JP2009118780A - Microorganism detection method and filtration apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism detection method that simply and quickly detects microorganisms, even in samples with impurities which are other than the microorganisms mixed therein. <P>SOLUTION: A vibrator 4 is operated by using a filtration apparatus 1 including a first filter 2 having a pore diameter of 1-10 μm; a housing 3 that stores the filter 2 and passes a liquid through the filter 2; and the vibrator 4 fixed on the housing 3. A liquid sample 50 containing cells and microorganisms to be detected, is filtered by the first filter 2, while applying a vibration of 0.1-100 Hz in a substantially perpendicular direction to the filtration surface, and a filtrate from which the cells are removed is recovered. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting microorganisms in a liquid coexisting with cells and a filtration apparatus used in the method.

  Microbial contamination in which microorganisms are mixed in the production facility may not only cause microbial contamination in the product, but also cause abnormalities in the production process. For example, if microorganisms that are not originally required for the reaction are mixed in the reaction tank or culture solution of the bioreactor, the manufacturing process becomes abnormal, and a product with a target quality cannot be obtained. Therefore, it is necessary to verify the microbial contamination by collecting samples and confirming the presence or absence of microbial contamination and the mixed microbial species.

Conventionally, for the verification of microbial contamination, a method of directly observing a collected sample as it is using a microscope or the like, and a method of culturing by adding the collected sample to a culture medium and observing the culture are widely used ( Non-patent document 1). The method of directly observing the collected sample can be verified in a short time because the process is simple, and the method of observing the culture can be verified with high accuracy because microorganisms are grown.
Translated by Yoji Mitsui, "Practical animal cell culture", Maruzen Co., Ltd., p104-105

However, in the method of observing the collected sample as it is, the detection sensitivity of microorganisms is low, and if contaminants such as cells other than microorganisms are mixed in the sample, it is difficult to clearly distinguish and detect them. There is a problem that the number of samples that can be observed is limited.
In addition, the method for observing the culture has a problem that the culture of the sample requires at least about two days, and the culture operation is complicated and cannot be verified quickly and easily.

  This invention is made | formed in view of the said situation, and makes it a subject to provide the detection method which can detect microorganisms simply and rapidly also with the sample in which contaminants other than microorganisms were mixed.

To solve the above problem,
The present invention is a method for detecting microorganisms in a liquid sample coexisting with cells, and a vibration of 0.1 to 100 Hz is applied to a filter having a pore diameter of 1 to 10 μm in a direction substantially perpendicular to the filtration surface. However, the present invention provides a microorganism detection method comprising a step of filtering the liquid sample with the filter and collecting the filtrate from which the cells have been removed.
The microorganism detecting method of the present invention preferably further includes a step of filtering the filtrate with a filter having a pore diameter of less than 1 μm and collecting the microorganisms on the filter.
In the microorganism detection method of the present invention, it is preferable that the filter having a pore diameter of 1 to 10 μm is filtered without reducing the pressure in the space on the side opposite to the filter surface side.
In the microorganism detection method of the present invention, the frequency of vibration applied to the filter having a pore diameter of 1 to 10 μm is preferably 1 to 20 Hz.
The present invention also relates to a filtration apparatus used in the microorganism detection method of the present invention, a filter having a pore diameter of 1 to 10 μm, a housing that contains the filter and allows liquid to pass through the filter, and an excitation that vibrates the housing. Provided is a filtration device characterized by comprising means.

  According to the present invention, microorganisms can be detected easily and quickly even in a sample in which contaminants other than microorganisms are mixed.

The present invention will be described in detail below.
<Filtration device>
FIG. 1: is a schematic block diagram which illustrates the filtration apparatus which concerns on this invention, (a) is a perspective view, (b) is a longitudinal cross-sectional view in the AA line of (a).
In the filtration device 1, the housing 3 accommodates the first filter 2 and allows liquid to pass through the first filter 2.

  The first filter 2 removes cells from a liquid in which cells and microorganisms coexist. The size of the microorganism is usually 1 to 5 μm, the size of the cell is usually larger than 10 μm, and the size of the animal cell suitable for use in a bioreactor is usually 20 μm or more. From such a viewpoint, in order not to allow cells to pass through, the pore size of the first filter 2 may be appropriately selected according to the type of microorganism to be detected within the range of 1 to 10 μm, and is 3 to 8 μm. Is preferred.

The material of the first filter 2 may be any material as long as it is stable with respect to the liquid to be filtered, and is usually used for separating microorganisms and biological components such as various synthetic resins such as polycarbonate and cellulose mixed ester. Things are fine.
Further, the thickness of the first filter 2 is not particularly limited, and may be appropriately selected according to the purpose. For example, in the case of removing animal cells, those having a size of about 50 to 300 μm are suitable.
And as the 1st filter 2, you may use a commercially available membrane filter.

The housing 3 includes a cylindrical inlet 31 for introducing a liquid sample to be filtered, a cylindrical outlet 33 for discharging the filtrate filtered by the first filter 2 to the outside, and the inlet 31 and the outlet 33. A cylindrical filtration chamber 32 serving as a liquid flow path is provided.
The filtration chamber 32 further includes an upper section 32a that serves as a flow path of the liquid sample to the first filter 2 and a lower section 32b that serves as a flow path for the filtrate obtained by filtration through the first filter 2 by the first filter 2. It is divided into and.
The first filter 2 has a disk shape and is arranged concentrically with the inlet 31 and the outlet 33.

The housing 3 can be divided into two vertically at the side portion of the filtration chamber 32, and the peripheral portion of the first filter 2 is sandwiched between the concave portions 320 formed on the divided surface. Therefore, the first filter 2 can be attached to and detached from the housing 3, and the first filter 2 can be replaced by dividing the housing 3. In the present invention, the first filter 2 is not necessarily replaceable. For example, the first filter 2 may be fixed to the housing 3 with an adhesive or the like, and the entire housing 3 may be replaced. As shown here, it is preferable to be replaceable because it is advantageous in terms of handling and cost.
Note that the method of fixing the first filter 2 to the housing 3 in a detachable manner is not limited to the above, and any known method may be used.

  The filtration surface 20 of the first filter 2 is surrounded by the housing 3 and is opened to the outside of the housing 3 only by the introduction port 31. Therefore, the filtration surface 20 is difficult for foreign matter such as microorganisms floating in the work space to adhere thereto, and if the opening end of the inlet 31 is sealed, the attachment of the foreign matter can be completely suppressed. .

The volumes of the upper section 32a and the lower section 32b are not particularly limited, and may be appropriately selected according to the purpose. When the filtration device 1 is used to detect microorganisms in a liquid sample for the purpose of verifying microbial contamination, the amount is preferably 10 to 100 ml in consideration of the amount of the liquid sample to be subjected to filtration. And it is preferable that the area of the filtration surface 20 of the 1st filter 2 is 1-20 cm < ² >.
Further, the volume ratio between the upper section 32a and the lower section 32b is not particularly limited.

  The inlet 31 of the injector 5 for introducing the liquid sample 50 can be inserted into the inlet 31. In addition, it is preferable that the inner diameter of the inlet 31 is smaller as long as the introduction of the liquid sample 50 is not prevented. Further, it is particularly preferable that the inlet 31 can be fitted with the inlet 51. In this way, when the liquid sample 50 is introduced into the housing 3, the liquid sample 50 can be reliably introduced without being scattered, and the introduction operation can be simplified. Furthermore, foreign substances such as microorganisms floating in the work space can be prevented from entering the housing 3 while the liquid sample 50 is being introduced.

  The inner diameter, outer diameter, and shape of the discharge port 33 are not particularly limited, and may be appropriately selected according to the purpose in consideration of handling of the filtrate, for example. For example, when the filtration device 1 is connected to a container that receives the filtrate, the shape and size of the discharge port 33 can be selected so as to fit into the opening of the container.

  The material of the housing 3 may be any material that is stable with respect to the liquid to be filtered. For example, various resins are suitable in consideration of ease of handling and strength. Especially, since the visual observation of the filtration surface 20 is easy, a transparent thing is preferable. Specifically, polypropylene etc. can be illustrated.

The vibrator 4 is fixed to the side surface 30 a of the outer surface 30 of the housing 3 as a vibrating means for applying vibration, and the first filter 2 can be vibrated through the housing 3.
The vibrator 4 may be any one that can vibrate at a low frequency of 100 Hz or less.
The vibrator 4 is fixed so that the vibration direction is substantially perpendicular to the filtration surface 20 of the first filter 2.

The number of vibrators 4 is not limited to one as shown here, but may be plural. The first filter 2 can be efficiently vibrated as the number of vibrators 4 increases, and the filtration device 1 can be manufactured at a lower cost as the number of vibrators 4 decreases.
Further, the fixed portion of the vibrator 4 on the outer surface 30 of the housing 3 is not limited to the side surface 30a as shown here, and may be the upper surface 30b or the lower surface 30c. However, the closer the fixed part is to the contact portion between the first filter 2 and the housing 3, the more efficiently the first filter 2 can be vibrated. Therefore, the vibrator 4 is fixed to the side surface 30a as shown in FIG. It is preferable.
Therefore, when a plurality of vibrators 4 are fixed, it is preferable to fix the first filter 2 to the side surface 30a of the housing 3 at equal intervals in order to efficiently vibrate the first filter 2.

  The method for fixing the vibrator 4 to the housing 3 is not particularly limited. For example, the vibrator 4 may be fixed via an adhesive. However, it is more convenient to fix the protrusion and the recess by detachably engaging with each other. Highly preferred. In addition, as described above, when the housing 3 can be divided into two, it is fixed by contacting only one of them, or by fixing to either one and only contacting the other. It is preferable to keep it.

The filtration device of the present invention is shown in FIG. 1 as long as it has a filter having a pore diameter of 1 to 10 μm, a housing that accommodates the filter and allows liquid to pass through the filter, and a vibration means fixed to the housing. It is not limited to things.
For example, here, the filtration surface 20 of the first filter 2 is shown as being opened to the outside of the housing 3 only by the inlet 31 as a filtration device, but the shape of the housing 3 is not limited to this, The housing 3 has a funnel shape, and the structure of the housing 3 may not exist in a direction facing the filtration surface 20.
Moreover, all of the inlet 31, the outlet 33, and the filtration chamber 32 may have a shape other than a cylindrical shape, and the radial cross section thereof may have other shapes such as a polygonal shape other than a circular shape.

The filtration device 1 is preferably used in combination with a collector equipped with the second filter so that the filtrate obtained by filtering with the first filter 2 can be immediately filtered with a second filter having a pore diameter of less than 1 μm. .
Thus, the front view of the state which combined the filtration apparatus 1 with the collector is shown in FIG.

  On the inner surface of the collector 6, a peripheral portion of the support plate 61 is provided with a second filter 62 and a flow path (not shown) for allowing the filtrate filtered by the second filter 62 to pass therethrough. Closely fixed. The support plate 61 is preferably disposed so that the second filter 62 installation surface is parallel to the grounding surface of the collector 6. And the support plate 61 should just be fixed to the inner surface of the collector 6 with an adhesive agent etc., for example.

  And the 2nd filter 62 is mounted on the support plate 61 so that the peripheral part may closely_contact | adhere to the inner surface of the collector 6. FIG. The second filter 62 may be sandwiched between the support plate 62 with a metal ring disposed, for example, in the vicinity of the peripheral edge thereof so that the second filter 62 can be stably held on the support plate 61.

As described above, the collector 6 is obtained by the support plate 61 and the filtration by the second filter 62 and the introduction section 6 a that becomes the flow path to the second filter 62 of the filtrate obtained by the filtration by the first filter 2. It is divided into a collection section 6b for collecting the filtrate.
The recovery section 6b is provided with a vent 64 that opens the section to the outside of the collector 6. The vent 64 has a cylindrical shape, and can be decompressed in the recovery section 6b by connecting to a decompression means (not shown) such as a suction pump.

  The shape and size of the opening 63 of the introduction section 6a are not particularly limited as long as the outlet 33 of the filtration device 1 can be inserted. And in order to stabilize the filtration apparatus 1, it is preferable that the said discharge part 33 can be fitted to the said opening part 63. FIG.

The support plate 61 includes a discharge pipe 611 that collects and discharges the filtrate filtered by the second filter 62. And the material of the support plate 61 should just be stable with respect to the liquid with which it uses for filtration, Glass, various resin, etc. are mentioned.
The support plate 61 is not limited to the one shown here. For example, the support plate 61 may not be provided with the discharge pipe 611 or may be a glass porous plate.

  The material of the collector 6 may be any material as long as it is stable with respect to the liquid to be filtered, as in the case of the support plate 61, but the filter obtained by filtration using the filtration surface of the second filter 62 or the first filter 2. Since the liquid and the filtrate obtained by filtration with the second filter 62 are easy to visually observe, a transparent one is preferable.

By setting the pore diameter of the second filter 62 to less than 1 μm, the microorganisms in the filtrate from which the cells have been removed by the first filter 2 are filtered and collected on the second filter 61.
The pore size of the second filter 62 can be appropriately selected depending on the type of microorganism to be detected, within a range of less than 1 μm.
The material of the second filter 62 may be any material that is stable with respect to the liquid to be filtered, and may be the same as the material of the first filter 2. The thickness of the second filter 62 is not particularly limited, and may be appropriately selected depending on the purpose. A commercially available membrane filter may be used as the second filter 62.

  If the collector 6 is provided with the 2nd filter with a hole diameter of less than 1 micrometer, things other than the form shown in FIG. 2 can be used suitably.

<Microbe detection method>
The microorganisms in the liquid sample coexisting with the cells can be detected using the filtration device 1 as follows.
When the vibrator 4 is operated, the vibration is transmitted to the first filter 2 through the housing 3. As a result, the first filter 2 is fixed to the housing 3 and vibrates integrally with the housing 3. When the liquid sample 50 is introduced onto the first filter 2 from the inlet 31 of the housing 3 while applying vibration to the first filter 2 in this way, the liquid sample 50 is filtered through the first filter 2. At this time, since the pore size of the first filter 2 is smaller than that of the cell and larger than that of the microorganism to be detected, the cell is filtered on the filter. At the same time, other foreign matters having a size larger than the hole diameter of the first filter 2 are also filtered out. And since the microorganisms of a detection target are contained in the obtained filtrate, what is necessary is just to collect | recover this filtrate and to use for the detection of microorganisms.

  The direction of vibration applied to the first filter 2 is substantially perpendicular to the filtration surface 20. By doing in this way, clogging of the first filter 2 is suppressed.

The frequency when the vibrator 4 is operated is 0.1 to 100 Hz, preferably 1 to 20 Hz, more preferably 5 to 10 Hz. By setting the frequency in the above range, a high effect of suppressing clogging of the first filter 2 can be obtained. Here, if the frequency exceeds 100 Hz, the cell may be damaged, and if it is less than 0.1 Hz, a sufficient filtration effect cannot be obtained.
The amplitude when the vibrator 4 is operated is not particularly limited, but is preferably 5 mm or less. By doing in this way, filtration can be performed still more efficiently.

When the liquid sample 50 is filtered by the first filter 2, it is preferable not to apply a pressure reducing operation for making the pressure in the lower section 32b smaller than the pressure in the upper section 32a. When the pressure in the lower compartment 32b is made smaller than the pressure in the upper compartment 32a, the filtration of the liquid sample 50 introduced into the upper compartment 32a is promoted immediately after the start of filtration. May clog.
Therefore, the filtration of the liquid sample 50 by the first filter 2 is preferably performed by natural filtration in which the liquid sample 50 is simply introduced onto the first filter 2 instead of suction filtration or vacuum filtration.

  The temperature of the liquid sample 50 may be appropriately selected according to the type of the liquid sample 50, but is usually preferably 40 ° C. or less, more preferably 30 ° C. or less, particularly preferably 20 ° C. so that cells and microorganisms are not destroyed. The following. The lower limit of the temperature may be any as long as the liquid sample 50 is not frozen.

  When the liquid sample 50 is filtered by the first filter 2, the vibrator 4 may be operated after the liquid sample 50 is introduced onto the first filter 2, but the first filter 2 is clogged. Since the suppressing effect is high, it is preferable to introduce the liquid sample 50 onto the first filter 2 after operating the vibrator 4 as described above.

  The filtrate collected by the filtration by the first filter 2 may be used for detection of microorganisms as it is, or may be used for detection of microorganisms after appropriate treatment such as concentration, for example. In the case of concentration, excessive heating treatment is not preferable for microorganisms, and concentration using a semipermeable membrane is preferable.

And it is especially preferable to collect | recover the microorganisms of a detection target on this 2nd filter by further filtering the filtrate collect | recovered by filtration with the 1st filter 2 with the 2nd filter of the hole diameter of 1 micrometer or less. In this way, the detection target microorganism can be detected quickly.
In this case, if the collector 6 is used in combination, the filtrate obtained by filtering with the first filter 2 can be immediately filtered with the second filter 62, and the operation is simple and suitable.

  In each process until the filtration by the second filter 62 is completed, it is preferable to suppress the destruction of cells and microorganisms. In particular, when the cells are destroyed, the constituent components of the cells may clog the filter used for filtration, particularly the second filter 62, and may cause process troubles such as filtration clogging. In order to suppress the destruction of cells and microorganisms, for example, a high frequency vibration exceeding 1 kHz is applied to a liquid sample containing these, or the liquid sample is heated to a high temperature exceeding 50 ° C. Without it, it ’s good.

  The microorganisms in the filtrate collected as described above and the microorganisms collected on the filter paper can be easily detected by a known technique using a microscope or the like.

  According to the microorganism detection method of the present invention, by selecting the pore size of the first filter, the direction and frequency of vibration applied to the first filter as described above, process troubles such as filtration clogging without destroying cells can be performed. It can be easily removed from the liquid sample without it. At the same time, cells having a size equal to or larger than that of cells can be easily removed. In this way, contaminants that are obstacles to direct detection of microorganisms using a microscope or the like can be easily removed, so even samples containing such contaminants can be directly detected without culturing microorganisms. Therefore, microorganisms can be detected simply and quickly. The microorganism detection method of the present invention is particularly suitable for a sample mixed with animal cells.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[Example]
Microorganisms were detected using the filtration device and the collector shown in FIG. The liquid sample subjected to detection contained animal cells as cells. In addition, a filtration device containing a cellulose mixed ester having a diameter of 4.7 cm, a thickness of 160 μm, and a pore diameter of 5 μm accommodated in a polypropylene housing as a first filter, and a second filter having a diameter of 2.5 cm, A collector provided with a polycarbonate membrane filter having a thickness of 10 μm and a pore diameter of 0.2 μm was used. Then, the liquid sample was introduced onto the first filter and filtered while the vibrator was vibrated at 8 Hz in a direction substantially perpendicular to the filtration surface of the first filter. . Subsequently, the obtained filtrate was immediately filtered with a second filter provided in the collector.
As a result, 10 ml of the liquid sample could be filtered in 34 seconds, and microorganisms could be detected on the second filter with a microscope.

[Comparative example]
Except for using a filtration device that does not include a vibrator, filtration was performed in the same manner as in the above example. As a result, it took 52 seconds to filter the liquid sample. Also the filtration rate became slow.

  The present invention can be used for verification of microbial contamination of production equipment and the like.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic block diagram which illustrates the filtration apparatus which concerns on this invention, (a) is a perspective view, (b) is a longitudinal cross-sectional view in the AA of (a). It is a front view which shows the state combined with the collector for collecting microorganisms of the filtration apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Filtration apparatus, 2 ... 1st filter, 20 ... Filtration surface, 3 ... Housing, 32 ... Filtration chamber, 32a ... Upper division, 32b ... Lower division, 4 ... Vibrator, 6 ... Collector, 61 ... Second filter, 50 ... Liquid sample

Claims (5)

A method for detecting microorganisms in a liquid sample coexisting with cells,
The step of collecting the filtrate from which the cells have been removed by filtering the liquid sample with the filter while applying vibration of 0.1 to 100 Hz to the filter having a pore diameter of 1 to 10 μm in a direction substantially perpendicular to the filtration surface. A method for detecting a microorganism, comprising:   The microorganism detection method according to claim 1, further comprising a step of filtering the filtrate with a filter having a pore diameter of less than 1 µm and collecting the microorganisms on the filter.   The microorganism detection method according to claim 1 or 2, wherein the filter having a pore diameter of 1 to 10 µm is filtered without reducing the pressure in the space opposite to the space on the filtration surface side of the filter.   The method of detecting a microorganism according to any one of claims 1 to 3, wherein the frequency of vibration applied to the filter having a pore diameter of 1 to 10 µm is 1 to 20 Hz. A filtration device used in the microorganism detection method according to any one of claims 1 to 4,
A filtration apparatus comprising: a filter having a pore diameter of 1 to 10 μm; a housing that accommodates the filter and allows liquid to pass through the filter; and a vibration means that vibrates the housing.

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