JPH0886771A - Method and device for quickly measuring number of living bacteria - Google Patents

Abstract

PURPOSE: To provide a method and a device for quickly measuring the number of living bacteria. CONSTITUTION: An electrolyte-containing culture medium 3 containing microorganisms is brought into intimate contact with the upper surface of a semiconductor device 2 having a thin-film insulating layer 1, and a constant voltage is applied between an electrode 4 and the semiconductor device 2 from a constant voltage source 5, so that as light rays from a light source 7 are scanned two-dimensionally by an optical scanner 8, currents that flow are measured by a current measuring device 6. The measured current values are converted into pHs by a computing unit 9 and the two-dimensional distribution of the pHs is measured, and the number of living bacteria is measured from spots where pH changes of 0.02 or more and 0.2 or less have occurred. The semiconductor device 2 uses a smooth semiconductor not more than 200μm thick and 200nm or less in surface roughness.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for rapidly measuring the viable cell count.

[0002]

2. Description of the Related Art In the industrial field where contamination by microorganisms such as foods, pharmaceuticals and cosmetics is a problem, microbial inspection to confirm the safety by measuring the number of viable microorganisms such as manufacturing process, quality control and hygiene inspection of food poisoning. Is commonly practiced.

Conventionally, as a method thereof, an agar plate medium containing a medium component corresponding to a microorganism to be measured is smeared or mixed with a fixed amount of an analyte such as food or a diluted product thereof, and a fixed period of time is applied. A so-called microbial culture inspection method has been widely used, in which colonies appearing after culturing in a constant temperature state are mainly visually measured to determine the viable cell count.

However, this method has a problem that it takes a few days for culturing, so that it cannot be promptly dealt with because of a waiting time until the result is known. For the purpose of solving this problem, various rapid microorganism measurement methods have been proposed, for example, adenosine-3-phosphate (ATP) is extracted from the microorganisms in a sample, and the amount is measured with a luminometer by causing a luminescence reagent to emit light. Bioluminescence method for measuring and calculating the number of microorganisms therefrom (Japanese Patent Laid-Open No. 163098/1990)
Etc. are known. In addition, the present inventors have so far
We have found that an optical scanning semiconductor pH sensor with spatial resolution can be used to detect viable bacteria on an agar plate (September 27, 1993, Autumn 54th Annual Meeting of the Society of Applied Physics, Japan). Shu, page 1066 27a-ZA
-4).

[0005]

However, the above-mentioned bioluminescence method has a low detection limit. For example, in the case of bacteria, at least about 10 ³ bacteria are required for detection. When it is not possible to measure or when multiple types of microorganisms are present, ATP is caused by the microorganisms.
Therefore, there is a problem in practical use, such as an inaccurate number of bacteria cannot be calculated because the contents of the are different.

Further, in the method using the optical scanning semiconductor pH sensor having the above-mentioned spatial resolution, since the distribution of pH change is two-dimensionally measured, it is effective in that the number of viable bacteria can be accurately measured. However, there was a problem that the sensitivity and resolution were insufficient to detect live bacteria in a short time.

Therefore, it was predicted that thinning the semiconductor substrate of the pH sensor would be an effective means for achieving high sensitivity and high resolution (September 2, 1993).
Proceedings of 54th JSAP Autumn Meeting, 1st
083 page 27p-ZC-46), the ratio of signal intensity to noise is so-called S /
It was not possible to realize a high sensitivity and rapid viable cell measurement with a low N ratio. In view of the above situation, the present invention has been made for the purpose of providing a method and an apparatus for rapidly measuring the viable cell count.

[0008]

Generally, in a structure composed of a semiconductor layer / insulator layer / electrolyte layer, a voltage is applied between the semiconductor and the electrolyte layer, and light energy is intermittent in a state where a depletion layer is formed in the semiconductor. , An alternating photoexcitation current will flow. On the other hand, the silanol group is for example, silicon nitride and silicon dioxide film surface an insulator (Si-O-H) by the state of the surrounding ^{pH, Si-O -, Si-} O-H,
Take Si-O-H ₂ ⁺ each structure. Due to the difference in these structures, the bending of the semiconductor band in contact with the insulator is different, and the voltage at which the photoexcitation current flows out differs even when the same optical energy is applied. Conversely, when this voltage (bias voltage) is constant, the surface The current value differs due to the difference in the possible structure. There is a strong correlation between this current value and the pH of the semiconductor surface. For example, according to the device of the present invention described later,
A voltage change of about 59 mV is observed. Therefore, the pH of a local region can be measured by irradiating the semiconductor surface with light with a constant bias voltage applied, and measuring the photoexcitation current at that portion. It can be measured as a two-dimensional image.

P having a spatial resolution having the above functions
As the H sensor, the pH of the solution in contact with the insulating layer is obtained by scanning in a two-dimensional direction (XY direction) while irradiating light from the semiconductor side of the semiconductor element including the insulating layer and the semiconductor layer in contact with the electrolyte-containing solution. What can measure the distribution is desirable,
For example, a silicon semiconductor layer can be used as the semiconductor layer, a silicon nitride layer and a silicon dioxide layer can be used as the insulating layer, and a potassium chloride solution or a sodium chloride solution can be used as the electrolyte-containing solution.

On the other hand, it is known that microorganisms secrete various substances extracellularly by their metabolism. Most of the substances are organic acids, alkaline organic substances, etc., and change the pH of the surrounding environment. Therefore, if it is a live bacterium, the surrounding pH is changed in the process of its growth and metabolism, and if it is a dead bacterium, it is not metabolized and does not grow, so there is almost no effect on the surrounding pH. Therefore, the number of viable bacteria can be measured by measuring the pH on the surface and the surroundings of the viable bacteria and the pH of the medium on which no microorganism is present and analyzing the distribution state thereof.

In order to measure live cells more rapidly, the amount of change in pH to be detected (hereinafter referred to as ΔpH) in the method for measuring the amount of change in pH corresponding to the substance produced by live cells as described above. Is preferably the smallest value that can be detected, but this change also differs depending on the type of microorganism and the medium used. The most suitable p that is sufficiently fast and can reliably detect and measure viable bacteria.
The change amount ΔpH of H is 0.02 or more and 0.2 or less, preferably 0.05 or more and 0.1 or less. It takes a long time for the amount of change in pH by viable bacteria to become larger than 0.2, and for E. coli, for example, it takes 10 hours or more, which is not quick. Further, when the amount of change in pH is 0.02 or less, the pH also changes due to variations in the state of the electrolyte-containing medium other than live cells, and the viable cell count cannot be accurately measured.

When measuring the above-mentioned pH change amount, a pH sensor having a spatial resolution is used as an optical scanning semiconductor p.
H sensor, the thickness of the semiconductor element is 200 μm or less,
It is preferably 100 μm or less, and the roughness of the semiconductor element surface, that is, the difference between the thickest part and the thinnest part of the semiconductor element is 200 nm or less, and preferably 10
It is desirable to use a smooth semiconductor element of 0 nm or less. When the thickness of the semiconductor element is 200 μm or more, when light is irradiated from the back surface of the semiconductor, electrons and holes excited by the light diffuse in the semiconductor element and the range of measuring the pH on the insulating layer becomes wide, As a result, changes in the pH distribution on the surface of the live bacteria and in the surroundings cannot be detected with high sensitivity, and bacteria cannot be detected within, for example, 8 hours.

Further, even when the surface of the semiconductor is rough and the thickness of the semiconductor layer is non-uniform, the attenuation factor of electrons and holes excited by light in the semiconductor layer is different, and the signal intensity on the insulating layer varies. As a result, noise is increased and sensitivity is significantly reduced. When the roughness of the semiconductor surface exceeds 200 nm, ΔpH that can be actually detected on the insulating layer becomes larger than 0.2 regardless of the energy level of the light irradiation device used to generate the photoexcitation current in the semiconductor. Rapid live bacteria detection is not possible. The thickness of the semiconductor element is 100μ
When the thickness is m, the surface roughness is preferably 100 nm or less, and when the thickness is 50 μm, the roughness is preferably 50 nm or less. That is, it is desirable that the ratio of the thickness of the semiconductor element to the roughness of the surface of the semiconductor element is approximately 1000: 1 or more.

FIG. 1 shows the construction of a rapid viable cell count measuring apparatus according to the present invention using an optical scanning semiconductor pH sensor. An electrolyte-containing culture medium 3 containing a microorganism to be measured is arranged on a semiconductor element 2 coated with an insulating thin film 1, and an electrode 4 is attached to the surface or inside of the electrolyte-containing culture medium 3. On the side of the semiconductor element 2 not coated with the insulating thin film 1, the light beam from the light source 7 is scanned by the optical scanning device 8 in the two-dimensional direction (X-Y direction) and irradiated. A constant voltage is applied between the electrode 4 and the semiconductor element 2 by the constant voltage power source 5, and the flowing current is measured by the current measuring device 6. The calculation device 9 calculates the current value measured by the current measurement device 6 as a two-dimensional pH distribution based on the measured current value and the optical scanning signal, and displays it on the display 10.

As the electrolyte contained in the culture medium 3, a potassium chloride solution, a sodium chloride solution or the like can be used. The amount of the electrolyte is such that the amount of change in pH can be measured to obtain conductivity, for example, 0.01 M to 0.5 M.
It can be a degree. As the semiconductor element coated with the insulating thin film, a silicon semiconductor having a silicon nitride layer and a silicon dioxide layer can be used. The method for forming the silicon dioxide layer on the semiconductor element may be a usual method, but for example, a silicon substrate may be formed in an oxygen stream at a temperature of 1000.
It can be formed by firing at about ° C. The silicon nitride layer can be further formed on the silicon dioxide layer by chemical vapor deposition. The thickness of the semiconductor layer can be 200 μm or less, and the thickness of the insulating thin film can be about 1 to 100 nm. An electrolyte-containing culture medium containing microorganisms, for example, having a thickness of about 1 mm is brought into close contact with the insulating thin film of this semiconductor element. In this case, it is desirable to bring the surface of the electrolyte-containing culture medium containing the microorganisms into contact with the insulating layer, but this is not the case when the thickness of the electrolyte-containing culture medium is thin. The electrode 4 may be any one as long as it is usually used, but a platinum electrode is particularly preferably used. The light source 7 may be any as long as it can generate a photoexcitation current to the semiconductor element 2, but a laser light source or the like that can focus on a spot of about 200 μm or less on the semiconductor element is preferable. It is preferable that the current measuring device 6 can measure a current of about 10 nA or more. Constant voltage power supply 5
Is a constant voltage between the semiconductor element 2 and the electrode 4 attached to the electrolyte-containing culture medium 3 containing microorganisms, for example, −10 V to +.
Any voltage capable of applying about 10 V may be used. Further, the optical scanning device 8 may be of a type in which the semiconductor element is moved with respect to a fixed light beam, or may be one in which deflection scanning of the light beam and movement of the semiconductor element are combined to perform optical scanning. Good.

[0016]

A pH sensor having a spatial resolution is used to measure the amount of change in pH corresponding to a substance produced by viable bacteria,
The viable cell count can be rapidly measured when the change amount of H is 0.02 or more and 0.2 or less. For example, in the case of Escherichia coli, it takes 24 to 48 hours to detect by a usual culture method, but it can be detected in about 8 hours by the method of the present invention. 8 hours for yeast, 24 hours for lactic acid bacteria, 1 for mold
Each can be detected in about 2 hours, and rapid measurement is possible.

[0017]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

(Example 1) [Measurement of E. coli] E. coli IFO 0330 using the apparatus shown in FIG.
The viable cell count of 1) was measured. The semiconductor element used was a silicon semiconductor having a thickness of 200 μm and a surface roughness of 200 nm, and an infrared semiconductor laser having an output of 5 mW and a wavelength of 830 nm was used as a light source. 1. It is a semiconductor device in the optical scanning device.
The area of 56 cm ² was scanned and measured. A constant voltage of 0.5 V was applied to the semiconductor element from a constant voltage power source. The electrolyte-containing culture media, used after adding sodium chloride to a 0.1M as an electrolyte in commercial coliform detection agar medium (Eiken Chemical Co., Ltd.), 2.56 cm ²
Inoculate so that about 10 E. coli are present per 3
It was kept at 7 ° C. The measured current value is expressed as a two-dimensional pH distribution by the computing device, and the pH at the center of the point where the pH change is circularly distributed compared to the surroundings,
The pH around the circle, i.e. the difference from the pH of the unchanged initial electrolyte-containing culture medium ([Delta] pH) was measured.

When the number of points where ΔpH is 0.02 or more and 0.2 or less in the range measured by scanning is taken as the number of viable bacteria, as shown in Table 1, The results were in good agreement with the numbers. Further, the measurement time was about 24 hours in the usual culture method, but the detection of Escherichia coli could be detected within 8 hours within the above ΔpH range, and rapid measurement was possible. The actual number of Escherichia coli was used after inoculating the agar medium and keeping it at 37 ° C. for 24 hours.
The percentage in the table is the ratio of the viable cell count measured by this device to the viable cell count of E. coli actually present on the agar plate used for the measurement, measured 5 times each, and the result is averaged to 1 The numbers are rounded off. Therefore, for example, if the measurement result of this device completely matches the actual viable cell count, 100%,
When only half is measured, it is 50%.

[0020]

[Table 1]

(Embodiment 2) [Thickness of semiconductor element] With the apparatus shown in FIG. 1, the thickness of the semiconductor element is variously changed to detect E. coli IFO 03301 under the same conditions as in the first embodiment. went. The thickness of the semiconductor element is adjusted to 600 μm.
The commercially available silicon of No. 1 was shaving and adjusted with a polishing device. The roughness of the surface of the semiconductor element was measured with a stylus roughness meter (Dectac) and was set to a level not exceeding 200 nm. Table 2 shows the results of the successive measurements.
E. coli could be detected within 8 hours at a size of less than μm, and rapid measurement was possible.

[0022]

[Table 2]

(Example 3) [Surface Roughness of Semiconductor Element] The apparatus shown in FIG. 1 was used to measure the surface roughness of the semiconductor element to be used under the same conditions as in Example 1. The method of changing the surface roughness is a combination of a so-called etching method of immersing a silicon substrate in a potassium hydroxide solution of about 1N and dissolving the surface, a method of polishing with sandpaper, and a method of mirror-finishing by mechanical polishing. Using. Finally, the thickness of the semiconductor element was set to 200 μm and the measurement was performed. Table 3 shows the time required for detecting E. coli and the ΔpH value at that time. When the roughness was 200 nm or less, E. coli could be detected within 8 hours. When the roughness was more than 200 nm, the lower limit of detectable ΔpH was large, and detection was not possible unless the pH change due to the substance produced by E. coli was large.

[0024]

[Table 3]

[0025]

According to the present invention, the viable cell count can be rapidly measured.

[Brief description of drawings]

FIG. 1 is a schematic view of a rapid viable cell count measuring apparatus using an optical scanning semiconductor pH sensor.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Insulating thin film, 2 ... Semiconductor element, 3 ... Electrolyte containing culture medium, 4 ... Electrode, 5 ... Constant voltage power supply, 6 ... Current measuring device, 7
... light source, 8 ... optical scanning device, 9 ... arithmetic device, 10 ... display

Claims (3)

[Claims] 1. A viable cell count measuring method for detecting a viable cell count by using a pH sensor having a spatial resolution to measure an amount of change in pH corresponding to a substance produced by a viable cell to detect the viable cell count.
A rapid measuring method for the viable cell count, wherein the amount of change in H is 0.02 or more and 0.2 or less. 2. A semiconductor element coated with an insulating thin film, an electrode, a constant voltage power source for applying a constant voltage between the electrode and the semiconductor element, and a current measuring device for measuring a current flowing between the electrode and the semiconductor element. An optical scanning pH sensor having a spatial resolution including a light source, and an optical scanning device that scans a light beam from the light source onto a surface of the semiconductor element that is not coated with an insulating thin film, and the optical scanning pH sensor A rapid viable cell count measuring device comprising an electrolyte-containing culture medium closely attached to an insulating thin film of a semiconductor element, wherein the electrode is attached to the electrolyte-containing culture medium. 3. The rapid viable cell count measuring method according to claim 2, wherein the semiconductor element is a smooth semiconductor having a thickness of 200 μm or less and a surface roughness of 200 nm or less.