JP2012037285A - Cell for measuring the number of microbes and instrument including the same for measuring the number of microbes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell for measuring the number of microbes and an instrument including the same for measuring the number of microbes, which allow reduction of measurement time even in the case that the number of microbes included in a specimen is small.SOLUTION: The cell for measuring the number of microbes includes a measurement chamber 6 for measuring the number of microbes included in a sample liquid, an inflow port 7 and an outflow port 8 provided for the measurement chamber 6, and a measuring electrode 12. A ceiling portion of the measurement chamber 6 is provided with a projecting wall 20 projecting toward a bottom portion, from the upstream side to the downstream side in a direction from the inflow port 7 to the outflow port 8. Both sides of a projecting lower end of the projecting wall 20 are formed into rising walls rising upward via edge parts, and a concentration electrode 21 is provided on a lower end surface of the projecting wall 20. The concentration electrode 21 is so formed that a height of the concentration electrode 21 from the bottom portion of the measurement chamber is reduced from the inflow port 7 toward the outflow port 8.

Description

  The present invention relates to a microorganism count measuring cell for measuring the number of microorganisms contained in a specimen and a microorganism count measuring apparatus using the same.

  The configuration of the conventional cell for measuring the number of microorganisms and the device for measuring the number of microorganisms using the same have the following configurations.

  That is, a conventional cell for measuring the number of microorganisms includes a measurement chamber that measures microorganisms contained in a sample solution, an inlet that allows the sample solution to flow into the measurement chamber, an outlet that allows the sample solution to flow out of the measurement chamber, and a measurement chamber. And a measuring electrode provided at the bottom of the.

  In addition, a conventional microorganism count measuring apparatus using a microorganism count measuring cell includes a measurement unit and an AC power supply unit connected to the measurement electrode of the microorganism count measuring cell. In the conventional microorganism count measuring apparatus, the AC power supply unit applies an AC voltage for collecting bacteria to the measurement electrode to trap the microorganism, and then applies a pulse voltage to the measurement electrode to destroy the trapped microorganism. To do. Thereafter, the AC power supply unit applies an AC voltage for measurement to the measurement electrode, measures the increase in conductance due to the outflow of cytoplasm from the destroyed microorganism, and calculates the number of microorganisms (see, for example, Patent Document 1). .

JP-A-11-127847

  The conventional configuration described above is very useful in that the number of microorganisms contained in the specimen can be measured. On the other hand, in the measurement, there has been an increasing demand for higher sensitivity, that is, to accurately measure the number of microorganisms even when the number of microorganisms contained in the sample is small.

In order to meet this demand, there is a problem that the measurement time becomes long.
That is, in the conventional microorganism measurement, microorganisms are destroyed, the increase in conductance due to the outflow of cytoplasm of the destroyed microorganisms is measured, and the number of microorganisms is calculated.

  At this time, in order to obtain a sufficient increase in conductance, it is necessary to collect an appropriate number of microorganisms. However, when the number of microorganisms contained in the sample solution is small, this collection takes a long time. As a result, the conventional microorganism count measuring apparatus has a problem that the entire measurement time is increased.

  In view of the above, an object of the present invention is to provide a microorganism count measuring cell and a microorganism count measuring apparatus having the same, which can shorten the measurement time even when the number of microorganisms contained in the sample is small. is there.

  In order to achieve this object, the microorganism count measurement cell of the present invention includes a measurement chamber, an inflow port, an outflow port, a measurement electrode, a protruding wall, a rising wall, and a concentration electrode. The measurement chamber measures the number of microorganisms contained in the sample solution. The inlet allows the sample liquid to flow into the measurement chamber. The outlet allows the sample liquid to flow out of the measurement chamber. The measurement electrode is provided at the bottom of the measurement chamber on the outlet side. The protruding wall is provided on the ceiling of the measurement chamber, and protrudes in the direction of the bottom from the upstream side corresponding to the outlet to the downstream side. The rising wall is formed on both sides of the lower end of the protruding wall and rises upward via the edge portion. The concentration electrode is formed so that the height from the bottom of the measurement chamber decreases from the inlet to the outlet at the lower end surface of the protruding wall.

  According to the present invention, since the time for collecting the number of microorganisms necessary for measurement can be shortened, the measurement time can be shortened as compared with the prior art.

1 is a block diagram showing the entirety of an embodiment of the present invention. The enlarged view of the measurement electrode. The principal part expanded sectional view at the time of the operation | movement. (A) (b) (c) is the principal part enlarged view of the measurement electrode at the time of the operation | movement. The principal part sectional view which looked at the time of the operation from the side. The principal part sectional view which looked at the time of the operation from the inflow side. Figure of the ceiling of the measurement room. The figure of the bottom part of the measurement chamber. The principal part sectional view which looked at the time of operation of other embodiments of the present invention from the side. The figure which shows the ceiling of the measurement chamber.

  Hereinafter, a bacterial count measuring apparatus (microorganism count measuring apparatus) according to an embodiment of the present invention will be described with reference to the accompanying drawings.

  FIG. 1 shows a bacterial count measuring device for measuring the number of microorganisms, for example bacteria. 1 in FIG. 1 is a measurement cell for measuring the number of bacteria.

  The measurement cell 1 is obtained by laminating a thin plate-like spacer 4 having a rectangular through hole 3 in the center on a rectangular substrate 2 and laminating a rectangular lid 5 on the spacer 4. It is. Thereby, a thin rectangular-shaped measurement chamber 6 is formed in the measurement cell 1 in the vertical direction.

  Further, an inlet 7 for allowing a sample solution containing bacteria to flow in is provided at one end side of the ceiling portion of the measurement chamber 6, and the sample solution is provided at the other end side of the ceiling portion of the measurement chamber 6. An outflow outlet 8 is provided. The inflow port 7 and the outflow port 8 are configured to protrude from the upper portion of the lid 5.

  Further, a sample reservoir 9 containing a sample solution is connected to the inflow port 7 via a water supply tube 10. On the other hand, a sample reservoir 9 is connected to the outlet 8 via a pump 11 and a water supply tube 10. Thereby, a return path for the sample liquid in the measurement chamber 6 to return to the specimen reservoir 9 is formed.

  Here, when the pump 11 is operated, the sample liquid stored in the specimen reservoir 9 flows into the measurement chamber 6 from the inlet 7 at the upper part of the lid 5 through the water supply tube 10. Then, the sample liquid flows from the left hand to the right hand in FIG. 1 in the measurement chamber 6, and then flows out from the measurement chamber 6 through the outlet 8, and then to the sample reservoir 9 through the pump 11. And return.

  A measurement electrode 12 for measuring the number of bacteria in the measurement liquid is provided at the bottom of the measurement chamber 6, that is, the upper surface of the substrate 2, and the bacteria in the measurement liquid are collected at the measurement electrode 12. The bacteria are destroyed and the number of bacteria in the sample solution is measured.

  The measurement electrode 12 is formed by vapor deposition of silver on the PET used as the base material of the substrate 2 and, for example, processed by a laser. As shown in FIG. 2, the measurement electrode 12 is composed of comb-shaped electrodes 12a and 12b. These comb-like electrodes 12a and 12b are in an opposed state in which they are extremely close to each other over a long path, and by applying an alternating voltage thereto, an electric field that generates positive dielectrophoresis between the two is provided. Form. Then, bacteria in the measurement liquid can be attracted and trapped in the gap portion where the electrodes 12a and 12b are opposed to each other by the dielectrophoresis phenomenon to collect the bacteria.

  Further, the comb-like electrodes 12 a and 12 b are each drawn from the measurement electrode 12 and connected to a connection portion 13 provided at an end portion of the substrate 2 as shown in FIG. 1. The comb-shaped electrodes 12 a and 12 b are connected to the measurement unit 14 and the measurement AC power supply unit (detection AC power supply unit) 15 through the connection unit 13.

  Then, the measurement AC power supply unit 15 applies an AC voltage to the measurement electrode 12 of the measurement cell 1, and the conductance of the measurement electrode 12 is measured by the measurement unit 14. Next, the measurement data is sent to the control calculation unit 16, and the control calculation unit 16 calculates and calculates the number of bacteria. Finally, the calculation result is displayed on the display unit 17.

  An operation unit 18 that gives instructions for these measurements is connected to the control calculation unit 16. A measurement AC power supply unit 15 is connected to the control calculation unit 16.

FIG. 3 is a diagram illustrating a state in which the measurement liquid flows in the measurement chamber 6.
In the present embodiment, since the thickness of the spacer 4 in FIG. 1 is, for example, 250 μm, the height of the measurement chamber 6 is 250 μm. In the measurement chamber 6 narrow in the vertical direction, from the inlet 7 in FIG. 1 toward the outlet 8, from the upstream side (left hand side in FIG. 3) to the downstream side (right hand side in FIG. 3). The measuring solution flows.

  A measurement electrode 12 is provided at the bottom of the measurement chamber 6. The measurement AC power supply unit 15 applies an appropriate AC voltage to the measurement electrode 12 to form an electric field that generates positive dielectrophoresis, thereby forming a trap region 19 for trapping bacteria. .

  The bacteria entering the trap region are attracted and trapped by the dielectrophoresis phenomenon and collected.

  FIG. 4 is a diagram for explaining the flow from the collection of bacteria to the measurement. 4A shows a state when bacteria are trapped on the measurement electrode 12, FIG. 4B shows a state when the trapped bacteria are destroyed, and FIG. 4C shows FIG. This shows a state when the measuring unit 14 is measuring the number of bacteria.

  Since this measurement method is a known method and described in the patent documents cited as prior art documents, a detailed description thereof will be omitted.

  When measuring the number of bacteria, first, the measurement AC power supply unit 15 applies an AC voltage for collection of bacteria to the measurement electrode 12 according to an instruction from the control calculation unit 16, and the electric field generated by this application causes a change in FIG. As shown in (a), bacteria are trapped on the measurement electrode 12.

  Next, when the measurement AC power supply unit 15 applies a pulse voltage for destroying the bacteria to the measurement electrode 12, the pulse voltage having a large energy is applied, and as shown in FIG. The cell membrane covering the cell is destroyed, and a large number of small holes are formed in the cell membrane. And as shown in FIG.4 (c), the cytoplasm of the content of bacteria elutes outside through this small hole.

  Since most of the eluted cytoplasm is a cytoplasm having high conductivity, the electrolyte concentration, that is, the conductance temporarily increases in the vicinity of the measurement electrode 12. The increased conductance is measured by the measurement AC power supply unit 15 by applying an AC voltage for measurement to the measurement electrode 12 and measured by the measurement unit 14. Thereafter, the increased conductance value is sent to the control calculation unit 16, and the number of bacteria is estimated from the maximum value of the increased conductance.

  Although the basic configuration and operation in the present embodiment have been described above, the main characteristic points of the bacterial count measuring apparatus according to the present embodiment will be described. That is, as shown in FIG. 5, the main feature of this embodiment is that the upstream side corresponding to the outlet 8 from the inlet 7 in FIG. That is, a projecting wall 20 projecting toward the bottom, that is, toward the substrate 2 is provided from the downstream side to the downstream side, and the concentration electrode 21 is provided on the lower end surface of the projecting wall.

  That is, in this embodiment, an alternating voltage is applied to the concentration electrode 21 to form an electric field that generates negative dielectrophoresis, that is, a block electric field 22 that blocks microorganisms from entering the protruding wall. Then, the bacteria in the measurement liquid flowing in from the inlet 7 are collected by being pushed down toward the bottom while blocking with the blocking electric field 22 and trapped in the measurement electrode 12 provided at the bottom of the measurement chamber 6. To do.

These contents will be described in more detail with reference to FIGS.
FIG. 5 is a cross-sectional view of the measuring cell 1 as viewed from the side from the upstream side to the downstream side corresponding to the outlet 8 from the inlet 7 of the measuring cell 1. FIG. 6 shows a cross-sectional view seen from the A1-A2 direction in FIG.

  As shown in FIG. 6, the projecting lower end of the projecting wall 20 is a right-angled edge portion 20 </ b> A on both sides. The both side surfaces of the protruding wall 20 are rising walls that rise upward via the edge portion 20A.

  A concentration electrode 21 is provided on the lower end surface of the protruding wall 20. As shown in FIG. 5, the concentration electrode 21 covers the entire lower end surface of the protruding wall 20.

  The concentration electrode 21 is formed along a continuous inclined surface in which the height from the bottom of the measurement chamber 6 decreases from the inlet 7 toward the outlet 8.

  Furthermore, a plurality of protruding walls 20 are provided in parallel on the ceiling of the measurement chamber 6 in parallel to the flow from the upstream side to the downstream side. In this embodiment, it is set as the structure by which the ceiling part of the measurement chamber 6 was covered by these protrusion walls 20, and it has a slit-shaped structure as shown in FIG.

  FIG. 7 is a diagram illustrating a state in which the ceiling of the measurement chamber 6 is looked up from the inside of the measurement chamber 6. As described above, for example, six protruding walls 20 are provided at predetermined intervals in the ceiling portion of the measurement chamber 6. And the concentration electrode 21 provided in the lower end surface gathers, and the concentration electrode part 23 is formed.

  In the concentration electrode part 23, the adjacent concentration electrodes 21 at the lower end of the protruding wall 20 are arranged with different polarities.

  More specifically, as shown in FIG. 5, the first, third, and fifth concentration electrodes 21 from the top in FIG. As shown, it is coupled at the ceiling of the measurement chamber 6 and connected to the ground via a connection terminal 24.

  On the other hand, the second, fourth, and sixth concentrating electrodes 21 from the top in FIG. 7 are coupled at the ceiling portion on the upstream side, connected to the concentrating AC power supply unit 26 via the connection terminal 25, and then connected to the ground. It is connected.

  Therefore, when an alternating voltage is applied to the concentration electrode portion 23, the adjacent concentration electrodes 21 at the lower end of the protruding wall 20 have different polarities.

  As shown in FIG. 1, the concentrated AC power supply unit 26 is connected to the control calculation unit 16 and operates in response to an instruction from the control calculation unit 16.

  FIG. 8 is a view showing the bottom of the measurement chamber 6. The measurement unit 12 shown in FIG. 2 is provided on the downstream side (right hand side in FIG. 8). As shown in FIG. 8, the comb-like electrode 12 a forming the measurement unit 2 is connected to the measurement AC power supply unit 15 via the connection terminal 27 provided on the connection unit 13 and then connected to the ground. Yes.

  The other comb-like electrode 12 b is connected to the ground via a connection terminal 28 provided on the connection portion 13.

The operation of the above configuration will be described in detail.
First, when measuring bacteria, the pump 11 in FIG. 1 is activated, and the measurement liquid stored in the sample reservoir 9 is caused to flow into the measurement chamber 6 via the inflow port 7. Then, as shown in FIG. 3, the measurement liquid flows on the measurement electrode 12 provided at the bottom of the measurement chamber 6.

  At this time, the measurement AC power supply unit 15 that has received an instruction from the control calculation unit 16 in FIG. 1 applies an AC voltage for collection to the measurement electrode 12. Then, an electric field that generates positive dielectrophoresis is created in the measurement electrode 12 by applying an alternating voltage, and a trap region 19 is formed as shown in FIG. Thereby, the bacteria that have entered the trap region 19 can be trapped and collected.

  Further, at this time, the concentrated AC power supply unit 26 applies an AC voltage that causes negative dielectrophoresis to the concentrated electrode 21.

  Then, as shown in FIG. 6, since both sides at the lower end of the protruding wall 20 are right-angled edge portions 20A, the negative electrode from the edge of the concentration electrode 21 provided on the lower end surface of the protruding wall 20 toward the bottom portion. The electric field that generates the dielectrophoresis of, ie, the blocking electric field 22 that blocks the microorganisms from entering the protruding wall side is formed.

  Since this electric field generates negative dielectrophoresis, it has a property opposite to that of the positive dielectrophoresis generated by the measurement electrode 12.

  In other words, the measurement electrode 12 exerts a force that attracts bacteria to the measurement electrode 12, but conversely, the concentration electrode 21 exerts a force that moves the bacteria away from the concentration electrode 21. As a result, the bacteria are blocked by the electric field and cannot enter into the electric field.

  Here, in the present embodiment, as shown in FIG. 7, the concentrated electrode 21 gathers on the ceiling portion of the measurement chamber 6 to form the concentrated electrode portion 23. Then, by applying an alternating voltage that generates negative dielectrophoresis to the concentrated electrode portion 23, an electric field that generates negative dielectrophoresis is created in the entire concentrated electrode portion 23. As a result, a plurality of electric fields are connected to form a block electric field surface 22A that covers the entire concentration electrode portion 23, and bacteria are not allowed to enter the block electric field surface 22A (in FIG. 6, above the block electric field surface 22A).

  Then, as shown in FIG. 5, bacteria contained in the measurement liquid flowing in from the inlet 7 are blocked by the blocking electric field surface 22A.

  At this time, as shown in FIG. 5, the height of the concentration electrode 21 from the bottom of the measurement chamber 6 is formed along a continuous inclined surface that decreases from the inlet 7 toward the outlet 8. . For this reason, the bacteria blocked by the blocking electric field surface 22A are pushed down toward the bottom and collected while being blocked by the blocking electric field surface 22A as they approach the outlet 8.

  Then, at the collected tip, that is, on the outlet 8 side of the concentration electrode 21, the measurement electrode 12 provided at the bottom of the measurement chamber 6 forms a trap region 19 for trapping bacteria. For this reason, bacteria (microorganisms) entering the trap region 19 can be efficiently attracted to the measurement electrode 12 and trapped.

  As a result, the time required for collecting the number of bacteria necessary for the measurement of the number of bacteria can be shortened, so that the entire measurement time can be shortened even when the number of bacteria in the sample solution is small.

  Furthermore, as shown in FIG. 5, the concentration electrode 21 has a height from the bottom of the measurement chamber 6 at the end on the outlet 8 side, and is provided at the bottom of the measurement chamber 6 on the downstream side of the concentration electrode 21. The height of the trap region 19 formed by the measurement electrode 12 is substantially the same.

  For this reason, the bacteria blocked by the blocking electric field surface 22A and guided to the bottom are surely guided to the trap region 19 and can be efficiently attracted and trapped on the measurement electrode 12.

  As a result, the time required for collecting the number of bacteria necessary for the measurement of the number of bacteria can be shortened compared to the conventional method, and the overall measurement time can be shortened.

  Further, as shown in FIG. 6, the protruding wall 20 provided with the concentration electrode 21 has a plurality of rows arranged in a slit structure with a predetermined interval therebetween. Thereby, a sample liquid can pass between the adjacent protruding walls 20 and the protruding walls 20, and the flow rate of the sample liquid can be increased. Therefore, the number of bacteria sent into the measurement chamber 6 can be increased. In addition, although the cross-sectional shape of the protrusion wall 20 can also be made into the trapezoid shape where the width | variety by the side of the concentration electrode 21 is narrow, in this embodiment, the protrusion wall 20 is made into the rectangular shape. Thereby, compared with trapezoid shape etc., the flow volume of the sample liquid which passes between the protrusion walls 20 can be enlarged.

  Even in this case, an electric field is generated from the edge of the concentration electrode 21 provided on the lower end surface of the protruding wall 20, and a block electric field surface 22A is formed as shown in FIG. For this reason, only bacteria are blocked by the blocking electric field surface 22A and guided toward the bottom.

Thereby, bacteria do not enter between the protruding wall 20 and the protruding wall 20.
As a result, the flow rate of the sample solution can be increased and the number of bacteria sent into the measurement chamber 6 can be increased. Therefore, the time for collecting the number of bacteria necessary for the measurement of the number of bacteria can be shortened, and the measurement time can be shortened as compared with the prior art.

(Other embodiments)
FIG. 9 shows a configuration of a bacterial count measuring cell according to another embodiment of the present invention. Here, instead of the protruding wall 20 formed along the continuous inclined surface shown in FIG. 6, a rectangular protruding wall 120 is provided, and a concentrated comb electrode portion composed of comb electrodes on the upstream side thereof. 129 is provided. Then, the height of the concentration electrode 121 from the bottom of the measurement chamber 106 is decreased stepwise from the inlet 107 toward the outlet 108.

  FIG. 10 shows a configuration of the concentration electrode 121, and a concentration comb electrode portion 129 is provided on the inlet 107 side of the concentration electrode 121. The concentrated comb electrode portion 129 is composed of comb-like electrodes 129a and 129b.

  The comb-like electrodes 129a and 129b are respectively connected to the concentration electrode 121 on the lower end surface 120a of the rectangular protruding wall 120 provided on the outflow port 108 side to form the concentration electrode portion 123.

  Also in the present embodiment, as shown in FIG. 9, bacteria in the sample liquid are guided to the trap region 119 at the bottom of the measurement chamber 106 by the block electric field surface 122A, and can be efficiently attracted and trapped on the measurement electrode 112. it can.

  As a result, the time for collecting the number of bacteria necessary for the measurement of the number of bacteria can be shortened, and the overall measurement time can be shortened.

  The present invention exerts an effect that the collection time can be shortened even when the number of microorganisms contained in the sample is small. Therefore, the present invention is widely used as a microorganism count measuring cell and a microorganism count measuring apparatus using the same. Expected.

DESCRIPTION OF SYMBOLS 1 Measurement cell 2 Substrate 3 Through-hole 4 Spacer 5 Lid 6 Measurement chamber 7 Inlet 8 Outlet 9 Specimen reservoir 10 Water supply tube 11 Pump 12 Measurement electrode 12a, 12b Comb-like electrode 13 Connection part 14 Measurement part 15 Measurement AC Power supply (detection AC power supply)
DESCRIPTION OF SYMBOLS 16 Control calculating part 17 Display part 18 Operation part 19 Trap area | region 20 Projection wall 20A Edge part 21 Concentration electrode 22 Block electric field 22A Block electric field surface 23 Concentration electrode part 24 Connection terminal 25 Connection terminal 26 Concentration alternating current power supply part 27 Connection terminal 28 Connection terminal 29 Concentrated comb electrode part 29a, 29b Electrode

Claims (7)

A measurement chamber for measuring the number of microorganisms contained in the sample solution;
An inlet for allowing the sample liquid to flow into the measurement chamber;
An outlet through which the sample liquid flows out of the measurement chamber;
A measurement electrode provided at the bottom on the outlet side in the measurement chamber;
A projecting wall that is provided on the ceiling of the measurement chamber and projects in the direction of the bottom from the upstream side to the downstream side corresponding to the outlet,
A rising wall formed on both sides of the lower end of the protruding wall and rising upward via an edge portion;
A concentration electrode formed on the lower end surface of the projecting wall such that the height from the bottom of the measurement chamber decreases from the inlet toward the outlet;
A cell for measuring the number of microorganisms. The concentration electrode is formed along a continuous inclined surface in which the height from the bottom of the measurement chamber decreases from the inlet toward the outlet.
The cell for measuring the number of microorganisms according to claim 1. The concentration electrode is formed on the outlet side so that the height from the bottom of the measurement chamber is substantially equal to the height of the trap region formed by the measurement electrode on the downstream side.
The cell for measuring the number of microorganisms according to claim 2. The concentration electrode is formed such that the height from the bottom of the measurement chamber decreases stepwise from the inlet to the outlet.
The cell for measuring the number of microorganisms according to claim 1. The ceiling portion is provided with a plurality of the protruding walls in parallel to the flow from the upstream side to the downstream side,
The cell for measuring the number of microorganisms according to any one of claims 1 to 4. The concentrating electrodes adjacent at the lower end of the protruding wall have different polarities from each other,
The cell for measuring the number of microorganisms according to claim 5. A cell for measuring the number of microorganisms according to any one of claims 1 to 6,
A specimen reservoir connected to the inlet of the cell for measuring the number of microorganisms;
A return path to the specimen reservoir connected to the outlet of the cell for measuring the number of microorganisms;
A measurement unit and a detection AC power supply unit connected to the measurement electrode of the cell for measuring the number of microorganisms
A control calculation unit connected to the measurement unit and the detection AC power source unit;
A concentration alternating current power source connected to the concentration electrode of the cell for measuring the number of microorganisms;
A microbe count measuring apparatus comprising:

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