JPH0823962A - Device for measuring number of viable microbes and spraying machine therefor - Google Patents

Abstract

PURPOSE:To provide a viable microbe number-measuring device capable of preventing the scatter of a reagent and rapidly spraying the reagent in a fine and suitable amount in a short retention time with a solution-supplying tool, capable of efficiently, accurately and simply spraying fine solution particles with a suction-vacuum chamber, capable of accurately using an expensive reagent in the fine amount by the use of the tool and the chamber, and capable of rapidly and simply measuring the number of viable microbes in high sensitivity. CONSTITUTION:This device for measuring the number of the viable microbes comprises a filtration device in which a membrane filter 4 for catching the viable microbes in a specimen is mounted, an ultrasonic sprayer in which a vibrator 3 having many small holes for spraying an ATP-extracting agent and a luminous reagent on a viable microbe-caught membrane filter 4 is disposed, and a biological luminous image- analyzing device for detecting luminous points on the membrane filter 4 subjected to a luminous treatment in high sensitivity to measure the number of the viable microbes. Especially, a specific chamber 2 and a specific solution-supplying tool 1 are disposed in the spraying machine.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for rapidly measuring the viable cell count in a sample. More specifically, the present invention relates to an improvement of a reagent sprayer in the apparatus and a reagent addition liquid supply tool used therefor.

[0002]

2. Description of the Related Art In the fields of foods, beverages, cosmetics, semiconductors, etc., it is extremely important to measure the viable cell count during the manufacturing process or in the products. Conventionally, the agar plate method was mainly used to detect the viable cell count, but there was a strong demand for a faster and simpler method, and in response to that request, in recent years,
A rapid detection device for microorganisms has been developed and used by skillfully combining the membrane filter method, bioluminescence emission technology, and weak light detection technology.

That is, the method was invented by the present applicant and filed earlier (Japanese Patent Application No. 3-40615, PCT / JP9).
(2/00145, Japanese Patent Application No. 5-328995, etc.) After filtering the sample with a hydrophilic membrane filter to capture viable bacteria, an extractant and then a luminescent reagent are sprayed, and a highly sensitive luminescence measurement system is used. This is a method of measuring the number of viable bacteria.
A convenient device for carrying out this method has already been marketed and used by the present applicant [RMDS "microorganism rapid detection device" (trade name) manufactured by Nippon Millipore Co., Ltd.]. With the advent of the above-mentioned rapid microorganism detecting device, the measurement of the viable cell count has become simpler, faster, and more accurate than ever before.

The Applicant has further found that hydrophobic membranes treated with a wetting agent are very good for use under certain conditions. That is, a liquid containing viable bacteria is filtered using a hydrophobic membrane or a hydrophilic membrane having strong water repellency to capture the cells in the filtration membrane, and an extraction reagent and a luminescent reagent are sprayed to contact the cells. The inventors have devised a method in which the detection sensitivity is remarkably increased and bacteria are immediately detected as clear bright spots without culturing after capture by filtration (Japanese Patent Application No. 5-44397).

In this method, when a small amount of the extraction reagent and the luminescent reagent are sprayed, the reagents remain in the form of ultrafine particles due to the surface tension and adhere to the attachment points of the live bacteria, and the extracted luminescent components do not diffuse and become ultrafine particles. It can be held by and can emit light. In this case, it is extremely important that these reagents are ultrafine particles having a diameter of 20 μm or less, and that they are ultratrace amounts of spray. Therefore, further improvement was required for the reagent atomization section.

That is, in the above apparatus, live adenosine-3-phosphate (hereinafter referred to as ATP) captured on the filter is used.
It is necessary to spray an extractant or a luciferin-luciferase luminescence reagent solution for extracting), and therefore various sprays and atomizers or conventional ultrasonic sprayers have been used for these sprays. However, when the viable cell count is small (10 ² or less / membrane), a more precise microscopic spray is required, and since the luminescent reagent used is expensive, the measurement effect can be obtained with as little spray as possible. The development of such a spraying method and spraying machine has been desired.

[0007]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, that is, an improvement of a spraying apparatus for reagents used in a rapid microorganism detecting apparatus for measuring the viable cell count, and further The object was to improve the method of adding a reagent to the spray device.

[0008]

In order to solve the above-mentioned problems, the present inventors have studied various sprayers, and as a result, have recently developed a high-performance ultrasonic atomizer (manufactured by Mikuni Co., Ltd., The inventors have found that an ultrasonic atomizer, Japanese Patent Application Laid-Open No. 4-150968) can be the most suitable atomizer for solving the problem if an improvement is made to the reagent addition method, and the present invention has been completed.

That is, according to the present invention, a filtration device equipped with a membrane filter for capturing live bacteria in a sample and a large number of small devices for spraying an ATP extractant and a luminescent reagent on the membrane filter capturing live bacteria are provided. Viable cell count consisting of an ultrasonic atomizer equipped with a vibrator with holes and a bioluminescence image analyzer that measures the viable cell count by detecting the light emission point on the membrane filter that has been subjected to light emission with high sensitivity It is an invention that provides a measuring device, and is particularly characterized in that a sprayer used in the device is equipped with a chamber and / or a liquid supply tool that exhibits excellent effects.

Hereinafter, the present invention will be described in detail. First, the overall configuration of the rapid viable cell count detection device (system) of the present invention will be described. The viable cell count rapid detection system of the present invention is roughly divided into three steps. The first step is a step of filtering the sample with a membrane filter to capture viable bacteria on the filter). The membrane filter used here is a conventionally used membrane filter for analyzing microorganisms, or the hydrophobic partition wall 4-shown in FIG.
A membrane filter 4 with a frame 4-3 having a large number of hydrophilic sections 4-1 surrounded by 2 (Japanese Patent Laid-Open No. 3-4061).
5, No. 5, PCT / JP92 / 00145), or a hydrophobic film treated with a wetting agent or the like is used. These membrane filters have the effects of efficiently concentrating and capturing live cells in a sample, capable of uniformly dispersing the live cells, and preventing diffusion dilution of ATP in the next step.

The second step is a step of adding an extractant for extracting ATP in live bacteria captured on the membrane filter in the above step onto the filter, and then adding a luminescent reagent thereto to cause luminescence. . AT used in this process
The present inventors have invented and applied for a P-extracting agent (Japanese Patent Laid-Open No. Hei 5 (1999) -58242).
(No. 328995) Volatile extractants having a boiling point of 120 ° C. or less, particularly methanol or ethanol are preferable, and commercially available luciferin-luciferase luminescent reagent is used as the luminescent reagent. This luciferin-luciferase luminescent reagent emits light by the oxidation of luciferin when contacted with ATP in the presence of magnesium ion and enzyme, and thus the viable cell count can be detected by measuring the luminescent bright spot number.

In the ATP extraction and luminescence in this step, it is necessary to add the above-mentioned extractant and luminescent reagent in the smallest possible amount, accurately, and easily in a short time.
That is, if the ATP extract is added more than necessary, the extracted ATP may be diluted, diffused or flow out in the vicinity, and the emission point may be blurred, and the remaining extractant may inhibit the emission. In order to prevent this, it takes time and effort to volatilize the extractant, so it must be avoided. Therefore, we tried to add an appropriate amount carefully with a sprayer or the like, or tried to spray an appropriate amount with the existing ultrasonic sprayer, but it was difficult to adjust the spraying amount for both, and the spraying unevenness was large, In addition, because of individual differences, it was not possible to obtain a sufficiently effective spray although skill was required. This point is the same in the addition of the luminescent reagent, and particularly in the case of the luminescent reagent used in the present invention, it is also expensive, so it is necessary to effectively add it in a small amount as industrially possible. The development of a spray addition method for

As described above, the present invention is, firstly, in response to this need, by inventing a spraying device most suitable for the method for rapidly detecting the viable cell count of the present invention. The spraying device is disclosed in Japanese Patent Laid-Open No. 4-1
This is an improvement of the ultrasonic atomizing device disclosed in Japanese Patent No. 50968. That is, the device of the above-mentioned Japanese Patent Application Laid-Open No. 11-242242 is a device for atomizing a liquid by utilizing elastic vibration generated by an ultrasonic vibrator having a structure in which a tongue-shaped vibrating plate is fixed to a piezoelectric vibrator. A large number of holes are provided in the diaphragm, and when liquid is dripped there, the liquid comes out through the holes onto the diaphragm and is atomized in fine particles. .

This ultrasonic atomizer has the effect that the particles of the fog become minute and uniform due to the action of the numerous holes in the vibrating section, and atomization is efficient and can be performed with low power consumption. The device also has the effect of becoming smaller. The atomizing device is adopted in the viable cell number rapid detection system of the present invention, because of its effects, in particular, the spray particles are fine and uniform, and efficient spraying is possible, and the entire device is downsized. This is because it has been confirmed that the above-mentioned point is more consistent with the performance required for the spraying device used in the viable cell count detection system of the present invention, as compared with the conventional spraying device.

However, the above-mentioned ultrasonic atomizing device of the above-mentioned Japanese Patent Laid-Open No. 10-134242 was not originally developed as a sprayer used in the rapid detecting device for the viable cell count of the present invention, and a conventional sprayer such as a nebulizer or a humidifier. Since it was intended for a specific application, such as an application or an ultrasonic color organ, some improvements were needed to accommodate the device system of the present invention.

[0016] One of them is that, in the case of spraying the extractant, it is necessary to spray a proper amount of a proper amount on the filter which has captured the viable bacteria, and a device for that is required. In order to improve the detection accuracy, spraying of the extractant requires the minimum amount necessary to extract ATP in viable bacteria. On the other hand, if the amount is too large, the ATP may be diluted, diffused or outflowed, or the emission point may be increased. Since the outline of the image becomes blurred and the detection sensitivity decreases, it must be avoided. Not only in the case of spraying a luminescent reagent, but also because the reagent is expensive and it is necessary to spray efficiently in a trace amount,
Furthermore, it can be said that the necessity of spraying a proper amount in a trace amount is higher.

Secondly, in order to improve the accuracy of the measurement, it is necessary to shorten the extraction and luminescence steps after capturing the viable bacteria as much as possible. For that reason, the spray liquid (extractant, luminescent reagent) is required. Therefore, there has been a need for an invention of a liquid supply tool for reducing the residence time (lead time) before spraying, preventing the reagent solution from scattering, and realizing uniform spraying. The liquid supply tool must have a structure in which the uniformity and adhesiveness of the contact surface with the vibration plate must be improved for efficient and uniform atomization, and the residual liquid amount must be as small as possible. .

In order to make the above two improvements, the present inventors first devised the chamber shown at 2 in FIGS. 1 and 2. As shown in the figure, this chamber 2 is a cylindrical body provided with a window 2-1 at the top for taking in and out the atomizing part such as a vibrator of a spraying device, and the opening at the bottom is a viable bacterium inside the cylinder. It is attached so that it can be installed on the membrane filter that has captured. And, preferably, the air in the chamber is sucked from the lower side of the bottom surface by a vacuum pump or the like to reduce the pressure. The degree of pressure reduction at this time is 5 to 50.
mmHg, preferably 10 to 30 mmHg.

The extractant or luminescent reagent finely atomized by the vibrating plate by the installation of this chamber and the suction from the bottom efficiently pours on the live bacteria on the membrane filter,
It is possible to exert each function with a small amount and an appropriate amount, and without a chamber the mist diffuses and the amount of chemical liquid increases,
In addition, efficient spraying becomes impossible.

Next, the inventors of the present invention invented a liquid supply tool for supplying an extractant and a luminescent reagent onto the diaphragm in order to improve the above. Conventionally, as a method of supplying a liquid onto the vibration plate of the ultrasonic atomizer, a method of dropping the liquid while controlling the liquid amount using a flow rate adjusting valve and a supply tube (JP-A-4-150968), A method of sucking up a liquid with a liquid-retaining material such as sponge and supplying it to the lower surface of the diaphragm (Japanese Patent Laid-Open No. 4-322290), in which a liquid-retaining material such as sponge is placed in a prismatic plastic case, and the lower portion is immersed in the liquid. The method of replenishing the vibrating plate on the upper end with the sucked up liquid (Japanese Patent Laid-Open No. 5-305257) has been devised and published by the developers of the ultrasonic atomizing device.

However, with these methods, it is difficult to accurately spray a small amount of an appropriate amount of the chemical liquid required by the present invention, and the retention time of the chemical liquid is short and there is no trouble such as scattering. could not. Therefore, as a result of studying the improvement of the supply port of the chemical liquid, the present inventors have found that when the chemical liquid supplied by the needle from the reagent bottle is dropped on the vibration plate of the vibrator, the liquid droplet is not dropped directly but once. The absorbent material sheet 1-1 absorbs it, and then averages it to obtain the vibrator 3
It was found that the intended effect can be obtained by supplying the liquid to the. And, as the liquid absorbing material, a small cylindrical body of sponge was initially used, but this causes problems in terms of residence time and residual liquid, and as a result of further improvement, the extractant of the present invention shown in FIG. The liquid supply tool 1 for suitably supplying the chemical solution of the luminescent reagent to the fog generating portion of the vibrator 3 has been invented.

In this liquid supply tool 1, a liquid absorbing material sheet 1-1 is formed by various methods on the bottom of a cylindrical support 1-2 having both ends having a cross-sectional shape corresponding to the shape of the fog generating portion of the vibrator 3. It has a structure bonded with. As the material of the cylindrical support 1-2, plastic, metal, or the like that is not corroded by the chemical liquid is used. Its outer diameter is 4 to 30 mm, preferably 6 to 12 mm, and its height is 4 to 30 mm, preferably 6 to 12 mm. Further, the wall of the cylindrical support may be provided with voids or may have a cage shape.

The surface of the sheet 1-1 attached to the bottom is preferably as flat as possible so that the liquid can be uniformly contacted with the diaphragm evenly. This sheet quickly absorbs the extractant and luminescent reagent used in the present invention,
Moreover, a sheet having excellent liquid absorbability capable of holding it is suitable. Therefore, as the sheet, a cellulosic or water-absorbing synthetic or semi-synthetic fiber sheet or nonwoven fabric used for filter paper or a liquid-absorbing natural or synthetic sponge sheet is used. As a specific example, a cellulosic filter manufactured by Nippon Millipore, AP-1
0 or the like is used. The thickness of this sheet is 0.3-3mm,
It is preferably 0.5 to 1 mm. Its planar shape needs to be a shape that matches the cross-sectional shape of the cylindrical support, but in the case of a cylindrical support, the diameter is 4 to 30 mmφ, preferably 6
The one having a diameter of up to 12 mm is used.

When the liquid supply tool 1 of the present invention is used, the liquid is hardly scattered as compared with the case where the chemical liquid is dropped directly on the vibrator, and the liquid is supplied almost uniformly to the entire fog generating portion of the vibrator. And since the liquid can be atomized accurately, there is no residual liquid,
A very small amount of spraying is possible. In addition, since the liquid seeps into and out of the sheet quickly, there is almost no lead time, and quick measurement can be performed according to the measurement operation.

The essential parts of the above-described sprayer of the present invention are shown in FIGS. In the figure, 1 is a liquid supply tool and 2 is a chamber. When sprayed using this sprayer and a liquid supply tool, a fine-grained AT having a particle size of 1 to 60 μm, preferably 5 to 20 μm, on the live bacteria captured on the membrane filter.
The P extractant and / or the luciferin luciferase-based luminescent reagent can be sprayed accurately and rapidly in the former amount of about 0.04 to 0.08 ml and in the latter case of about 0.02 to 0.04 ml.

The filter sprayed with the luminescence reagent as described above is the bioluminescence image analysis apparatus which is the third step of the present invention [eg, ARGUS-50 / manufactured by Hamamatsu Photonics KK].
CL or Japan Millipore RMDS],
The bright spot is imaged and the viable cell count is measured. This bioluminescence image analysis system can process weak luminescence with high sensitivity as compared with a conventional measuring device, and can process and analyze data quickly. Therefore, by adopting this, the filter and reagent of the present invention can be improved. It was possible to realize an extremely excellent method for measuring live bacteria in synergy with the effect of the sprayer.

The outline of the system is as shown in FIG. The system shown in FIG. 6 two-dimensionally detects the luminescence arranged as close as possible to the filter holder 11, the light-shielding housing 16 and the filter for supporting the filter 4 after being treated with the extraction reagent and the luminescence reagent. A taper fiber 17, an ultra-high-sensitivity television camera 18 including an optical amplifier and an image pickup tube, a camera controller 19, an image processor 20, and a data analysis device 2
1. It is composed of a TV monitor 22.

The measurement is carried out by the light-emitting treated filter 4
Place the filter holder 11 holding (the filter holding the viable bacteria) in close contact with the tapered fiber surface,
30-18 photons two-dimensionally using ultra-sensitive TV camera, camera controller and image processor
The light is accumulated for 0 seconds, for example, 120 seconds, the light emission from the fungus body is imaged, and the image processing is performed by the data analysis device to eliminate the minute light emission noise and display only the strong light emission from the live bacteria on the TV monitor. By this processing, the light emission other than the bacterial cells is erased as noise, and thus the measured number of bright spots substantially coincides with the viable cell count.

[0029]

The present invention will be described in the following examples.

Example 1 One platinum loop E. coli (Escherichia coli IFO) was added to a test tube containing 5 ml of SCD medium (manufactured by Nippon Pharmaceutical Co., Ltd.).
13898) was inoculated and cultured overnight at 37 ° C., then diluted with Hepes buffer (pH 7.75) to about 100 cells / ml, and 0.2 ml thereof was used as a sample solution. Hydrophobic P
VDF film (HVHP.47mmφ, Japan Millipore Co., Ltd.)
After making a hydrophilic filter by immersing it in a small amount of methanol and washing it thoroughly with water, add 10 ml
Hepes buffer solution (manufactured by Sigma) was added, and 0.2 ml of the sample solution was further added, followed by suction filtration. 15 ml of the same buffer
After washing 3 times with, remove the membrane filter from the filter.

Next, the above-mentioned membrane filter was set in the spraying device shown in FIGS. 1 and 2, and the ATP extraction reagent (manufactured by Nippon Millipore Co., Ltd.) and the luminescence reagent (manufactured by Nippon Millipore Co., Ltd.) were set as follows. Spray)). The extraction reagent and the luminescent reagent are filled in the reagent bottle 5 and attached to the manifold 7. This set has two series of extraction reagents and luminescence reagents. The manifold 7 is provided with needles 8 on the upper and lower sides, one is a reagent dropping 8-a, the other is a reagent introducing 8-b and a gas intake needle 8-c for feeding liquid at gas pressure. is there.

Since the entire manifold 7 has a function of turning upside down, when the reagent bottle is attached, 8
This can be done with the needles -b and 8-c on the lower side. An electromagnetic valve 6 is further attached to the manifold 7, and the valve opens and closes under the control of the control mechanism.
The liquid-feeding gas (such as nitrogen gas) is controlled to quantitatively feed the reagent.

When the attachment of the reagent bottle 5 is completed, the manifold is rotated so that the reagent bottle is located above, and then the liquid supply tool 1 is placed on the vibrator (3T-UVA manufactured by Mikuni).
The vibrator 3 is provided with a guide 3-1 that allows the liquid supply tool 1 to be installed and held at a predetermined position.

The liquid supply tool 1 of the present invention has a thickness of 1 mm and a diameter of 7.
A 5 mm cellulose-based filter 1-1 is welded. The height of the support 1-2 is 8 mm.

Next, the chamber 2 of the present invention is attached to the chamber holder 9. The chamber holder 9 can be opened greatly to the left and right when the chamber is attached, and holds the chamber with an appropriate strength. This chamber 2
It has a diameter of 40 mm and a height of 200 mm and is made of polycarbonate resin. The diameter and height can be changed depending on the size of the filter. The chamber holder 9 has an integral structure with the chamber holder mount 10, and the chamber holder mount has a mechanism that moves vertically by about 5 to 20 mm by air pressure (or motor drive).

The membrane filter 4 treated in the previous step is attached to a filter holder 11 made of a corrosion-resistant metal (or plastic), which is composed of a filter attachment ring and a filter holder. The filter holder 11 is installed on the base plate 12. The base plate 12 is provided with one or more suction holes 13, and suction is performed at a pressure of 10 to 30 mmHg. Then, a Teflon rail 14 for filter feeding is installed on this plate, and by placing a membrane filter on this rail, it can be moved to a predetermined position.

The filter holder 11 mounted with the membrane filter 4 placed on the rail 14 is sent to below the chamber 2. Chamber 2 is filter holder 1
1 can be lifted upwards so that it is directly below the chamber, and when the filter 4 is directly below the chamber 2,
The chamber 2 can be lowered to its original position.

Next, the electromagnetic valve 6 is opened, nitrogen gas is supplied into the reagent bottle 5, and methanol as an ATP extraction reagent is dripped into the 0.08 cc liquid supply tool 1. These processes can be automated or continuous by providing a control means and an operable means. The liquid supply sheet (cellulosic filter) 1-1 of the liquid supply tool 1 immediately absorbs the reagent, while the electromagnetic valve 6
At the same time as the opening, the vibrator 3 starts to operate, and the reagent uniformly held on the liquid supply material sheet 1-1 of the liquid supply tool 1 is immediately atomized into a mist and sprayed downward. Since the inside of the chamber is sucked down from the suction hole 13 and has a negative pressure of 20 mmHg, the atomized extraction reagent hardly adheres to the wall of the chamber and is sprayed evenly on the surface of the membrane filter 4 in a trace amount.

After the extraction reagent is sprayed as described above, the membrane filter is dried again, and then 0.04 cc of the luminescent reagent is sprayed by the same apparatus and method as described above and dried, and the next biological image luminescence is performed. Call the system. The biological image emission analysis system is shown in FIG. 6 and is as described above, and RMDS manufactured by Nippon Millipore was used.
As a result of the measurement, 18 to 22 viable bacteria (bright spots) were measured.

Example 2 Saccharomyces Saccharomyc
es cerevisiae, IFO 0209) was cultured overnight in a glucose-peptone medium (manufactured by Eiken Chemical Co., Ltd.), and then 10 times in a phosphate buffer (pH 7.5).
0.5 of test solution obtained by diluting to ² cells / ml
ml was used as the sample liquid. A hydrophilic PVDF membrane filter (HVWP.47 mmφ, manufactured by Nippon Millipore Co., Ltd.) shown in FIG. 5 having a section surrounded by a large number of hydrophobic partition walls was attached to a filter, and a membrane filter obtained by filtration and collection was subjected to Example 1 In the same manner as described above, spraying of 0.08 cc of the extractant reagent and 0.04 cc of the luminescent reagent was carried out, and 45-60 bright spots were measured when measured by the same RMDS as in Example 1.

Example 3 Escherichia coli (Escherichh) cultured in the same manner as in Example 1 was obtained by aseptically filtering 350 ml of commercial canned beer (manufactured by Company A).
ia coli IFO 13898) is diluted with Hepes buffer (pH 7.75) to about 100 cells / ml, and 0.2 ml of the diluted solution is a polycarbonate membrane filter (trade name: Isopore 0.4 μm, 47 mmφ, Japan Millipore Co., Ltd.). After filtering using S.A., it was placed on a SCD agar (manufactured by Nippon Pharmaceutical Co., Ltd.) plate and cultured at 37.degree. C. for 6 hours. After that, the membrane filter was removed, the extraction reagent 0.04 cc and the luminescence reagent 0.02 cc were sprayed by the same apparatus and method as in Example 1, and the measurement was performed by RMDS in the same manner as in Example 1. The bright spot was measured.

[0042]

When the liquid to be sprayed is supplied to the ultrasonic atomizer which atomizes the liquid by elastic vibration, the liquid supply tool of the present invention is used to scatter the liquid (ATP extractant, luminescent reagent). As a result, troubles and losses due to the above can be prevented, and the retention time of the liquid is short, and the liquid can be evenly supplied to the atomization generating portion on the vibration plate. Then, by installing a chamber and further suctioning and depressurizing from below, it is possible to easily and accurately add the atomized liquid particles to the live bacteria on the membrane filter without individual differences. was gotten.

The above-mentioned effects synergize with each other, and in a technical field in which control and management of viable bacteria in the manufacturing process and products are important, for example, pharmaceuticals, foods, beverages, cosmetics, semiconductors, etc. It can be said that the effect of being able to be used for, and being able to measure the viable cell count rapidly, easily and with high sensitivity is extremely large.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a spraying device used in the present invention.

FIG. 2 is a perspective view of a spraying device used in the present invention.

FIG. 3 is a cross-sectional view of a chamber of a spraying device used in the present invention and a suction portion thereof.

FIG. 4 is a perspective view of a liquid supply tool of the spraying device used in the present invention.

FIG. 5 is a plan view (A) showing an example of a membrane filter used in the present invention and a partially cutaway enlarged view (B) thereof.

FIG. 6 is a schematic diagram of a bioluminescence image analysis system used in the present invention.

[Explanation of symbols]

 1 Liquid Supply Tool 1-1 Liquid Absorbing Material Sheet 1-2 Cylindrical Support of Liquid Supply Tool 2 Chamber 2-1 Chamber Window 3 Vibrator 3-1 Guide 4 Membrane Filter 4-1 Membrane Filter Section 4-2 Partition wall of membrane filter 4-3 Membrane filter holder 5 Reagent bottle 6 Electromagnetic valve 7 Manifold 8 Needle 8-a Reagent dropping needle 8-b Reagent introducing needle 8-c Gas pressure intake needle 9 Chamber holder 10 Chamber Holder mount 11 Filter holder 12 Base plate 13 Suction hole 14 Rail 16 Shading housing 17 Tapered fiber 18 Ultra-sensitive TV camera 19 Camera controller 20 Image processor 21 Data analysis device 22 TV monitor

Claims (9)

[Claims] 1. A sprayer used for spraying an extractant and a luminescent reagent in a device for rapidly measuring the number of viable bacteria in a sample, wherein a vibrator provided with a large number of small holes is ultrasonicated. A sprayer for viable cell count measuring apparatus, which vibrates to atomize the liquid supplied onto the vibrator. 2. The sprayer for a viable cell count measuring apparatus according to claim 1, wherein a chamber is provided for efficiently suspending the fine particulate extractant or luminescent reagent atomized by the sprayer on the membrane filter. 3. The sprayer for a viable cell count measuring apparatus according to claim 2, wherein suction and decompression are performed from the bottom of the chamber. 4. The raw material according to any one of claims 1 to 3, wherein a liquid supply tool, which is obtained by adhering a liquid absorbing material sheet to a bottom portion of a tubular support, is attached to a liquid supply portion of a vibrator of a sprayer. A sprayer for bacteria count device. 5. The sprayer for a viable cell count measuring apparatus according to claim 4, wherein the tubular support of the liquid supply tool is made of plastic, and the liquid absorbing material sheet is a cellulosic filter. 6. An ultrasonic wave vibrates a filtering device equipped with a membrane filter for trapping viable bacteria in a specimen and a vibrator provided with a large number of small holes used for spraying an extracting agent and a luminescent reagent. And a bioluminescence image analyzer for measuring the viable cell count by highly sensitively detecting a light emission point on a membrane filter that has been subjected to a light emission treatment and a sprayer that atomizes the liquid supplied onto the vibrator. Bacteria count measuring device. 7. The viable cell count measuring device according to claim 6, wherein the membrane filter is a hydrophilic membrane filter having a hydrophilic filtration membrane section surrounded by a large number of hydrophobic section walls. 8. A sprayer comprising the chamber according to claim 2 and the liquid supply tool according to claim 4, and the suction and decompression according to claim 3 being performed. The viable cell count measuring device according to claim 6. 9. The bioluminescence image analysis device is a device comprising a filter holder, a light-shielding housing, a taper fiber, an ultra-sensitive television camera, a camera controller, an image processor, a data analysis device and a television monitor. Viable cell count measuring device.