JP2012034595A - Apparatus for counting number of microorganisms - Google Patents

Abstract

An object of the present invention is to provide an apparatus for measuring the number of microorganisms capable of shortening the collection time even when the number of microorganisms contained in a sample is small.
The bacterial count measuring apparatus of the present invention is provided in a measurement chamber 6 for measuring microorganisms contained in a sample solution, an inlet 7 provided in the measurement chamber 6, and in the measurement chamber. The measurement cell 1 having the outlet 8 through which the sample solution flows out and the measurement electrode 12 provided at the bottom of the measurement chamber 6, the measurement unit 14 connected to the measurement electrode 12 of the measurement cell 1, and the AC power supply unit 15. Then, the AC power supply unit 15 applies the first AC voltage V1 to the measurement electrode 12 to trap microorganisms, and then applies the pulse voltage V2 to the measurement electrode 12 to destroy the trapped microorganisms. Then, the 2nd alternating voltage V3 was applied to the measurement electrode 12, and it was set as the structure which measures the number of microorganisms, and made the 2nd alternating voltage V3 smaller than the 1st alternating voltage V1.
[Selection] Figure 1

Description

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

A conventional microorganism count measuring apparatus has the following configuration.
That is, the conventional microorganism count measuring apparatus includes a measurement cell, a measurement unit connected to the measurement electrode of the measurement cell, and an AC power supply unit. The measurement cell includes a measurement chamber for measuring microorganisms contained in the sample liquid, an inlet provided in the measurement chamber through which the sample liquid flows in, an outlet provided in the measurement chamber through which the sample liquid flows out, and a measurement chamber. And a measuring electrode provided at the bottom. Then, the AC power supply unit applies a first AC voltage to the measurement electrode to trap microorganisms, and then applies a pulse voltage to the measurement electrode to destroy the trapped microorganisms. Thereafter, the AC power supply unit applies a second AC voltage to the measurement electrode, measures an increase in conductance due to cytoplasmic outflow from the destroyed microorganism, and calculates the number of microorganisms (see, for example, Patent Document 1).

JP-A-11-127847

The above conventional example is very useful in that the number of microorganisms contained in a sample 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 measurement of microorganisms, the microorganisms are destroyed, the increase in conductance due to the outflow of the cytoplasm of the destroyed microorganisms is measured, and the number of microorganisms is calculated. At this time, the blank response accompanied by the increase in conductance Will also occur.

  In order to avoid the effect of this blank response and perform measurement appropriately, it is necessary to collect more bacteria and increase the conductance increase due to bacterial destruction, but the number of microorganisms contained in the sample This collection takes a lot of time because there are few. As a result, the conventional microorganism count measuring apparatus has a problem that the entire measurement time is increased.

  Accordingly, an object of the present invention is to provide a microorganism count measuring apparatus that can shorten the measurement time even when the number of microorganisms contained in a specimen is small.

  In order to achieve this object, the present invention includes a measurement cell, a measurement unit, and an AC power supply unit. The measurement cell has a measurement chamber for measuring microorganisms contained in the sample solution, and a measurement electrode provided in the measurement chamber. The measurement unit and the AC power supply unit are connected to the measurement electrode of this measurement cell. The AC power supply unit applies a first AC voltage to the measurement electrode to trap the microorganism, and then applies a pulse voltage to the measurement electrode to destroy the trapped microorganism, and then to the measurement electrode Then, a second AC voltage smaller than the first AC voltage is applied, and the number of microorganisms is measured in the measurement unit.

According to the present invention, the collection time can be shortened even when the number of microorganisms contained in the sample is small.
That is, in the present invention, since the second AC voltage for measuring the conductance is smaller than the first AC voltage, the increase in conductance due to the blank response can be reduced, and the blank response The influence can be suppressed.

Therefore, measurement is possible even when the increase in conductance due to destruction of microorganisms is small, and measurement is possible even if the number of microorganisms in the specimen is small.
As a result, the time for collecting the microorganisms contained in the specimen can be shortened, so that the overall measurement time can be shortened.

1 is a block diagram showing the entirety of an embodiment of the present invention. The expansion perspective view of the measurement electrode part. The principal part expanded sectional view at the time of the operation | movement. (A) (b) (c) is the principal part enlarged view at the time of the operation | movement. The figure which shows the voltage waveform at the time of the operation | movement. The figure which shows the blank response at the time of the operation | movement. The figure which shows the blank response at the time of the operation | movement, and a measured value. The principal part enlarged view at the time of the operation | movement.

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. Thus, a rectangular parallelepiped measuring chamber 6 is formed inside the measuring cell 1 and is thin in the vertical direction (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 flows out to the other end side of the ceiling portion of the measurement chamber 6. An 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 the sample reservoir 9 through the pump 11. Return to
A measurement electrode 12 for measuring the number of bacteria in the measurement liquid is provided on the bottom of the measurement chamber 6, that is, on the upper surface of the substrate 2. At the measurement electrode 12, bacteria in the measurement liquid are collected, the bacteria are destroyed, and the number of bacteria in the sample liquid is measured.

  The measurement electrode 12 is formed by vapor-depositing silver on PET used as the base material of the substrate 2 and processing it with a laser, for example. 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 opposed to each other over a long path, and by applying an alternating voltage thereto, an electric field is generated between them, and the dielectrophoresis phenomenon Thus, bacteria in the measurement liquid can be trapped and collected in the gap portion where the electrodes 12a and 12b face each other.

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 AC power supply unit 15 through the connection unit 13.
The 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. An 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 this 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 (vertical direction), from the inlet 7 in FIG. 1 toward the outlet 8, the upstream side (left hand side in FIG. 3) to the downstream side (right hand side in FIG. 3). The measuring solution flows toward.

A measurement electrode 12 is provided at the bottom of the measurement chamber 6. As a result, in the region where the bacteria in the measurement solution are trapped by the measurement electrode by the dielectrophoretic force generated by the electric field formed by the measurement electrode 12, that is, in the trap region 19, the bacteria passing therethrough are trapped and collected.
Here, the operation from the collection of bacteria to the measurement in the present embodiment will be described with reference to FIG. 1, FIG. 4, and FIG.

First, FIG. 4A to FIG. 4C are diagrams for explaining the state of bacteria collected on the measurement electrode 12. 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.
FIG. 5 shows the state of the voltage applied to the measurement electrode 12. P1 in FIG. 5 indicates a collection period, P2 indicates a bacteria destruction period, and P3 indicates a conductance measurement period.

In the present embodiment, for example, measurement of a test solution containing 100 E. coli per milliliter will be described as an example.
First, in the collection period P1, 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 through 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 AC power supply unit 15 that has received an instruction from the control calculation unit 16 in FIG. 1 applies the first AC voltage V1 for collection to the measurement electrode 12, as shown in FIG. Then, a trap region 19 is formed on the measurement electrode 12 by the electric field generated by the application of the first AC voltage V1, as shown in FIG. 3, and the bacteria of the measurement liquid passing through the trap region 19 undergo dielectrophoresis. It is trapped on the measuring electrode 12 by force and collected.

Thus, when the number of bacteria contained in the measurement solution is as very small as 100 per milliliter, conventionally a long collection time of several hours has been required.
In the bacterial count measuring apparatus of the present embodiment, the higher the voltage of the first AC voltage V1 for collecting bacteria, the larger the electric field, and the higher the trap region 19. For this reason, bacteria can be trapped efficiently, and as a result, the collection time can be shortened compared with the past.

In the present embodiment, as shown in FIG. 5, the first AC voltage V1 for collecting bacteria is applied to the measurement electrode 12 with a magnitude of, for example, 7 Vpp and 100 kHz.
Then, as shown in FIG. 4A, bacteria are collected on the measurement electrode 12. At this time, as shown in the lower part of FIG. 5, in the collection period P1, the conductance gradually increases due to the bacteria gathered on the measurement electrode 12, and at the end of the collection period P1, the collected collection conductance GT0 and Become. However, it is difficult to measure the number of bacteria contained in the measurement liquid from the change in conductance up to GT0. The reason is that since the number of bacteria contained in the measurement liquid is very small, the change up to GT0 is very slight, and the conductance change due to the temperature change of the measurement liquid is dominant, This is because it is very difficult to measure only the conductance change caused by bacteria.

  Next, the bacteria collected on the measurement electrode 12 are destroyed during the bacteria destruction period P2. At this time, the pulse voltage V2 for destroying the bacteria is applied to the measurement electrode 12 by the AC power supply unit 15 for 10 ms with a magnitude of, for example, 20 Vpp and 100 kHz. Then, in the measurement electrode 12, as shown in FIG. 4 (b), the cell membrane covering the outside of the bacteria is destroyed by applying a high energy pulse voltage, and many small holes are formed in the cell membrane. As shown in FIG. 4 (c), the cytoplasm of the bacteria is eluted outside through the small holes.

Since most of the eluted cytoplasm of the bacteria is a cytoplasm having high conductivity, the electrolyte concentration temporarily increases in the vicinity of the measurement electrode 12. This simultaneously means an increase in conductance measured at the measuring electrode 12.
Finally, in the conductance measurement period P3, the AC power supply unit 15 stops applying the pulse voltage V2 for destroying bacteria, and the second AC voltage V3 for measurement is, for example, 2.5 Vpp and 100 kHz. Apply to measurement electrode 12 for 3 seconds. Then, since the cytoplasm of the bacteria was eluted, the electrolyte concentration rapidly increased in the vicinity of the measurement electrode 12 at the start of the measurement period P3. That is, the conductance in the vicinity of the measurement electrode 12 is increased.

Then, the conductance is measured by the measurement unit 14 in FIG. 1 using the measurement electrode 12 and sent to the control calculation unit 16 as the conductance increase GTP.
The control calculation unit 16 refers to a table (not shown) of the conductance increase GTP and the number of bacteria prepared in advance based on the sent conductance increase GTP. The number of bacteria is calculated, and the display unit 17 displays the result.
In the present embodiment, for example, 1 × 10 2 (cfu / ml) is displayed on the display unit 17.

The main characteristic point in this embodiment is that the second AC voltage V3 for measurement is smaller than the first AC voltage V1 for collecting bacteria.
The purpose is to suppress the influence of the blank response as a disturbance when measuring the conductance.

First, this blank response will be described below.
FIG. 6 is a diagram for explaining the influence of this blank response. Here, pure water is used as a sample solution, and a first AC voltage V1 for collecting bacteria, a pulse voltage V2 for destroying bacteria, and a second AC voltage V3 for measuring conductance are sequentially applied to the measurement electrode 12. The obtained conductance increase value GTP is shown on the vertical axis.

In FIG. 6, the horizontal axis represents that the second AC voltage V3 for measurement was measured with two types of voltages of 5 Vpp and 2.5 Vpp. Each of these measurements was performed seven times, and the average values were represented as blank values B1 and B2 in a bar graph, and the variation values E1 and E2 were represented using thin lines.
Here, when pure water is used as the sample liquid, since the pure water is not energized, the blank values B1 and B2 are supposed to be zero. However, in reality, reactions of blank values B1 and B2 and variation values E1 and E2 have come out as blank responses.

The reason for this has not yet been elucidated, but it is thought that the following is probably occurring in the vicinity of the measurement electrode 12.
That is, when destroying bacteria, a pulse voltage V2 for destroying bacteria is applied to the measurement electrode 12, and this pulse voltage is applied to the first alternating voltage V1 for collecting bacteria or the second voltage for measurement. Since the amplitude is larger than the sine wave AC voltage of the AC voltage V3 and it is a rectangular wave, it has much larger energy than the sine wave. Due to this large energy, an electric field strong enough to destroy the trapped bacteria is formed on the comb-shaped electrodes 12a and 12b constituting the measurement electrode 12.

At this time, as shown in FIG. 8, a strong electric field is concentrated on the edge portions of the comb-shaped electrodes 12a and 12b, and the metal ions of the comb-shaped electrodes 12a and 12b are formed from the edge portions. It is thought that it is eluted in pure water one after another, and conductance appears as a blank response through this metal ion.
Thereafter, when an AC voltage V3 for conductance measurement is applied in this state, a current flows in the pure water through the eluted metal ions, and the local pure water is near the comb-shaped electrodes 12a and 12b. This will cause an increase in temperature.

Then, due to this temperature rise, the conductance of pure water further increases, and it is presumed that the conductance is measured as shown in FIG.
In the measurement of the number of bacteria in this embodiment, the increase in conductance due to the destruction of bacteria is measured, and the number of bacteria is calculated based on the value. Therefore, the increase in conductance due to factors other than the conductance of bacteria is This is a very unfavorable state. That is, it is preferable that the blank response in FIG. 6 is eliminated during measurement.

As shown in FIG. 6, when the AC voltage V3 for measuring conductance is increased to 5 Vpp, the blank value B1 indicating the increase in conductance increases and the variation value E1 also increases.
Conversely, when the AC voltage V3 is reduced to 2.5 Vpp, the blank value B2 is reduced and the variation value E2 is also reduced.

That is, it can be seen that the current flowing through the metal ions changes according to the magnitude of the AC voltage V3, and the blank values B1 and B2 and the variation values E1 and E2 change.
Therefore, in the present embodiment, the measurement AC voltage V3 is made smaller than the first AC voltage V1 for collecting bacteria to suppress the influence of the blank response.
Moreover, FIG. 7 is a figure explaining the influence of this blank response, In addition to the blank response of FIG. 6, the value which measured E. coli was added.

In the actual measurement example of Escherichia coli, the same first AC voltage V1 for collecting bacteria and the pulse voltage V2 for destroying bacteria as in the two examples (5 Vpp and 2.5 Vpp) measured in the blank were used. Then, the AC voltage V3 for measuring the conductance is measured at 2.5 Vpp, the average value of the three measurements is represented as a measured value R1 by a bar graph, and the variation value E3 is represented by a thin line.
In FIG. 7, the thin line indicating the variation value E1 of the blank response at 5 Vpp overlaps with the variation value E3 of E. coli.

That is, in this case, the actual measurement of E. coli is indistinguishable from the blank response, and in order to carry out the measurement appropriately, it is necessary to collect more bacteria and measure the increase in conductance.
In other words, it can be seen that in the measurement of 5 Vpp, it is necessary to spend more time on collecting the bacteria.

On the other hand, the size of the fine line of the variation value E2 of the blank measurement at 2.5 Vpp is suppressed as compared with the thin line indicating the variation of the blank response at 5 Vpp. As a result, there is no place overlapping with the thin line indicating the measured variation value E3 of E. coli, that is, the actual measurement of E. coli in this case indicates that an appropriate measurement has been performed.
Therefore, in this embodiment, since the 2nd alternating voltage used for conductance measurement is made smaller than the 1st alternating voltage at the time of a trap, the electric current which flows into the metal ion mentioned above can be made small. As a result, the temperature increase of the sample solution can be suppressed, the influence of the blank response can be suppressed, and the number of bacteria can be accurately measured.

As a result, since the influence of the blank response could be suppressed, the measurement can be performed even when the increase in conductance due to the destruction of the bacteria is small, and the number of bacteria can be accurately measured even if the number of microbacteria to be collected is small. .
As a result, even when the number of bacteria contained in the sample solution is small, the time for collecting the bacteria on the measurement electrode 12 can be shortened, so that the entire measurement time can be shortened.

  As described above, the present invention is expected to be widely used as an apparatus for measuring the number of microorganisms because it has the effect of shortening the collection time even when the number of microorganisms contained in the sample is small. .

DESCRIPTION OF SYMBOLS 1 Measurement cell 2 Board | substrate 3 Through-hole 4 Spacer 5 Cover body 6 Measurement chamber 7 Inlet 8 Outlet 9 Sample reservoir 10 Water supply tube 11 Pump 12 Measurement electrode 12a, 12b Comb-shaped electrode 13 Connection part 14 Measurement part 15 AC power supply Section 16 Control operation section 17 Display section 18 Operation section 19 Trap region V1 First AC voltage V2 Pulse voltage V3 AC voltage P1 Bacteria collection period P2 Destruction period P3 Measurement period GTP conductance increase B1, B2 Blank value E1, E2 Variation value

Claims (3)

A measurement cell having a measurement chamber for measuring microorganisms contained in the sample liquid, and a measurement electrode provided in the measurement chamber;
A measurement unit and an AC power supply unit connected to the measurement electrode of the measurement cell;
With
The AC power supply unit applies a first AC voltage to the measurement electrode to trap microorganisms, then applies a pulse voltage to the measurement electrode to destroy the trapped microorganisms, and then Applying a second AC voltage smaller than the first AC voltage to the measurement electrode, and measuring the number of microorganisms in the measurement unit;
Microbe count measuring device. The measurement cell has an inlet that allows the sample liquid to flow into the measurement chamber, and an outlet that allows the sample liquid to flow out of the measurement chamber.
The microorganism count measuring apparatus according to claim 1. A sample reservoir for storing the sample solution is connected to the inlet, and a return path to the sample reservoir is provided at the outlet.
The microorganism count measuring apparatus according to claim 2.

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