JP2012228183A - Biological activity measuring device and method for estimating minimum growth inhibiting concentration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biological activity measuring device capable of quickly eliminating heat (hot heat or cold heat) carried from the outside along with the installation of samples into the device and capable of shortening a time required for measuring a metabolic activity or a proliferating activity of the biological cells principled by detecting the metabolic heat of biological cells.SOLUTION: The heat (hot heat or cold heat) taken into a constant temperature chamber 10 when installing a sample container 17 can be quickly eliminated by heating/cooling the sample container 17 by applying DC current to a sensor 7 such that the temperature difference between the sample chamber 17 and a constant temperature container 13 becomes small based on a temperature measuring result of the sensor 7 (peltiert element) installed while being sandwiched by the sample container 17 and the constant temperature container 13. Consequently, The measurement of metabolic heat shown by microorganism cells can be dramatically quickly carried out compared to a conventional method.

Description

  The present invention relates to a biological activity measuring device based on the principle of measuring heat generated with the metabolic activity of biological cells, and more particularly to a biological activity measuring device that can shorten the time required for measurement.

  Conventionally, (1) optical method, (2) impedance measurement method, (3) agar plate culture method, etc. are known as means for tracking the growth of microorganisms. Among the above, the optical method (1) is limited to a sample that transmits light, and the impedance measurement method (2) is limited to a sample having electrical conductivity. There are certain restrictions on the concentration of microorganisms that can be measured, that is, the dynamic range. Therefore, in general, the agar plate culture method (3), which was founded 200 years ago by Robert Koch, Germany, is employed. However, the agar plate culture method based on the culture of microorganisms on a petri dish consumes an enormous amount of plastic petri dishes, and the culture time is very long (usually 24 hours or 48 hours). There is a problem that it is too wasteful in terms of labor required for culturing microorganisms.

  As a device for solving the problems in the conventional method for measuring microbial activity as described above, an apparatus described in Patent Document 1 below is known. This device is based on the detection of heat generated by metabolic activity of microbial cells, and can obtain highly reliable information that faithfully quantifies the metabolic activity of microbial cells. In addition, the measurement can be carried out very easily as compared with the agar plate culture method (3) using a large amount of petri dish.

Japanese Patent No. 1903288

  By the way, in order to use an apparatus for detecting metabolic heat of microbial cells as shown in Patent Document 1, it is necessary to prepare and prepare a target microbial cell sample and then introduce and install it in the apparatus. is there. At that time, it is inevitable that the thermal equilibrium state in the apparatus is disturbed by heat (hot or cold) brought into the apparatus from the outside together with the microorganism cell sample. That is, as long as the introduced heat does not completely disappear even though the measurement device is based on the detection of metabolic fever exhibited by microbial cells, the device signal displayed by the measurement device reflects the metabolic fever derived only from microbial cells. In other words, it does not show the exact metabolic activity of microbial cells. Usually, it takes about 3 hours even if it is short after the sample is installed, and about 10 hours if it is long, and the measurement is started after the heat brought in from the outside is completely lost. It is. This time depends on factors such as the state and properties of the sample, and the material and structure of the measuring apparatus on which the sample is installed.

  The present invention has been made in view of such circumstances, and an object thereof is to quickly erase heat (hot or cold) brought in from the outside when a sample is placed in the apparatus, etc. It is an object of the present invention to provide a biological activity measuring apparatus capable of shortening the time required for measuring the metabolic activity or proliferation activity of a biological cell based on the principle of detection.

  A first aspect of the present invention relates to a biological activity measurement apparatus that measures heat generated by biological cells contained in a sample. This biological activity measurement device is provided with a thermostat and a plurality of measurement cells that are arranged in the thermostat, are formed by a block of a high thermal conductivity material, and contain a sample container in which a sample to be measured is placed. A constant temperature container, a first surface that contacts the constant temperature container, and a second surface that is in contact with the constant temperature container and is disposed between the constant temperature container and the sample container inside each of the plurality of measurement cells. A plurality of thermoelectric elements each generating an electromotive force according to a temperature difference from the surface, a measuring unit for measuring each electromotive force generated by the plurality of thermoelectric elements, and applying to the plurality of thermoelectric elements A voltage output unit that outputs each DC voltage, a switch unit that connects the plurality of thermoelectric elements to the measurement unit or the voltage output unit, and the thermoelectric element alternately to the measurement unit and the voltage output unit Connect Controlling the switch unit, controlling the measurement unit to measure the electromotive force during a period in which the thermoelectric element and the measurement unit are connected, and the electromotive force measured in the measurement unit is the thermostatic container If the temperature of the sample container is lower than the voltage, the voltage is output so as to output a DC voltage having a polarity that releases heat from the second surface during a period in which the thermoelectric element and the voltage output unit are connected. When the output unit is controlled and the measured electromotive force indicates that the temperature of the sample container is higher than that of the thermostatic container, the second surface is connected during a period in which the thermoelectric element and the voltage output unit are connected. And a control unit that performs a series of thermal control processes for controlling the voltage output unit so as to output a DC voltage having a polarity that absorbs heat in each of the plurality of thermoelectric elements.

  Preferably, the control unit determines the level of the DC voltage output from the voltage output unit according to the level of the electromotive force measured by the measurement unit, and the time interval connecting the thermoelectric element and the voltage output unit. And at least one of the lengths of time during which the DC voltage is output from the voltage output unit during a period in which the thermoelectric element and the voltage output unit are connected.

  Preferably, the control unit performs the thermal control process when the electromotive force measured in the measurement unit falls within a predetermined range indicating that the temperature difference between the constant temperature container and the sample container is sufficiently small. finish.

Preferably, the control unit controls the switch unit to connect the plurality of thermoelectric elements to the measurement unit when a predetermined time elapses after the thermal control process is completed, and The measurement unit is controlled to measure the electromotive force of the thermoelectric element.
Preferably, the biological activity measuring device includes a group of electromotive force data indicating the electromotive force of the plurality of thermoelectric elements measured after the lapse of the predetermined time, and a plurality of samples accommodated in the plurality of measurement cells. Based on a group of concentration data indicating the concentration of the substance having antimicrobial action added to each sample in the container, a coefficient of a predetermined function representing a relationship between the electromotive force of the thermoelectric element and the concentration of the substance is calculated. A regression analysis unit that is calculated by a regression analysis method; and a concentration calculation unit that calculates the concentration of the substance when the electromotive force is zero in the predetermined function having the coefficient calculated by the regression analysis unit. Have.

  Preferably, the control unit controls the switch unit and the measurement unit so that the group of electromotive force data is obtained each time a plurality of predetermined times have elapsed after the thermal control process is completed, The regression analysis unit calculates the coefficient for each of the plurality of predetermined times based on the group of electromotive force data and the group of concentration data obtained each time the plurality of predetermined times elapses. The concentration calculation unit calculates the concentration of the substance when the electromotive force is zero based on the coefficient calculated by the regression analysis unit for each of the plurality of predetermined times, and calculates the concentration. Based on the plurality of concentrations, an average value of the concentrations is further calculated.

  The second aspect of the present invention relates to a minimum growth inhibition concentration estimation method for estimating the minimum concentration required for a substance exhibiting antimicrobial activity to inhibit the growth of microorganisms. This minimum growth inhibition concentration estimation method prepares a plurality of microorganism culture samples to which the substances of different concentrations are added, and arranges a plurality of sample containers containing the plurality of microorganism culture samples inside a thermostatic chamber. Measuring the electromotive force according to the temperature of the sample container respectively generated in a plurality of thermoelectric elements disposed in contact with the plurality of sample containers inside the thermostat, and the plurality of the measured plurality Based on the group of electromotive force data indicating the electromotive force of the thermoelectric element, and the group of concentration data indicating the concentration of the substance added to each of the plurality of microorganism culture samples, A step of calculating a coefficient of a predetermined function representing a relationship with the concentration of the substance by a regression analysis method, and the predetermined function having the calculated coefficient when the electromotive force is zero And a step of calculating the concentration of the serial material.

  According to the present invention, a DC voltage is applied to a thermoelectric element installed to measure the temperature difference between the sample container and the thermostatic container, and the sample container is heated or reduced so that the temperature difference between the sample container and the thermostatic container is reduced. By cooling, it is possible to quickly erase the heat (hot or cold) brought in from the outside when the sample is installed in the apparatus, and the time required for measurement can be shortened.

It is a figure which shows an example of the whole structure of a biological activity measuring apparatus. It is a top view which shows an example of the external appearance of the thermostat container seen from the upper side. It is a figure which shows an example of a structure of a switch part, a voltage output part, and a measurement part. It is a figure showing the time change of the measured value by the sensor from immediately after putting a measuring object sample in a thermostat. It is the figure which showed the example of the time change of the measured value of the metabolic fever performed by the biological activity measuring apparatus shown in FIG. 1 about ten microorganism culture samples from which the density | concentration of an antimicrobial substance differs. It is the figure showing the relationship between the measured value of the 12th hour in the graph shown in FIG. 5, and the density | concentration of the antimicrobial substance contained in a culture sample. It is the figure showing the relationship between the measured value of the 10th hour in the graph shown in FIG. 5, and the density | concentration of the antimicrobial substance contained in a culture sample. It is the figure showing the relationship between the measured value of the 8th hour in the graph shown in FIG. 5, and the density | concentration of the antimicrobial substance contained in a culture sample. It is the figure which put together and represented the regression curve shown in FIGS. 6-8.

<First Embodiment>
First, an outline of the biological activity measurement apparatus according to the first embodiment of the present invention will be described.
The biological activity measuring apparatus according to the present embodiment continuously detects metabolic heat released by microbial cells during the growth process by the Seebeck effect exhibited by a sensor (thermoelectric element) that is a heat detector, thereby proliferating microbial cells. Activity is measured quantitatively. When a microorganism culture sample is installed in the apparatus for starting this measurement, extra heat (hot or cold) is brought into the apparatus from the outside. However, the biological activity measuring apparatus according to the present embodiment uses the above sensor. Using the Peltier effect shown by, the heat brought in from the outside is erased. This makes it possible to quickly detect only the heat derived from the metabolic activity of the target microbial cell by the Seebeck effect of the sensor.

  In the biological activity measuring apparatus according to this embodiment, the sensor (thermoelectric element) in contact with the sample container measures the electromotive force (Seebeck voltage) generated by the Seebeck effect, and the sample container is heated or cooled by the Peltier effect. And a circuit for outputting a voltage to the sensor. These circuits are switched by a switch circuit and connected to the sensor. The biological activity measuring apparatus repeatedly measures the Seebeck voltage of the sensor according to the difference between the sample temperature and the reference temperature after the microbial cell sample is introduced and installed in the apparatus. The biological activity measurement apparatus switches the connection destination of the sensor from the measurement circuit to the voltage output circuit during this measurement, outputs a DC voltage to the sensor, and heats or cools the sample container by the Peltier effect of the sensor.

  When the temperature of the sample container in contact with the sensor is higher than the reference temperature, the biological activity measuring device sets the polarity of the DC voltage so that the surface in contact with the sample container absorbs heat by the Peltier effect and outputs it to the sensor. Reduce the temperature. On the other hand, when the temperature of the sample container is lower than the reference temperature, the biological activity measuring device outputs a DC voltage having the opposite polarity to the above to the sensor to increase the temperature of the sample. In addition, the biological activity measuring device measures the Seebeck voltage (the temperature of the sample container and the reference) by measuring the level of the DC voltage output to the sensor, the time interval for outputting the DC voltage to the sensor, the length of time for outputting the voltage, etc. Adjust according to temperature difference.

  The biological activity measurement apparatus repeats the above control until the difference between the temperature of the sample container and the reference temperature is sufficiently small, so that the heat (hot or cold) brought together with the microbial sample when the microbial cell sample is installed in the apparatus. ). In addition, the biological activity measurement device is designed to reduce the difference between the temperature of the sample container and the reference temperature quickly, the level of the DC voltage applied to the sensor, the time interval for applying the DC voltage, and the length of time for applying the DC voltage. Adjust the thickness appropriately according to the measured Seebeck voltage. As a result, even if a sample with a temperature different from the reference temperature is installed in the device, these temperature differences can be quickly eliminated, so that measurement of the temperature based on the original metabolic heat can be started in a short time. Is possible.

  Next, the biological activity measurement apparatus according to the present embodiment will be described in detail with reference to the drawings.

FIG. 1 is a diagram illustrating an example of the overall configuration of the biological activity measurement apparatus according to the present embodiment.
The biological activity measuring device shown in FIG. 1 includes a thermostatic bath 10, a thermostatic container 13, a plurality of sensors S, a switch unit 20, a voltage output unit 30, a measuring unit 40, and a system control device 50.

  The thermostat 10 is a device that keeps the internal temperature constant. For example, the thermostatic chamber 10 is covered with a heat insulating material on the outside, and a mechanism (pipe or the like) for circulating a liquid (water or the like) is turned inside. By supplying the fluid whose temperature is adjusted by the heating / cooling device to the circulation mechanism, the inside of the thermostatic chamber 10 is adjusted to a desired temperature. In the example of FIG. 1, the thermostatic chamber 10 includes a box body 12 having a space therein and a lid portion 11 that seals an opening on the upper side.

  The thermostatic container 13 is a container formed of a block of a material having high thermal conductivity (for example, a metal such as aluminum), and includes a plurality of small chambers (measuring cells) for accommodating a sample container 17 in which a sample to be measured is placed. ) Is provided. The thermostatic container 13 is disposed in the thermostatic bath 10, and the whole is kept at a constant temperature.

FIG. 2 is a plan view showing an example of the appearance of the thermostatic container 13 as viewed from above. In the example of FIG. 2, 17 cylindrical measurement cells (C1 to C16, CR) for accommodating the sample container 17 are formed in the block of the thermostatic container 13. These measurement cells are arranged at symmetrical positions with respect to the center of the block of the thermostatic container 13. The upper opening of the measurement cell is closed by the lid 14 after the sample container 17 is arranged.
The central measurement cell CR can accommodate a sample container containing a reference material for comparing the temperature with another sample to be measured.

  The sensor S is a thermoelectric element having a function of exchanging heat energy and electric energy, and is installed in each of the plurality of measurement cells (C1 to C16, CR). The sensor S is placed between the sample container 17 and the constant temperature container 13. In the example of FIG. 1, the sensor S is laid on the inner bottom of the measurement cell, and the sample container 17 is placed thereon. The sensor S generates an electromotive force according to the temperature difference between the surface (first surface) that contacts the thermostatic container 13 and the surface (second surface) that contacts the sample container 17 (Seebeck effect).

The switch unit 20 connects the plurality of sensors S to the measurement unit 40 or the voltage output unit 30 according to the control of the system control unit 50.
The voltage output unit 30 outputs DC voltages to be applied to the plurality of sensors S according to the control of the system control device 50.
The measurement unit 40 measures the electromotive force due to the Seebeck effect generated by the plurality of sensors S according to the control of the system control device 50.

FIG. 3 is a diagram illustrating an example of the configuration of the switch unit 20, the voltage output unit 30, and the measurement unit 40.
In FIG. 3, reference signs S <b> 1, S <b> 2, S <b> 3. Sensors S1, S2, S3,... Are arranged in different measurement cells, and each detects the temperature of one sample.

The voltage measurement circuit 40-i (i = 1, 2, 3...) Is a circuit belonging to the measurement unit 40, measures the electromotive force generated by the Seebeck effect in the sensor Si, and sends the measurement result to the system controller 50. Output.
For example, as shown in FIG. 2, the voltage measurement circuit 40-i amplifies a minute electromotive force (Seebeck voltage) generated by the sensor Si, and converts an output signal of the amplification circuit 41 into a digital signal. An analog-digital conversion circuit 42 is included. The analog-digital conversion circuit 42 performs an analog-digital conversion operation according to the control of the system control device 50, for example.

The voltage output circuit 30-i (i = 1, 2, 3...) Is a circuit belonging to the voltage output unit 30, and outputs a DC voltage to the sensor Si according to the control of the system control device 50. When a voltage is applied by the voltage output circuit 30-i, the sensor Si heats or cools the sample container 17 by the Peltier effect. The direction of heat flow (heat dissipation / heat absorption) of the sensor Si is determined according to the polarity of the output voltage of the voltage output circuit 30-i. The system control device 50 controls the level and polarity of the output voltage, the voltage output interval, and the output time in the voltage output circuit 30-i.
For example, as shown in FIG. 2, the voltage output circuit 30-i includes a digital-analog conversion circuit 32 that converts a digital signal supplied from the system control device 50 into an analog signal, and amplifies the analog signal so as to be positive or negative. The amplifying circuit 31 is output as a direct current voltage.

  The switch circuit 20-i (i = 1, 2, 3,...) Is a circuit belonging to the switch unit 20, and is controlled by the system control device 50 according to the input terminal of the voltage measurement circuit 40-i or the voltage output circuit 30-i. Connect the output terminal to the terminal (+,-) of the sensor Si.

  The system control device 50 is a device that comprehensively controls the operation of the biological activity measurement device, and includes, for example, a computer that executes processing according to a program. The computer of the system controller 50 includes, for example, a memory that stores program codes, a processor that sequentially reads program codes stored in the memory and executes processing, a bus that connects the processor and the memory, a bus controller that controls the bus, A cache memory interposed between the processor and the memory is included. The system control device 50 also includes various device controllers for the computer to exchange data with peripheral devices such as the hard disk device 70 and the display device 60, and a network interface circuit for communicating with other devices via the network. May be included.

The system control device 50 includes a control unit 51 as a component by cooperation between hardware such as a computer and a program.
The control unit 51 performs a process (thermal control process) of alternately repeating voltage measurement and voltage application so that the temperature difference between the sample container 17 and the thermostatic container 13 approaches zero for each of the sensors S1, S2, S3. Do.
That is, the control unit 51 controls the switch circuit 20-i so that the sensor Si (i = 1, 2, 3,...) Is alternately connected to the voltage measurement circuit 40-i and the voltage output circuit 30-i. In a period in which the sensor Si and the voltage measurement circuit 40-i are connected, the control unit 51 controls the voltage measurement circuit 40-i so as to measure the electromotive force (Seebeck voltage) of the sensor Si. When the electromotive force measured in the voltage measurement circuit 40-i indicates that the temperature of the sample container 17 is lower than that of the constant temperature container 13, the control unit 51 is a period in which the sensor Si and the voltage output circuit 30-i are connected. , The voltage output circuit 30-i is controlled so that the sensor Si outputs a direct current voltage that releases heat at the contact surface (second surface) with the sample container 17. On the other hand, when the electromotive force measured in the voltage measurement circuit 40-i indicates that the temperature of the sample container 17 is higher than that of the thermostatic container 13, the control unit 51 connects the sensor Si and the voltage output circuit 30-i. During this period, the voltage output circuit 30-i is controlled such that the sensor Si outputs a direct current voltage that absorbs heat at the contact surface (second surface) with the sample container 17.

  When performing this thermal control process, the control unit 51 determines the level of the DC voltage output from the voltage output circuit 30-i, the sensor according to the level of the electromotive force of the sensor Si measured in the voltage measurement circuit 40-i, A time interval for connecting Si and the voltage output circuit 30-i (that is, a time interval for voltage output to the sensor Si), and a direct current from the voltage output circuit 30-i during a period of connecting the sensor Si and the voltage output circuit 30-i. The length of time for which the voltage is output is appropriately adjusted so that the temperature difference between the sample container 17 and the thermostatic container 13 quickly approaches zero. For example, the control unit 51 increases the DC voltage level, shortens the time interval of voltage output to the sensor Si, or outputs the DC voltage output time as the temperature difference between the sample container 17 and the thermostatic container 13 increases. The temperature difference is increased quickly by increasing the degree of heating or cooling by the sensor Si.

  In addition, the controller 51 detects that the electromotive force of the sensor Si measured in the voltage measurement circuit 40-i falls within a predetermined range indicating that the temperature difference between the thermostatic container 13 and the sample container 17 is sufficiently small. Then, the thermal control process for the sensor Si is terminated. When the thermal control processing of all the sensors (S1, S2, S3...) Is completed, it is considered that the extra heat brought in from the outside has been erased. Therefore, the control unit 51 determines the metabolic activity of the microbial cells contained in the sample. Start the original measurement to examine the fever involved. That is, the control part 51 connects each sensor to the measurement part 40 by the switch part 20, and starts the measurement of each electromotive force.

  Here, in the biological activity measuring apparatus shown in FIG. 1 having the above-described configuration, an operation for erasing excess heat (hot and cold) brought into the apparatus before the start of measurement will be described.

  First, the user opens the lid 11, arranges the sample container 17 in each measurement cell of the thermostatic container 13, closes the lid 11, and seals the inside of the thermostatic chamber 10. Next, the user operates an input device (switch, button, keyboard, etc.) (not shown) of the system control unit 50 to execute a heat control process for erasing excess heat brought into the thermostat 10. .

  When the control unit 51 receives an instruction to start the thermal control process, the switch 51 switches the sensors Si (i = 1, 2, 3,...) Alternately to the voltage measurement circuit 40-i and the voltage output circuit 30-i. The circuit 20-i is controlled. The control unit 51 controls the voltage measurement circuit 40-i so as to measure an electromotive force (Seebeck voltage) generated in the sensor Si during a period in which the sensor Si and the voltage measurement circuit 40-i are connected. The electromotive force of the sensor Si generated according to the temperature difference between the sample container 17 and the constant temperature container 13 is amplified by the amplifier circuit 41, then converted into a digital signal by the analog-digital conversion circuit 42, and sent to the control unit 51. To be acquired. When acquiring the electromotive force data from the voltage measurement circuit 40-i, the control unit 51 determines whether the temperature of the sample container 17 is higher or lower than the temperature of the thermostatic container 13 based on the measured polarity of the electromotive force. .

When the temperature of the sample container 17 is lower than the temperature of the thermostatic container 13, the control unit 51 causes heat to be released from the sensor Si to the sample container 17 during a period in which the sensor Si and the voltage output circuit 30-i are connected. A DC voltage having a set polarity is output from the voltage output circuit 30-i. The sensor Si to which the DC voltage of the voltage output circuit 30-i is applied absorbs heat from the contact surface (first surface) with the constant temperature container 13 by the Peltier effect, and contacts with the sample container 17 (second surface). Heat is released at the surface). When the sample container 17 is heated by the sensor Si, part of the cold heat of the sample container 17 brought in from the outside is erased, and the temperature difference between the sample container 17 and the thermostatic container 13 is reduced.
On the other hand, when the temperature of the sample container 17 is higher than the temperature of the thermostatic container 13, the control unit 51 absorbs the heat of the sample container 17 by the sensor Si during the period in which the sensor Si and the voltage output circuit 30-i are connected. In this way, a DC voltage with the polarity set is output from the voltage output circuit 30-i. The sensor Si to which the DC voltage of the voltage output circuit 30-i is applied absorbs heat from the contact surface (second surface) with the sample container 17 by the Peltier effect, and contacts with the constant temperature container 13 (first surface). Heat is released at the surface). When the sample container 17 is cooled by the sensor Si, a part of the heat of the sample container 17 brought in from the outside is erased, and the temperature difference between the sample container 17 and the constant temperature container 13 is reduced.

  Actually, the heat transfer is not performed instantaneously but always takes a certain time, so that the heat brought in from the outside is not offset by one heating or cooling by the sensor Si. Therefore, the control unit 51 alternately repeats the temperature measurement using the Seebeck effect of the sensor Si and the heating / cooling using the Peltier effect by the thermal control process described above, and the temperature difference between the sample container 17 and the thermostatic container 13. Is gradually reduced.

In this thermal control process, the control unit 51 determines the level of the DC voltage output from the voltage output circuit 30-i to the sensor Si, the time interval for outputting the DC voltage, and the output time of the DC voltage per time. You may adjust according to the measurement result of the temperature difference by the measurement circuit 40-i.
For example, when the electromotive force of the sensor Si measured in the voltage measurement circuit 40-i is large (when the temperature difference is large), the control unit 51 increases the DC voltage, and as the electromotive force decreases (the temperature difference decreases). The output level of the voltage output circuit 30-i may be adjusted so that the DC voltage becomes smaller (as it becomes smaller).
In addition, when the electromotive force of the sensor Si measured in the voltage measurement circuit 40-i is large, the control unit 51 increases the output time of the DC voltage per time, and the output time decreases as the electromotive force decreases. As such, the switch circuit 20-i and the voltage output circuit 30-i may be controlled.
Alternatively, when the electromotive force of the sensor Si measured in the voltage measurement circuit 40-i is large, the control unit 51 shortens the time interval for outputting the DC voltage, and the time interval becomes longer as the electromotive force becomes smaller. As described above, the switch circuit 20-i and the voltage output circuit 30-i may be controlled.
By appropriately performing these controls, the control unit 51 quickly converges the temperature difference between the sample container 17 and the thermostatic container 13 to near zero while preventing the sample container 17 from being heated or cooled more than necessary. Let

  When the measured value of the electromotive force of the sensor Si is reduced by the thermal control process and included in a predetermined range close to zero, the control unit 51 has a sufficiently small temperature difference between the sample container 17 and the thermostatic container 13. It judges that it is a thing and complete | finishes a thermal control process. Since the heat brought in from the outside is almost erased by the processing so far, the control unit 51 starts the original measurement operation for detecting the metabolic heat of the microbial cells in the sample.

FIG. 4 is a diagram showing the time change of the measured value by the sensor S immediately after the sample to be measured is put in the thermostat 10, and shows the difference between the case where the thermal control process described above is performed and the case where it is not performed. A curve CV1 is a graph when the thermal control process is not performed, and a curve CV2 is a graph when the thermal control process is performed.
In the example of FIG. 4, since the sample container 17 containing the sample having a temperature higher than the reference temperature (the temperature of the thermostatic container 13) is introduced and installed in the thermostatic chamber 10, the temperature of the sample container 17 decreases with time. It approaches the reference temperature. When the heat control process is not performed, as shown in the curve CV1, the heat brought into the thermostat 10 is attenuated to the reference temperature with time based on the heat conduction side of Newton, but the heat control process is performed. By doing so, the rate of attenuation is increased, as shown by curve CV2. That is, if the thermal control process is not performed, the sensor output (Seebeck voltage) does not become zero even after 80 minutes have elapsed, but if the thermal control process is performed, the sensor output becomes zero in about 20 minutes.

As described above, according to the biological activity measurement apparatus according to the present embodiment, the sample container is based on the temperature measurement result of the sensor 7 (thermoelectric element) installed between the sample container 17 and the thermostatic container 13. Heat supplied to the interior of the thermostat 10 when the sample container 17 is installed by supplying a DC voltage to the sensor 7 so that the temperature difference between the thermostat 17 and the thermostatic container 13 is reduced to heat and cool the sample container 17 (see FIG. (Hot / cold) can be quickly erased. Therefore, measurement of metabolic fever exhibited by microbial cells can be performed significantly faster than in the past.
In addition, by providing the same number of independent circuits as described above for the number of samples, even when a plurality of samples having different temperature histories are measured, each sample can be measured quickly.

<Second Embodiment>
Next, a second embodiment of the present invention will be described.
The second embodiment relates to a method for estimating a minimum growth inhibitory concentration in a biological activity measuring device that measures biological activity based on metabolic fever.
The minimum growth inhibitory concentration is the minimum concentration at which a substance exhibiting antimicrobial action inhibits the growth of microorganisms, and is an index evaluated particularly in the new development of a substance exhibiting antimicrobial action. In the conventional agar plate culture method, there is a problem that the agar plate culture must be repeated by changing the concentration of the substance exhibiting antimicrobial action until a significant result is obtained. By measuring the metabolic fever of these samples in parallel and processing the data, it is possible to estimate the minimum growth inhibition concentration in a short time with less labor.

In the method for estimating the minimum growth inhibition concentration according to the present embodiment, first, the growth of microorganisms is performed on a plurality of microorganism culture samples (solid, liquid, or a mixture of both) to which target antimicrobial substances are added at different concentrations. The metabolic fever released with each is measured.
That is, a plurality (for example, 6 to 52) of microorganism culture samples to which substances having antimicrobial activity are added at different concentrations are prepared, and the temperature of a plurality of sample containers containing the plurality of microorganism culture samples is adjusted. For example, it is measured in parallel by a biological activity measuring apparatus as shown in FIG. Thereby, for each of the microbial samples having different concentrations of the antimicrobial substance, the metabolic heat corresponding to the amount of growth of the microbial cells in the process of growth is measured.

When the metabolic fever of each microbial culture sample is measured at an arbitrary incubation time, metabolic fever is no longer generated by the numerical analysis method based on the relationship between the metabolic fever measurement data of each sample and the concentration of the antimicrobial substance ( The concentration of the antimicrobial substance (which prevents microbial growth) is estimated as the minimum inhibitory concentration.
That is, metabolic heat measurement data and antimicrobial substance concentration data of each sample obtained when a predetermined culture time (for example, 8 hours or 12 hours from the start of culturing of microorganisms) has elapsed. Based on the above, a coefficient of a predetermined function (for example, a power function) representing the relationship between the metabolic fever measurement data and the concentration data is calculated by a regression analysis method or the like. Then, in a predetermined function having the calculated coefficient, the concentration of the antimicrobial substance (minimum growth inhibition concentration) when the metabolic heat value indicated by the measurement data is zero is calculated.
In this case, the average value and the error of the minimum growth inhibition concentration at a specific temperature can be calculated by calculating and averaging the minimum growth inhibition concentration by the above-described method at each of a plurality of times during the growth.

  Hereinafter, a configuration example of the system control device 50 when the minimum growth inhibition concentration is estimated in the biological activity measurement device shown in FIG. 1 will be described. Other configurations of the biological activity measurement apparatus according to the present embodiment are the same as those of the biological activity measurement apparatus according to the first embodiment.

The control unit 51 of the system control device 50 detects the sensor Si (i = 1, 1) after a predetermined time elapses after the above-described thermal control process ends (that is, after the heat brought into the thermostatic chamber 10 is erased). 2, 3) and the voltage measurement circuit 40-i are controlled, and the voltage measurement circuit 40-i is controlled so as to measure the electromotive force of the sensor Si.
For example, the control unit 51 obtains a group of electromotive force data from the sensors S1, S2, S3,... Every time a plurality of predetermined times have elapsed since the thermal control process is completed (for example, every predetermined time). The switch unit 20 and the measurement unit 40 are controlled.

  Further, the system control device 50 includes a regression analysis unit 52 and a concentration calculation unit 53 as components by cooperation of hardware such as a computer and a program.

The regression analysis unit 52 includes a group of electromotive force data indicating the electromotive force of the sensors S1, S2, S3,... Measured after a lapse of a predetermined time, and a plurality of sample containers accommodated in a plurality of measurement cells of the thermostatic container 13. 17 is a coefficient of a predetermined function that represents the relationship between the electromotive force of the sensor S and the concentration of the antimicrobial substance based on a group of concentration data indicating the concentration of the antimicrobial substance contained in each of the samples (microbe culture samples) Is calculated by regression analysis (such as least squares). For example, the regression analysis unit 52 calculates the coefficient of the above function for each of a plurality of predetermined times based on a group of electromotive force data and a group of concentration data obtained each time a plurality of predetermined times elapses.
As a function for calculating coefficients in the regression analysis unit 52, for example, a polynomial (power function) such as a quadratic equation or a cubic equation, a hyperbolic function, or the like can be used.

  The concentration calculation unit 53 calculates the concentration of the antimicrobial substance (minimum growth inhibition concentration) when the electromotive force of the sensor S becomes zero in a predetermined function having the coefficient calculated by the regression analysis unit 52. For example, the concentration calculation unit 53 determines the antimicrobial substance in the case where the electromotive force of the sensor S is zero based on the coefficient of the predetermined function calculated by the regression analysis unit 52 for each of a plurality of predetermined times. The density is calculated, and the average value and error of the density are further calculated.

  Next, an operation for obtaining an estimated value of the minimum growth inhibition concentration in the bioactivity measuring apparatus having the above-described configuration will be described.

  First, a user prepares a plurality of microbial culture materials prepared under the same conditions except that the concentration of the antimicrobial substance is different, and installs them in each measurement cell of the thermostatic container 13 of the thermostatic bath 10. And the system control device 50 is operated to execute the thermal control process. The control unit 51 executes a heat control process for erasing heat brought into the thermostatic chamber 10. After the thermal control process is completed, the control unit 51 controls the switch unit 20 and the measurement unit 40, for example, at regular intervals, and acquires a group of electromotive force data from the sensors S1, S2, S3,. The control unit 51 stores the acquired group of electromotive force data in a storage device (for example, the hard disk device 70).

When the group of electromotive force data obtained by the sensors S1, S2, S3,... Is acquired and stored in the storage device, the regression analysis unit 52 determines the electromotive force and the concentration based on the group of electromotive force data and the group of concentration data. A coefficient of a predetermined function (one coefficient or a coefficient having a plurality of sets) is calculated by a regression analysis method and stored in a storage device. Based on the coefficient calculated by the regression analysis unit 52, the concentration calculation unit 53 calculates the concentration (minimum growth inhibition concentration) when the electromotive force becomes zero in the function having the coefficient, and stores it in the storage device.
The regression analysis unit 52 may calculate a coefficient each time a group of electromotive force data is acquired by the sensors S1, S2, S3,... May be calculated. The concentration calculation unit 53 may calculate the minimum growth inhibition concentration every time the coefficient is calculated by the regression analysis unit 52, or after the coefficient is calculated a plurality of times by the regression analysis unit 52, the minimum is determined for each. The growth inhibitory concentration may be calculated.

  When a predetermined time elapses after the thermal control process, the control unit 51 ends the electromotive force data by the switch unit 20 and the measurement unit 40. The concentration calculation unit 53 calculates an average value and an error of the minimum growth inhibition concentration based on the calculated minimum growth inhibition concentrations.

FIG. 5 is a diagram showing an example of a temporal change in the measured value of metabolic fever performed by the biological activity measurement apparatus according to the present embodiment for 10 microorganism culture samples having different concentrations of antimicrobial substances. The time-dependent change of the measured value from the time to the 14th hour is shown. The horizontal axis represents the culture time, and the vertical axis represents the measured value of metabolic fever. Here, the measured value of metabolic fever is a voltage value obtained by detecting the electromotive force of the sensor S in the measuring unit 40, and the unit of voltage (μV) is an example.
In the example of FIG. 5, measurement is simultaneously performed on 10 culture samples of the same microbial species having different concentrations of antimicrobial substances. Curves A0 to A9 are graphs of measurement results for each culture sample. The concentrations of antimicrobial substances are 0% for curve A0, 1% for curve A1, 2% for curve A2, 3% for curve A3, and curve A4. Is 4%, curve A5 is 5%, curve A6 is 6%, curve A7 is 7%, curve A8 is 8%, and curve A9 is 9%.
Since the measured value of metabolic fever by the sensor S corresponds to the amount of growth of the microbial cells, the curves A0 to A9 representing the change with time are also referred to as growth curves in microbial culture.
Referring to FIG. 5, the slope of the growth curve decreases as the concentration of the antimicrobial substance added to the microorganism culture sample increases. This indicates that the higher the concentration of the antimicrobial substance, the stronger the growth of microbial cells is suppressed.

FIG. 6 is a graph showing the measured values (y0, y1,..., Y9) at the 12th hour in the graph shown in FIG. 5 from the curves A0 to A9 and the relationship with the concentration of the antimicrobial substance contained in the culture sample. FIG. It can be seen from the graph of FIG. 6 that the measured value corresponding to the amount of microbial growth decreases as the concentration of the antimicrobial substance increases.
The thin solid line in FIG. 6 is a curve of a function obtained by the multiple regression analysis technique so as to be in good agreement with the graph of measured values (y0, y1,..., Y9). This function is used when there is a theoretically valid function, but a quadratic equation, a cubic equation (power function), a linear hyperbolic function, or the like may be used. A curve indicated by a thin solid line in FIG. 6 is a regression curve obtained by regression analysis using a linear hyperbolic function. The regression analysis unit 52 calculates the coefficient of this regression curve.

  The regression curve shown in FIG. 6 decreases as the concentration on the horizontal axis increases, and the measured voltage on the vertical axis becomes zero at a certain concentration. The concentration of the antimicrobial substance on which the vertical axis becomes zero represents the minimum growth inhibition concentration. The concentration calculation unit 53 calculates the concentration when the measured voltage becomes zero in the regression curve obtained by the regression analysis. In the example of FIG. 6, the minimum growth inhibition concentration is 9.72%.

  As shown in FIG. 6, since one minimum growth inhibition concentration can be calculated from a group of measured values (electromotive force data) obtained for one culture time, if this is performed for each of a plurality of culture times, a plurality of minimum growths can be obtained. The inhibitory concentration can be calculated. The concentration calculation unit 53 calculates an average value and an error of the minimum growth inhibition concentration based on the plurality of minimum growth inhibition concentrations calculated for a plurality of culture times.

FIG. 7 shows the relationship between the measured value and the concentration of the antimicrobial substance in the form of a graph as shown in FIG. Is. 8 is a graph showing the relationship between the measured value and the concentration of the antimicrobial substance in the growth curve shown in FIG. It is.
The thin solid lines in FIGS. 7 and 8 are regression curves obtained by the same regression analysis as in FIG. When the minimum growth inhibitory concentration is calculated from this regression curve, a result of 9.58% is obtained for the graph at 10 hours of culture shown in FIG. 7, and 9.38% is obtained for the graph at 8 hours of culture shown in FIG. .

FIG. 9 is a diagram in which the regression curves shown in FIGS.
In the example of FIG. 9, the three regression curves intersect at almost the same concentration. However, in actual measurement, there is a measurement error in each, so they do not necessarily overlap. Therefore, the concentration of the antimicrobial substance when the electromotive force of the sensor S becomes zero (the metabolic heat becomes zero) is calculated for each of FIGS. A high minimum growth inhibitory concentration. That is, a group of measured values obtained from the sensors S1, S2, S3... Are measured at different n culture times, and the average value and error of the n minimum growth inhibition concentrations calculated based on the measured values are calculated. By doing so, the minimum growth inhibition concentration considering the measurement error can be obtained. 6 to 8, n = 3, the average value of the minimum growth inhibition concentration is 9.56%, and the accuracy (standard deviation) is 0.17%.
The accuracy (error) of the average value may be expressed by, for example, standard error or percentage error in addition to the standard deviation. In the above example, the standard error is 0.10% and the percent error is 1.03%.

As described above, according to the method for estimating the minimum growth inhibition concentration according to the present embodiment, a plurality of microbial culture samples to which antimicrobial substances having different concentrations are added are prepared, and the plurality of microbial culture samples are placed. A plurality of sample containers 17 are arranged in the thermostatic chamber 10. Sensors (thermoelectric elements) S1, S2,... Are arranged in contact with the plurality of sample containers 17 inside the thermostat 10, and the temperature of the sample container 17 generated in each sensor (thermoelectric elements) S1, S2,. The measuring unit 40 measures the electromotive force according to the above. In the regression analysis unit 52, based on the group of electromotive force data obtained by the measurement of the measurement unit 40 and the group of concentration data indicating the concentration of the antimicrobial substance added to each of the plurality of microorganism culture samples, the sensor A coefficient of a predetermined function representing the relationship between the electromotive force and the concentration of the antimicrobial substance is calculated by the regression analysis method. The concentration calculation unit 53 calculates the concentration of the antimicrobial substance when the electromotive force of the sensor becomes zero in the predetermined function having the calculated coefficient.
Therefore, the time, labor, and resources required to obtain the estimated value of the minimum growth inhibition concentration can be greatly reduced as compared with the case of using the conventional agar plate culture method in which a large number of culture samples are repeatedly cultured.

  Further, according to the method for estimating the minimum growth inhibition concentration according to the present embodiment, the minimum growth inhibition concentration for each culture time is calculated based on a group of electromotive force data measured for each of a plurality of culture times, and the calculation is performed. Since the average value and the error of the minimum growth inhibition concentration are calculated based on the plurality of minimum growth inhibition concentrations, a more reliable estimated value of the minimum growth inhibition concentration can be obtained.

  Although several embodiments of the present invention have been described so far, the present invention is not limited to the above-described embodiments, and includes various variations.

  For example, in the first embodiment described above, the switch circuit 20-i (i = 1, 2, 3,...) Connected to the sensor Si is the amplifier circuit 31 of the voltage output circuit 30-i or the voltage measurement circuit 40-. Although it is configured independently from the i amplifier circuit 41 (FIG. 3), the present invention is not limited to this example. In another embodiment of the present invention, the current flowing from the sensor Si to the amplifier circuit 31 by increasing the output impedance of the amplifier circuit 31 by appropriately controlling semiconductor elements such as transistors constituting the output stage of the amplifier circuit 31 is obtained. You may block it. Similarly, the current flowing from the sensor Si to the amplifier circuit 41 may be cut off by appropriately controlling semiconductor elements such as transistors constituting the input stage of the amplifier circuit 41 to increase the input impedance of the amplifier circuit 41.

  In the second embodiment described above, when the electromotive forces of the sensors S1, S2, S3... Are measured in a plurality of culture times in order to calculate the minimum growth inhibition concentration, the plurality of culture times are always constant. It is not necessary, and may be arbitrarily changed according to, for example, the type of microorganism used in the test or the culture temperature.

  In the above-described embodiment, the system control device 50 including the control unit 51, the regression analysis unit 52, and the concentration calculation unit 53 may be configured by a single computer, or may be configured by a plurality of computers connected to be communicable. Also good. In addition, the control unit 51, the regression analysis unit 52, and the concentration calculation unit 53 may be configured with all functions based on a program, or at least some of the functions may be configured with hardware such as a logic circuit. .

  The present invention can be used in various fields in which microbial cells are involved. For example, development of pharmaceuticals, preservative treatment of foods and cosmetics, production of foods using microorganisms, quality control in food distribution, environment including soil It can be widely used in fields such as the management of microorganisms in the environment and the evaluation of the effects of physical factors such as radiation on cells. Moreover, it can be used not only for microbial cells but also for fields involving the cultivation of various biological cells such as some animal and plant cells.

DESCRIPTION OF SYMBOLS 10 ... Constant temperature bath, 11, 14 ... Cover part, 12 ... Box, 13 ... Constant temperature container, 17 ... Sample container, 20 ... Switch part, 20-1, 20-2 ... Switch circuit, 30 ... Voltage output part, 30 -1, 30-2 ... voltage output circuit 31, 41 ... amplifier circuit, 32 ... digital-analog conversion circuit, 40 ... measurement unit, 40-1, 40-2 ... voltage measurement circuit, 42 ... analog-digital conversion circuit , 50: System control device, 51: Control unit, 52: Regression analysis unit, 53: Concentration calculation unit, 60: Display device, 70: Hard disk device

Claims (6)

A biological activity measuring device for measuring heat generated by biological cells contained in a sample,
A thermostat,
A thermostatic container provided with a plurality of measurement cells that are arranged in the thermostatic bath, are formed of blocks of a high thermal conductivity material, and contain a sample container in which a sample to be measured is placed;
Temperatures of a first surface in contact with the constant temperature container and a second surface in contact with the sample container that are sandwiched between the constant temperature container and the sample container in each of the plurality of measurement cells. A plurality of thermoelectric elements each generating an electromotive force according to the difference;
A measuring unit for measuring each electromotive force generated by the plurality of thermoelectric elements;
A voltage output unit that outputs a DC voltage to be applied to the plurality of thermoelectric elements;
A plurality of thermoelectric elements, each of which is connected to the measurement unit or the voltage output unit;
The switch unit is controlled to connect the thermoelectric element to the measurement unit and the voltage output unit alternately, and the electromotive force is measured during a period in which the thermoelectric element and the measurement unit are connected. And when the electromotive force measured in the measurement unit indicates that the temperature of the sample container is lower than that of the thermostatic container, the second time is applied during a period in which the thermoelectric element and the voltage output unit are connected. When the voltage output unit is controlled to output a direct-current voltage having a polarity for releasing heat from the surface, and the measured electromotive force indicates that the temperature of the sample container is higher than that of the thermostatic container, A series of thermal control processes for controlling the voltage output unit to output a DC voltage having a polarity that absorbs heat on the second surface during a period in which the thermoelectric element and the voltage output unit are connected, For each element And a control unit for performing,
A biological activity measuring device. The control unit, according to the level of electromotive force measured in the measurement unit,
The level of the DC voltage output from the voltage output unit,
A time interval for connecting the thermoelectric element and the voltage output unit, and
A length of time for outputting the DC voltage from the voltage output unit during a period in which the thermoelectric element and the voltage output unit are connected;
Adjusting at least one of the
The biological activity measuring apparatus according to claim 1. The control unit ends the thermal control process when the electromotive force measured in the measurement unit falls within a predetermined range indicating that the temperature difference between the constant temperature container and the sample container is sufficiently small.
The biological activity measuring apparatus according to claim 2. The control unit controls the switch unit to connect each of the plurality of thermoelectric elements to the measurement unit when a predetermined time elapses after the thermal control process is completed, and the plurality of thermoelectric elements Controlling the measurement unit to measure the electromotive force;
A group of electromotive force data indicating the electromotive force of the plurality of thermoelectric elements measured after the lapse of the predetermined time, and resistance added to the samples in the plurality of sample containers accommodated in the plurality of measurement cells, respectively. A regression analysis unit that calculates a coefficient of a predetermined function representing a relationship between the electromotive force of the thermoelectric element and the concentration of the substance by a regression analysis method based on a group of concentration data indicating a concentration of the substance exhibiting a microbial action; ,
In the predetermined function having the coefficient calculated in the regression analysis unit, a concentration calculation unit that calculates the concentration of the substance when the electromotive force becomes zero,
The biological activity measuring apparatus according to claim 3. The control unit controls the switch unit and the measurement unit so that the group of electromotive force data is obtained each time a plurality of predetermined times have elapsed after the thermal control process is finished,
The regression analysis unit calculates the coefficient for each of the plurality of predetermined times based on the group of electromotive force data and the group of concentration data obtained each time the plurality of predetermined times elapses. ,
The concentration calculation unit calculates the concentration of the substance when the electromotive force is zero based on the coefficient calculated by the regression analysis unit for each of the plurality of predetermined times, and the calculated plurality Further calculating the average value of the concentration based on the concentration of
The biological activity measuring apparatus according to claim 4. A method for estimating a minimum growth inhibition concentration in which a substance having an antimicrobial action estimates a minimum concentration necessary for inhibiting the growth of microorganisms,
Preparing a plurality of microorganism culture samples to which the substances of different concentrations are added, and arranging a plurality of sample containers containing the plurality of microorganism culture samples in a thermostatic chamber;
Measuring electromotive force according to the temperature of the sample container, respectively generated in a plurality of thermoelectric elements disposed in contact with the plurality of sample containers inside the thermostat;
Based on the group of electromotive force data indicating the measured electromotive force of the plurality of thermoelectric elements and the group of concentration data indicating the concentration of the substance added to each of the plurality of microorganism culture samples, Calculating a coefficient of a predetermined function representing the relationship between the electromotive force of the thermoelectric element and the concentration of the substance by a regression analysis method;
Calculating the concentration of the substance when the electromotive force is zero in the predetermined function having the calculated coefficient;
A method for estimating the minimum growth inhibition concentration.