JP2012231765A - Method for determining dirt - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly and precisely determining whether dirt in a building is mold or not in situ.SOLUTION: The method for determining whether the dirt in a building is mold or not in situ includes: a sample collection step of collecting a sample from the dirt; a quantification step of quantifying abundance of a coenzyme in the sample; and a determination step of determining whether the dirt is mold or not based on the abundance of the coenzyme.

Description

  The present invention relates to an indoor dirt determination method for a building. More specifically, the present invention relates to a method for determining whether or not indoor dirt is mold.

  If the indoor dirt of a building is microorganisms, particularly mold, the contamination may increase and removal may be difficult unless immediate measures are taken. Moreover, the measures for removing the dirt differ depending on whether the dirt is caused by microorganisms or not. For this reason, it is necessary to determine whether the dirt is caused by microorganisms.

  Generally, when investigating microorganisms in a sample collected from a test object, the sample is inoculated into a medium and cultured for several days. For example, in Patent Document 1, microorganisms floating in the air are captured on the membrane filter by sucking air through a heat-resistant membrane filter, and the microorganism captured on the membrane filter is captured on the medium. A method for rapidly measuring a microorganism is disclosed in which ATP in the cultured microorganism is detected by a bioluminescence reaction after culturing.

JP 2008-161143 A

  However, the process of inoculating the sample in the medium and culturing requires at least about 5 days. For this reason, it takes about one week at the minimum from taking a sample until a judgment result is obtained. For this reason, when it is determined whether the indoor dirt of the building is mold by the conventional method, it takes a long time to judge, and if the dirt is caused by mold, the response is delayed and it is difficult to remove the dirt. There was a case.

  In addition, in the culture, when the selection of the medium is not appropriate, it cannot be detected as a colony despite the presence of microorganisms in the sample, the microorganisms that should be detected are not detected, In some cases, it was misjudged.

  Accordingly, an object of the present invention is to provide a method for quickly and correctly determining whether or not indoor dirt is mold.

  The present invention is a method for determining whether or not indoor dirt is mold, a sample collecting step for collecting a sample from the dirt, a quantitative step for quantifying the amount of coenzyme in the sample, and a coenzyme And a determination step of determining whether the dirt is mold based on the abundance of.

  According to the method of the present invention, it is possible to determine whether or not indoor dirt is mold in a short time on site. For this reason, it is possible to determine on the spot an appropriate measure for removing the dirt. In addition, since the method of the present invention does not include a step of culturing microorganisms, a correct determination result can be obtained without erroneous determination due to inappropriate selection of a medium.

  The coenzyme comprises nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). One or more compounds selected from the group are preferred. In the determination step, when the total amount of the selected compounds per unit area is less than the predetermined value A1, it is determined that the stain is not mold, and when the amount is greater than the predetermined value A2, It is determined that the dirt is mold, and if it is a predetermined value A1 or more and less than A2, it is preferable to determine whether the dirt is mold by observing the sample with a microscope.

  By using NAD, NADP, NADH, or NADPH as a coenzyme, it can be rapidly quantified. Further, in the determination step, it is possible to quickly determine whether or not the stain is mold by making a determination based on the total amount of the selected compound per unit area. Furthermore, even if it is difficult to determine whether or not dirt is mold based on the amount of NAD, NADP, NADH or NADPH, whether or not it is mold on the spot by performing microscopic observation Can be determined.

  The coenzyme may be adenosine triphosphate (ATP). In the determination step, if the amount of ATP per unit area is less than the predetermined value B1, it is determined that the dirt is not mold, and if it is greater than or equal to the predetermined value B2, the dirt is mold. When the determination is made and the predetermined value B1 or more and less than B2, it is preferable to determine whether or not the dirt is mold by observing the sample with a microscope.

  By using ATP as a measurement target as a coenzyme, it can be rapidly quantified. Further, in the determination step, it is possible to quickly determine whether the dirt is mold by determining whether the dirt is mold according to the amount of ATP per unit area. Furthermore, even if it is difficult to determine whether the dirt is mold based on the amount of ATP present, it is possible to determine whether it is mold on-site by performing microscopic observation.

  According to the present invention, it is possible to provide a method for quickly and correctly determining whether or not indoor dirt is mold.

FIG. 6 is a schematic diagram illustrating one embodiment of a template. It is the schematic which shows one Embodiment of the expansion-contraction pole for template sticking. It is the schematic which shows one Embodiment of the expansion pole for sample collection. It is the schematic which shows one Embodiment of a test paper. 3 is a flowchart illustrating an embodiment of an NAD method. It is a flowchart which shows one Embodiment of ATP method. 10 is a graph showing the results of Experimental Example 2. A: Lucifer II, B: Lucifer 250 plus, C: Lucifer 250 plus and Lucifer ATP elimination reagent set, D: Lucifer HS set without ATP elimination reagent, E: Lucifer HS set. 10 is a graph showing the results of Experimental Example 3. 10 is a graph showing the results of Experimental Example 6.

  Hereinafter, preferred embodiments will be described with reference to the drawings as the case may be. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. In addition, the drawings are exaggerated for easy understanding, and the dimensional ratios do not necessarily match those described.

  The method for determining whether or not indoor dirt is mold is based on a sample collection step for collecting a sample from the dirt, a quantification step for quantifying the coenzyme in the sample, and the mold contamination based on the amount of coenzyme present. A determination step of determining whether or not.

  First, the sample collection step will be described. In one embodiment, a sample is taken from dirt on indoor walls, ceilings, floors, and the like. The sample is preferably taken from a certain area. This makes it easy to compare the abundance of the coenzyme obtained in the quantitative step described later with a reference value as a value per unit area.

In order to collect a sample from a certain area, it is preferable to use a sample collection frame called a wipe inspection frame or template. A template is a paper frame with a central part cut out. The template is pressed against the surface to be inspected to specify a sample collection range, and a sample is collected from the area specified by the frame, and is generally commercially available. . A commercially available template has an opening of 100 cm ² and specifies the sample collection range while pressing it against the surface to be inspected, and the sample can be collected by wiping the specified area with absorbent cotton or the like. It is common. However, as a result of investigations by the inventors, it has become clear that a normal 100 cm ² template is too wide for the determination method of the present embodiment. The determination in the determination step to be described later, in order to perform more accurate is preferably the opening of the template is 1～25Cm ^2, more preferably 1~9cm ^2, 4cm ² is particularly preferred.

FIG. 1 is a schematic diagram illustrating one embodiment of a template. As shown in FIG. 1, the template 100 includes an opening 20 that specifies a sample collection range and a hole 30 for collecting the template, and is bent along a fold line 40. The periphery of the hole 30 is reinforced. The hole 30 is used when collecting the template from the surface to be inspected after collecting the sample. Even when the template is pasted out of reach, for example, the template can be easily recovered from the surface of the object to be measured by hooking the hook attached to the tip of the telescopic pole into the hole 30 and peeling it off. Become. The entire surface 10 of the template 100 is an adhesive layer. The opening 20 has an area of 1 to 25 cm ² .

  Since a commercially available template does not have an adhesive layer, it is necessary to collect a sample while pressing it with one hand. For this reason, samples could only be collected within reach. In addition, it was necessary to use a stepladder etc. where it was out of reach such as the ceiling. On the other hand, by using the template of the above embodiment, it is not necessary to press the template with one hand, so that it is easy to collect a sample from a place out of reach. In order to collect a sample in an unreachable place, a template can be attached to the surface to be inspected using a telescopic pole described below.

  FIG. 2 is a schematic view showing an embodiment of a template sticking telescopic pole. The template pasting extension pole 200 includes an extension pole 210 and a jig 220 fixed to the tip of the extension pole 210 by a fixing band 215. A template is set on the surface 230 of the jig 220 and pressed against the surface to be inspected to attach the template. Thereby, even if it is a place out of reach, a template can be easily attached.

  In one embodiment, a sterile cotton swab can be used for sampling. In order to collect a sample efficiently, it is preferable to wet a cotton swab with sterilized water during sample collection. Instead of sterile water, a suitable sterilized liquid such as phosphate buffered saline may be used. In order to make an accurate determination, a sample of the surface to be inspected is collected by thoroughly tracing the range specified by the template using a cotton swab. When collecting a sample from an unreachable range, use a sample collection telescopic pole with a cotton swab attached to the tip of the telescopic pole. FIG. 3 is a schematic view showing an embodiment of a sample collection telescopic pole. The sample collection telescopic pole 300 includes the telescopic pole 210 and a jig 310 fixed to the tip of the telescopic pole 210 by a fixing band 215. A cotton swab 320 is connected to the jig 310 and a sample is taken from the range specified by the template.

In one embodiment, a sample collection pad can be used to collect a sample. FIG. 4 is a schematic view showing an embodiment of a test paper having a sample collection pad. The test paper 400 includes a sample collection pad 420 bonded to the surface of the support 410. The surface 430 of the sample collection pad 420 has an area of 1 to 5 cm ² . A sample is collected by pressing the sample collection pad 420 a predetermined number of times against the surface to be inspected. The sample collection pad 420 is preferably pressed to a different position each time so that the sample collection positions do not overlap. Thereby, a sample can be taken from a certain area without using a template. In order to collect a sample efficiently, it is preferable to wet the sample collection pad 420 with sterilized water during sample collection. Instead of sterile water, a suitable sterilized liquid such as phosphate buffered saline may be used. For example, when a sample collection pad having an area of 1.68 cm ² is used, the number of times of pressing against the surface to be inspected for sample collection is preferably 1 to 30 times, more preferably 5 to 20 times. preferable.

  Next, the quantitative step will be described. In the quantification step, the amount of coenzyme present in the sample collected in the sample collection step is quantified.

  In one embodiment, the coenzyme is one or more compounds selected from the group consisting of NAD, NADP, NADH and NADPH. Hereinafter, the determination method in which the coenzyme is NAD, NADP, NADH, or NADPH may be referred to as “NAD method”.

  When β-D-glucose and glucose-1-dehydrogenase are reacted with NAD or NADP, D-gluconolactone, NADH and NADPH are produced. Subsequently, when a tetrazolium salt and diaphorase are allowed to act on NADH and NADPH produced by the above reaction, and NADH and NADPH present in the sample, the tetrazolium salt is converted into a formazan salt. Based on the color of this formazan salt, the total abundance of NAD, NADP, NADH and NADPH in the sample can be quantified. Therefore, in the present embodiment, β-D-glucose, glucose-1-dehydrogenase, tetrazolium salt and diaphorase are reacted with the sample to correspond to the total abundance of NAD, NADP, NADH and NADPH in the sample. A color of formazan salt is obtained. In this case, glucose-1-dehydrogenase uses both NAD and NADP as substrates. Diaphorase uses both NADH and NADPH as substrates.

  In this embodiment, a sample is collected using the sample collection pad. Subsequently, the above reaction is performed by dropping a solution of β-D-glucose, glucose-1-dehydrogenase, tetrazolium salt, and diaphorase onto the pad from which the sample was collected, and NAD, NADP, NADH, and NADPH in the sample are subjected to the above reaction. The color of the formazan salt is obtained according to the total abundance, and the total abundance of NAD, NADP, NADH and NADPH is quantified based on the color intensity. In the case where NAD, NADP, NADH or NADPH is not present in the sample, the formazan salt cannot be colored. The quantification may be performed using a color difference meter, but it is also possible to obtain a calibration curve by using the above-mentioned reaction using a known concentration of NAD, NADP, NADH or NADPH that has been serially diluted in advance, and quantify it visually. it can. The quantification of the total abundance of NAD, NADP, NADH, and NADPH according to the present embodiment can be easily performed in the field in a short time.

  In the said embodiment, glucose-1-dehydrogenase may use only NAD as a substrate. The diaphorase may be one using only NADH as a substrate. In this case, the total abundance of NAD and NADH in the sample is quantified as a coenzyme.

  Moreover, in the said embodiment, glucose-1-dehydrogenase may use only NADP as a substrate. Further, the diaphorase may use only NADPH as a substrate. In this case, the total abundance of NADP and NADPH in the sample is quantified as a coenzyme.

  The quantification of the total abundance of NAD, NADP, NADH, and NADPH can be performed using, for example, HY-RiSE (trade name, Merck).

  In one embodiment, the coenzyme is ATP. Hereinafter, the determination method in which the coenzyme is ATP may be referred to as “ATP method”. Luciferin, a luminescent substrate, is converted to oxyluciferin by luciferase in the presence of ATP and magnesium ions, and emits light. This light emission amount is proportional to the ATP amount.

  In this embodiment, a sample is collected using the above template and a sterilized cotton swab. Subsequently, the swab from which the sample is collected is immersed in a reaction solution containing luciferin, magnesium ions and luciferase and stirred. As a result, light emission corresponding to the amount of ATP present in the sample can be obtained. If no ATP is present in the sample, no luminescence is obtained. The amount of luminescence can be measured using a luminometer. The luminometer can be brought into the field and is preferably a small portable type. For example, Lumitester C-110 (trade name, Kikkoman Corporation) is preferably used. It is preferable to obtain a calibration curve by performing the above-described reaction using ATP having a known concentration that has been serially diluted in advance to emit light. Using the calibration curve, the amount of ATP present in the sample can be determined from the amount of luminescence. The quantification of ATP according to the present embodiment can be easily performed in the field in a short time.

  The quantification of the above ATP can be carried out using, for example, Lucipak II (trade name, Kikkoman Corporation), which is an ATP quantification kit.

  NAD and ATP are metabolites of microorganisms. Therefore, when NAD or ATP is detected from the sample according to the above embodiment, it can be determined that microorganisms are present on the surface to be inspected. This microorganism is not limited to mold. However, it is very rare that indoors generate a large amount of microorganisms other than mold to form dirt. Therefore, in most cases, when NAD or ATP is detected from a sample according to the above embodiment, it can be determined that mold is present on the surface to be inspected.

  Next, the determination step will be described. In the determination step, it is determined in the field whether the indoor dirt is mold based on the abundance of the coenzyme in the sample determined in the determination step.

  In the case of the NAD method, if the total abundance of NAD, NADP, NADH and NADPH in the sample is less than the predetermined value A1 per unit area of the surface to be inspected, it is determined that the dirt is not mold and the predetermined value In the case of A2 or more, it is determined that the dirt is mold. On the other hand, when it is greater than or equal to the predetermined value A1 and less than A2, it may be difficult to make a deterministic determination. For this reason, it is determined by microscopic observation of a sample whether dirt is mold. The microscope is preferably a small portable type that can be brought into the field. As a result of observing with a microscope, when the presence of spores and hyphae is recognized, it can be determined that the mold is mold.

  In one embodiment, the predetermined values A1, A2 are as follows. When HY-RiSE (trade name, Merck) is used as the NAD quantification kit, and the sample is collected and colored by pressing 20 times against the surface to be inspected, the amount of color development is determined visually and the degree of color development is determined. Correspondingly, it is classified into 0-3 stages as follows. Those with almost no color development are classified as stage 0. Those whose peripheral part is light purple and slightly discolored are classified as the first stage. Those in which the central part changes to dark purple and the peripheral part also changes to purple are classified as the second stage. Those in which the central portion is clearly dark purple and the surroundings are also purple are classified as the third stage. When expressed as a numerical value, the 0th stage is represented as 0, the 1st stage is represented as 1, the 2nd stage is represented as 2, and the 3rd stage is represented as 3. In the present embodiment, the predetermined value A1 is 1 (first stage), and the predetermined value A2 is 3 (third stage).

  In the case of the ATP method, if the amount of ATP present in the sample is less than the predetermined value B1 per unit area of the surface to be inspected, it is determined that the dirt is not mold, and if it is greater than the predetermined value B2, the dirt Is determined to be mold. On the other hand, if the amount of ATP present in the sample is a predetermined value B1 or more and less than B2, it may be difficult to make a deterministic determination. For this reason, it is determined by microscopic observation of a sample whether dirt is mold. As a result of observing with a microscope, when the presence of spores and hyphae is recognized, it can be determined that the mold is mold.

In one embodiment, the predetermined values B1, B2 are as follows. Lumitester C-110 (trade name, Kikkoman Corporation) calibrated as in Experimental Example 1 described later is used. A sample collected from 4 cm ^{2 of the} surface to be inspected is caused to emit light using Lucipak II (trade name, Kikkoman Co., Ltd.), which is an ATP quantification kit, and the amount of luminescence is measured using Lumitester C-110 (trade name, Kikkoman Corporation). taking measurement. In the present embodiment, the predetermined value B1 is 100, and the predetermined value B2 is 10,000.

  As the microscope, for example, a small inverted microscope (DSM-1 type, Daiko Science Co., Ltd.) can be used. It is preferable to connect a digital camera system connected to a personal computer instead of the eyepiece of the microscope and display the microscope image on a portable monitor such as a notebook personal computer display. As the digital camera system, for example, Moticam 2000 (trade name, Shimadzu Rika Co., Ltd.) can be used. Thereby, a microscope image can be confirmed by a plurality of people. The magnification of the microscope is preferably 20 to 1200 times.

  When observing a sample with a microscope, a part of the sample used in the NAD method or ATP method may be dropped on a slide glass and observed, or a sample for microscopic observation may be newly collected from the surface to be inspected. . When a new sample is collected, if the surface to be inspected is a material that is difficult to peel off, the sample may be attached to the adhesive surface of the cellophane tape by applying and peeling the cellophane tape on the surface to be inspected. In this case, the cellophane tape to which the sample is attached can be attached to a slide glass and observed with a microscope. Also, if the surface to be inspected is made of a material that can be easily peeled off and the surface may be damaged if sampled with cellophane tape, sample the surface to be inspected with a cotton swab moistened with sterile water. It can be applied on a slide glass and observed with a microscope.

  When the presence of a coenzyme such as NAD or ATP is detected in the sample in the above quantification step, it can be determined that live mold exists in the sample. On the other hand, in observation with a microscope, it is possible to determine whether mold is alive or not based on the observed spore and mycelial morphology.

  Examples of molds that can be detected by this method include those belonging to the genus Aspergillus, Alternaria, Ketomium, Cladosporium, Eurotium, Penicillium, Fusarium, Rhizopus, and Trichoderma.

  FIG. 5 is a flowchart illustrating an embodiment of the NAD method. In the present embodiment, first, a sample is collected from the surface to be inspected, and the total abundance of NAD, NADP, NADH, and NADPH in the sample is quantified (step S1). If the total amount of NAD, NADP, NADH, and NADPH is less than the predetermined value A1 per unit area of the surface to be inspected (A in step S2), it is determined that the dirt is not mold (step S2). S3) When the value is equal to or greater than the predetermined value A2 (B in Step S2), it is determined that the dirt is mold (Step S4). On the other hand, when the total abundance of NAD, NADP, NADH, and NADPH in the sample is a predetermined value A1 or more and less than A2 per unit area of the surface to be inspected (C in Step S2), microscopic observation is performed (Step S5). ). As a result of microscopic observation, when the presence of spores and mycelia was confirmed in the sample (YES in Step S6), it was determined that the dirt was mold (Step S4), and the presence of spores and mycelia was not confirmed. In this case (NO in step S6), it is determined that the dirt is not mold (step S3).

  FIG. 6 is a flowchart illustrating an embodiment of the ATP method. In the present embodiment, a sample is collected from the surface to be inspected, and the amount of ATP present in the sample is quantified (step S1). As a result of quantification, when the amount of ATP present is less than the predetermined value B1 per unit area of the surface to be inspected (A in step S2), it is determined that the dirt is not mold (step S3), and is greater than or equal to the predetermined value B2. In the case (B in Step S2), it is determined that the dirt is mold (Step S4). On the other hand, when the amount of ATP present in the sample is a predetermined value B1 or more and less than B2 per unit area of the surface to be inspected (Step S2C), microscopic observation is performed (Step S5). As a result of microscopic observation, when the presence of spores and mycelia was confirmed in the sample (YES in Step S6), it was determined that the dirt was mold (Step S4), and the presence of spores and mycelia was not confirmed. In this case (NO in step S6), it is determined that the dirt is not mold (step S3).

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples of the present invention. However, the present invention is not limited to these examples, and various modifications can be made without departing from the technical idea of the present invention. Can be changed.

(Experimental example 1)
(Create a calibration curve)
A calibration curve for the luminescence amount of ATP was prepared using Lumitester C-110 (trade name, Kikkoman Corporation). First, a standard in which the concentration of ATP is 0, 2 × 10 ⁻¹² , 2 × 10 ⁻¹¹ , 2 × 10 ⁻¹⁰ , 2 × 10 ⁻⁹ , 2 × 10 ⁻⁸ , 2 × 10 ⁻⁷ (M). A solution was prepared. Then, these standard solutions were light-emitted according to the instruction manual using Lucifer 250 plus (trade name, Kikkoman Corporation) which is an ATP quantification kit. Subsequently, the luminescence amount (relative value) of each solution was measured using Lumitester C-110 (trade name, Kikkoman Corporation). The measurement results are shown in Table 1.

(Experimental example 2)
(Measurement of light emission based on ATP in the sample)
Using a plurality of commercially available ATP assay kits, Aspergillus niger spores suspended in a known concentration were used as samples, and the amount of luminescence based on ATP in the samples was measured. First, Aspergillus niger spores were suspended in sterilized water, and a solution that was serially diluted in several steps within a range of 1 × 10 ⁴ to 1 × 10 ⁷ cells / ml was prepared as a mold sample.

  Subsequently, using a plurality of ATP determination kits, light emission corresponding to the amount of ATP present in each sample was obtained, and the light emission amount was measured. The kit uses Lucipack II, Lucifer 250 Plus, Lucifer 250 Plus and Lucifer ATP Eliminating Reagent Set, Lucifer HS Set without ATP Eliminating Reagent, and Lucifer HS Set (both trade names, Kikkoman Corporation) The operation was performed according to the instruction manual. The amount of luminescence was measured using Lumitester C-110 (trade name, Kikkoman Corporation) used in Experimental Example 1.

  FIG. 7 is a graph plotting the amount of luminescence based on the amount of ATP present. A value measured by Lumitester C-110 (trade name, Kikkoman Corporation) was used as the light emission amount. A to E in the graph are the results using the following kits, respectively. A: Lucipack II, B: Lucifer 250 plus, C: Lucifer 250 plus and Lucifer ATP elimination reagent set, D: Aluc elimination HS set from Lucifer HS set, E: Lucifer HS set.

(Experimental example 3)
(Color development based on NAD in sample)
Using a commercially available NAD quantification kit, Aspergillus niger spores suspended at a known concentration were used as samples, and formazan salt color development based on the amount of NAD present in the samples was obtained. First, Aspergillus niger spores were suspended in sterile water and serially diluted to a concentration of 1 × 10 ⁴ cells / ml to 1 × 10 ⁷ cells / ml to obtain a mold sample.

  Subsequently, using the NAD quantification kit, color development corresponding to the amount of NAD present in each sample was obtained. HY-RiSE (trade name, Merck) was used as the NAD quantification kit. The amount of color development was visually determined and classified into 0 to 3 stages according to the degree of color development as follows. Those with almost no color development were classified as the 0th stage. Those whose peripheral part was light purple and slightly discolored were classified as the first stage. Those in which the central portion changed to dark purple and the peripheral portion changed to purple were classified as the second stage. Those in which the central portion was clearly changed to dark purple and the surroundings were also changed to purple were classified as the third stage.

  FIG. 8 is a graph plotting the amount of color development based on the amount of NAD present in each sample. The color development amount was classified according to the degree of color development. The 0th stage was represented as 0, the 1st stage was represented as 1, the 2nd stage was represented as 2, and the 3rd stage was represented as 3.

(Experimental example 4)
(Judgment by ATP method)
First, a sample was collected from indoor dirt. A template having a 2 × 2 cm opening was affixed to the wall surface. A template obtained by cutting out a part of a full-surface adhesive sticker (trade name SSZ-33RY, Sumitomo 3M Co., Ltd.) into a 2 × 2 cm size was used. Subsequently, a swab of Lucipak II (trade name, Kikkoman Co., Ltd.), which is an ATP quantification kit, was wetted with sterilized water.

  Subsequently, the swab with the sample attached was returned to the main body of Lucipack II, and the main body was shaken down several times according to the instruction manual, and the reagent of Lucipack II was dropped into the measurement tube. Next, the undissolved reagent was thoroughly shaken to dissolve, the measurement tube was removed from the main body, and the amount of luminescence was measured using Lumitester C-110 (trade name, Kikkoman Corporation) used in Experimental Example 1. Quantification of the ATP abundance was performed on various indoor soils.

(Judgment by microscopic observation)
A sample was taken from a region almost identical to the surface to be inspected measured by the ATP method, and observed with a microscope. First, a template having an opening of 2 × 2 cm was attached to the wall surface. A template obtained by cutting out a part of a full-surface adhesive sticker (trade name SSZ-33RY, Sumitomo 3M Co., Ltd.) into a 2 × 2 cm size was used. Subsequently, using a cotton swab that was sterilized and moistened with phosphate buffered saline, the area specified by the template was thoroughly traced to obtain a sample. As a cotton swab, a wipe check II (trade name, Eiken Chemical Co., Ltd.) was used. Subsequently, the cotton swab with the sample attached was applied on a slide glass and observed with a microscope. As the microscope, a small inverted microscope (DSM-1 type, Daiko Science Co., Ltd.) connected with a digital camera system (Moticam 2000, trade name, Shimadzu Rika Co., Ltd.) was used. The microscope image was displayed on the display of a notebook computer. If spores and hyphae were observed, the indoor dirt was determined to be mold.

  On the surface to be inspected, in the sample whose luminescence amount measured with Lumitester C-110 (trade name, Kikkoman Co., Ltd.) is less than 100, the presence of spores and hyphae was confirmed even by microscopic observation. There wasn't. In addition, in the sample having a measured light emission amount of 10,000 or more, the presence of spores and mycelia was confirmed by microscopic observation. On the other hand, in samples with a measured light emission amount of 100 or more and less than 10,000, the presence or absence of spores or mycelia may or may not be recognized. Table 2 shows the measurement value of the light emission amount based on the amount of ATP present and the result of determination by microscopic observation. The numbers in the table mean the number of corresponding samples.

  The determination by the ATP method and the determination by microscopic observation were able to be performed in a short time at a site where the contamination to be inspected exists.

(Experimental example 5)
(Determination by NAD method)
HY-RiSE (trade name, Merck) was used as the NAD quantification kit. According to the instruction manual, the pad portion of the test paper of HY-RiSE was moistened with a wetting liquid, and the sample was collected by pressing it against indoor soil to be examined 20 times. The area of the pad portion was 1.68 cm ² . Subsequently, the substrate solution and the enzyme solution were dropped on the pad portion to which the sample was attached, and left in the dark for 30 minutes. Subsequently, the total amount of NAD, NADP, NADH, and NADPH in the sample was visually determined based on the amount of color of the formazan salt as in Experimental Example 3, and was quantified by classifying into 0 to 3 stages. The above quantification was performed on various indoor soils.

(Judgment by microscopic observation)
In the same manner as in Experimental Example 3, a sample was taken from a region almost identical to the surface to be inspected measured by the NAD method, and observed with a microscope. If spores and hyphae were observed, the indoor dirt was determined to be mold. In the sample where the total abundance of NAD, NADP, NADH and NADPH on the surface to be examined was less than the first stage, the presence of spores and mycelia was not confirmed even under microscopic observation. In addition, in the samples in which the total amount of NAD, NADP, NADH and NADPH was in the third stage or higher, the presence of spores and mycelia was confirmed by microscopic observation. On the other hand, in samples where the total abundance of NAD, NADP, NADH and NADPH is not lower than the first stage and lower than the third stage, the presence or absence of spores and mycelia may or may not be recognized. Table 3 shows the color development amount based on the total abundance of NAD, NADP, NADH, and NADPH and the result of determination by microscopic observation. The numbers in the table mean the number of corresponding samples.

  The above-described determination by the NAD method and determination by microscopic observation could be performed in a short time at the site where the contamination to be inspected exists.

(Experimental example 6)
(Comparison between NAD method and ATP method)
The relationship between the total amount of NAD, NADP, NADH, and NADPH quantified and the amount of ATP was examined in each sample of indoor dirt determined whether or not it was mold in Experimental Examples 3 and 4. FIG. 9 is a graph plotting the total abundance of NAD, NADP, NADH, and NADPH and the ATP abundance in each sample. There was a good correlation between the two results.

  According to the present invention, it is possible to provide a method for quickly and correctly determining whether or not indoor dirt is mold.

  10, 230, 430 ... face, 20 ... opening, 30 ... hole, 40 ... crease, 100 ... template, 200 ... telescopic pole for template application, 210 ... telescopic pole, 215 ... fixed band, 220, 310 ... jig, 300 ... telescopic pole for sample collection, 320 ... cotton swab, 400 ... test paper, 410 ... support, 420 ... sample collection pad.

Claims (3)

A method for judging whether or not indoor dirt is mold,
A sample collection step for collecting a sample from dirt;
A quantification step to quantify the amount of coenzyme present in the sample;
A determination step of determining whether the soil is mold based on the amount of coenzyme present. The coenzyme is one or more compounds selected from the group consisting of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. ,
In the determination step, the abundance per unit area of the coenzyme is:
If it is less than the predetermined value A1, it is determined that the dirt is not mold,
If the value is equal to or greater than the predetermined value A2, the dirt is determined to be mold,
2. The method according to claim 1, wherein when the sample is less than or equal to a predetermined value A <b> 1 and less than A <b> 2, whether or not the dirt is mold is determined by microscopic observation of the sample. The coenzyme is adenosine triphosphate;
In the determination step, the abundance per unit area of adenosine triphosphate is
If it is less than the predetermined value B1, it is determined that the dirt is not mold,
If it is equal to or greater than the predetermined value B2, the dirt is determined to be mold,
2. The method according to claim 1, wherein when the sample is less than or equal to a predetermined value B <b> 1 and less than B <b> 2, it is determined whether or not the dirt is mold by microscopic observation of the sample.

Images