JP2007159520A - Floated bacteria-collecting container and method for collecting and counting bacteria floated in air with the container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a floated bacteria-collecting container on whose inner surface a water-soluble organic solvent having a viscosity and a non-volatile property in extents not scattered by the blowing of air is coated so that the bacteria floated in air can quickly and economically be collected/counted in a relatively short time, and to provide a method for collecting/counting the bacteria with the floated bacteria-collecting container. <P>SOLUTION: This collection container 2 for blowing air containing bacteria therein to collect the bacteria is characterized by coating the inner surface of the container 4 having an opening 6 also used as an air-blowing port with a water-soluble organic solvent having a viscosity and a non-volatile property in extents not scattered by the blowing of air to form a bacterium-collecting layer 8. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a collection container for airborne bacteria and a method for collecting and counting the floating bacteria in the Miyako using the container. In the present specification, “bacteria” includes fungi and the like in addition to bacteria.

  In food factories and the like, it is necessary to keep air clean for product quality control, and it is important to know the number of bacteria in clean air.

    As a method of collecting and counting such bacteria, conventionally, a large amount of air containing airborne bacteria is blown onto the collection plate using an air sampler, and the bacteria are inertial in the medium formed on the upper surface of the collection plate. It was made to collide and it culture | cultivated as it was and the colony of a microbe was counted (patent document 1).

    In addition, a method is known in which aerial microorganisms are captured using a filter, cell membranes of microorganisms are dissolved, ATP (adenosine-3-phosphate) in the cells is extracted, and quantified using a bioluminescence phenomenon ( Patent Document 2).

    On the other hand, as a method for quickly measuring bacteria already collected on a filter, a method for fluorescent staining of bacteria is known (Patent Document 3).

Furthermore, as a floating bacteria collection device, a portable device is known in which a petri dish in which an agar medium is formed is installed inside the outside air suction port, and outside air is sucked and blown onto the agar medium ( Patent Document 4).
JP 2000-300246 JP-A-3-112495 JP-A-10-323197 JP 2002-153259

    The method of Patent Document 1 can be performed at the lowest cost among the above-mentioned three methods, but requires about 48 hours for the cultivation of bacteria. Therefore, if it is found that the air is contaminated 48 hours after collecting the bacteria, the product produced during that time is inferior in quality and has a large loss.

    On the other hand, the method of Patent Document 2 (ATP bioluminescence method) takes several hours to count and is relatively fast, but the measuring device is expensive.

    The method of Patent Document 3 is suitable for counting bacteria collected on the filter within a reasonable time and cost range, but how to collect airborne bacteria on the filter. Is a problem. When collecting and counting airborne bacteria in the indoor environment, the number of bacteria in such air is small (less than 1 per liter), so it is possible to measure the real value of reliable bacteria without going through the culture process. Needs to collect bacteria from a large volume of air of 100L to 1,000L or more. However, there has never been a general means to connect from collection / recovery to counting.

    Therefore, the present applicant conducted extensive research to find a means for collecting and recovering the floating bacteria sucked by the air sampler. In paragraph 0004 of Patent Document 4 above, as a method of using the collection device, a method of washing agar in which bacteria are planted and putting this liquid into an ATP bioluminescence device is introduced.

    Therefore, the applicant made two sets of test bodies in which 100 L of air was sprayed on an agar medium formed in a petri dish with an air sampler, and the first test body was washed once with 5 mL of water, and the second test. The body was washed once with 5 mL of water and cultured as it was. If all the bacteria can be recovered by washing, the bacteria will not be counted even if they are cultured afterwards, but for bacteria, 16-19 colonies in the first specimen and the second test. 4-5 colonies were observed in the body. Regarding fungi, 16 to 20 colonies were observed in the first specimen, and 5 to 9 colonies were observed in the second specimen. From these test results, it was found that when bacteria were collected on an agar medium, it was difficult to transfer all of the collected bacteria to a filter for fluorescent staining.

    In terms of ease of transfer, it may be possible to put a liquid such as water in the petri dish and spray the air, but if a large amount of air is sprayed, the collected liquid will evaporate or splashes containing bacteria will be generated. Bacteria collection efficiency deteriorates due to scattering. In addition, it is possible to collect bacteria by solidifying with gelatin instead of agar medium, and then recovering the bacteria by heating and liquefying. It was found that the liquid could not pass through the membrane filter, and it was difficult to count the bacteria.

    Accordingly, the present invention provides a container for a water-soluble organic solvent having a viscosity and non-volatility that does not scatter when air is blown so that airborne bacteria can be collected and counted economically in a relatively short time. And a method for collecting and counting bacteria using the collection container.

A first means is a collection container for capturing bacteria by blowing air containing bacteria therein.
The trapping layer 8 is formed by applying a water-soluble organic solvent having a viscosity and non-volatility that does not scatter when air is blown to the inner surface of the container body 4 having an opening that also serves as an air blowing port. Forming.

  This means proposes a collection container which is an instrument for collecting and collecting airborne bacteria. As the material for capturing bacteria, if it is an insoluble solid such as the above-mentioned agar, it will be difficult to wash away bacteria from the surface and collect it. On the other hand, if it is simply a liquid filled in the container, There is a possibility of spilling outside. Therefore, in the present invention, a collection container with high recovery efficiency and easy handling is provided by first applying a water-soluble organic solvent to the inner surface of the container body.

  The “container body” is preferably a container having a wide-mouthed opening such as a petri dish or a beaker.

  The “bacterial catching layer” may be formed on the entire inner surface of the container body, but may be formed only on the inner surface of the bottom of the container body in order to prevent unwashing when collecting bacteria. Moreover, a microbe capture layer is formed by apply | coating an organic solvent to the container body inner surface. “Coating” means that the organic solvent is applied to the inner surface of the container body in a layered manner, and is not limited to the case where the solvent is actually applied.

  The “organic solvent” is a water-soluble solvent so that the collected bacteria can be washed away with washing water. It is desirable that this organic solvent easily pass through a filter used for fluorescent staining or the like at room temperature. In order to ensure filter permeability, an organic solvent that has been permeated through the filter in advance may be used. It is also necessary to avoid organic solvents that inhibit the growth of airborne bacteria or have a bactericidal action. Furthermore, it is necessary to use a viscous liquid so that even if a large amount of air is blown, it does not scatter. Examples of such solvents include glycerin, lactic acid, polyethylene glycol and the like. In particular, since glycerin is almost neutral, it is suitable as a material for collecting bacteria. Furthermore, the organic solvent needs to be non-volatile.

The second means has the first means, and
The entire upper surface of the bottom wall 5 of the container body 4 is a horizontal flat surface,
The microbe-capturing layer 8 is a mixture of the non-volatile water-soluble organic solvent mixed with a volatile organic solvent having little persistence, and is put into the container body 4 and vaporizes the volatile organic solvent. The uniform thickness is formed over the entire top surface of the bottom wall 5.

  In this means, after the liquid mixture of the non-volatile organic solvent and the volatile organic solvent is put into the container body, the latter is evaporated, and the upper surface of the flat bottom wall of the container body is caused by the action of gravity. A thin and uniform coating film is uniformly formed. When the volatile solvent is vaporized, the container body is supported so that the upper surface of the bottom wall is horizontal. Examples of the volatile organic solvent having little persistence include ethanol and methanol. For example, if about 1 mL of a mixture of glycerin and ethanol mixed at a ratio (volume ratio) of 1: 9 is applied to the bottom wall of the petri dish, a good microbe-capturing layer can be obtained. If the same amount of pure glycerin solution is applied, it is difficult to wash away at the time of washing. Further, when it is attempted to apply 0.1 mL of pure glycerin liquid, it is difficult to apply the liquid thinly on the target surface, and when unpainted, the quality (collecting ability) of the collection container varies. This means can avoid such inconvenience.

The third means has the first means or the second means,
And the said container body 4 is formed with a synthetic resin material, Furthermore,
The said bactericidal layer 8 is formed by apply | coating what added a small amount of nonionic surfactant to the said non-volatile water-soluble organic solvent to the container body 4 inner surface.

  In this means, a surfactant is used to make the organic medium easily compatible with the synthetic resin material. As the surfactant, a nonionic surfactant that is less toxic to microorganisms is used. Examples of suitable surfactants include polysorbate 20 and polysorbate 80.

The fourth means is a method for collecting airborne bacteria.
A process of sucking air containing airborne bacteria and blowing it into the collection container 2 which is one of the first to third means, attaching the bacteria to the organic solvent, and capturing it;
The method comprises a step of putting sterilized washing water into the collection container 2 and draining and collecting the captured bacteria together with the organic solvent.

  By comprising in this way, the microbe collected in the collection container is collect | recovered immediately, without culturing, and a count operation can be started. A conventionally known air sampler can be used for air suction. According to this configuration, it is not necessary to culture compared to the conventional method in which airborne bacteria collide with air and collide with a filter to capture and culture, so that it does not take time to start counting. In this specification, “sterilized cleaning water” (or sterilized water) is sterilized against physiological water containing about 0.85% sodium chloride in addition to sterilized water. It shall contain sterile physiological saline that has been treated. Also, “running out” means taking out widely in a fluid state, and includes sucking up with a dropper or the like.

  After capturing and collecting the airborne bacteria by this means, counting may be performed by a conventionally known appropriate method. A preferable method in terms of cost is the above-described fluorescent staining method. For example, a method for quantifying the above-mentioned adenosine triphosphate (APT), filendotoxin, peptidoglycan, etc. derived from bacteria is known. In addition to the usual counting operation, DNA can be extracted from the collected airborne bacteria and subjected to DNA-DNA hybridization to detect and count specific species of bacteria.

The fifth means is a method for counting airborne bacteria,
A process of sucking air containing airborne bacteria and blowing it into the collection container 2 which is one of the first to third means, attaching the bacteria to the organic solvent, and capturing it;
A process of putting sterilized washing water in the collection container 2 and draining and collecting the captured bacteria together with the organic solvent,
The process of permeating the washing water containing the collected bacteria through the filter,
A process of counting the bacteria that have migrated to the filter surface by fluorescent staining.

  In this means, combining the above-mentioned bacteria collection method with a conventionally known fluorescent staining method, the work can be completed in about 30 minutes from collection to counting, and a measuring device is installed in a factory without a counting facility. Bring it in and take measurements on site.

The method according to the sixth means is a method for counting airborne bacteria,
A process of sucking air containing airborne bacteria and blowing it into the collection container 2 according to any one of claims 1 to 3, attaching bacteria to an organic solvent, and capturing the organic solvent;
Injecting sterilized water containing nutrients into the collection container 2 that has captured bacteria,
After this addition, the process of putting the collection container 2 into the thermostatic chamber 18 until the bacteria wake up from the dormant state;
A process of collecting the liquid in the collection container 2 taken out from the thermostatic bath 18 and draining and collecting the collected bacteria together with the organic solvent;
The process of allowing the collected liquid containing bacteria to permeate through the filter,
A process of counting the bacteria that have migrated to the filter surface by fluorescent staining.

  In the above-described fluorescent staining method, it is difficult to detect a dormant microbial cell, that is, a spore bacterium or mold. Therefore, in the present invention, before the collection process, the collection container is placed in the thermostat 18 and is awakened from the sleep state and then collected and counted. Examples of “nutrients” include glucose and sucrose.

The collection container according to the first means has the following effects.
O The trapping layer 8 is formed by coating an organic solvent on the inner surface of the container body 4, so that the solvent does not flow out or scatter by blowing air, and is easy to handle and carry, and is easy to produce.
○ Since the organic solvent is water-soluble, it is easier to remove from the collection container and has better filter permeability than the case where agar or the like that is insoluble in water is used as the collection material, and the bacteria recovery efficiency is good.
○ Because the trapping part is layered, it can be formed with a small amount of organic solvent, and the amount of water required to dissolve it can be small, so most of the trapped bacteria can be taken out from the collection container.

The method according to the second means has the following effects.
-Since the latter is evaporated after applying the solution which melt | dissolved the non-volatile organic solvent in the volatile organic solvent to the target surface, a coating film can be formed uniformly without remaining on the target surface.
○ Since the coating film can be evenly thinned, it is easy to dissolve all the coating film and the recovery efficiency is increased, and the organic solvent can be saved, and further, the washing water for dissolving this can be reduced.

The method according to the third means has the following effects.
○ Because non-ionic surfactants are used to make organic media easy to adapt to synthetic resin materials, cheaper synthetic resin containers can be used than glass containers, which is economical.
○ Since plastic containers can be disposable, work efficiency is good.
○ Since the organic solvent can be applied thinly on the inner surface of the synthetic resin container, all of the organic solvent can be washed away, and the bacteria recovery rate is good.

  According to the method for collecting floating bacteria according to the fourth means, it is not necessary to cultivate the airborne bacteria as compared with the conventional method in which the suspended bacteria collide with air with a filter to capture and culture. , It doesn't take time to start counting.

The bacteria counting method according to the fifth means has the following effects.
○ Because the fluorescent staining method is used, it is less expensive than the ATP method, and results can be obtained quickly.
○ The equipment to be used is a collection container, air sampler, and fluorescence detector. These are large enough to be carried around. You can bring in a fluorescence tester and provide a service to measure on the spot.

  According to the method according to the sixth means, since the collection container capturing the bacteria is placed in the thermostatic layer, the dormant cells can be germinated and counted.

  FIG. 1 shows a collection container 2 according to the present invention.

  The collection container 2 is formed of a container body 4 and a bacteria collection layer 8.

  The container body 4 is a petri dish type in which the peripheral wall stands up from the periphery of the flat bottom wall 5 and has an opening 6 that is an air blowing port. The container body is a plastic disposable container.

  The capturing layer 8 is formed on the upper surface of the bottom wall of the container body 4. This microbe-capturing layer is formed by applying a water-soluble organic solvent having viscosity and non-volatility to the upper surface of the bottom wall.

  As a preferred example, 0.1% polysorbate 80, 9.9% glycerin, and 90% ethanol are mixed in a volume ratio to prepare a mixed solution. This ethanol mixed solution is preferably filtered through a membrane filter in advance. As shown in FIG. 2, 1 mL of the mixed solution is taken into a pipetter 10 and poured into a container body 4 placed on a horizontal mounting surface (not shown), and ethanol is evaporated to evaporate the bottom of the container body. The trapping layer 8 having a uniform thickness is formed over the entire upper surface of the wall 5.

  Next, a general method for counting airborne bacteria will be described with reference to the drawing (see FIG. 5).

  First, as shown in FIG. 3, the collection container 2 of FIG. 1 is set on the air sampler 12. Then, air is sucked in to collect bacteria in the collection layer 8 of the collection container 2.

  Next, as shown in FIG. 4, the collection container 2 is taken out, sterile physiological saline or the like is injected into the container body 4, and the organic solvent together with the captured bacteria is dissolved in the saline. When this dissolved water is filtered through a membrane filter 14 fixed with a holder (not shown), the solvent passes through the filter, but the bacteria remain on the filter. The membrane filter may be set in the fluorescence detector 16 and counted.

表１は本実施形態の方法と通常の培養法とで、同一場所から採取した空気内の細菌を計数した結果を示している。第１回、第２回の結果ともに、通常の培養法よりも本発明の方法の方が検出した菌数が大きい。このことから、本発明では従来の方法で検出されていない菌まで計数しているものと考えられる。

Table 1 shows the results of counting bacteria in the air collected from the same place by the method of the present embodiment and the normal culture method. In both the first and second results, the number of bacteria detected by the method of the present invention is larger than that of the normal culture method. From this, in this invention, it is thought that the bacteria which are not detected with the conventional method are counted.

  Next, when counting the spore-like bacteria or mold, the bacteria are collected in the collection container 2 as shown in FIG. 3 and then sterilized in the collection container 2 containing substances such as glucose that are nourishing for the bacteria. Add water. Then, as shown in FIG. 6, the collection vessel in which the bacteria are collected is put in the thermostatic bath 18 and kept warm for several hours until the germination state is reached (see FIG. 5). Then, the collection container 2 is taken out from the thermostat, the liquid in the collection container is poured out, the bacteria contained in the liquid are collected on the membrane filter, and placed in the fluorescence detector. In this embodiment, since the temperature is maintained until germs germinate, it takes more time than in the first embodiment, but a measurement result can be obtained more quickly than in a normal method of culturing until the germs form colonies.

It is sectional drawing of the collection container which concerns on this invention. It is a figure which shows the manufacturing process of the container of FIG. It is a figure which shows the 1st process of the counting method of the floating bacteria based on this invention. It is a figure which shows the 2nd process of the method. 3 is a flowchart of a method according to the present invention. It is a figure which shows the process of the modification of the method.

Explanation of symbols

2 ... Collection container 4 ... Container body 6 ... Opening part 8 ... Bacteria collection layer
10 ... Pipetta 12 ... Air sampler 14 ... Membrane filter
16 ... Fluorescence detector 18 ... Constant bath

Claims (6)

In a collection container for capturing bacteria by blowing air containing bacteria inside
The trapping layer 8 is formed by applying a water-soluble organic solvent having a viscosity and non-volatility that does not scatter when air is blown to the inner surface of the container body 4 having an opening that also serves as an air blowing port. A floating bacteria collection container characterized by being formed. The entire upper surface of the bottom wall 5 of the container body 4 is a horizontal flat surface,
The microbe-capturing layer 8 is a mixture of the non-volatile water-soluble organic solvent mixed with a volatile organic solvent having little persistence, and is put into the container body 4 and vaporizes the volatile organic solvent. The suspended bacteria collection container according to claim 1, wherein the container is formed to have a uniform thickness over the entire upper surface of the bottom wall 5. The container body 4 is formed of a synthetic resin material,
The said microbe-capturing layer 8 was formed by apply | coating what added a small amount of nonionic surfactant to the said non-volatile water-soluble organic solvent to the container body 4 inner surface, It is characterized by the above-mentioned. Or the floating bacteria collection container of Claim 2. A process of sucking air containing airborne bacteria and blowing it into the collection container 2 according to any one of claims 1 to 3, attaching bacteria to an organic solvent, and capturing the organic solvent;
A method for collecting airborne bacteria, comprising a step of putting sterilized washing water into the collection container 2 and draining and collecting the collected bacteria together with an organic solvent. A process of sucking air containing airborne bacteria and blowing it into the collection container 2 according to any one of claims 1 to 3, attaching bacteria to an organic solvent, and capturing the organic solvent;
A process of putting sterilized washing water in the collection container 2 and draining and collecting the captured bacteria together with the organic solvent,
The process of permeating the washing water containing the collected bacteria through the filter,
And a method of counting airborne bacteria, characterized by comprising a step of fluorescently staining and counting the bacteria that have migrated to the filter surface. A process of sucking air containing airborne bacteria and blowing it into the collection container 2 according to any one of claims 1 to 3, attaching bacteria to an organic solvent, and capturing the organic solvent;
A process of adding sterilized water containing nutrients to the collection container 2 that has captured bacteria;
After this addition, the process of putting the collection container 2 into the thermostatic chamber 18 until the bacteria wake up from the dormant state;
A process of collecting the liquid in the collection container 2 taken out from the thermostatic bath 18 and draining and collecting the collected bacteria together with the organic solvent;
The process of allowing the collected liquid containing bacteria to permeate through the filter,
And a method of counting airborne bacteria, characterized by comprising a step of fluorescently staining and counting the bacteria that have migrated to the filter surface.

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