JP6143329B2 - Method and apparatus for detecting food bacteria using electrical impedance - Google Patents

Description

  The present invention relates to a method and apparatus for detecting food bacteria that can easily and easily detect bacteria in food, and more specifically to a method and apparatus for detecting food bacteria using electrical impedance measurement.

  Food and drink intake is indispensable in animals including humans. Despite advances in food processing and storage technologies, it is still difficult to completely prevent “food poisoning”, which is an addiction caused by consumption of food and drink. Every year, food poisoning events occur frequently, and there is a great interest in food safety.

  Based on the cause, food poisoning is caused by natural poisoning (puffer fish poisonous, mushroom poisoning, etc.), food poisoning due to food spoilage (such as food degradation products), and bacteria (staphylococci, Salmonella, Vibrio parahaemolyticus) Etc.) or food poisoning caused by breeding. Among these food poisonings, food poisoning by bacteria (ie, bacterial food poisoning) is caused by contamination or propagation of bacteria in food, but is usually invisible. It is difficult without going through the process.

  Occurrence of food poisoning caused by food and drink provided in facilities that supply a large amount of food such as canteens, lunch centers, supermarkets, convenience stores, etc., causes vomiting, diarrhea, abdominal pain, high fever, etc. to a large number of people Therefore, particularly serious problems are likely to occur. In addition, general bacteria that are not normally infected also become infectious when they exceed a certain number. Bacterial growth also occurs in all foods and beverages such as rice, bread, cakes, seaweed salad, fish, minced meat, vegetable juice.

  As one of the techniques for measuring the growth of microorganisms, a technique called impedance method has been known for a long time. Impedance is an electrical resistance value at an alternating current. When an electrode is inserted into a liquid medium mixed with microorganisms and cultured while a weak current is applied, the resistance value changes due to the growth of the microorganisms. This is a method of measuring the number of microorganisms by detecting this change in resistance value (Non-Patent Document 1).

  The two-electrode method using a current-carrying electrode as a measurement electrode has a problem that the reproducibility of the measured impedance is poor, and an impedance method using four electrodes for measuring the voltage drop of the solution during current-carrying has been proposed (non-patented) References 2 and 3). The apparatus shown in Non-Patent Document 2 using the four-electrode method is an E. coli method using a culture cell having a volume of about 1.5 ml and an electrode having a diameter of 0.7 mm. An example in which E. coli is measured is shown, and the bacterial culture has an electrical resistance of about 120 ohm-cm, and the change in impedance due to culture is described to be about 4% after 5-8 hours of culture. .

  As a technique for quantifying bacteria contained in a liquid to be measured, DEPIM (Dielectrophoretic Impedance Measurement Method) combining dielectrophoresis and impedance measurement is known. DEPIM applies an alternating voltage of a predetermined frequency to electrodes in a bacterial suspension, collects bacteria between electrode gaps, and quantifies the number of bacteria by measuring the change in impedance between the electrodes at this time. (Patent Document 1).

JP 2011-200152 A

Tsuneoka et al., Automatic detection of bacterial growth by electrical method, Clinicopathology, 27 (7), 563-567, 1979 Kiyoshi et al., Electrical impedance measurement of culture solution under bacterial culture, Medical Electronics and Biotechnology, Vol. 19, No. 1, 35-39 (Feb. 1981) Miike et al., Change in electrical impedance during yeast growth and fermentation, IEICE Transactions, Vol. J69-C, No. 10, 1134-1340, 1986

  As a means for preventing food poisoning, general preventive measures (for example, keeping cooking utensils such as knives and mana plates clean, keeping fingers of people involved in cooking clean, etc.) are taken. However, these means are indirect means based on experience that it is possible to avoid adhesion of bacteria to food and drink when such a thing is performed. It is not a direct means of preventing food poisoning by confirming contamination and / or propagation and discarding or treating bacteria if contaminated and / or breeding. And if food poisoning occurs, a health center survey (cultivation of bacteria attached to food and drink that seems to be the cause) will be necessary, and the provision of food and drink will be stopped and the causative bacteria detected and confirmed The impact is greater, such as being necessary.

Therefore, there is a demand for means for confirming whether bacteria are mixed and / or propagated in food and drink. However, devices and methods used in inspection facilities such as health centers are complex and expensive. The impedance method has been conventionally used in laboratories and the like as a method for measuring the growth of microorganisms and the number of bacteria, but was not an apparatus and method that can be easily measured in a cafeteria or a lunch center.
Food poisoning has become a very big problem, and it is possible for a person other than an expert to detect and / or identify bacterial contamination in food and drink in a place that is not a specialized inspection facility such as a cafeteria or a lunch center. There was a need for a possible method and apparatus.

  An object of the present invention is to provide a method and an apparatus capable of detecting bacteria by a simple operation even in a place that is not such a specialized inspection facility and a person other than a specialist.

In the present invention, a low-salt concentration bacterial culture medium in which a test sample (for example, food) is suspended is placed in a measurement container of a predetermined size and cultured, and the same test sample is placed in a reference container of a predetermined size. A method and apparatus for detecting bacteria in a test sample (for example, food) by culturing in the same medium containing a suspended antibacterial agent and measuring changes in electrical impedance in both solutions. .
The present invention is also as follows.
(1) In a method for detecting bacteria in a sample by measuring changes in electrical impedance based on bacterial growth, the following steps:
a. Prepare a measurement solution in which the sample is suspended in a low-salt bacterial culture medium, and suspend the same sample in the same low-salt bacterial culture medium that contains an antibacterial agent at a concentration that inhibits bacterial growth. The step of preparing the reference solution, where the salt concentration of the low salt concentration bacterial culture medium in which the sample is suspended is a salt concentration corresponding to a salt concentration of 0.2% NaCl or less in the measurement solution and the reference solution. Have been prepared,
b. Measuring the electrical impedance of each of the measurement liquid and the reference liquid using an electrode composed of four electrodes, and c. By measuring the impedance change (for example, temperature change of the culture solution) not caused by bacterial amplification using the reference solution in the culture environment, and measuring the electrical impedance change of the measurement solution, Measuring process,
A method for detecting bacteria in a sample comprising:
(2) The method for detecting bacteria according to (1), wherein the sample is a food (3) The initial value of the electrical impedance value of the measurement solution is at least 70 ohms (1) or (2) How to detect bacteria.
(4) The method for detecting bacteria according to any one of (1) to (3), wherein the measurement solution and the reference solution are at least 10 ml or more.
(5) The impedance detection unit of the electrode is disposed so as to be located in each intermediate part of the measurement liquid and the reference liquid (intermediate part of the liquid upper layer and the liquid lower layer), and the length of the impedance detection part of the electrode is The method for detecting a bacterium according to any one of (1) to (4), wherein the bacterium is 10 mm or less.
(6) The method for detecting bacteria according to any one of (1) to (5), wherein the electrode has a diameter of 1.0 mm to 3.0 mm.
(7) The method for detecting a bacterium according to any one of (1) to (6), wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium.
(8) In the above (7), the bacterium is at least one bacterium selected from the group consisting of Escherichia coli, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Bacillus cereus, and Clostridium botulinum. A method for detecting the described bacteria.
(9) A cell with electrodes used to detect bacteria in a sample,
Container 5 for containing the medium containing the sample,
A lid 1 having an opening 2 that can be fitted to the top of the container;
Four electrodes 4 that are arranged in a straight line from the center of the lid portion to both side surfaces and that extend from the lid to a substantially central portion in the vertical direction of the container, and the four electrodes A non-conductive electrode support portion 3 that can be fitted into the opening, and the electrode support portion is the impedance detection portion 6 only in the vicinity of the tip of the electrode. Covering the electrode so as to be exposed in the medium,
A cell with an electrode.
(10) The electrode-attached cell according to (9), wherein the container has a cylindrical shape and a capacity of at least 10 ml.
(11) The electrode-attached cell according to (9) or (10), wherein the electrode is a rod-shaped electrode having a diameter of 1.5 mm to 3.0 mm.
(12) The cell with an electrode according to any one of (9) to (11), wherein the sample is food.
(13) A storage structure 35 having at least two storage portions 34 for storing the electrode-attached cell according to any one of (9) to (12), wherein the storage structure is a sample A cell with a measurement electrode containing a medium (measuring solution) containing, and a cell with a reference electrode containing a medium (reference solution) containing a sample and an antibacterial agent can be stored.
A current supply unit for supplying a current to the electrode of the electrode-equipped cell, and a measurement unit for measuring an impedance of the measurement liquid and the reference liquid to detect an impedance change;
A device for detecting bacteria based on changes in electrical impedance.
(14) The apparatus according to (13), further including an indicator unit that turns on the lamp based on the detection result of bacteria.
(15) The electrode according to any one of (9) to (12) for detecting bacteria in a sample using the method according to any one of (1) to (8) With cell.
(16) The apparatus according to any one of (13) and (14), wherein the bacteria in food are detected using any one of the methods (1) to (8).

  By using the method for detecting a bacterium of the present invention, it is possible to quickly detect a bacterium in a food, for example, during processing of a food or within a cooking time. Since the method and apparatus of the present invention are simple and simple, they can be used at food processing or cooking sites, can be easily operated even by non-experts, and can accurately detect bacteria in food.

It is an exploded view of the cell with an electrode used for the method and apparatus of this invention. The state at the time of measuring impedance by setting a cell with an electrode in a detection device is shown. It is sectional drawing of the cell with an electrode of the state which put the measurement liquid or the reference liquid, and was covered. It is a perspective view of one mode of the device of the present invention. It is an exploded view of the apparatus shown in FIG. 1 shows a block diagram of one apparatus configuration of the present invention. The electric impedance change measured using the measurement liquid and the reference liquid is schematically shown. The lower row shows the difference. It is the result of examining the influence of the salt concentration on the impedance. It is the figure which showed the change of the length of an impedance detection part. It is the result of culturing Escherichia coli and measuring the impedance change. It is the graph which re-plotted the result of FIG. The result of the electrical impedance change in the measurement liquid and the reference liquid measured using the apparatus shown in FIG. 4 is shown. A shows the change in electrical impedance in each solution, and B shows the difference. The result of having detected E. coli using the apparatus described in FIG. 4 is shown. The result of having detected Salmonella using the apparatus of FIG. 4 is shown.

  In this specification, when “A to B” is described, it is used in the meaning including A as the lower limit and B as the upper limit.

  The detection method and apparatus of the present invention can be used without particular limitation as long as it is an object containing bacteria (or microorganisms), but is preferably used for food. As used herein, “food” refers to unprocessed foods, processed foods such as processed meat and processed fish, processed foods such as prepared foods and dairy products, cooked foods, and provided to customers. Means uncooked or cooked dishes, beverages or other things that people take as food. In the case where the food is a beverage, it can be used as diluted with a medium that is a diluent. If the food is a solid, it can be crushed using a container in a medium that is a diluent, but in the case of a harder solid, for example, a diluent or physiological saline is moistened. It can also be used by using a wiping material that wipes the surface of food with wiping gauze, absorbent cotton or the like, or finely crushed with a mixer or stomacher.

  Bacteria that can be detected in the present invention are not limited, but, for example, Gram-negative bacteria or Gram-positive bacteria, in particular, Salmonella, Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Bacillus cereus, Clostridium botulinum, etc. Food poisoning bacteria are preferred as detection targets of the present invention.

The medium for culturing a low salt concentration bacteria used in the present invention is a liquid medium having a salt concentration corresponding to 0.10% NaCl or less, which is suitable for culturing bacteria. Medium includes, for example, milk casein, animal meat, heart muscle, gelatin, soy protein and other peptones, extracts such as meat extract and yeast extract, substances required by bacteria such as amino acids, vitamins, serum and tissues, bile acids It consists of substances that selectively inhibit the growth of specific bacterial species, such as salts and sodium dodecyl sulfate. Commercially available liquid medium suitable for bacterial culture, for example, LB medium, meat extract broth medium, casein / soybean mixed peptone medium, heart fusion medium, brain fusion medium, tryptone medium with reduced or eliminated salt It can be used without particular limitation.
The salt concentration of the culture medium for low salt concentration bacteria used in the present invention is a salt concentration corresponding to 0.10% NaCl or less, and preferably 0.08% or less.
In the present invention, the salt concentration of the medium in the measurement container containing the measurement sample is 0.0045% to 0.2% equivalent to NaCl by using the low salt concentration bacterial culture medium. An impedance resistance value suitable for measurement can be obtained. The salt concentration of the medium in the measurement container containing the measurement sample is preferably 0.01 to 0.1%, more preferably 0.04 to 0.08% NaCl.

  In the present invention, as a control, an antibacterial agent-containing medium for low salt concentration bacterial culture containing a sample is used. Antibacterial-containing low salt concentration bacterial culture medium (referred to as a reference solution in the present invention) containing a sample to be used as a control contains the same or lower salt concentration as the measurement solution containing an antibacterial agent at a concentration that inhibits bacterial growth. What is necessary is just to suspend the same sample as a measurement liquid in the culture medium for bacteria culture. Such a reference solution can be prepared by adding an antibacterial agent to a low-salt concentration bacterial culture medium in which the sample is suspended so that it contains an antibacterial agent at a concentration that inhibits bacterial growth. The sample may be prepared by suspending the sample in a low salt concentration bacterial culture medium containing the agent. As a control in the measurement of electrical impedance, a medium that is not contaminated with bacteria is usually used. However, if the measurement target is a sample that affects the electrical impedance of foods, etc. Since impedance is affected, measurement may be difficult. In particular, in a measurement system in which a sufficient impedance resistance value cannot be obtained, since the change in impedance value accompanying cell proliferation is small, the influence of measurement errors increases, and appropriate bacteria cannot be detected. In the present invention, in the control, by adding an antibacterial agent in advance and performing simultaneous measurement in a state where the number of bacteria does not increase, more accurate detection is achieved by removing the influence of the impedance value due to the sample (for example, food) itself and temperature change It can be performed.

  The antibacterial agent used in the present invention can be used without particular limitation as long as it does not inhibit the growth of bacteria to be measured and does not greatly affect the electrical impedance, and a normal commercially available antibacterial agent can be used. Examples include, but are not limited to, polyhexamethylene, biguanide, hydrochloride, ampicillin, kanamycin, novobiocin, methylnaphthoquinone, triazine, isoprathiolane, iprodione, and siabendazole. The concentration of the antibacterial agent contained in the medium in the control container is not particularly limited as long as it is not less than the concentration that inhibits the growth of bacteria, but a concentration that almost completely inhibits the growth of bacteria is preferable.

In the measurement method of the present invention, a sample (for example, a sample in a low salt concentration bacterial culture medium containing an antibacterial agent) and a reference solution (a medium in which a sample (for example, food) is suspended in a medium for low salt concentration bacterial culture) are suspended. The same amount of the medium in which the food) is suspended is preferably the same, and in order to efficiently detect bacteria in the sample (for example, food) in a short time, it is at least 10 ml, preferably 20 ml, more preferably 30 ml. It is.
The measurement container and the reference container used in the present invention are preferably the same material and the same capacity. The capacity of the container is preferably such that 10 to 40 ml of a measurement solution and a reference solution can be placed in order to efficiently measure bacteria in a sample (eg food) in a short time, preferably at least 10 ml or more. 20 ml or more, more preferably 30 ml or more. The upper limit of the volume is not particularly limited, but it is preferably 100 ml or less, particularly 50 ml or less because the apparatus becomes compact.
In the method of the present invention, as will be described below, in order to generate an appropriate impedance resistance value and reduce noise, the impedance detection part of the electrode is placed in the middle part of the solution (the upper part of the solution and the lower part of the solution). It is preferable to use a measuring container and a reference container having a capacity corresponding to the amount of the measuring liquid and the reference liquid.

In the detection method of the present invention, the food to be measured, which is a sample, is suspended in a low-salt bacterial culture medium used for measurement and diluted stepwise (for example, 10 times each) using the same medium. And measuring the electrical impedance change based on bacterial growth for each, and measuring bacteria in the sample (eg food). In addition, the number of bacteria mixed in a sample (for example, food) can be determined by comparing the change in electrical impedance based on the growth of bacteria in each culture solution diluted stepwise.
For example, using 10 g of food as a sample, 90 ml of culture medium is used as a diluent, and about 100 ml of measurement medium (initial measurement liquid) is obtained. Since it can be measured as it is, it can reduce the sampling loss of bacteria in the target food and increase the amount of collected bacteria. Hard to occur. Further, dilute the initial measurement solution in a stepwise manner with a medium, for example, collect 5 ml, add 45 ml of the medium and dilute it ten times stepwise to create a measurement solution in which the bacterial concentration is diluted stepwise. Can do. By simultaneously measuring these stepwise diluted measurement solutions in addition to the initial measurement solution, the number of bacteria mixed in the food can be determined.
In this way, by using a large amount of solution, equipment such as a pipette that handles a small amount of 1 ml or less becomes unnecessary, and a person other than a specialist can easily perform measurement outside a specialized facility.

In the present invention, the electrical impedance is measured using an electrode composed of four electrodes. The electrode is preferably a rod-like electrode, and the diameter is preferably 1.0 mm or more, and more preferably 1.5 mm or more. The electrode having a diameter of 1.0 mm to 3.0 mm, more preferably 1.5 to 3.0 mm, and particularly about 1.5 mm is disadvantageous in that the design and processing of the apparatus becomes easy when the diameter is large, but the impedance resistance is low. A 2 mm rod electrode is preferred.
The area of the impedance detection part of the electrode arranged in the culture medium for bacteria culture is preferably not too large in order to avoid noise during measurement. Specifically, the length of the electrode of the impedance detection unit placed in the culture medium is preferably 10 mm or less, more preferably 5 mm or less, and still more preferably 2 to 3 mm.
As the electrode, a stainless steel electrode, a platinum wire electrode, a gold wire electrode, a carbon rod electrode or the like can be used, and a stainless steel electrode is preferable.

  The electrical impedance is measured while applying a weak voltage to the electrodes arranged in the liquid medium. The reference voltage is preferably 70 to 1600 mV, particularly preferably 900 to 1100 mV. When it is less than 70 mV or 1600 mV, noise occurs. If the current value in the liquid medium is excessive, it inhibits the growth of bacteria, and is preferably 5 mA or less, more preferably 1 mA or less.

The solution resistance (electrical impedance) measured in the method of the present invention is at least 70 ohms, preferably from about 100 to about 200 ohms. In the method of the present invention, since a decrease in solution resistance of about 20% can be confirmed by bacterial growth, bacteria can be detected with very high accuracy.
The measurement (culture) time can be appropriately changed depending on the number of bacteria in the food and the type of bacteria, but the measurement is usually completed within 5 hours, preferably within 2 hours. When the number of bacteria in the food is large, a decrease in impedance can be confirmed in a short time, and detection can be completed in a short time.
When the method of the present invention is used, if the number of bacteria in the food is 10000 cfu / 10 g or more, it can be detected by measurement within 5 hours.

  Further, in the present invention, by selecting a medium suitable for the growth of a specific bacterial species as a medium for culturing a low salt concentration bacterium, it is possible to detect not only general living bacteria but also a specific bacterial species such as Escherichia coli. Is possible. In addition to the coliform group, these bacteria can be efficiently detected by using a low salt concentration culture medium suitable for Salmonella, Vibrio parahaemolyticus, Staphylococcus aureus and the like.

  The change in electrical impedance can be measured by monitoring the value obtained by subtracting the electrical impedance of the reference solution from the electrical impedance of the measurement solution at the same time, but the average of the electrical impedance in the reference solution up to a certain point in time Measurement can also be performed by obtaining a value and monitoring the value obtained by subtracting the value from the electrical impedance of the measurement liquid. By measuring the electrical impedance of the reference solution, influences based on causes other than cell growth can be eliminated, and bacterial growth can be accurately measured (FIG. 7).

  A schematic diagram of the structure of the cell with electrodes of the present invention is shown in FIGS. FIG. 1 is an exploded view of an electrode-equipped cell, and FIG. 2 shows a state when impedance is measured with the electrode-equipped cell set in a detection device. The electrode-equipped cell includes a container 5 in which a measurement liquid or a reference liquid is placed, a lid 1 and an electrode part (electrode 4 and electrode support part 3). An opening 2 is provided in the center of the lid, and the electrode is fixed by inserting an electrode support into the opening. The electrode 4 is composed of four electrodes and is arranged in a straight line from the center of the lid to both side surfaces. The distance between each electrode can be adjusted to obtain an appropriate impedance depending on the size of the container. For example, in the case of the container 5 having a diameter of about 40 mm, the distance between the electrodes can be about 10 mm. As shown in FIG. 2, four electrode contacts for connecting a power source to the electrodes are positioned in the opening 2 of the lid.

  Most of the electrode (4) is covered with an electrode support (3) made of a non-conductive material, thereby preventing the thin electrode from being broken. In order to increase the impedance resistance, it is generally preferable that the electrode is thin. However, when a person who is not an inspection specialist handles a simple device at a site such as a kitchen, there is a risk that the thin electrode may break and the operation is difficult. difficult. Therefore, it is preferable to use an electrode having a diameter of 1.5 mm or more. The impedance detection part (near the lower end 6 of the electrode 4 in FIG. 1) exposed from the electrode support part is preferably shorter in order to obtain good impedance resistance, preferably 10 mm or less, and particularly preferably 5 mm or less. 2 to 3 mm is more preferable.

  In the present invention, by using the electrode support part, it is possible to arbitrarily adjust the area of the impedance detection part of the electrode to obtain a good impedance, and the position of the impedance detection part of the electrode is set to the measurement liquid and the reference liquid. It can be arranged so as to be located in an intermediate part (in particular, only in the vicinity of the intermediate part) between the upper layer and the lower layer. Since the impedance detection part of the electrode is located in the middle part of the measurement / reference solution, it is possible to avoid the influence of impurities (sediment and suspended matter) that affect the impedance measurement derived from the sample (for example, food). That is, when food or the like is used as a sample, food suspension is generated in the upper layer of the measurement solution and the reference solution, and food precipitate is generated in the lower layer, so by placing the detection unit in the middle of the solution, Measurement inhibition is prevented and stable measurement can be realized.

  FIG. 3 shows a state in which the measurement liquid or the reference liquid is put and covered. The electrode 16 is supported by the electrode support 15, and the impedance detector near the tip is located in the middle of the solution 13 in the container 12. The contact 14 at the other end of the electrode is located in the central opening of the lid 11 and is connected to the contact (23 in FIG. 4) of the detection device to be supplied with a constant current from the outside.

  FIG. 4 is a perspective view of an example of the detection apparatus of the present invention. In FIG. 4, the measurement container and the reference container are configured so that five pairs can be set, but there is no particular limitation and only one pair may be used. Since one measurement can be performed in one pair, it is preferable that the measurement can be performed in 2 to 5 stations when measuring serially diluted products at the same time. The device lid 21 is provided with a terminal 23 that can come into contact with the contact of the upper electrode of the electrode-attached cell, and in order to ensure the contact between the electrode upper terminal 14 of the electrode-attached cell and the terminal 23 of the body lid, 21 is formed with a storage portion 22 that matches the shape of the lid of the cell. When the lid is closed, an electric current flows through the electrodes, and impedance measurement starts (state 26 under measurement). The device is connected to a PC and can be controlled by the PC. An indicator 27 is arranged on the front surface of the main body 25 so that when an impedance change occurs, it can be displayed according to the degree. FIG. 6 is a block diagram showing the configuration of one embodiment of the apparatus of the present invention.

  FIG. 5 is an exploded view of the apparatus of FIG. A heater 36 and a bathtub 37 for keeping the culture medium at 37 ° C. are disposed under the storage portion 34 for storing the cell 33 with electrodes.

EXAMPLES Hereinafter, although this invention is demonstrated from an Example, this invention is not limited to a following example.

(Example 1) Influence of impedance on impedance by salt concentration Using an electrode-equipped cell having the same structure as that shown in FIG. Bacterial growth was confirmed under conditions using a commercially available normal LB medium, but the impedance value was small (less than 20 ohms), the change in impedance value by culture was very small, and there was an influence of noise. A change in impedance value that can be used for detection could not be confirmed. Therefore, examination was performed using a medium LB medium having a reduced salt concentration. Measurement conditions: Reference voltage 2000 mV, constant current: 1 mA
Composition of measurement solution: (a) 0.9% NaCl aqueous solution (saline), (b) 0.5% NaCl aqueous solution (0.5% NaCl), (c) 0.1% NaCl aqueous solution, (d) LB Medium (tryptone 10 g, yeast extract 5 g, NaCl 5 g / 1000 ml), (e) salt-free LB medium (tryptone 10 g, yeast extract 5 g / 1000 ml) (f) BPW medium (peptone 10 g, NaCl 5 g, disodium hydrogen phosphate 3.5 g , Dipotassium hydrogen phosphate 1.5 g / 1000 ml) (g) salt-free BPW medium (1% tryptone water)
Each solution was prepared and 40 ml was put into a measurement container (40 ml container: diameter = 38 mm), an electrode was set in the solution, and the solution was placed in an incubation cover (37 ° C.) and left as it was for 1 hour. Thereafter, measurement was performed for 5 minutes.
As a result, it was confirmed that the resistance value increased as the salt concentration decreased. When the resistance value was measured, a good resistance value (about 70 ohms or more) could be obtained under conditions (c, e and g) of 0.1% NaCl concentration or less.

  Using a NaCl aqueous solution for determining the range of the salt concentration, the salt concentration was decreased from 0.1%, and the impedance value was measured (measurement conditions: reference voltage 1600 mV, constant current: 1 mA). The results are shown in FIG. It was found that when the salt concentration was less than or equal to 0.1% NaCl, a good resistance value was exhibited.

(Embodiment 2) Change in resistance value depending on electrode length The length of the impedance detection unit (voltage detection unit and constant current unit (part 6 in FIG. 1)) of the electrode is changed to affect the resistance value during measurement. investigated. As shown in the following table and FIG. 9, measurements were performed by changing the lengths of the voltage detection unit and the constant current unit (measurement conditions: reference voltage 1600 mV, constant current 1 mA, electrode diameter 2 mm).

  As a result, in either case, it can be confirmed that when the electrode is lengthened, the resistance value is decreased. In addition, when the constant current electrodes (two of the four electrodes) are lengthened, It was confirmed that noise was generated. Therefore, it has been found that better resistance can be obtained when the electrode area (length) is all unified and the area is made as small as possible.

(Example 3) Measurement of impedance change by culture Using 1% tryptone water (manufactured by Nacalai Tesque) as a medium, the change in impedance value by culture was measured as follows.
(3-1) Measurement of impedance change A common agar medium was coated with Escherichia coli and cultured at 37 ° C overnight. Next, the bacteria were scraped from the petri dish using platinum thread and dissolved in 5 ml of LB medium. 45 ml of 1% tryptone water medium was added to a 50 ml tube, and 5 ml of the bacterial cell solution was added thereto to obtain a 10-fold diluted solution (1). Thereafter, serial dilution up to 10 ⁶ times was performed (diluted solutions (2) to (6)). As the blank, a medium containing only the medium and the bacteria was prepared, and a reference solution in which 45 μl of the antibacterial agent Supermill 88 was added thereto was prepared. 40 ml of each solution was transferred to a measurement container (cell with electrode), the electrode was set, the lid was closed, the container and the electric probe were connected, and an incubation cover (37 ° C.) was covered. Measurement was started under the measurement conditions of a reference voltage of 1200 mV and a constant current of 1 mA, and the cells were cultured for about 5 hours, and changes in impedance values were measured. The results are shown in FIG.
(3-2) Measurement of the number of bacteria (cfu) 100 μl of the diluted bacterial solution (4) to (6) (10 ⁴ and 10 ⁶ times diluted) was dispensed into each dish, and a normal agar medium was added to each dish. After pouring and solidifying, the cells were cultured overnight at 30 ° C., and the number of colonies was counted the next day. As a result, the number of bacteria of the diluted solution (6) (10 ⁵ times diluted solution) was 4.15 × 10 ³ .

In the above measurement result (FIG. 10), FIG. 11 shows the data plotted again with the numerical value for 5 minutes after the lapse of 40 minutes from the start of measurement as the initial value 0. By performing such data processing, the growth of the bacteria, that is, the presence of the bacteria could be confirmed with higher accuracy. From these results, the time when the impedance change occurred was faster as the bacterial concentration was higher, and the fluctuation was about 20% of that of Blank. In addition, the noise seen during culture was very small (about 1%). From this result, it was found that the present invention can be detected with a bacterial count of about 10 ³ orders / ml in 5 hours. Furthermore, it was found that the number of bacteria in the sample can be determined by the time when the impedance change occurs.

(Example 4) Influence of resistance value by medium composition The composition of the culture medium for measurement was changed, and the influence on the resistance value at the time of measurement was examined. As the medium, 0.4 to 1% casein peptone aqueous solution (manufactured by Nacalai Tesque) and 0.1 to 0.5% beef extract aqueous solution (manufactured by Nacalai Tesque) were measured, and the impedance was measured (measuring condition: standard) Voltage 1600 mV, constant current 1 mA).
As a result, a resistance value of a solution of 100 ohms or more was obtained under all conditions. In the beef extract, there was little variation in the resistance value depending on the concentration.

Example 5 Measurement of Electrical Impedance Change Using the apparatus having the structure shown in FIGS. 4 and 5, the electrical impedance change was measured as follows.
A colony was taken from the bacteria (E. coli (NBRC 3301) or Salmonella (NBRC 100797)) inoculated on a normal agar medium, and the medium solution (Cat. Fst-1000P / M Daiichi Kosho) was prepared in advance according to the vendor's manual. ) It was dissolved in 40 ml and cultured overnight using a thermostat set in advance at 37 ° C. to obtain a preculture solution.
20 ml of the cultured preculture was added to 180 ml of the medium solution and mixed well. 20 ml of the mixed solution was added to 180 ml of the previously dispensed medium solution and mixed well. This operation was repeated 4 times to prepare a dilution series. Dispense the prepared admixture into a measuring container 4 x 40 ml for each dilution, and add half of the dispensing solution (2) to the antibacterial solution (polyhexamethylene biguanide hydrothiol so that each becomes a pair. Two drops (about 80 μl) of Cat. Fst-K, Daiichikosho, which is mainly composed of a ride, were added. After preparing each solution, attach a lid with an electrode, set antibacterial agent not added (A ch: Broth ch) on the front side of the measuring device, and antibacterial agent on the far side (B ch: Reference ch), and lower the arm Then press the start switch to measure.
Changes in electrical impedance were measured using a medium solution (no antibacterial agent added and with an antibacterial agent) prepared using a fungus (E. coli (NBRC 3301)) and the number of bacteria adjusted to 1.5 x 10 ⁵ cfu / mL The results are shown in FIG. A shows the change in electrical impedance when no antibacterial agent is added (Broth ch) and when there is an antibacterial agent (Reference ch), and B shows the difference between them.

(Example 6) Initial concentration of microorganism and determination time Using data obtained by the measurement according to the method of Example 5, positive determination points were calculated. Ch data was subtracted. An average value of 62 to 67 minutes of the subtracted data was calculated and used as an offset value. The offset value was subtracted from the data after 68 minutes to obtain a measured value. A point where the measured value exceeded 6 ohms and continued for 10 minutes was defined as a positive point. Data was plotted using the positive points and the number of bacteria obtained from each data. The number of bacteria was calculated as follows.
100 μl of the 5th from the prepared culture dilution series was weighed onto 3 ordinary agar media, and the solution was evenly smeared using a conage bar. The culture was carried out overnight using a thermostatic chamber set in advance at 37 ° C. After culturing, the number of colonies was counted, and the number of bacteria in the measurement solution was calculated.
The results for E. coli are shown in FIG. 13, and the results for Salmoreella are shown in FIG. From these results, it was found that 10 ⁵ cfu / mL can be positive for bacteria in about 3 hours, and that even when the number of bacteria is on the order of 100, it can be confirmed by measurement for 10 hours. Thus, it can be seen that the number of bacteria can be accurately measured using the method and apparatus of the present invention.

  By the method and apparatus of the present invention, it is possible to detect bacterial contamination in food easily and quickly.

DESCRIPTION OF SYMBOLS 1 Lid 2 Opening part 3 Electrode support part 4 Electrode 5 Container 6 Impedance detection part 11 Lid 12 Main body 13 Measuring solution 14 Electrode terminal 15 Electrode support body 16 Electrode 21 Device main body cover 22 Cell lid storage part 23 Contact 24 Cell 25 with an electrode Device Main body 26 Closed lid 27 Indicator 31 Device main body lid 32 Cell lid storage part 33 Cell with electrode 34 Cell storage part 35 Storage structure 36 Heater 37 Bathtub 38 Main body cover

Claims (16)

In a method for detecting bacteria in a sample by measuring changes in electrical impedance based on bacterial growth, the following steps:
a. Prepare a sample solution in which a part of the sample is suspended in a low-salt bacterial culture medium, and contain the same sample in the same low-salt bacterial culture medium that contains an antibacterial agent at a concentration that inhibits bacterial growth . Preparing a reference solution in which another part is suspended, wherein the salt concentration of the low salt concentration bacterial culture medium in which the sample is suspended is 0.2% NaCl in the measurement solution and the reference solution. It is prepared to have a salt concentration corresponding to the following:
b. A step of measuring the electrical impedance of each of the measurement liquid and the reference liquid using a rod-shaped electrode comprising four electrodes, wherein the rod- shaped electrode is a medium in which 2 mm to 10 mm at the tip of the electrode which is an electrical impedance detection unit Covered with a non-conductive material so as to be exposed in, and c. A step of measuring bacteria in a sample by measuring an impedance change not caused by amplification of bacteria in a culture environment using the reference solution and measuring a change in electrical impedance of the measurement solution;
A method for detecting bacteria in a sample comprising:   The method for detecting bacteria according to claim 1, wherein the sample is food.   The method for detecting bacteria according to claim 1 or 2, wherein an initial value of an electrical impedance value of the measurement liquid is at least 70 ohms.   The method for detecting bacteria according to any one of claims 1 to 3, wherein the measurement solution and the reference solution are at least 10 ml or more. How the impedance detecting section of the electrode is arranged so as to be positioned in respective intermediate portions of the test solution and reference solution, for detecting the bacterium according to any one of claims 1 to 4.   The method for detecting bacteria according to claim 1, wherein the electrode has a diameter of 1.0 mm to 3.0 mm.   The method for detecting a bacterium according to any one of claims 1 to 6, wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium. Wherein the bacterium is E. coli, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Campylobacter, Listeria, Clostridium perfringens, Bacillus cereus, and the bacterium according to claim 7 is at least one microorganism that is selected from the group consisting of botulinum How to detect. A cell with electrodes used to detect bacteria in a sample,
A container for the medium containing the sample,
A lid having an opening that can be fitted to the top of the container;
Four rod-like electrodes constituting four electrodes, which are arranged in a straight line from the center of the lid portion toward both side surfaces and extend from the lid to a substantially central portion in the vertical direction of the container, and the four electrodes A non-conductive electrode support that can be fitted into the opening, wherein the electrode support is an impedance detector and the tip of the electrode is 2 mm to 10 mm in the medium in the container. Covering the electrode to be exposed in,
A cell with an electrode.   The electrode-equipped cell according to claim 9, wherein the container has a cylindrical shape and a capacity of at least 10 ml. The diameter of the electrode is 1.5Mm～3.0Mm, electroded cell according to claim 9 or 10.   The cell with an electrode according to any one of claims 9 to 11, wherein the sample is food. A storage structure having at least two storage units for storing the electrode-attached cells according to any one of claims 9 to 12, wherein the storage structure is a medium containing a sample (measuring solution) A cell with a measurement electrode containing, and a cell with a reference electrode containing a medium (reference solution) containing a sample and an antibacterial agent can be stored.
A current supply unit for supplying a current to the electrode of the electrode-equipped cell, and a measurement unit for measuring an impedance of the measurement liquid and the reference liquid to detect an impedance change;
A device for detecting bacteria based on changes in electrical impedance. Furthermore, the apparatus of Claim 13 including the indicator part which lights a lamp | ramp based on the detection result of bacteria.   The cell with an electrode according to any one of claims 9 to 12, for detecting bacteria in a sample using the method according to any one of claims 1 to 8.   15. An apparatus according to claim 13 or 14 for detecting bacteria in a sample using the method according to any one of claims 1-8.

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