JP2007289004A - Method for measuring viable cell number, apparatus for measuring viable cell number and slime monitoring apparatus and slime controlling agent addition system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring viable cell number, which quickly measures the measuring viable cell number of microorganisms in high sensitivity. <P>SOLUTION: The method for measuring viable cell number is a method for measuring viable cell number in a sample from an oxygen amount consumed by microorganisms with which in obtaining an oxygen consumption by impressing a voltage to a Clark type oxygen electrode and detecting an oxygen concentration signal, a measurement cell is charged with sterile water saturated with dissolved oxygen, a voltage is impressed on the electrode, an initial oxygen concentration signal (Cal-1) of sterile water is detected, the impression of the voltage is stopped, the measurement cell is charged with a sample saturated with dissolved oxygen, a voltage is impressed on the Clark type electrode, an initial oxygen concentration signal (Sam-1) of the sample is detected, the impression of the voltage is stopped, oxygen is consumed by microorganisms in the sample for a given time, a voltage is impressed on the electrode, an oxygen concentration signal (Sam-2) after oxygen consumption is detected, the impression of the voltage is stopped, the measurement cell is charged with sterile water saturated with dissolved oxygen, a voltage is impressed on the electrode, an oxygen concentration signal (Cal-2) of sterile water after the measurement is detected and the viable cell number is calculated by using each of the obtained oxygen concentration signals. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is, for example, a viable cell count measuring method for quickly and highly sensitively measuring the viable cell count of microorganisms in various industrial waters such as water in each process of the pulp and paper industry and cooling water for cooling towers, and slime control using the same. Agent automatic addition system and slime monitor.

  Conventionally, in the industry where various process waters such as process water in the pulp and paper industry and cooling water for cooling towers are used, slime damage caused by microorganisms in the service water has become a problem. It has caused evil. Here, the slime refers to viscous mud substances generated by microbial factors in the industrial wastewater, and in particular, in the pulp and paper industry, various adverse effects such as paper breakage, eyeballs, stains, and operational deterioration. It is a cause of.

  Therefore, in the paper pulp industry, in order to prevent such slime failure, a method of adding a slime control agent containing a bactericide or the like to an aqueous system such as effluent is widely adopted. In this method, the drainage before and after the addition of the slime control agent is collected, and the effect is confirmed by measuring the number of viable bacteria in the sample. It is necessary to increase or decrease. Until now, methods such as plate culture have been employed for the measurement of the viable cell count. However, when the number of viable bacteria is measured by such a method, since a culture time of at least 48 hours is usually required, it is difficult to take measures to increase the slime control agent when the effect is insufficient. It had been. In other words, it is important to measure the number of viable bacteria in a sample as quickly as possible in order to enable immediate response when adding a slime control agent. It was.

  Several proposals have been made on methods for rapidly measuring the number of viable bacteria in liquid samples containing various types of water. For example, a method has been proposed in which the number of viable bacteria in a liquid sample is determined by measuring the dissolved oxygen concentration in the liquid sample using an oxygen electrode by utilizing the property that aerobic bacteria consume oxygen by respiration (patent) Reference 1 and Patent Reference 2). These methods use a Clark-type oxygen electrode as the oxygen electrode, apply voltage continuously between the platinum electrode (anode) and the silver electrode (cathode), and measure the rate of decrease in dissolved oxygen. The number of viable bacteria in a sample is obtained.

  However, in these known methods, in addition to oxygen consumption by microorganisms, oxygen is self-consumed due to the electrode reaction at the platinum electrode, and thus the measured values reflect both oxygen consumptions. For this reason, until now, it has been impossible to increase the detection sensitivity and accuracy of the viable count to the required level.

JP-A-56-140898 JP 63-15150 A

  An object of the present invention is to use a viable cell count measurement method capable of quickly measuring a viable cell count of a microorganism with high sensitivity and high accuracy, a viable cell count measurement apparatus for performing the measurement method, and the measurement method. A slime monitoring method and a slime control agent addition system are provided.

  As a result of intensive studies to solve the above problems, the present inventors applied voltage to the Clark-type oxygen electrode and detected the oxygen concentration signal by detecting the oxygen concentration signal. The voltage is applied intermittently every time the oxygen concentration signal is detected (ie, the voltage is applied and the oxygen concentration signal is After the detection, the application of the voltage is stopped immediately, and then, when another oxygen concentration signal is detected, the voltage is applied again) (intermittent application method). It has been found that the self-consumption of oxygen induced by the reaction can be suppressed to a minimum, and as a result, the detection sensitivity and accuracy of the viable cell count can be greatly improved. Furthermore, it has been found that the detection sensitivity and accuracy of the viable cell count can be further improved by performing sequential calibration using sterile water before and after measuring the sample to be measured. And the present invention was completed from these knowledge.

（３）前記無菌水は、抗菌剤、または抗菌剤および洗浄剤を精製水に含有させたものである、前記（１）または（２）記載の生菌数測定方法。
（４）酸素電極への電圧印加時間が１回あたり１〜６分間である、前記（１）〜（３）のいずれかに記載の生菌数測定方法。
（５）前記（１）〜（４）のいずれかに記載の生菌数測定方法に用いる測定装置であって、密閉可能な測定セルと、該測定セル内の液体に溶存酸素を飽和させる酸素飽和手段と、クラーク型酸素電極に電圧を印加するための電圧印加手段と、電圧印加により生じた酸素濃度信号を検知する検知手段と、前記測定セル内の液温を制御する温度制御手段とを備える、ことを特徴とする生菌数測定装置。
（６）前記試料に代えて無菌水を供給する無菌水供給手段をも備える、前記（５）記載の生菌数測定装置。
（７）前記（１）〜（４）のいずれかに記載の生菌数測定方法による測定を所定時間ごとに行ない、該測定で得られた酸素消費量の増減によってスライム量を監視する、ことを特徴とするスライムモニター方法。
（８）前記（１）〜（４）のいずれかに記載の生菌数測定方法において測定された酸素消費量に基づきスライムコントロール剤の添加量を制御する機構を備える、ことを特徴とするスライムコントロール剤添加システム。 That is, the present invention has the following configuration.
(1) A method for measuring the number of viable bacteria in a sample from the amount of oxygen consumed by microorganisms, wherein the oxygen consumption is determined by applying a voltage to a Clark-type oxygen electrode and detecting an oxygen concentration signal. The following operations (i) to (viii) are performed in this order, and calculation is performed using the obtained oxygen concentration signals (Sam-1), (Sam-2), (Cal-1), and (Cal-2). A method for measuring the number of viable bacteria.
(I) An operation of filling the measurement cell with the sterilized water after saturating the dissolved oxygen concentration in the sterilized water with the contact with the atmosphere blocked.
(Ii) An operation of stopping the application of the voltage after applying the voltage to detect the initial oxygen concentration signal (Cal-1) of sterile water.
(Iii) After saturating the dissolved oxygen concentration in the sample, filling the measurement cell with the sample in a state where contact with the atmosphere is blocked.
(Iv) An operation of stopping the application of the voltage after applying the voltage and detecting the initial oxygen concentration signal (Sam-1) of the sample.
(V) An operation in which the microorganisms in the sample consume oxygen for a predetermined time.
(Vi) An operation of stopping the application of the voltage after applying the voltage and detecting the oxygen concentration signal (Sam-2) after oxygen consumption.
(Vii) An operation in which the concentration of dissolved oxygen in sterile water is saturated again, and then the sterile cell is filled with the sterile water in a state where contact with the atmosphere is blocked.
(Viii) An operation of stopping the application of the voltage after detecting the oxygen concentration signal (Cal-2) of sterile water after the sample measurement by applying a voltage.
(2) Oxygen consumption is calculated by substituting the obtained oxygen concentration signals (Sam-1), (Sam-2), (Cal-1) and (Cal-2) into the following formula (1). The method for measuring the viable cell count according to (1), which is obtained as a consumption rate.

(3) The method for measuring the number of viable bacteria according to (1) or (2) above, wherein the sterile water contains an antibacterial agent or an antibacterial agent and a cleaning agent in purified water.
(4) The viable cell count measurement method according to any one of (1) to (3), wherein the voltage application time to the oxygen electrode is 1 to 6 minutes per time.
(5) A measuring device for use in the viable cell count measuring method according to any one of (1) to (4), wherein the measuring cell is capable of being sealed, and oxygen is used to saturate dissolved oxygen in the liquid in the measuring cell. A saturation means, a voltage application means for applying a voltage to the Clark oxygen electrode, a detection means for detecting an oxygen concentration signal generated by the voltage application, and a temperature control means for controlling the liquid temperature in the measurement cell. A viable cell count measuring device characterized by comprising.
(6) The viable cell count measurement apparatus according to (5), further including aseptic water supply means for supplying aseptic water instead of the sample.
(7) The measurement by the viable cell count measurement method according to any one of (1) to (4) is performed every predetermined time, and the slime amount is monitored by increasing or decreasing the oxygen consumption obtained by the measurement. The slime monitor method characterized by this.
(8) A slime comprising a mechanism for controlling the amount of slime control agent added based on the oxygen consumption measured in the viable cell count measurement method according to any one of (1) to (4). Control agent addition system.

  According to the present invention, the viable cell count is obtained by determining the oxygen consumption consumed by the microorganisms, instead of culturing as in the prior art, and the viable cell count is obtained. In addition, it is possible to measure the viable cell count with high sensitivity and high accuracy because it is not applied continuously as in the past, but it is applied intermittently and sequentially calibrated using sterile water. Has the effect of being able to In addition, since the viable count can be measured with high sensitivity (detection sensitivity) and high accuracy in this way, the area of the electrode in the measuring apparatus used for the measurement can be reduced, and the apparatus can be downsized. realizable. In addition, the increase in sensitivity and accuracy reduces the load on the electrical system such as an amplifier circuit, thereby reducing the cost. Further, by intermittently applying the voltage, aging (consumption) of the silver electrode that is the counter electrode of the platinum electrode can be reduced, and the life of the device including the electrode can be extended.

  Furthermore, according to the present invention, it is possible to quickly and highly sensitively measure the number of viable bacteria with high accuracy, so that it is possible to easily and reliably grasp the occurrence of slime in various wastewater, and slime damage It is also possible to obtain an effect that a slime control agent for preventing water can be added at an extremely accurate timing while seeing its effect.

<Viable count method>
The method for measuring the number of viable bacteria of the present invention is a method for measuring the number of viable bacteria from the amount of oxygen consumed by microorganisms in a sample to be measured.

In the viable cell count measurement method of the present invention, the following (i) to (viii) are performed in this order when the oxygen consumption amount is determined by applying a voltage to the Clark oxygen electrode and detecting the oxygen concentration signal. To do. That is,
(I) After saturating the dissolved oxygen concentration in sterile water, filling the measuring cell with the sterile water in a state where contact with the atmosphere is blocked;
(Ii) an operation of stopping the application of the voltage after detecting the initial oxygen concentration signal (Cal-1) of sterile water by applying a voltage;
(Iii) after saturation of the dissolved oxygen concentration in the sample, filling the measurement cell in a state where the sample is not in contact with the atmosphere;
(Iv) An operation of stopping the application of the voltage after applying the voltage and detecting the initial oxygen concentration signal (Sam-1) of the sample;
(V) an operation for consuming oxygen to microorganisms in the sample for a predetermined time;
(Vi) an operation of stopping the application of the voltage after detecting the oxygen concentration signal (Sam-2) after the consumption of oxygen by applying a voltage;
(Vii) An operation of filling the measurement cell with the sterile water after the saturated oxygen concentration in the sterile water is saturated again, in a state where contact with the atmosphere is blocked.
(Viii) an operation of stopping the application of the voltage after detecting the oxygen concentration signal (Cal-2) of sterile water after the sample measurement by applying a voltage;
Are performed in this order.

  In the above operations (i) to (viii), the voltage is not always applied continuously as in the prior art, but is an intermittent application method in which the voltage is intermittently applied every time each oxygen concentration signal is detected. Apply voltage. Thereby, the self-consumption of oxygen induced by the electrode reaction can be suppressed to the minimum, and as a result, the detection sensitivity and accuracy can be improved. The method for measuring the number of viable bacteria of the present invention is greatly different from the conventional method for measuring the number of viable bacteria in which a voltage is applied by a continuous application method using a Clark-type oxygen electrode in terms of the voltage application method.

  Among the operations (i) to (viii) above, the operations (i), (ii) and (vii), (viii) are performed before and after a series of operations ((iii) to (vi)) using a sample. The oxygen concentration signals (Cal-1) and (Cal-2) when using sterile water are detected. In the method for measuring the viable cell count of the present invention, these oxygen concentration signals (Cal-1) and The measurement results are sequentially calibrated using (Cal-2). By adopting the above-described intermittent application method and performing sequential calibration using sterile water, the detection sensitivity and accuracy of measurement can be greatly improved. The viable cell count measurement method of the present invention is greatly different from the conventional viable cell count measurement method in that sequential calibration is performed using sterile water as described above. Note that the calibration of the measurement results using the oxygen concentration signals (Cal-1) and (Cal-2) means that the oxygen concentration signals (Cal-1) and (Cal-2) are used in the calculation for obtaining the oxygen consumption. (For example, see formula (1) and formula (2) described later).

  In the viable count method of the present invention, the voltage application time to the oxygen electrode is preferably 1 to 6 minutes per time, more preferably 1.5 to 3 minutes per time. . Here, the voltage application time per time means each voltage application time (the voltage is applied when the oxygen concentration signal is obtained by the operations (ii), (iv), (vi), and (viii)). And the time from when the voltage application is stopped). If the voltage application time per time is longer than the above range, the self-consumption of oxygen derived from the electrode reaction may not be sufficiently suppressed, whereas if the voltage application time per time is shorter than the above range, This is not preferable because it tends to be difficult to detect the oxygen concentration signal.

In the operations of (i), (iii) and (vii), filling the measurement cell with the sample or sterile water in a state where contact with the atmosphere is blocked means that the measurement cell is filled with the sample or sterile water and sealed. (Specifically, there is only a liquid phase consisting of a sample or sterile water in a sealed measurement cell, and there is no gas phase such as air). When the measurement cell is filled with a sample or sterile water and sealed, several washing operations are performed by filling the measurement cell or the sterile water and then draining (hereinafter, this operation is referred to as co-washing). After repeating the process, it is desirable that the measurement cell is sealed in a state where it overflows and fills in the measurement cell.
In the operations (i), (iii) and (vii), as a method of saturating the dissolved oxygen concentration in the sample or sterile water, that is, a method of equilibrating the dissolved oxygen concentration, for example, oxygen or Air may be bubbled for a predetermined time.

In each of the operations (ii), (iv), (vi) and (viii), the magnitude of the applied voltage is preferably −500 to −1000 mV, more preferably −600 to −700 mV. . In addition, the magnitude | size of an applied voltage may differ for every said operation, and may be the same.
In the operations (ii), (iv), (vi), and (viii), it is desirable to detect the oxygen concentration signal after the output is stabilized after the voltage is applied. The output is stabilized usually after about 1 minute from the start of application, and it is sufficient to detect the oxygen concentration signal after such a period of time. In addition, when applying a voltage in each of the operations (ii), (iv), (vi) and (viii), it is more accurate to obtain a more accurate measurement result by stirring the sample or sterile water in the measurement cell with a stirrer or the like. Is preferable in obtaining. The stirring speed at this time is not particularly limited, but is preferably 100 to 2000 rpm.

  In the operation (v), that is, the operation for consuming oxygen to the microorganisms in the sample (hereinafter, this operation is also referred to as oxygen consumption (cultivation)), the time for the microorganisms in the sample to consume oxygen (oxygen consumption time (culture) Time)) may be set as appropriate, but it is usually 5 to 120 minutes, preferably 6 to 90 minutes. In addition, at the time of oxygen consumption (culture) in the operation (v), it is preferable to stir the sample or sterile water in the measurement cell with a stirrer or the like in order to obtain a more accurate measurement result. The stirring speed at this time is not particularly limited, but is preferably 100 to 2000 rpm.

  During each of the above operations, it is preferable to control the temperature of the sample or sterile water to be 20 to 50 ° C, more preferably 30 to 45 ° C. In particular, at the time of oxygen consumption (culture) in the operation (v), it is important to keep the liquid temperature constant because the oxygen consumption of microorganisms may be affected by the sample liquid temperature.

  As the sterilized water, purified water containing an antibacterial agent or an antibacterial agent and a cleaning agent is preferable. Of course, the present invention is not limited to this, and purified water can be used as it is as sterile water, and if it contains at least an antibacterial agent, it is not purified water but distilled water, ion-exchanged water. Industrial water, tap water, etc. can also be used.

  Examples of the antibacterial agent that can be contained in the sterile water include benzalkonium chloride, sodium azide, 2-methyl-4-isothiazolin-3-one, and 5-chloro-2-methyl-4-isothiazoline-3. -One, 1,2-benzisothiazolin-3-one, parahydroxybenzoate, 1,4-bis (bromoacetoxy) -2-butene, 1,2-bis (bromoacetoxy) ethane, methylenebisthiocyanate, etc. Of these, one or two or more of these can be used in combination. The content of these antibacterial agents is not particularly limited, but for example, 20 to 2000 ppm as an active ingredient may be contained in sterile water, and preferably 300 to 1000 ppm.

  Examples of the detergent that can be contained in the sterile water include surfactants such as polyoxyethylene (10 mol) polyoxypropylene (2 mol) lauryl ether, sodium dodecylbenzenesulfonate, tartaric acid, citric acid, apple Hydroxycarboxylic acids such as acids, organic phosphonic acids such as 1-hydroxyethylidene-1,1-diphosphonic acid, polycarboxylic acids such as sodium polyacrylate and sodium polymaleate, inorganic acids such as sulfamic acid and phosphoric acid, etc. Of these, one or a combination of two or more can be used. Although content of these cleaning agents is not specifically limited, For example, 10-5000 ppm should just be contained as an active ingredient in aseptic water, Preferably it is good to contain 100-3000 ppm.

  The method for measuring viable cell count of the present invention calculates oxygen consumption using the oxygen concentration signals (Sam-1), (Sam-2), (Cal-1) and (Cal-2) obtained by the above operation. To do. An arithmetic expression for calculating the oxygen consumption amount using the four oxygen concentration signals may be set as appropriate according to the purpose or the like. For example, the oxygen consumption amount is obtained by using the obtained oxygen concentration signal (Sam− By substituting 1), (Sam-2), (Cal-1) and (Cal-2) into the following formula (1), it can be obtained as an oxygen consumption rate (%). As an arithmetic expression that can be employed in addition to the expression (1), for example, the following expression (2) can be given.

  In the viable cell count measurement method of the present invention, the viable cell count determined from the correlation formula between the oxygen consumption rate (%) thus determined and the viable cell count value separately measured by a plate culture method or the like. By using the factor converted into, the number of viable bacteria in the sample can be automatically measured.

  As a sample to be measured in the method for measuring the viable cell count of the present invention, for example, water for various processes such as each process water of the pulp and paper industry and cooling water for a cooling tower may be used as it is, or as necessary. It may be used after appropriately diluted at an appropriate magnification.

  The viable cell count measuring method of the present invention can be carried out using the viable cell count measuring apparatus of the present invention described later. Details of the case of using the viable count apparatus of the present invention described later will be described in the section <Viable count apparatus>. Of course, the method for measuring the viable cell count of the present invention can also be performed using an apparatus other than the viable cell count measuring apparatus of the present invention described later.

<Viable count device>
The viable cell count measuring device of the present invention is a measuring device used in the above-mentioned viable cell count measuring method of the present invention. For this purpose, a sealable measuring cell and oxygen for saturating dissolved oxygen in the liquid in the measuring cell. A saturation means, a voltage application means for applying a voltage to the Clark oxygen electrode, a detection means for detecting an oxygen concentration signal generated by the voltage application, and a temperature control means for controlling the liquid temperature in the measurement cell. I have.
Hereinafter, an embodiment of the viable cell count measuring apparatus of the present invention will be specifically described with reference to the drawings. However, the viable count apparatus of the present invention is not limited to the following embodiment.

FIG. 1 is a schematic diagram showing the configuration of an embodiment of the viable cell count measuring apparatus of the present invention. As shown in FIG. 1, this viable cell count measuring apparatus is provided with a temperature control region 31 and a dissolved oxygen detector 20 in this order along the flow path direction of the sample. A sterile water tank 11 is connected to the sample flow path in the temperature control region 31 via a sterile water supply valve (V1).
In FIG. 1, P <b> 1 is a pump for sending the sample to the temperature control region 31, and P <b> 2 is a pump for sending the sample or sterile water to the dissolved oxygen detector 20. Delivery of the sample and sterile water is controlled by opening and closing the sterile water supply valve (V1) and the sample supply valve (V2), respectively.
When the air valve (V3) is opened, air can be supplied. By opening the air valve (V3), air can be bubbled through the sample or sterile water to saturate the dissolved oxygen. That is, in this embodiment, the air valve (V3) serves as oxygen saturation means.

In FIG. 1, a temperature control region 31 indicated by a broken line is temperature-controlled by temperature control means (not shown). Specific examples of the temperature control means include a thermostatic bath and a heat exchange jacket.
P3 is a pump for discharging the sample or sterile water from the system, and the discharge of the sample and sterile water is controlled by opening and closing the drain valve (V4).
The oxygen concentration signal from the dissolved oxygen detector 20 is detected by the calculation means 12 (for example, a built-in computer) and subjected to calculation processing. That is, in this embodiment, the calculation means 12 becomes a detection means.

FIG. 2 is a schematic diagram showing the configuration of the dissolved oxygen detector of the apparatus shown in FIG. In FIG. 2, reference numeral 21 denotes a measurement cell which is filled with a sample or sterile water from the flow path on the pump (P2) side and drains the sample or sterile water from the flow path on the drain valve (V4) side. . It is important that the measurement cell 21 can be sealed so that oxygen in the air does not dissolve in the sample or sterile water. Further, the measurement cell 21 is accommodated in a temperature control region 32 in order to keep the inside of the cell at a constant temperature.
The measurement cell 21 is provided with a Clark oxygen electrode 22 and voltage application means (not shown) for applying a voltage to the Clark oxygen electrode 22. This voltage application means (not shown) is capable of intermittent voltage application (for example, can be arbitrarily switched between on and off), which is a feature of the viable cell count measurement method of the present invention. There is no particular limitation, and a known voltage application means can be used.
In the measurement cell 21, a stirrer piece 23 for stirring the liquid is placed inside the bottom as a stirring means, and a magnetic stirrer 24 for rotating the stirrer piece 23 is installed outside the bottom. . The stirring means in the measurement cell 21 is not limited to the stirrer piece 23 and the magnetic stirrer 24. For example, a stirring blade 25 as shown in FIG. 4 and a motor (not shown) for driving the stirring blade 25 are provided. You can also.
Further, when the stirring means is provided, for example, as shown in FIG. 4, the stirring means (stirring blade 25) is opposed to the tip 22a of the oxygen electrode 22 (preferably facing the front of the tip 22a. It is desirable to install it so that it is located. Thereby, the pressure which the front-end | tip part 22a of the oxygen electrode 22 receives is reduced, and the measurement precision of oxygen concentration can be improved more.

  In FIG. 2, a temperature control region 32 indicated by a broken line is a region including the measurement cell 21, and controls the liquid temperature in the measurement cell 21 by temperature control means (not shown). By this temperature control, it is possible to maintain the liquid temperature of the sample or sterile water at a desired temperature. In particular, it is important to keep the liquid temperature constant at the time of oxygen consumption (during the operation (v)), where the oxygen consumption of microorganisms may be affected by the sample liquid temperature. It is desirable to do it reliably. Specific examples of the temperature control means include a thermostatic bath and a heat exchange jacket.

Hereinafter, the usage pattern of the viable cell count measuring apparatus of the present invention will be specifically described.
First, sterile water is injected from the sterile water supply valve (V1) to fill the measurement cell 21 and each flow path in the system, and the measurement cell 21 and each flow path are washed. Subsequently, the sterile sterile water is drained from the measurement cell 21 and each flow path in the system from the drain valve (V4). Next, new sterile water was injected from the sterile water supply valve (V1) to fill the measurement cell 21 and each flow path in the system, and at the same time, the air valve (V3) was opened to saturate dissolved oxygen in the sterile water. Thereafter, the air valve (V3) is closed to shut off contact with the atmosphere, and in this state, sterile water is filled into the measurement cell 21. Next, voltage application and stirrer stirring are started at the oxygen electrode 22, and after the output is stabilized, the oxygen concentration signal (Cal-1) is recorded. Then, the voltage application is stopped, and washing is performed with sterile water.

  Next, the sample is injected into the measurement cell 21 from the sample supply valve (V2), and the co-washing is repeated several times. Thereafter, the sample is injected into the measurement cell 21 from the sample supply valve (V2), and at the same time, the air valve (V3) is opened to saturate the dissolved oxygen in the sample, and then the air valve (V3) is closed to the atmosphere. In this state, the measurement cell 21 is filled with the sample. Next, voltage application and stirrer stirring are started at the oxygen electrode 22, and after the output is stabilized, the oxygen concentration signal (Sam-1) is recorded, and then voltage application is stopped. The sample in the measurement cell 21 is stirred for a predetermined time, voltage application is started again at the oxygen electrode 22, and after the output is stabilized, the oxygen concentration signal (Sam-2) is recorded. The sample in 21 is discharged.

  Next, aseptic water is injected from the sterile water supply valve (V1) to fill the measurement cell 21 and each flow path in the system, the measurement cell 21 and each flow path are washed, and the measurement cell 21 is discharged from the drain valve (V4). Drain sterile water from each channel in the system. Subsequently, new sterile water was injected from the sterile water supply valve (V1) to fill the measurement cell 21 and each flow path in the system, and at the same time, the air valve (V3) was opened to saturate dissolved oxygen in the sterile water. Thereafter, the air valve (V3) is closed to shut off contact with the atmosphere, and in this state, sterile water is filled into the measurement cell 21. Next, voltage application and stirrer stirring were started at the oxygen electrode 22, and after the output was stabilized, the oxygen concentration signal (Cal-2) was recorded. Then, voltage application was stopped, and the measurement cell 21 and each flow path were connected to sterile water. The series of steps is completed after washing.

  Thereafter, using the oxygen concentration signals (Cal-1), (Cal-2), (Sam-1) and (Sam-2), and the viable cell count factor obtained separately by a plate culture method or the like, The number of viable bacteria in the sample is calculated by the calculation means 12 (built-in computer or the like) and displayed on a display (not shown).

<Slime monitoring method>
The slime monitoring method of the present invention performs the measurement by the above-mentioned method for measuring viable cell count of the present invention every predetermined time, and monitors the amount of slime by increasing or decreasing the oxygen consumption obtained by the measurement. According to such a slime monitoring method of the present invention, it is possible to easily and reliably grasp the occurrence of slime in various wastewaters, for example, when adding a slime control agent to prevent slime failure. This makes it possible to reliably prevent slime failure efficiently. In the slime monitoring method of the present invention, the interval for performing the measurement by the viable count method is not particularly limited, and may be set as appropriate according to the type of the target sample.

<Slime control agent addition system>
The slime control agent addition system of the present invention includes a mechanism for controlling the addition amount of the slime control agent based on the oxygen consumption measured in the above-mentioned viable cell count measurement method of the present invention. Hereinafter, an embodiment of a slime control agent addition system of the present invention will be described with reference to the drawings.

  FIG. 3 is a block diagram schematically showing an embodiment of the slime control agent addition system of the present invention. As shown in FIG. 3, first, industrial water such as white water and raw material slurry in a paper mill is sent to a viable cell count measurement device in order to monitor the number of bacteria. In the viable cell count apparatus, each oxygen concentration signal described above is detected to calculate the oxygen consumption rate in the industrial water according to the viable cell count measuring method of the present invention, and the oxygen consumption signal (oxygen consumption rate) is calculated. Arithmetic processing is performed. The result of this arithmetic processing is sent to a control and monitoring device (specifically, for example, a personal computer can be used). Based on the result, slime control is performed according to the level of oxygen consumption rate (viable cell count). A command to change the addition amount of the agent to an appropriate amount is sent to the slime control agent automatic addition device. In accordance with this command, the slime control agent is added in an appropriate amount according to the oxygen consumption rate (viable cell count) in the automatic slime control agent addition device. In this way, the slime control agent addition system of the present invention can prevent slime failure by maintaining the number of viable bacteria in white water or raw material slurry at a predetermined preferable level.

  The slime control agent that can be used in the method for adding a slime control agent of the present invention is not particularly limited, but one or more of bactericides and antibacterial agents can be used as the slime control agent. It is also possible to use a bactericide and an antibacterial agent in combination as a slime control agent. Examples of bactericides include 2,2-dibromo-3-nitrilopropionamide, 1,4-bis (bromoacetoxy) -2-butene, 1,2-bis (bromoacetoxy) ethane, 2,2-dibromo- Examples include 2-nitroethanol. Examples of the antibacterial agent include those described above as antibacterial agents that can be contained in sterile water, among which 2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazoline Preferable examples include -3-one, 1,2-benzisothiazolin-3-one, and methylene bis thiocyanate.

  The slime monitoring method and the slime control agent addition system of the present invention can be applied to any scene where slime can be generated in water, such as various process water in the paper pulp industry and cooling water in a cooling tower. Especially in the pulp and paper industry, slime can cause various harmful effects such as paper breaks, eyeballs, stains, and reduced operability. Therefore, the slime monitoring method and the slime control agent addition system of the present invention are extremely applicable. It is effective.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to a following example.
The number of viable bacteria by the plate culture method was measured as follows. That is, first, the water sample was serially diluted 10 times with sterilized water. 0.1 ml of this diluted solution was smeared on a standard agar medium and cultured at 32 ° C. for 48 hours. And the number of viable bacteria in 1 mL of water samples was calculated | required from the number of formed colonies.

(Example)
White water under the wire (save all) was collected at a newsprint manufacturing factory and used as a water sample. The pH of this water sample was 4.5, and the number of viable bacteria was measured by a plate culture method and was 10 ⁷ order. As a result of investigating the bacterial species, it was confirmed that the Pseudomonus genus had the largest number of 10 ⁷ orders, followed by the Bacillus genus 10 ⁵ orders. All of these species belong to aerobic bacteria.

A stock sample solution (10 ⁷ order) was serially diluted with sterilized water to prepare sample solutions diluted to the order of 10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ , respectively. Using these sample solutions and water sample stock solutions as measurement targets (samples), the oxygen consumption rate by microorganisms in each sample was measured using the viable cell count measuring apparatus of the present invention shown in FIGS. 1 and 2.

  Specifically, first, aseptic water (purified water added with 300 ppm of sodium azide) is injected by opening the sterile water supply valve (V1) to fill the measurement cell and each flow path in the system, After washing each flow path, the valve (V1) was closed, and the drain valve (V4) was opened to drain dirty sterile water from the measurement cell and each flow path in the system. Subsequently, the sterile water supply valve (V1) is opened to inject new sterile water (similar to the above) to fill the measurement cell and each flow path in the system, and at the same time, the air valve (V3) is opened to open the sterile water. After the dissolved oxygen was saturated, the air valve (V3) was closed to shut off contact with the atmosphere, and in this state, sterile water was filled in the measurement cell. Next, a voltage of −650 mV was applied at the oxygen electrode, and at the same time, stirring was started at 500 rpm with a stirrer. After 3 minutes later, the output stabilized and an oxygen concentration signal (Cal-1) was detected, and then the voltage application was stopped. . At this time, the voltage application time from applying the voltage to stopping was 3 minutes. Thereafter, sterile water in the measurement cell was drained.

  Next, the sterile water supply valve (V1) and the air valve (V3) are closed, the sample supply valve (V2) is opened and a water sample is injected into the measurement cell, and then the valve (V2) is closed and the drain valve (V4) ) Was opened, the water sample in the measurement cell and each flow channel was discharged, and the operation of filling the water sample in the measurement cell and each flow channel (co-washing) was repeated twice. Thereafter, the sample supply valve (V2) is opened to inject the water sample to fill the measurement cell and each flow path in the system, and at the same time, the air valve (V3) is opened to saturate the dissolved oxygen in the water sample. The air valve (V3) was closed to shut off contact with the atmosphere, and in this state, the water sample was filled in the measurement cell. Next, a voltage of −650 mV was applied at the oxygen electrode, and simultaneously stirring was started at 500 rpm by a stirrer. After 3 minutes, the output stabilized and an oxygen concentration signal (Sam-1) was detected, and then the voltage application was stopped. . At this time, the voltage application time from applying the voltage to stopping was 3 minutes.

  Subsequently, the water sample in the measurement cell was stirred at 250 rpm for 60 minutes while keeping the liquid temperature at 40 ° C., and oxygen was consumed by the microorganisms. After stirring, a voltage of -650 mV was applied again with an oxygen electrode, and at the same time stirring was started with a stirrer at 500 rpm. After 3 minutes, the output stabilized and an oxygen concentration signal (Sam-2) was detected, and then voltage was applied. Stopped. At this time, the voltage application time from applying the voltage to stopping was 3 minutes. Then, the drain valve (V4) was opened and the water in the measurement cell was discharged.

  Next, aseptic water (same as above) is injected by opening the sterile water supply valve (V1) to fill the measurement cell and each flow path in the system, and after washing the measurement cell and each flow path, the valve ( V1) was closed, and the drain valve (V4) was opened to drain dirty aseptic water from the measurement cell and each flow path in the system. Subsequently, the sterile water supply valve (V1) is opened to inject new sterile water (similar to the above) to fill the measurement cell and each flow path in the system, and at the same time, the air valve (V3) is opened to open the sterile water. Of dissolved oxygen. Next, a voltage of −650 mV was applied at the oxygen electrode, and at the same time, stirring was started at 500 rpm with a stirrer. After 3 minutes later, the output stabilized and an oxygen concentration signal (Cal-2) was detected, and then the voltage application was stopped. . At this time, the voltage application time from applying the voltage to stopping was 3 minutes.

The oxygen concentration signals (Cal-1), (Cal-2), (Sam-1) and (Sam-2) thus obtained are sent to the calculation means (built-in computer) 12 and the above-described equation ( The calculation based on 1) was performed, and the oxygen consumption rate was displayed on the display. The results are shown in Table 1. In the table, in order to compare the detection sensitivities, the relative values when the measured value in the bacterial count order 10 ³ is 1 are also shown.

(Comparative example)
In the same measuring apparatus as in the example, except that the voltage is always applied (continuously applied), the same as in the example (that is, “voltage application is stopped” in a series of operations in the example) All the operations were omitted and the operation was continued with the voltage applied.) The oxygen consumption rate by microorganisms in each sample was measured for the same measurement object as in the example. The results are shown in Table 2. In the table, in order to compare the detection sensitivities, the relative values when the measured value in the bacterial count order 10 ³ is 1 are also shown.

From the results of Tables 1 and 2, it can be seen that the measurement method of the present invention clearly shows a change in signal value with respect to each bacterial count order and has a high detection sensitivity as compared with the method of applying voltage continuously. . In the method of applying voltage continuously, the detection limit is 10 ⁴ to 10 ⁵ order as the number of viable bacteria, whereas in the measurement method of the present invention, the number of viable bacteria is measured at a high significance level up to 10 ³ order. I can confirm that I can do it.

It is the schematic which shows the structure of the viable cell count measuring apparatus of this invention. It is the schematic which shows the structure of the dissolved oxygen detection part in the viable count apparatus of this invention. It is a block diagram showing roughly one embodiment of the slime control agent addition system of the present invention. It is the schematic which shows another structure of the dissolved oxygen detection part in the viable cell count measuring apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Aseptic water tank 12 Calculation means 20 Dissolved oxygen detection part 21 Measurement cell 22 Oxygen electrode 22a Oxygen electrode front-end | tip part 23 Stirrer piece 24 Magnetic stirrer 25 Stirrer blades 31 and 32 Temperature control area

Claims (8)

A method for measuring the number of viable bacteria in a sample from the amount of oxygen consumed by microorganisms, wherein the oxygen consumption is determined by applying a voltage to a Clark-type oxygen electrode and detecting an oxygen concentration signal. i) to (viii) are performed in this order, and the obtained oxygen concentration signals (Sam-1), (Sam-2), (Cal-1) and (Cal-2) are used for calculation. A method for measuring the number of viable bacteria.
(I) An operation of filling the measurement cell with the sterilized water after saturating the dissolved oxygen concentration in the sterilized water with the contact with the atmosphere blocked.
(Ii) An operation of stopping the application of the voltage after applying the voltage to detect the initial oxygen concentration signal (Cal-1) of sterile water.
(Iii) After saturating the dissolved oxygen concentration in the sample, filling the measurement cell with the sample in a state where contact with the atmosphere is blocked.
(Iv) An operation of stopping the application of the voltage after applying the voltage and detecting the initial oxygen concentration signal (Sam-1) of the sample.
(V) An operation in which the microorganisms in the sample consume oxygen for a predetermined time.
(Vi) An operation of stopping the application of the voltage after applying the voltage and detecting the oxygen concentration signal (Sam-2) after oxygen consumption.
(Vii) An operation in which the concentration of dissolved oxygen in sterile water is saturated again, and then the sterile cell is filled with the sterile water in a state where contact with the atmosphere is blocked.
(Viii) An operation of stopping the application of the voltage after detecting the oxygen concentration signal (Cal-2) of sterile water after the sample measurement by applying a voltage. The oxygen consumption is calculated as the oxygen consumption rate by substituting the obtained oxygen concentration signals (Sam-1), (Sam-2), (Cal-1) and (Cal-2) into the following equation (1). The method for measuring the number of viable bacteria according to claim 1 to be obtained.

  The method for measuring the number of viable bacteria according to claim 1 or 2, wherein the sterile water is an antibacterial agent or an antibacterial agent and a cleaning agent contained in purified water.   The method for measuring the viable cell count according to any one of claims 1 to 3, wherein the voltage application time to the oxygen electrode is 1 to 6 minutes per time.   It is a measuring apparatus used for the viable cell count measuring method in any one of Claims 1-4, Comprising: The measuring cell which can be sealed, Oxygen saturation means which saturates dissolved oxygen in the liquid in this measuring cell, Clark type A voltage applying means for applying a voltage to the oxygen electrode; a detecting means for detecting an oxygen concentration signal generated by the voltage application; and a temperature control means for controlling the liquid temperature in the measurement cell. To measure the number of viable bacteria.   6. The viable cell count measurement apparatus according to claim 5, further comprising aseptic water supply means for supplying aseptic water instead of the sample.   A slime monitor characterized in that the measurement by the method for measuring the viable cell count according to any one of claims 1 to 4 is performed at predetermined intervals, and the slime amount is monitored by increasing or decreasing the oxygen consumption obtained by the measurement. Method. A slime control agent addition system comprising a mechanism for controlling the addition amount of the slime control agent based on the oxygen consumption measured in the method for measuring the viable cell count according to any one of claims 1 to 4.

Images