JP2013172665A - Method for grasping attaching characteristics of microorganism, and method for evaluating test environment of microorganisms - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evaluate a test environment of microorganisms by considering effects given to measurement results of the microorganisms by the attaching characteristics of the microorganisms at the inner wall surface of a spraying chamber device, etc., used for the measurement of the amount of the microorganisms.SOLUTION: A method for evaluating a testing environment of microorganisms is provided by executing as evaluation processes, (S2) an installing process of installing a collided bacteria-collecting means, (S4) a sterilizing process of sterilizing an evaluation objective surface, (S6) a spraying process of performing spray for bacteria, (S8) a culturing process of culturing the bacteria collected from the collided bacteria-collecting means and the evaluation objective surface, (S10) a counting process of counting the number of surface-collided bacteria (=A) by the collided bacteria-collecting means, (S12) a collection of the attached bacteria at an exposed surface, and a counting process of counting the number of attached bacteria (=B), (S14) a calculation process of calculating the ratio of the attached number of bacteria to the number of bacteria collided with the surface(=B/A), and (S16) an evaluation process of evaluating the ratio (=B/A).

Description

  The present invention relates to a method for grasping microorganism adhesion characteristics and a method for evaluating microorganism test environments, and more particularly to a method for grasping microorganism adhesion characteristics and a method for evaluating microorganism test environments for evaluating a test environment such as a chamber apparatus used in a bioaerosol test. .

  In recent years, the necessity of microbial tests for estimating the amount of microorganisms floating in the air (airborne bacteria) has been increasing. Such a microbial test is required in terms of hygiene management, for example, in the medical field such as the food industry and hospitals, and is required in terms of quality control in the pharmaceutical manufacturing industry. Specific methods of the microorganism test for estimating the amount of microorganisms floating in the air include, for example, a falling bacteria test method, an air sampler method, and an impinger method.

  An evaluation apparatus for evaluating the performance and characteristics of a microorganism detection apparatus (such as an adhesive sheet) used for such microorganism collection has also been developed. In such an evaluation apparatus, for example, a microorganism detection apparatus to be evaluated is installed in a closed space such as a chamber apparatus, and a microorganism (or a microorganism suspension) is sprayed in the closed space, and the microorganism detection apparatus The reliability of the microorganism test is evaluated and the performance of the microorganism detection apparatus is evaluated by inspecting a predetermined amount of microorganisms collected.

  For example, Patent Document 1 discloses a novel method for measuring the amount of microorganisms, in particular, a method for simply sampling and counting airborne microorganisms. Specifically, (1) the step of collecting microorganisms present in the air on the pressure-sensitive adhesive sheet, (2) the step of bringing the microorganism-collecting surface of the pressure-sensitive adhesive sheet into contact with the surface of the medium, (3) Disclosed is a method for detecting microorganisms floating in the air, which includes a step of observing and counting microorganisms that have proliferated through the adhesive sheet.

  Further, for example, Patent Document 2 discloses an environment evaluation method capable of performing evaluation of microorganisms floating in the space with higher accuracy. Specifically, an environment in which microorganisms are collected in an evaluation chamber separated from the external space by an isolation wall, and then removed particles are supplied to sterilize and remove microorganisms, and the collected microorganisms are measured. In the evaluation method, there is disclosed an environmental evaluation method characterized in that a spore-forming spore-forming bacterium, such as Bacillus subtilis, is used as the microorganism in order to prevent the natural disappearance of the microorganism.

  Further, for example, Patent Document 3 discloses an environment evaluation apparatus and an environment evaluation method that can perform evaluation of minute substances floating in a space with higher accuracy. Specifically, the evaluation room isolated from the external space by the isolation wall, the fine substance supply means for supplying microorganisms into the evaluation room, and the fine substance removal means for supplying removal particles for removing microorganisms into the evaluation room; And an environmental evaluation apparatus having a minute substance collection means for collecting microorganisms in the evaluation chamber, wherein a plurality of air supply holes are provided on almost the entire surface of the isolation wall except at least the floor surface. An environment evaluation apparatus is disclosed in which air flows into the apparatus.

JP 2007-135476 A JP 2008-022764 A JP 2008-022765 A

However, in the conventional method for evaluating a microbial test environment described above, in the microbial test process as an evaluation method, the inner wall surface of a chamber device (hereinafter also referred to as “a spray chamber device”) used for a bioaerosol test. Since the impact of impacting fungi (for example, airborne fungi such as bacteria and fungi) or fungi attached to the inner wall on the test results is not managed, the stability of the bioaerosol test environment and the validity of the evaluation results ( (Reliability) and evaluation accuracy cannot be ensured. More specifically, the state of attachment of fungi to the inner wall surface of the spray chamber device is not managed, and thus the state of attachment of fungi has various effects on the implementation of one or more evaluations. It is not considered to be able to give.
The techniques described in Patent Documents 1 to 3 also do not disclose until specific examination and management of the state of attachment of fungi to the inner wall surface of the spray chamber apparatus.

Therefore, the present invention has been made in view of the above-mentioned conventional problems, and can quantitatively measure the adhesion characteristics of microorganisms on the inner wall surface of a spray chamber apparatus or the like used for measuring the amount of microorganisms. Thus, an object of the present invention is to provide a method for evaluating a microbial test environment in which the microbial test environment can be evaluated in consideration of the influence of the adhesion characteristics on the measurement results of microorganisms.
Another object of the present invention is to provide a method for grasping microorganism adhesion characteristics that can quantitatively measure the adhesion characteristics of microorganisms on the inner wall surface of a spray chamber apparatus or the like used for measuring the amount of microorganisms.

  In order to solve the above-mentioned problem, the method for evaluating a microorganism test environment according to the present invention includes a step of installing a collision bacteria collecting means for collecting microorganisms that have collided with a collection surface in a first region of an evaluation object surface; A step of cleaning a surface of a second region different from the first region of the evaluation target surface, a spraying step of spraying microorganisms on the evaluation target surface under specified conditions, and the collision bacteria collection Culturing microorganisms collected by the means and microorganisms collected from the surface of the second region to form first and second colony groups, and capturing the collision bacteria from the first colony groups. Calculating the number of microorganisms collected by the collecting means as the number of surface-impacting bacteria, calculating the number of microorganisms attached to the surface of the second region from the second colony group, and the above-mentioned The number of surface-attached bacteria and the number of surface-impacting bacteria A step of grasping the microbial attachment characteristics of the evaluation target surface based, characterized in that it comprises a and a step of evaluating the microorganisms test environment with the evaluation target surface on the basis of microbial attachment characteristics described above grasped.

  By adopting such a configuration, it is possible to evaluate the microorganism test environment by grasping the microorganism adhesion characteristics on the surface to be evaluated.

  Further, the method for evaluating a microorganism test environment according to the present invention includes a step of installing a collision bacteria collecting means for collecting microorganisms that have collided with a collection surface in a partial region of the evaluation target surface, and a microorganism under specified conditions. A first spraying step for spraying the surface to be evaluated, a step of performing a first air sampling for estimating a first colony formation rate of the microorganisms sprayed in the first spraying step, and the above Removing the collision bacteria collecting means from the surface to be evaluated and forming a clean surface on the surface of the partial area after the removal; and a second method of spraying microorganisms on the surface to be evaluated under defined conditions A spraying step, a step of performing a second air sampling for estimating a second colony formation rate of the microorganism sprayed in the second spraying step, a microorganism collected by the collision bacteria collecting means, and the above Remove microorganisms collected from the clean surface. A step of forming first and second colony groups by culturing each of them, a step of calculating the number of microorganisms collected from the first colony group by the collision bacteria collection means as the number of surface collision bacteria, and The step of calculating the number of microorganisms attached to the clean surface from the second colony group as the number of surface-attached bacteria, and the number of surface-impacting bacteria and the number of surface-attached bacteria are the first and second colony formation rates, respectively. A step of correcting and grasping the microorganism adhesion characteristics of the surface to be evaluated based on the corrected number of surface-impacting bacteria and the corrected number of surface-attached bacteria or the ratio of these two numbers; And a step of evaluating a microbial test environment having the evaluation target surface on the basis of adhesion characteristics.

  By adopting such a configuration, it becomes possible to evaluate the microbial test environment (more precise evaluation) by a technique in which the installation area where the collision bacteria collecting means is installed and the area of the clean surface where the attached bacteria adhere are made the same area. .

  A particle counter for measuring the number of particles (corresponding to the number of microorganisms) floating in the unit volume of air in the first and second air sampling, and a predetermined amount of the air is sprayed on the medium to colonize microorganisms on the medium. An air sampler (air sampler for measuring the number of microorganisms that form colonies out of microorganisms suspended in a unit volume of air), and the microorganisms form colonies from the measured values Obtain colony formation rate. Thereby, it is easy to correct the difference in colony formation rate (formability) due to the difference in the spraying conditions of microorganisms and to compare them according to a common standard.

  In addition, the method for grasping the microorganism adhesion characteristics according to the present invention includes a step of installing a collision bacteria collecting means for collecting microorganisms that have collided with the collection surface in the first region of the evaluation object surface, A step of cleaning a surface of a second region different from the first region, a spraying step of spraying microorganisms on the evaluation target surface under defined conditions, and a microorganism collected by the collision bacteria collecting means And microorganisms collected from the surface of the second region to form first and second colonies, respectively, and collected from the first colonies by the collision bacteria collecting means A step of calculating the number of microorganisms as a surface-collision bacteria number, a step of calculating the number of microorganisms attached to the surface of the second region from the second colony group, and the number of surface-attached bacteria, and the above-mentioned Based on the number of surface-impacting bacteria, Characterized in that it comprises the steps of grasping the microbial adhesion properties of the surface, the

  Such a configuration is advantageous because it makes it possible to grasp the microorganism adhesion characteristics on the surface to be evaluated.

  Furthermore, the method for grasping the microorganism adhesion characteristics according to the present invention includes a step of installing a colliding bacteria collecting means for collecting microorganisms that collide with the collection surface in a partial region of the evaluation target surface, and a microorganism under specified conditions. A first spraying step for spraying the surface to be evaluated, a step of performing a first air sampling for estimating a first colony formation rate of the microorganisms sprayed in the first spraying step, and the above Removing the collision bacteria collecting means from the surface to be evaluated and forming a clean surface on the surface of the partial area after the removal; and a second method of spraying microorganisms on the surface to be evaluated under defined conditions A spraying step, a step of performing a second air sampling for estimating a second colony formation rate of the microorganism sprayed in the second spraying step, a microorganism collected by the collision bacteria collecting means, and the above Microbes collected from the clean surface A step of forming each of the first and second colony groups by culturing, and a step of calculating the number of microorganisms collected from the first colony group by the collision bacteria collecting means as the number of surface collision bacteria, The step of calculating the number of microorganisms attached to the exposed surface from the second colony group as the number of surface-attached bacteria, and the number of surface-collision bacteria and the number of surface-attached bacteria are the first and second colony formation rates, respectively. And the step of grasping the microbial adhesion characteristics of the evaluation target surface based on the corrected number of surface impacting bacteria and the corrected number of surface attached bacteria or the ratio of these two numbers. It is characterized by.

  By adopting such a configuration, it becomes possible to evaluate (more accurate evaluation) the microorganism adhesion characteristics by a technique in which the installation area where the collision bacteria collecting means is installed and the exposed surface area where the attached bacteria adhere are the same area. . Moreover, the difference in the spraying conditions in the two-stage bacterial spraying is corrected, and the condition is good.

  It is desirable to use at least one of a pressure-sensitive adhesive sheet for measuring surface-attached bacteria, a stamp medium, a Petri film (microorganism culture sheet), or the like as the collision bacteria collection means. In addition, it is desirable to use at least one of a pressure-sensitive adhesive sheet for measuring surface-attached bacteria, a stamp medium, a petri film, a sampling method, and the like for collection from the clean surface of the partial area. Further, other means (methods) having functions equivalent to these may be used.

According to the method for evaluating a microbial test environment of the present invention, in measuring the amount of microorganisms, for quantitatively grasping the microbial adhesion characteristics possessed by the inner wall surface of the microbial test environment (for example, a spray chamber device) to be evaluated. Since the process is performed, there is an effect that a given microbial test environment can be evaluated in consideration of the influence of the microbial adhesion property on the microbial test.
In addition, according to the method for grasping the microorganism adhesion characteristics of the present invention, there is an effect that the microorganism adhesion characteristics on the inner wall surface of a spray chamber apparatus or the like used for measuring the amount of microorganisms can be measured quantitatively.

It is process drawing which showed the evaluation method of the microorganism test environment which concerns on the 1st Embodiment of this invention according to process. It is explanatory drawing which shows the external appearance of the microorganism test environment which is the evaluation object of the evaluation method of the microorganism test environment which concerns on embodiment of this invention. It is explanatory drawing which shows the state (there is almost no rebound) of the collision microbe collected by the collision microbe collection means in the evaluation method of the microbe test environment which concerns on embodiment of this invention. The figure (A) is a figure explaining the bacteria adhesion state (partially attached and partly bounces) on the evaluation surface, and the figure (B) explains the state where bacteria are partly attached corresponding to the characteristics of the evaluation surface. FIGS. 6A and 6B are explanatory diagrams for explaining the collection of bacteria attached to the evaluation surface. It is process drawing which showed the evaluation method of the microorganism test environment which concerns on other embodiment of this invention according to process.

  In the method for evaluating a microorganism test environment according to the present invention, in order to accurately evaluate a microorganism (or fungi, etc.) detection device, it is sufficient to adjust the jet velocity during the spraying of the microorganism to be within a specified range. In addition to this, attention is paid to the fact that it is necessary to consider the adhesion state (adhesion characteristics) of microorganisms to the inner wall surface of a chamber apparatus or the like which is a test environment.

  That is, if bacteria adhere to the inner wall surface of a chamber apparatus or the like, in the case of a spray test, there is an effect of reducing the number of bacteria floating in the spray chamber apparatus. In addition, when such a spray test is continuously performed a plurality of times, there is an effect that bacteria attached to the inner wall surface increase the number of bacteria floating in the spray chamber apparatus as the number of tests is repeated. Therefore, in the case of the microbial spray test, it is necessary to manage the microbial test environment so that the above-described influence does not impair the stability of the spray test. In particular, when a microorganism test is performed using a specific bacteria detection device, the test is performed at an initial time point and a calibration time point in maintenance after the bacteria detection device is actually used for a specified period. Therefore, it is important to maintain the stability of the spray test and to improve the reliability of the test result by managing the test environment as described above.

  In the method for evaluating a microbial test environment according to the present invention, the method for managing the test environment is a method for evaluating the microbial test environment. More specifically, the ratio of the number of bacteria A (the number of surface-impacting bacteria) that can be directly collected on the surface to be evaluated such as the inner wall surface of the spray chamber apparatus to the number of bacteria B (the number of surface-attached bacteria) attached to the surface ( = B / A), and this ratio is numerically managed. In addition, about the measurement of this bacteria count A (surface collision bacteria count), a well-known method can be used. As this known method, for example, there is a method in which the adhesive surface of the pressure-sensitive adhesive sheet is exposed and placed on the aforementioned evaluation target surface before spraying the bacteria, and collected and counted during the spraying of the bacteria. In addition, there is a method in which a medium surface of a general standard medium (including a stamp medium in a category) is exposed and placed on the evaluation target surface before the bacteria are sprayed, and collected and counted during the spraying of the bacteria. Furthermore, the method of counting using a Petri film can also be used. In any case, what is installed on the evaluation target surface is preferably one that does not affect the shape of the evaluation target surface as much as possible (for example, a sheet-like one).

  On the other hand, for the above-mentioned measurement of the number of bacteria B (the number of bacteria attached to the surface), a known method for collecting bacteria attached to the surface after the spraying of bacteria can be used. More specifically, a method in which adherent bacteria are collected with an adhesive sheet after spraying of bacteria, a method of collecting in a stamp medium after spraying of bacteria, a method of collecting with a wiping method, and the like are typically available. It is. In the method for evaluating a microbial test environment according to the present invention, what is sprayed in the spraying process (microorganisms) includes not only the above-mentioned bacteria but also fungi (spores, molds, mold spores, etc.). And

Hereinafter, embodiments of a method for evaluating a microbial test environment and a method for grasping microbial adhesion characteristics according to the present invention will be described in detail with reference to the drawings.
FIG. 1 is a process diagram showing a method for evaluating a microbial test environment according to the first embodiment of the present invention for each process. In the figure, the evaluation method of the microorganism test environment of the present embodiment includes, as an evaluation process, an installation process of the collision bacteria collecting means (step S2), an exposed surface formation process (step S4), and a bacteria spraying process ( Step S6), a step of culturing the sprayed bacteria (Step S8), a step of counting the number of surface-collision bacteria (= A) by the collision bacteria collection means (Step S10), collection of adherent bacteria on the exposed surface, and Counting process (Step S12) of the number of adherent bacteria (= B), calculating process (Step S14) of the ratio (= B / A) of the number of adherent bacteria and the number of surface-impacting bacteria, and the number of adhered bacteria showing the microorganism adhesion characteristics And the ratio (= B / A) of the ratio of the number of bacteria colliding with the surface (step S16), and the evaluation process of the microbial test environment, for example, the process of evaluating air cleanliness, airborne particulates, etc. (step S18) And these processes ( Step S2～S18) is preferably carried out in this order.

  FIG. 2 is an explanatory diagram showing an appearance of a microbial test environment that is an evaluation target of the microbial test environment evaluation method according to the embodiment of the present invention. The microbial test environment evaluated by the microbial test environment evaluation method of this embodiment is the spray chamber apparatus 1 for bioaerosol test shown in FIG. Usually, in the spray test using the spray chamber apparatus 1, the microparticles 12 of the bacterial suspension are sprayed upward from a sprayer (nebulizer) 11 installed at the center of the space 2 in the spray chamber apparatus 1. Thereafter, the fine particles are uniformly diffused into the chamber space 2 by the air currents of a plurality of fans (not shown) installed in the chamber apparatus 1. Further, the spray chamber apparatus 1 is provided with a particle counter (not shown) for counting the number of fine particles and an air sampler for collecting floating bacteria on the surface of the agar medium. The collected fungi are cultured, and one fungus grows into one colony. The number of bacteria collected on the agar medium is calculated from the number of colonies (colony group).

FIG. 3 is an explanatory view showing the state of the collision bacteria collected by the collision bacteria collection means 4 in the microorganism test environment evaluation method according to the embodiment of the present invention. Here, the “collision bacteria” are bacteria that are sprayed from the nebulizer 11 to become airborne bacteria and collide with the target surface (the collection surface 4 and the evaluation surface 5). Part of the collided bacteria rebounds on the target surface, and part adheres to the target surface (see 3 and 3a in FIG. 4 described later).
In FIG. 3, reference numeral 2 is a chamber apparatus space, reference numeral 3 is spray bacteria collected and collected in the chamber apparatus space, reference numeral 4 is a collecting surface of a collision bacteria collecting means for collecting spray bacteria, and reference numeral 5 is The evaluation surfaces including the installation surface of the collision bacteria collecting means and the adhesion surface to which the spray bacteria adhere are shown.

Hereinafter, the evaluation method of the microbial test environment of this embodiment will be described for each step with reference to FIGS. 1 to 3. First, in the installation process (step S2) of the collision bacteria collection means, the collision bacteria collection means 13 (for example, collision bacteria is formed on a part of the inner wall surface (surface to be evaluated) of the spray test chamber apparatus 1 shown in FIG. A pressure-sensitive adhesive sheet, stamp medium, or Petri film (microbe culture sheet) can be installed with the adhesive surface or medium surface exposed. This adhesive surface or culture medium surface is considered to adhere almost all the floating bacteria that collide due to the viscosity of the surface. The installation position of the collision bacteria collection means 13 is selected at an appropriate location such as the front-side inner wall surface, the side-side inner wall surface, and the floor surface of the chamber space 2 (the installation position is not particularly limited).
Next, in the exposed surface formation step (step S4) for attaching the attached bacteria, the inner wall surface (evaluation target surface) of the spray test chamber apparatus 1 shown in FIG. A part of the surface is kept clean and exposed so as to be another evaluation target surface.

Next, in the bacteria spraying step (step S6), the bacterial suspension is sprayed from a sprayer (nebulizer) 11 installed in the center of the spray test chamber device 1 in accordance with defined conditions.
Moreover, in the collection culture process (step S8), the colliding bacteria collection means (adhesive sheet, stamp medium, Petri film, etc.) 13 installed above is collected, and the bacteria are divided and grown in an incubator to grow into colonies. In addition, an adhesive sheet for measuring surface-attached bacteria or a stamp medium is adhered to the surface to be evaluated of the inner wall surface of the chamber apparatus 1 or the attached bacteria are collected using a wiping method or the like (partial process procedure is shown in FIG. 4). Shown), the bacteria are divided and proliferated in an incubator, and the captured bacteria are grown into colonies of a size that can be observed with the naked eye. The culture is performed, for example, at 32 ° C. for about 16 hours (not limited to this).

  Next, in the step of calculating the number of surface-collision bacteria (= A) by the collision bacteria collection means 13 (step S10), the number of bacterial colonies is counted and the bacteria count A is calculated. For example, the number of colonies (colony group) corresponds to the number of bacteria A if cultured under conditions where one bacterium is cultured and cell division is performed to form one colony.

  Similarly, in the step of calculating the number of adherent bacteria (= B) on the evaluation target surface (step S12), the number of bacterial colonies in the portion exposed to the surface is counted. For example, if one bacterium is cultured and cell division is performed to form one colony, the number of colonies (colony group) corresponds to the number of bacteria B attached.

  Next, in the calculation step (step S14) of the ratio (= B / A) between the number of surface-adherent bacteria and the number of surface-impacting bacteria, the above-mentioned number of adhered bacteria (= B) is calculated as the above-mentioned number of surface-impacting bacteria (= A). The quotient (adhesion property) divided by is calculated. Next, in the evaluation step (step S16) of the ratio (= B / A) between the number of surface-attached bacteria and the number of surface-impacting bacteria, the number of surface-impacting bacteria and the number of surface-attached bacteria are evaluated as the microorganism attachment characteristics. In addition, an evaluation is made on the quotient (that is, adhesion characteristics, or coefficient of restitution (A−B) / A) obtained by dividing the number of attached bacteria (= B) by the number of surface collision bacteria (= A). For example, the resilience coefficient can be compared with a predetermined constant to determine strength, and the resilience coefficient can be classified into a plurality of classes based on the magnitude of the value.

  Finally, the microbe test environment evaluation step (step S18) is performed to evaluate the microbe test environment using the spray test chamber apparatus 1 as the test environment. In the evaluation in the microbe test environment evaluation process (step S18), for example, in the case of a spray test, the above coefficient of restitution (or adhesion characteristics) has an effect of reducing the number of bacteria floating in the spray chamber apparatus. Consider.

  In evaluating the microbial test environment by performing the above-described series of process steps S2 to S18, it is possible to perform the evaluation by the same process a plurality of times and pay attention to the change in the adhesion characteristics during this time. As described above, even when the spray test is continuously performed a plurality of times, the coefficient of restitution (adhesion characteristics) has an effect of increasing the number of bacteria floating in the spray chamber apparatus as the number of tests is repeated. Consider that. By referring to the evaluation result of the microbial test environment, in the case of the microbial spray test, the microbial test environment can be managed so that the stability of the spray test is not impaired by the above-described influence.

  In the above-described bacteria spraying step (step S6), what is sprayed is not limited to bacteria, and in addition to this, it is possible to spray fungi including spores, yeasts, molds, mold spores and the like. is there. Further, in the evaluation in the above-described microbe test environment evaluation step (step S18), separately, the number of spray particles (the number of bacteria) in the air of a unit volume measured using a particle counter or the like, or an air sampler is used. Evaluation factors of the test environment including the number of microorganisms that formed colonies among the microorganisms that were suspended in the unit volume of air collected (for example, the colony formation rate indicating how many microorganisms are activated) ).

  FIG. 4 is an explanatory diagram illustrating the attached bacteria collecting step in the attached bacteria collecting step (step S8) from the evaluation target surface. FIG. 2A shows a state of the chamber space 2 of the spray chamber apparatus 1 when the bacteria spraying step (step S6) is being performed. There are spray bacteria 3 that collide with and adhere to the evaluation target surface 5 and spray bacteria 3a that collide with the evaluation target surface 5 but bounce back. FIG. 5B shows the state of the chamber space 2 immediately after the bacteria spraying step. In FIG. 2C, the collection surface 4 which is an adhesive sheet for measuring surface-attached bacteria or a stamp medium is brought into close contact with the upper surface of the evaluation target surface 5 which is a part of the inner wall surface of the spray chamber apparatus 1. It shows a state. Furthermore, (D) of the figure peels off the collection surface 4 that is an adhesive sheet for measuring surface-attached bacteria or a stamp medium from the upper surface of the evaluation target surface 5 that is a part of the inner wall surface of the spray chamber device 1. The state where the surface-attached bacteria 3 that are part of the spray bacteria are collected on the collection surface 4 is shown.

  According to the evaluation method of the microorganism test environment according to this embodiment, the adhesion characteristics of the evaluation surface are evaluated by specifically counting the number of bacteria attached to the evaluation surface 5 which is the inner wall surface of the spray chamber apparatus 1. Therefore, there is an effect that the microbial test environment to be evaluated can be evaluated in a form that can guarantee the validity (reliability) of the test results and the test accuracy. Since the microorganism test in the environment is a known one such as the measurement of airborne microorganisms described in Patent Document 1 described above, the description thereof is omitted.

(Other embodiments)
FIG. 5 is a process diagram showing a method for evaluating a microbial test environment according to another embodiment of the present invention for each process. The evaluation method of the microorganism test environment according to the present embodiment includes an installation area in which the colliding bacteria collecting means are installed on the inner wall surface of the spray chamber apparatus 1 and an inner wall surface (an evaluation target surface) area to which the attached bacteria are attached. Corresponds to a situation where an evaluation method for making the same region (a more precise evaluation is possible) should be performed.

  In the step shown in FIG. 5, first, in the step of installing the collision bacteria collecting means (step S22), the collision bacteria collection means (for example, an adhesive sheet, stamp medium, or Petri film (microorganism culture sheet that can collect the collision bacteria) is collected. ) Etc.) with the adhesive surface or medium surface exposed. Next, the first spraying step (step S24) of bacteria (microorganisms) is performed under predetermined conditions, and the bacteria are dropped on the collision bacteria collecting means and the like. At this time, the number of floating bacteria (or density of floating bacteria) in the chamber sprayed in the first spraying process by the first air sampling is measured. For example, the measurement can be performed using a particle counter (not shown), an air sampler, or the like (a known measuring device). The particle counter has a function of counting the number of airborne bacteria in the air per unit volume in the chamber. The air sampler collects airborne bacteria by spraying a predetermined amount (volume) of air in a chamber onto an agar medium having a diameter of about 9 cm, for example. The collected bacteria are then cultured to form colonies, and the number of colonies (corresponding to the number of bacteria that become colonies) obtained from unit volume of air (including bacteria that do not become colonies) is calculated. . Then, from the ratio of the number of colonies obtained from the unit volume of air to the number of airborne bacteria contained in the air per unit volume measured by the particle counter (including bacteria that do not become colonies) Bacterial colony formation rate (colony forming ability) C1, which is the proportion of bacteria that become colonies among the airborne microbes, is calculated (S26).

  Further, in the step of forming a clean surface on the evaluation target surface (step S28), in order to remove the collision bacteria collecting means from the chamber and to attach spray bacteria to the same area after the removal, the surface of the area is previously stored. A cleaning process for removing the (evaluation target surface) (a process for removing bacteria from the evaluation target surface so as to leave nothing on the evaluation target surface) is performed (S28).

  Subsequently, a second spraying process is performed in which a second spray of bacteria is performed under predetermined conditions (step S30). At this time, the second air sampling is performed in the same manner as the first air sampling (S26). As a result, the number of floating bacteria (or density of floating bacteria) sprayed in the chamber in the second spraying process is measured, and the colony formation rate (colony forming ability) C2 of bacteria in the second spraying process is calculated (S32). ).

  Note that the order of steps S22 to S26 and steps S28 to S32 of the execution procedure described above may be interchanged.

  The collision bacteria collection means (for example, an adhesive sheet, a stamp medium, a Petri film, etc.) 13 is collected, and the bacteria are divided and grown in an incubator to grow into colonies. In addition, an adhesive sheet for measuring surface-attached bacteria, a stamp medium, a Petri film, etc. are adhered to the evaluation target surface in the same area after removing the collision bacteria collecting means, or the attached bacteria are collected using a wiping method or the like. Gather. The collected adherent bacteria are divided and proliferated in an incubator to grow into colonies. For example, the culture is performed at 32 ° C. for about 16 hours (S34).

  Next, in the step of calculating the number of surface collision bacteria (= A) by the collision bacteria collection means 13 (step S36), the number of bacterial colonies collected and cultured by the collision bacteria collection means 13 is counted. Since the number of collected bacteria corresponds to the number of colonies, the number A of surface-impacting bacteria is determined by the number of colonies.

  Similarly, in the calculation step (step S38) of the number of adherent bacteria (= B) on the exposed surface, the number of bacterial colonies collected and cultured from the evaluation target surface is counted. Since the number of collected bacteria corresponds to the number of colonies, the number B of adherent bacteria is determined by the number of colonies.

Next, correction C for the calculated number of bacteria is performed in consideration of the difference in the recovery rate of bacteria due to the difference in spraying conditions in the first spraying process and the second spraying process (S40). That is, the surface collision bacteria count A is corrected by the bacterial colony formation rate C1 (see step S26) in the first spraying process. Similarly, the number B of surface-attached bacteria is corrected by the bacterial colony formation rate C2 (see step S32) in the second spraying process.
For example, when the count number A of colonies of the number of surface-impacting bacteria is 12.3 [cfu / cm ² ] on the surface of the medium where bacteria are collected, the colony formation rate C1 of bacteria is 0.098 [CFU / PC]. Then, the correction value A ′ is 124.5 [cfu / cm ² ]. Further, when the count number B of the colonies of the number of surface-attached bacteria is 1.6 [cfu / cm ² ] on the surface of the medium collecting the bacteria, the bacterial colony formation rate C2 is 0.14 [CFU / PC]. Then, the correction value B ′ is 11.4 [cfu / cm ² ]. Here, [cfu] is the number of colonies, and [PC] is the count value of the particle counter.

  Next, the ratio (= B '/ A') between the corrected number of surface-attached bacteria and the number of surface-impacting bacteria is calculated (step S42). Furthermore, in the next evaluation process (step S44), the number of surface-attached bacteria and the number of surface-impacting bacteria are evaluated as microorganism adhesion characteristics. In addition, the ratio (B '/ A') between the number of surface-attached bacteria and the number of surface-impacting bacteria, that is, the adhesion characteristic, or the coefficient of restitution ((A'-B ') / A') is evaluated. For example, the resilience coefficient can be compared with a predetermined constant to determine strength, and the resilience coefficient can be classified into a plurality of classes based on the magnitude of the value.

  Finally, a microbe test environment evaluation process is performed to evaluate the microbe test environment using the spray test chamber apparatus 1 as a test environment (step S46). In the evaluation in the evaluation process of the microorganism test environment, for example, in the case of a spray test, it is considered that the restitution coefficient (or adhesion characteristics) has an effect of reducing the number of bacteria floating in the spray chamber apparatus.

  As described above, according to the embodiment of the present invention, the bacteria of the bacteria that collide with the evaluation target surface such as the inner wall surface of the spray chamber apparatus (the bacteria that bounce on the surface and the bacteria that do not bounce and adhere to the surface). By determining the ratio (= B / A) of the number A (the number of surface-impacting bacteria) and the number of bacteria B (the number of surface-adhering bacteria) that adheres without rebounding to the surface, and observe the adhesion ratio of these bacteria It can be determined whether or not the microbial test environmental conditions such as adhesion characteristics are fluctuating. Thereby, it is possible to know whether or not the test environment is stable, and the condition is good. The reflection ratio (= (A−B) / A) may be used as a parameter.

  In addition, although the spray test chamber apparatus is used in the Example of this invention, the spraying of microorganisms should just be performed suitably on the surface of a target object, and a chamber apparatus is not essential. Although the present invention provides a method of wall surface state management for improving the stability of the spray test, the stability management object may be a wall surface or a surface, and is not limited to the inner wall surface of the spray test chamber apparatus. Further, the surface is not limited to a flat surface, and there is no problem even if there is some unevenness as long as there is little influence on the adhesion of microorganisms by spraying. For example, if you want to know the adhesion characteristics of a wide surface of furniture, install the furniture in a chamber device, spray microorganisms (preferably spore bacteria), and use this method to easily determine the adhesion characteristics of microorganisms on a wide surface. You can investigate.

  In an embodiment of the present invention, a microorganism is used. If it is not a microorganism (preferably a spore fungus that forms a colony and is highly resistant to environmental changes) but is a non-microbial particle that does not form a colony, the entire adhesive sheet should be observed under a microscope. Particles are counted, but such an artificial counting operation is difficult. Alternatively, an expensive observation counting device is required. In addition, the collection efficiency varies even when adopting a method of collecting fine particles from the surface into a liquid and concentrating them with a centrifuge, and many facilities such as a turbidimeter, a microscope, and a liquid particle counter are required. It is. In this regard, there is an advantage that simple equipment can be used if the number of microorganism colonies that can be observed with the naked eye is counted and the number of microorganisms is calculated as in this embodiment.

  DESCRIPTION OF SYMBOLS 1 Spray test chamber apparatus, 2 chamber apparatus space, 3 spray microbe, 4 collection surface, 5 evaluation object surface, 6 surface adhesion microbe, 11 sprayer (nebulizer), 13 collision microbe collection means, S2-S18, S22- S46 Evaluation process

Claims (14)

A step of installing a colliding bacteria collecting means for collecting microorganisms colliding with the collecting surface in the first region of the evaluation target surface;
Cleaning a surface of a second region different from the first region of the evaluation target surface;
A spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Culturing microorganisms collected by the collision bacteria collecting means and microorganisms collected from the surface of the second region, respectively, to form first and second colony groups;
Calculating the number of microorganisms collected by the collision bacteria collection means from the first colony group as the number of surface collision bacteria,
Calculating the number of microorganisms attached to the surface of the second region from the second colony group as the number of surface-attached bacteria;
Grasping the microbial adhesion characteristics of the surface to be evaluated based on the number of surface-attached bacteria and the number of surface-impacting bacteria;
Evaluating a microbial test environment having the evaluation target surface based on the grasped microbial adhesion characteristics;
A method for evaluating a microbial test environment, comprising:   The microorganism testing environment evaluation method according to claim 1, wherein bacteria are used as the microorganisms to be sprayed in the spraying step, or fungi including spore, yeast, mold, and mold spores are used.   It is characterized by using a pressure-sensitive adhesive sheet, a stamp medium, a Petri film, or a sampling method for measuring surface-attached bacteria for collecting microorganisms from at least the collision bacteria collecting means and the surface of the second region. The method for evaluating a microbial test environment according to claim 1 or 2. A step of installing a colliding bacteria collecting means for collecting microorganisms that collided with the collecting surface in a partial region of the evaluation target surface;
A first spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Performing a first air sampling for estimating a first colony formation rate of the microorganisms sprayed in the first spraying step;
Removing the collision bacteria collecting means from the evaluation target surface and forming a clean surface on the surface of the partial region after the removal;
A second spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Performing a second air sampling for estimating a second colony formation rate of the microorganisms sprayed in the second spraying step;
Culturing the microorganisms collected by the collision bacteria collecting means and the microorganisms collected from the clean surface, respectively, to form first and second colonies, and
Calculating the number of microorganisms collected by the collision bacteria collection means from the first colony group as the number of surface collision bacteria,
Calculating the number of microorganisms attached to the clean surface from the second colony group as the number of surface-attached bacteria;
The surface collision bacteria count and the surface adhesion bacteria count are corrected by the first and second colony formation rates, respectively, and the corrected surface collision bacteria count and the corrected surface adhesion bacteria count or these two Grasping the microbial adhesion characteristics of the evaluation target surface based on the ratio of the number of bacteria,
Evaluating a microbial test environment having the evaluation target surface based on the grasped microbial adhesion characteristics;
A method for evaluating a microbial test environment, comprising:   5. The microorganism test environment according to claim 4, wherein bacteria are used as the microorganisms to be sprayed in the first and second spraying steps, or fungi including spores, yeasts, molds, and mold spores are used. Evaluation method.   A pressure-sensitive adhesive sheet for measuring surface-attached bacteria, a stamp medium, a Petri film, or a sampling method is used for collecting microorganisms from at least one of the collision bacteria collection means and the clean surface of the partial area. The method for evaluating a microbial test environment according to claim 4 or 5.   The first and second air samplings include a particle counter that counts the number of microorganisms floating in a unit volume of air, and an air sampler that sprays a predetermined amount of the air onto the medium to form microbial colonies in the medium. The method for evaluating a microbial test environment according to any one of claims 4 to 6, wherein the microbial test environment is evaluated. A step of installing a colliding bacteria collecting means for collecting microorganisms colliding with the collecting surface in the first region of the evaluation target surface;
Cleaning a surface of a second region different from the first region of the evaluation target surface;
A spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Culturing microorganisms collected by the collision bacteria collecting means and microorganisms collected from the surface of the second region, respectively, to form first and second colony groups;
Calculating the number of microorganisms collected by the collision bacteria collection means from the first colony group as the number of surface collision bacteria,
Calculating the number of microorganisms attached to the surface of the second region from the second colony group as the number of surface-attached bacteria;
Grasping the microbial adhesion characteristics of the surface to be evaluated based on the number of surface-attached bacteria and the number of surface-impacting bacteria;
A method for grasping microbial adhesion characteristics, comprising   9. The method for grasping microorganism adhesion characteristics according to claim 8, wherein bacteria are used as microorganisms to be sprayed in the spraying step, or fungi containing spores, yeasts, molds, and mold spores are used.   It is characterized by using a pressure-sensitive adhesive sheet, a stamp medium, a Petri film, or a sampling method for measuring surface-attached bacteria for collecting microorganisms from at least the collision bacteria collecting means and the surface of the second region. The method for evaluating a microbial test environment according to claim 1 or 2. A step of installing a colliding bacteria collecting means for collecting microorganisms that collided with the collecting surface in a partial region of the evaluation target surface;
A first spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Performing a first air sampling for estimating a first colony formation rate of the microorganisms sprayed in the first spraying step;
Removing the collision bacteria collecting means from the evaluation target surface and forming a clean surface on the surface of the partial region after the removal;
A second spraying step of spraying microorganisms on the evaluation target surface under defined conditions;
Performing a second air sampling for estimating a second colony formation rate of the microorganisms sprayed in the second spraying step;
Culturing the microorganisms collected by the collision bacteria collecting means and the microorganisms collected from the clean surface, respectively, to form first and second colonies, and
Calculating the number of microorganisms collected by the collision bacteria collection means from the first colony group as the number of surface collision bacteria,
Calculating the number of microorganisms attached to the clean surface from the second colony group as the number of surface-attached bacteria;
The surface collision bacteria count and the surface adhesion bacteria count are corrected by the first and second colony formation rates, respectively, and the corrected surface collision bacteria count and the corrected surface adhesion bacteria count or these two Grasping the microbial adhesion characteristics of the evaluation target surface based on the ratio of the number of bacteria,
A method for grasping microbial adhesion characteristics, comprising   12. The microorganism adhesion property according to claim 11, wherein bacteria are used as the microorganisms to be sprayed in the first and second spraying steps, or fungi including spores, yeasts, molds, and mold spores are used. How to grasp.   It is characterized by using a pressure-sensitive adhesive sheet, a stamp medium, a Petri film, or a sampling method for measuring surface adhering bacteria for collecting microorganisms from at least the collision bacteria collecting means and the clean surface of the partial area. The method for grasping the microorganism adhesion characteristics according to claim 11 or 12.   The first and second air samplings include a particle counter that counts the number of microorganisms floating in a unit volume of air, and an air sampler that sprays a predetermined amount of the air onto the medium to form microbial colonies in the medium. The method for grasping microorganism adhesion characteristics according to any one of claims 11 to 13, wherein the method is used.

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