JP2012183066A - Method for quickly determining microorganism and capturing system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly determining microorganisms, capable of efficiently capturing airborne microorganisms, capturing not only general bacteria but also thermoduric microorganisms such as thermoduric acidophilic bacteria and thermoduric fungi and quickly determining the microorganisms by using a quick determination method, and to provide a microorganism capturing system suitable for the method.SOLUTION: The method for the quick determination of microorganisms includes the capture of microorganisms floating in air by the top of a heat-resistant membrane filter by sucking the air through the membrane filter, the culture of the microorganisms captured by the top of on the membrane filter on a culture medium and the detection of ATP in the cultured microorganism by a bioluminescence reaction; wherein suction is performed by an ejector 4 using compressed air. The microorganism capturing system 1 has the ejector 4 to suck surrounding air from an inducing flow channel using compressed air supplied from a compressor and a holder 13 connected to the inducing flow channel of the ejector 4 and supporting the membrane filter 12.

Description

  The present invention relates to a method for rapidly measuring microorganisms floating in the air and a microorganism capturing apparatus suitable for carrying out the method.

  2. Description of the Related Art Conventionally, as a device for capturing floating microorganisms, a collision-type floating microorganism capturing device is known that collides an air flow with an agar medium and captures the floating microorganisms in the medium (for example, Patent Document 1).

  In addition, as a method for rapidly measuring microorganisms in liquid, the liquid containing microorganisms is filtered through a filtration membrane, captured on the filtration membrane, incubated for a short time, and then sprayed with a liquid luminescent reagent to contact the microorganism and its vicinity. A rapid measurement method is known in which ATP (adenosine triphosphate) of microorganisms is optically detected by a bioluminescence method (for example, Patent Document 2).

JP 2001-149064 A JP-A-11-137293

  However, the conventional collision type floating microorganism capture device is considered that most of the suspended microorganisms in the air flow are not captured by being bounced on the medium and discharged together with the exhaust gas, and the capture efficiency is low. It took a long time and it took at least about 5 days to make a decision. In addition, conventional collision-type floating microorganism capture devices can measure general bacteria and fungi, but cannot measure heat-resistant microorganisms such as heat-resistant acidophilic bacteria and heat-resistant molds, which are particularly problematic in food production areas. Heat-resistant acidophilic bacteria and heat-resistant molds need to give a so-called heat shock (for example, 70 ° C., 10 minutes) in order to enable measurement. When the agar medium is heated at such a high temperature, the agar medium is Because it melts.

  In the conventional rapid measurement method, microorganisms in the liquid are targeted, and suspended microorganisms in the air are not targeted.

  Therefore, the present invention can capture airborne microorganisms in the air efficiently and can capture not only general bacteria but also heat-resistant microorganisms such as heat-resistant acidophilic bacteria and heat-resistant molds using a rapid measurement method. And it aims at providing the microorganisms rapid measurement method which can detect rapidly, and the microorganisms capture apparatus suitable for implementation of this method.

  In order to achieve the above object, the method for rapidly measuring microorganisms according to the present invention captures microorganisms floating in the air by sucking the air through a heat resistant membrane filter, and the membrane filter. After culturing the microorganisms captured on the filter on the medium, ATP in the cultured microorganisms is detected by a bioluminescence reaction, and air suction is performed by an ejector using compressed air.

  In the rapid measurement method, heat shock may be applied to the heat-resistant microorganisms captured on the membrane filter by heat-treating the membrane filter on which the microorganisms are captured before the culture.

  The microorganism capture device according to the present invention is a microorganism capture device that captures microorganisms floating in the air on the membrane filter by sucking air through a heat-resistant membrane filter, from a compressor. It has an ejector that sucks ambient air from the trigger flow path using compressed air, and a holder that is connected to the trigger flow path of the ejector and supports the membrane filter.

  Preferably, the holder has a cup-shaped portion, and a membrane filter is supported on the bottom of the cup-shaped portion.

  Furthermore, the microorganism capturing apparatus further includes a chamber connected to the trigger flow path and containing the membrane filter, the chamber introducing a trigger flow path side connection port connected to the trigger flow path and external air. It is preferable to include a connection port portion for introducing outside air, and the holder that supports the membrane filter between the trigger channel side connection port portion and the outside air introduction port portion.

  According to the method for rapidly measuring microorganisms according to the present invention, by placing a membrane filter in a forced intake channel and capturing floating microorganisms, it is possible to efficiently capture floating microorganisms and reduce the culture time. The rapid measurement method based on the bioluminescence reaction of ATP can greatly reduce the total detection time, and can also detect specific microorganisms such as heat-resistant acidophilic bacteria and heat-resistant molds by heat-treating the membrane filter.

  In addition, if air is sucked by an ejector that uses compressed air, the compressed air is usually used in food factories and the like, and suspended microorganisms can be captured using the compressed air, thereby reducing costs.

It is a side view showing a 1st embodiment of a microorganisms capture device concerning the present invention in a partial section. It is a side view which shows 2nd Embodiment of the microorganisms trapping apparatus which concerns on this invention in a partial cross section.

  The best mode for carrying out the present invention will be described below with reference to FIGS. FIG. 1 shows a first embodiment of a microorganism capturing apparatus.

  The microorganism capture device 1 of the first embodiment includes a flow rate control regulator 3 to which an air tube 2 that supplies compressed air from a compressor (not shown) is connected, an ejector 4 that is connected to the regulator 3, and an ejector 4 The vacuum filter 7, the valve 8, the flow rate indicator 9, the flow rate sensor 10, the holder adapter 11, and the holder adapter 11 connected to the aseptic filter 5 connected to the discharge side of the gas outlet, and the inducement flow path 6 of the ejector 4. A holder 13 that is freely supported and supports the membrane filter 12 is provided. The ejector 4 includes a diffuser (not shown) inside, and when the diffuser ejects air at a high speed by introducing compressed air, the air is induced from the trigger channel 6 and discharged through the sterile filter 5. .

  The holder 13 includes a pair of clamping pieces 13a and 13b that can clamp the membrane filter 12 by screw coupling. The sandwiching pieces 13 a and 13 b have an internal channel that communicates with the trigger channel 6. The clamping piece 13a has a nozzle portion 13a1 that can be inserted into the adapter holder 11, and the clamping piece 13b has a cup-shaped portion 13b1. The inducement flow path 6 of the ejector 4 opens at the bottom of the cup-shaped portion 13b1, and the membrane filter 12 is supported at the bottom of the cup-shaped portion near the opening. Since the membrane filter 12 is hermetically sealed by the holder 13, the inducing flow path 6 of the ejector 4 is configured to intake air only through the membrane filter 12.

  The membrane filter 12 has heat resistance that can withstand heat treatment when heat shock is applied to heat resistant microorganisms such as heat resistant acidophilic bacteria and heat resistant mold. As a material of such a membrane filter 12, a cellulose mixed ester can be mentioned as a suitable example. The membrane filter 12 has a narrow aperture of generally 0.1 to 10 μm and can be appropriately selected according to the size of the microorganism to be captured. As this type of membrane filter 12, for example, one sold by Advantech Toyo Co., Ltd. can be used.

  It has been experimentally confirmed that the suction amount (liter) and the suction time are in a proportional relationship when sucking at a constant flow rate under the control of the regulator 3, and to obtain the desired suction amount, measure the suction time. It ’s fine. However, since the membrane filter 12 is used in a dry state, the suction time is adjusted so that the captured microorganisms are not killed by being exposed to the dry state for a long time. Experimentally, it has been confirmed that microorganisms on the membrane filter 12 are alive in a dry suction of about 15 minutes. The flow rate can be set as appropriate.

  By connecting the aseptic filter 5 to the exhaust part from the ejector 4, it is possible to prevent the microorganisms floating in the compressed air from the compressor (not shown) from being discharged.

  In the microorganism capturing apparatus 1 having the above-described configuration, when compressed air is sent from a compressor (not shown) through the air tube 2, the ejector 4 generates a negative pressure in the inducing channel 6 and sucks air through the membrane filter 12. As a result, microorganisms are captured by the membrane filter 12, so that suspended microorganisms can be captured more efficiently than in the conventional collision method.

  Further, since the holder 13 has a cup-shaped portion, even if microorganisms bounced off by the membrane filter 12 exist, they collide with the inner wall of the cup-shaped portion and are again sucked by the membrane filter 12, so that there is little capture leakage. Become.

  The microorganism capturing apparatus according to the present invention collects airborne microorganisms in the air using compressed air from a compressor. In food factories and pharmaceutical factories where rapid measurement of airborne microorganisms is particularly necessary, compressors are usually installed, and compressed air is used to drive various devices. Therefore, since the microorganism capturing apparatus 1 of the present invention can collect floating microorganisms using equipment already installed in this kind of factory or the like, the cost of the microorganism capturing apparatus itself can be reduced.

  Next, a second embodiment of the microorganism capturing device will be described with reference to FIG. Components similar to those in the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  The microorganism capturing device 20 of the second embodiment includes a chamber 21 connected to the trigger channel 6. The chamber 21 is formed by connecting two divided bodies 21a and 21b by screw connection, and supports the membrane filter 12 between the divided bodies 21a and 21b. That is, a holder is formed and the membrane filter 12 is disposed inside. The one divided body 21a that can be accommodated in the inner wall is provided with an inducement flow path side connection port portion 22 connected to the inducement flow path 6, and the other divided body 21b is introduced with outside air for introducing external air. A connection port portion 23 is provided.

  The microorganism capturing apparatus 20 of the second embodiment is arranged outside the measurement zone G, and connects the hose 24 to the port drawn from the measurement zone to the outside air introduction connection port 23, while connecting the regulator 3. The compressed air supply hose 2 is connected to.

  When compressed air is introduced into the hose 2, the air in the measurement area G sucked through the hose 24 is filtered by the membrane filter 12 in the chamber 21 by the action of the ejector 4, and passes through the trigger channel 6. A diffuser (not shown) in the ejector 4 is discharged through a sterile filter 5.

  According to the microorganism capturing apparatus 20 of the second embodiment, when measuring floating microorganisms in an area where there are almost no microorganisms such as a clean room, the microorganism capturing apparatus 20 can be measured without bringing it into the area. There is no risk of contaminating the area, and the number of microorganisms in the area can be measured purely.

  Next, an embodiment of a rapid measurement method for microorganisms according to the present invention will be described.

  The membrane filter 12 that captures the suspended microorganisms by the microorganism capturing device of the first embodiment or the second embodiment is removed from the holder, and the standard agar medium (PCA), antibiotic-added glucose / peptone agar medium (GPA), or Place on a medium such as antibiotic-added potato dextrose agar medium (PDA) and incubate for a predetermined time. For example, when measuring the number of viable bacteria, culture is performed at 36 ° C. for 20 hours using PCA, and when measuring the number of fungi, culture is performed at 25 ° C. for 44 hours using GPA.

  When measuring heat-resistant acidophilic bacteria and heat-resistant molds, heat treatment (for example, 70 ° C., 10 minutes) is performed on the membrane filter 12 before culturing, so that heat-resistant acidophilic bacteria and heat-resistant molds are obtained. Apply a so-called heat shock to activate. Although the heat processing conditions for giving a heat shock are suitably set according to the kind of microorganisms used as object, generally it is 70-75 degreeC and 5 to 10 minutes.

  After carrying out the prescribed culture, a luciferin-luciferase luminescence reagent is added, and ATP (adenosine triphosphate) in the living cells of the microorganism is caused to emit light by a bioluminescence (bioluminescence) reaction, which is applied to a bioluminescence measuring device, and the microorganism Count colonies. As the bioluminescence measuring apparatus, a known apparatus can be used, and for example, a bioluminescence image analysis system commercially available from Japan Millipore Corporation can be used.

  In the above embodiment, suction by an ejector has been exemplified. However, in the method for rapidly measuring microorganisms according to the present invention, suction using a vacuum pump or other suction means is also possible.

  According to the measurement method as described above, since microorganisms floating in the air can be efficiently captured, it is possible to significantly reduce the number of measurement days by combining bioluminescence measurement with a bioluminescence reaction. . According to experiments, for example, it has been confirmed that determination can be made in about 24 hours for general bacteria and about 48 hours for fungi. In addition, it is possible to measure heat-resistant acidophilic bacteria and heat-resistant molds in the air that have been difficult to measure in the past.

Test Example A test was conducted on an example of the method of the present invention and a conventional comparative example. The test conditions are as follows. Sampling was performed in a room that was not microbially controlled.

[Example]
The membrane filter in which suspended microorganisms were captured by the microorganism capturing apparatus shown in FIG. 1 was placed on the medium, and the microorganisms were cultured on the medium, and measurement was performed using a bioluminescence image analysis system manufactured by Nippon Millipore Corporation.

・ Membrane filter; manufacturer MILLIPORE
Part number EZHAGG474
Small diameter 0.45μm
Material Cellulose mixed ester ・ Aspiration air volume 100L 6 times ・ Aspiration time 3 minutes each ・ Medium medium A: Standard agar medium
B: Glucose with antibiotics and peptone agar medium-Culture conditions A: 36 ° C, 20 hours
B: 25 ° C., 44 hours [Comparative Example]
Microorganisms captured on the medium by air suction were cultured using a collision type microorganism capturing device (product name: Merck Air Sampler, product number: MAS100) manufactured by MERCK, and the number of colonies was counted.

-Medium A: Standard agar medium
B: Glucose / peptone agar medium supplemented with antibiotics • Aspirated air volume 100 L 3 times • Aspirate time 1 minute each • Culture conditions A: 36 ° C., 3 days
B: 25 ° C., 5 days Test results are shown in Table 1 below.

  From the results in Table 1, it was found that the Examples can capture microorganisms more efficiently and can be measured in a shorter period of time than the Comparative Examples.

1,20 Microbe capture device 4 Ejector 13 Holder 21 Chamber

Claims (4)

Microorganisms suspended in air by sucking air through a membrane filter having heat resistance are captured on the membrane filter, and the microorganisms captured on the membrane filter are cultured on a medium and then cultured. ATP is detected by bioluminescence reaction,
The method for quickly measuring microorganisms, wherein the suction is performed by an ejector using compressed air. A microorganism capturing device that captures microorganisms floating in the air on the membrane filter by aspirating air through a heat resistant membrane filter, using the compressed air from the compressor to surround the trigger channel The microorganism capturing apparatus comprising: an ejector that sucks air; and a holder that is connected to an inducement flow path of the ejector and supports the membrane filter. The microorganism capturing apparatus according to claim 2, wherein the holder has a cup-shaped portion, and a membrane filter is supported on the bottom of the cup-shaped portion. The chamber further includes a chamber that is connected to the trigger flow path and accommodates the membrane filter, and the chamber includes a trigger flow path side connection port portion connected to the trigger flow path, and an outside air introduction connection port for introducing external air. The microorganism capturing apparatus according to claim 2, further comprising: a portion, and the holder that supports the membrane filter between the trigger flow channel side connection port portion and the outside air introduction port portion.

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