 by the action of the temperature adjusting mechanism 30, and the temperature of the collection carrier 2 quickly rises to 37 ° C. Since the collection carrier 2 employed in the second embodiment is also a temperature-sensitive resin material having an upper critical solution temperature higher than 25 ° C. and lower than 37 ° C., the temperature rises from 25 ° C. to 37 ° C. Phase transition to sol form. As a result of this step, 1 mL of the bacterial suspension liquid sample is collected in the collection device 1 ′, in other words, in the space formed by the container wall 3 ′ and the filter 4.

  In the ATP elimination step (c), the collection device holder 22 in which the collection device 1 ′ is installed is conveyed to a position below the ATP elimination agent dispenser 24, and 30 μL of the ATP elimination agent is added to the above bacterial suspension. Addition and mixing (hereinafter, the reagent addition operation by the dispenser is performed in the same manner, but detailed operations such as transport are omitted, and only the dispensing is described). The collection device holder 22 on which the collection device 1 ′ is installed is conveyed to a position above the eye plate holder 23, and the eye plate holder 23 on which a sterilized eye plate (not shown) is installed is conveyed to an upper position. A filter 4 is formed by fitting the filter 4 and the eye plate on the lower surface of the collecting device 1 ′. By standing for 30 minutes in this state, ATP other than viable bacteria is eliminated. As a result of this step, 1.03 mL of a reaction solution from which ATP other than viable bacteria has been eliminated is obtained in the collection device 1 ′.

  In the filtration step (d), the suction mechanism is operated to depressurize the lower surface of the filter, and the reaction liquid is sucked and filtered through the electromagnetic valve 41 to the waste liquid trap 42 at the eye plate of the filter 4. After filtration is completed, the suction mechanism is stopped and the lower surface of the filter is returned to atmospheric pressure. As a result of this step, a collection device 1 ′ containing a filter 4 that captures viable bacteria in the reaction solution is obtained.

  Prior to the ATP extraction step (e), the eye plate holder 23 is transported to a lower position, the filter 4 on the lower surface of the collection device 1 'is separated from the eye plate, and the configuration of the filtration mechanism is released.

  In the ATP extraction step (e), 100 μL of the ATP extraction reagent is added to the upper surface of the filter 4 using the ATP extraction reagent dispenser 25 and mixed at room temperature for 20 seconds. Since the lower surface of the filter 4 is at atmospheric pressure, no filtration is performed, and since the lower surface of the filter is not in contact with the eye plate, the liquid remains on the filter 4 due to surface tension. As a result of this step, 100 μL of a sample solution in which ATP contained in live bacteria is dissolved is obtained on the filter.

  In the ATP recovery step (f), 30 μL of the sample solution is sucked using the ATP recovery pipettor 26. As a result of this step, 30 μL of 100 μL of the sample solution in which ATP contained in the living bacteria is dissolved is obtained as a recovered liquid in the pipette. The utilization efficiency of the recovered liquid is 0.3.

  Prior to the ATP measurement step (g), 0.2 mL of ATP luminescent reagent is injected into the test tube installed in the test tube holder 28 using the luminescent reagent dispenser 27, and the amount of baseline luminescence is measured. Next, using a 200 amol ATP standard sample, the amount of luminescence from the standard sample is measured in the same manner. Hereinafter, the effective light emission amount and sensitivity of the standard sample are obtained by the same procedure as in the first embodiment.

  In the ATP measurement step (g), 30 μL of the recovered solution is additionally injected into a test tube containing the ATP luminescent reagent using the ATP recovery pipettor 26, and the amount of sample luminescence is measured in the same manner. Hereinafter, according to the same procedure as in the first embodiment, the effective light emission amount of the sample, the ATP amount in the collected liquid, the ATP amount contained in the viable bacteria in the sample, the average viable cell count in the sample, and the gas sample of unit volume The average number of viable bacteria contained in is determined. As a result of this step, the ATP content in the sample solution is measured, and the test result is obtained as the number of airborne bacteria per unit volume.

  The greatest difference between the present embodiment and the first embodiment is the presence or absence of automation from the bacteria recovery step (b) to the ATP measurement step (g). Therefore, according to the second embodiment, in addition to the same effects as the first embodiment, by performing automation, high sensitivity, simplification, labor saving, prevention of human error, improvement of reproducibility, A special effect such as can be expected.

<Third Embodiment>
The third embodiment of the present invention is basically the same as the first and second embodiments, except that a synthetic polymer is used as the temperature sensitive resin material of the collection carrier 2 in the collection device 1. Different.

The temperature-sensitive resin material used in this embodiment is a synthetic polymer that undergoes a gel-sol phase transition near room temperature, and the following two types of materials are compared and studied.
(A) Meviol Gel MB-10 manufactured by Meviol Co., lower critical solution temperature (LCST) of about 20 ° C.
(B) 10: 1 copolymer of N-acryloylglycinamide (NAGAm) and N-methacryloyl-N′-biotinylpropylenediamine (MBPDA) (hereinafter abbreviated as NAGAm / MBPDA), upper critical solution temperature (UCST) approx. 22 ° C.

  NAGAm / MBPDA synthesized according to the method described in Non-Patent Document 3 is used.

  FIG. 10 shows the results of the property evaluation after preparing the collection carrier 2 containing 4% by weight of the above-mentioned two kinds of synthetic polymers, performing autoclave sterilization and ATP decomposition at 120 ° C. for 20 minutes. After maintaining the condition of (1) in FIG. 10 for overnight or more, the temperature characteristics are changed in the order of conditions (2) and (3), and the change in the gel / sol state is observed. That is, whether the synthetic polymer that has been changed by changing the condition from 15 ° C. to 37 ° C. returns to the initial 15 ° C. state when the condition is changed to 15 ° C. again. As shown in FIG. 10, MB-10 is gel and sol under the conditions (2) and (3), respectively, and this process is as defined by LCST (sol below transition temperature and gel above transition temperature). ) Phase transition.

  However, it is a soft gel under the condition (1) in FIG. 10, and there is a problem that there is a deviation from hysteresis or time-dependent LCST.

  On the other hand, NAGAm / MBPDA is in the form of gel and sol under the conditions of (1), (3), and (2) in FIG. 10 and is in the phase defined by UCST (gel below transition temperature, sol above transition temperature). Showing metastasis.

  Therefore, it can be determined that NAGAm / MBPDA is superior to MB-10 from the viewpoint of temperature characteristics. Similar results are observed when the concentration of NAGAm / MBPDA is 1% to 10% by weight.

  Next, filtration characteristics are evaluated for NAGAm / MBPDA. Filtration characteristics are as follows. The collection carrier 2 is heated to 37 ° C. to be in a sol state, and pressure filtration is performed with a syringe filter having a pore size of 0.45 μm and vacuum filtration is performed with a membrane filter having a pore size of 0.45 μm. As a result, although NAGAm / MBPDA could be filtered under pressure, it would not pass through the filter as it was (4 wt% sol) even if it was vacuum filtered.

  Therefore, as a result of diluting the sol with pure water and examining the possibility of vacuum filtration, it did not pass through 1/2 dilution (2% by weight), but reduced pressure by performing 1/3 dilution (1.3% by weight). It turns out that filtration is possible. In addition, by increasing the dilution factor and decreasing the viscosity of the sample solution, passive filtration using a water absorption pad or the like provided on the back of the filter can be employed instead of suction filtration.

  As a result of the above examination, a temperature-sensitive resin based on a synthetic polymer can be applied to the present invention as the collection carrier 2 of the collection device 1, and particularly NAGAm / MBPDA is 1 to 10% by weight, particularly preferably about 4% by weight. It can be seen that the trapping carrier 2 contained in% shows an upper critical solution temperature of 15 ° C. or higher and 37 ° C. or lower and can be used suitably.

  The main difference between the third embodiment and the first embodiment is that the temperature is set to 15 ° C. in the bacteria collection step (a), and the temperature of the collection carrier 2 is set to 15 ° C. In addition to collecting the bacteria after the preparation, the collection carrier 2 that has been solated in the filtration step (d) is preliminarily diluted and then filtered, or pressure filtration is performed without dilution.

  According to this embodiment, the synthetic polymer used as the temperature-sensitive resin material has high stability, can obtain homogeneous characteristics with good reproducibility, and can reduce the cost by mass production. In addition, the autoclaving enables sterilization of viable bacteria derived from the material, and the autoclaving enables removal of the material-derived ATP (without using an ATP scavenger).

<Summary of Embodiment>
The collection device has the effect of exhibiting the same collection performance as the collection carrier in the prior art by collecting airborne bacteria using the collection carrier as a gel. Moreover, the collection carrier can be handled in substantially the same manner as the bacterial suspension by phase-transforming the collection carrier into a sol and diluting as it is or as necessary. For this reason, the process of taking out the bacteria from the collection carrier can be omitted. Therefore, there is no loss due to microbial spillage or microbial damage in the extraction process.

  Moreover, since the temperature conditions in the collection and recovery of the bacteria are mild (15 ° C. to 37 ° C.), problems such as damage and death due to the heat of the bacteria do not occur. Therefore, an effect that extremely high recovery efficiency is achieved can be expected.

  In addition, it is possible to replace the complicated extraction process with a simple process of temperature change or a combination of temperature change and dilution, and effects such as automation, system configuration simplification, speedup, reproducibility improvement, pollution risk reduction, etc. There is.

  In addition, since the bacterial suspension has a small amount of liquid, the concentration of bacteria is high and the utilization efficiency of bacteria is high. And by filtering the bacterial suspension and capturing the bacteria in the bacterial suspension on the filter, the liquid volume becomes substantially zero, and the concentration and utilization efficiency of the bacteria are remarkably high. .

  It is possible to eliminate the influence of water-soluble and small particle size contaminants, and it is highly reliable. Therefore, there is an advantage that bacteria can be automatically measured with high sensitivity, high accuracy, and so on. Moreover, the measurement accuracy and sensitivity in the inspection of airborne bacteria are high, and an effect that the result can be obtained simply and quickly can be expected.

  Since the collection device according to the present invention has a high collection efficiency of viable bacteria, can avoid damage and killing of bacteria, and can provide a collection carrier with high collection efficiency of viable bacteria, it has high sensitivity, high reliability, and simple It can be applied to quick viable count measurement. Moreover, since the collection | recovery of the living microbe from a collection support | carrier and filtration concentration can be performed simply and efficiently by using this device, it can be applied suitably also to the automatic measurement of the number of live bacteria.

It is sectional drawing which shows schematic structure of the collection device 1 by the 1st Embodiment of this invention. It is a schematic block diagram of the collection apparatus 11 by 1st Embodiment. It is a flowchart which shows the measurement procedure of the microbe by 1st Embodiment. It is a figure which shows the light emission measurement value by the ATP eraser composition of the model collection support | carrier by 1st Embodiment. It is the temperature and filtration characteristic by the gelatin composition of the model collection support | carrier by 1st Embodiment. It is the temperature by the glycerol composition of the model collection support | carrier by 1st Embodiment, and a filtration characteristic. FIG. 6 is a diagram showing a comparison of recovery characteristics between the first embodiment and a conventional example. It is sectional drawing which shows schematic structure of the collection device 1 'by 2nd Embodiment. It is a figure which shows schematic structure of the automatic measuring apparatus 21 by 2nd Embodiment. It is a characteristic comparison of the collection support | carrier by the synthetic polymer by 3rd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,1 'Collection device 2 Collection carrier 3 Container 3' Container wall 4 Filter 11 Collection apparatus 11a Upper member 11b Lower member 12 Intake nozzle 13 Holder 14 Temperature control mechanism 15 Air gap 16 Pump 17 Exhaust duct 18 Exhaust filter 21 Automatic Measuring device 22 Collection device holder 23 Eye plate holder 24 ATP remover dispenser 25 ATP extraction reagent dispenser 26 ATP recovery pipettor 27 Luminescent reagent dispenser 28 Test tube holder 29 Controller 30 Temperature control mechanism 31 Sterilization and cleaning mechanism 41 Solenoid valve 42 Waste liquid trap 43 Suction pump 44 Photon counter (a) Bacteria collection step (b) Bacteria collection step (c) ATP elimination step (d) Filtration step (e) ATP extraction step (f) ATP recovery process (g) ATP measurement process

Claims (12)

A collection device for collecting microorganisms in the atmosphere,
A first member containing a polymer that undergoes a phase transition between gel and sol in a temperature range of 15 degrees to 37 degrees;
A container for housing the first member,
The polymer is gelatin, and the gelatin is contained in the first member at a weight ratio of 5% to 10% with respect to the first member .
Capturing device moisture content of the first member, characterized in der Rukoto 30 wt% or more.   The collection device according to claim 1, wherein the first member includes an alcohol having no bactericidal action.   The collection device according to claim 2, wherein the alcohol is any one of glycerol, ethylene glycol, and propylene glycol.   The collection device according to claim 2, wherein the alcohol is glycerol, and the glycerol is contained in the first member in a weight ratio of 30% to 60% with respect to the first member.   The collection device according to claim 1, wherein the container has a filter on a bottom surface thereof. The collection device according to claim 1, wherein the gelatin concentration is 7.5 wt% or more and 10 wt% or less. 2. The collection device according to claim 1, wherein the first member includes an ATP erasing agent, and the content of the ATP erasing agent is 0.03% or more. A holding member for holding a first member containing a polymer that undergoes a phase transition between gel and sol within a temperature range of 15 degrees to 37 degrees;
A temperature control unit for controlling the temperature of the first member;
An air flow control unit for flowing air to the first member,
The polymer is gelatin, and the gelatin is contained in the first member at a weight ratio of 5% to 10% with respect to the first member .
Analytical system water content of the first member, characterized in der Rukoto 30 wt% or more. The analysis system according to claim 8 , wherein the temperature control unit switches between heating and cooling of the first member before and after the air flow control unit causes air to flow through the first member. In the first member, the gel-like polymer is placed on a filter for filtering the sol,
Furthermore, it comprises an eye plate holding part for holding an eye plate that is combined with the filter to form a filtration mechanism,
The analysis system according to claim 8 , wherein the filtration mechanism filters the polymer that has been solated by the temperature control unit. The analysis system according to claim 8, wherein the gelatin concentration is 7.5 wt% or more and 10 wt% or less. The analysis system according to claim 8, wherein the first member contains an ATP erasing agent, and the content of the ATP erasing agent is 0.03% or more.

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