JPH0343069A - Method for measuring live cell of microbial cell - Google Patents

Abstract

PURPOSE:To remarkably shorten measuring time, enable direct automatic measurement of live cells and measure the live cells useful for food production plants, etc., by reacting a specific chemical substance with a sample which is an object of measurement containing microorganisms and irradiating the sample with light at a specific wavelength. CONSTITUTION:A chemical substance, reactive with enzymes or coenzymes present in live cells of microorganisms and producing a fluorescent substance in the cells is reacted with a sample which is an object of measurement containing the microorganisms, mixed and brought into contact therewith for a prescribed time. The sample is then irradiated with light at a wavelength required to excite the fluorescent substance produced in the cells. Light emitted from the individual microorganisms in the sample is subsequently measured as the number of spots at that time. Furthermore, e.g. homovanilic acid, are preferably used as the aforementioned chemical substance.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for measuring living microbial cells, and particularly to raw materials used in food manufacturing plants and pharmaceutical manufacturing platforms.
Concerns methods for product quality control and sterilization equipment (measurement of living cells, which is advantageously applied to performance confirmation, etc.).

[Conventional technology]

In food manufacturing and pharmaceutical manufacturing plants, microbial measurements are performed to control the quality of raw materials and products and to check the performance of sterilization equipment.

The most commonly used conventional method for measuring microorganisms, especially for measuring living cells, is the agar culture method. In this method, a sample is dispersed on or within agar into which microbial nutrients have been dissolved, and colonies are formed on or within the agar by culturing at an appropriate temperature, and the number of colonies is counted. The number of viable cells is determined by this method.

However, this method involves culturing, which causes colonies to form! However, the measurement time required is at least 10 hours or more, depending on the type of bacteria, and several days depending on the type of bacteria, which often impedes the quality control of a single product.

Therefore, many studies have recently been conducted on methods for rapid measurement of microorganisms, and among them, the pyoluminetsenne method has been put into practical use. In the Pioluminetsenne method, A
A well-known method is to utilize the reaction between TP (adenosine triphosphate) and lumferin/vVferase, which is a firefly bioluminescent enzyme.

This method uses AT, a type of coenzyme contained in living cells.
When luciferin luciferase acts on P, photons are released, and by measuring the amount of photons, the number of viable bacteria can be indirectly determined. However, this method requires the release of ATP contained in the cells to the outside of the cells1, which requires cumbersome pretreatment operations such as solubilizing the cells with a surfactant, etc. Since this is an indirect method of estimating the number of viable bacteria by counting the total amount of photons inside, it is not possible to obtain an absolute value that indicates whether or not each individual microorganism is alive, and the reliability of the measurement may be affected. There was a limit to its applicability to samples with low concentrations of S, which were problematic.

The microbial measurement methods required in the food and pharmaceutical fields are:
It must be able to be used immediately for controlling sterilization equipment, product quality control, etc. To achieve this, it must be highly sensitive, provide rapid results, and have the potential for automation. No method has been found that serves the purpose.

[Problem to be solved by the invention]

The conventional method has the following problems.

■ The agar culture method requires a very long measurement time ■ The bioluminescence method is a method that indirectly estimates the number of cells by counting the total number of photons emitted from the cell suspension, and it is not possible to estimate the number of viable bacteria. It cannot be directly counted and there is a problem with the reliability of measurement.

The present invention aims to solve the above-mentioned problems and provide a method that can directly measure the number of individual viable bacteria with high sensitivity and quickly.

[Means to solve the problem]

In the present invention, a chemical substance that reacts with enzymes or coenzymes present in living cells of microorganisms and generates fluorescent substances within the cells is applied to a sample to be measured containing microorganisms, and after a certain period of mixed contact, intracellular Measurement of live microbial cells characterized by irradiating a sample with light of a wavelength necessary to excite the fluorescent substance generated in the sample, and measuring the light emitted from each microorganism in the sample as a number of points. It's a method.

[Effect]

Table 1 shows the combinations of chemical substances that react with enzymes or coenzymes present in living cells to produce fluorescent substances within the cells.

table Examples of the present invention are shown below.

〔Example〕

Figure 1 shows the configuration of a prototype device for rapid detection and automatic measurement of living cells. In FIG. 1, reference numeral 1 denotes a light source for irradiating cells with light, and a mercury lamp with an output of 100 W is used. 2 is a collector lens for concentrating the mercury lamp, 3
is an excitation filter, which is used to select the wavelength necessary to cause living cells to emit light. When fluorescein diacetate was used, an excitation filter that transmits a wavelength range of 450 to 490 nm was selected based on preliminary experimental results.

Although a mercury lamp was used as a light source, a laser can also be used as a light source if it can excite fluorescent substances generated within cells, and in this case, the excitation filter 5 is unnecessary. Mirror 4 is an objective lens, and in this experiment, a 20x magnification lens was used. Reference numeral 6 denotes a glass mounting plate, which is used to pass the sample through and perform continuous measurements; however, when continuous measurement is not required, the sample may be placed on a slide glass and measured directly. 14 is a glass container for mixing the fluorescein diacetic acid and the sample, 15 is a magnetic nut with a heater, and 16 is a rotor for stirring and mixing the fluorescein diacetate and the sample while keeping them at a predetermined temperature. be. P is the injection line for the microbial sample to be measured, P2 is the injection line for fluorescein diacetate,
By contacting and mixing in the glass container 14 for a predetermined period of time at a predetermined temperature, fluorescein diacetate comes to produce fluorescein, which is a fluorescent substance, by the action of an enzyme in living cells. This sample is passed through P, to SE/L/
At this time, when irradiated with light of 450 to 490 nm, living cells that have accumulated fluorescein within the cells emit fluorescence with a main wavelength of 520 nm. The absorption filter 7 allows wavelengths in this vicinity to pass, and the lens 8
The luminescent cells are imaged by the camera 9 via the camera 9. Reference numeral 10 denotes an image processing device capable of outputting an image of luminescent cells existing within an arbitrarily settable brightness level, and the image processed image is displayed on a monitor 12. By connecting the image-processed signal to the particle counter 11, the number of cells in any brightness range can be counted.

Among them, instead of image processing, the light emitted from the cells in the cell is directly received by the light receiver through the absorption filter 7 and the lens 8.
Pulse counter 1 that counts the output of the receiver
Even in combination with 1, the number of viable bacteria can be measured. 13 is a data processing and analysis device which calculates and outputs the number of cells within a predetermined brightness range and the brightness distribution of cells.

Heat sterilizers are frequently used in food sterilization, and control of the button, heating temperature, and amount of heated steam blown is often problematic. Information on the number of viable bacteria can be quickly fed back to the heating control of the sterilizer, making it extremely useful for product quality control.

Next, a test example using this device will be shown.

(1) Baker's yeast cells used in the test were cultured for 48 hours and concentrated using a centrifuge (at 00 Orpm).

5 minutes), the cells were collected, washed with pH 7.01/15M phosphate buffer, and used.

The dead bacteria were heat-treated at 121°C for 5 minutes, and a mixture of live bacteria and dead bacteria at a ratio of 1:1 was used as a cell sample.

(2) Fp-olecein diacetic acid solution Add fluorescein diacetate to acetone.

Adjusted to 1η/mtv concentration, pH 7,01/15
Further dilute with M phosphate buffer to 0.2! rq/-
The concentration was set to

(3) Action pH Add fluorescein diacetate solution at a ratio of 1:1 to cells adjusted to a concentration of 10' to 107 cells/-, and add 1N
HCL or 1N Na OX (at pH 4, 5
, 6, 7, 8, and 10 were prepared, respectively.

(4) Working temperature of cell fluid and fluorescein diacetate solution,
Action time Magnet Nutara with heater Action 1 temperature is 35
The temperature was kept constant at 5°C, and the action time was varied at 5°C, 10°C, 20°C and 40.60 minutes.

f5), i! Experiments were conducted with and without vigorous mixing of samples containing IIl cell solution and fluorescein di#4 acid solution with strong stirring using a magnetic stirrer equipped with a heater and a 1-cycle heater.

(6) Experimental results (influence of pH) Samples maintained at pH 4, 5, 6, 7, 8, and 10 at a temperature of 35°C were examined for the presence or absence of viable bacteria luminescence for 60 minutes without stirring. As a result, luminescence was confirmed in the pH range of 5 to 8, but the luminescence was weak under the conditions of pH 4, button and 10.

(ii) Effect of action time Next, the action time was set to 5.10, 20 for the pH&O sample.
, 40, and 60 minutes, the light emission started after 10 minutes, and after 40 minutes, the amount of light emission reached a substantially stable region.

(iii) Effect of stirring For the sample with pH & 0, the reaction time was changed to 5°10.20.40 60 minutes for the sample with and without stirring. After 5 minutes had elapsed, it was already brightly emitting light, and after 10 minutes, it was possible to measure all living cells, indicating that the effect of stirring and mixing is very large in performing rapid measurements.

Figure 2 shows the results of measuring viable bacteria by image processing under conditions of pH 6.0 and action time of 10 minutes. In FIG. 2, the horizontal axis shows the intensity of cell light emission (brightness), and the vertical axis shows the ratio of the number of cells measured to the total number of cells that received light, and shows the brightness distribution of living cells. In this case, the number of live cells measured is 50, and it can be seen that these 50 live cells are within the brightness range of 50 to 125. Dead cells do not emit light, so they are not detected or measured. Note that this image processing device can display brightness in 0 to 255 steps depending on the brightness of the cells. From this, set the brightness level to 50 to 125 in advance.
If the luminescent cells within this range are counted, the number of viable bacteria will be automatically determined.

〔Effect of the invention〕

The effects of the present invention are: ■ Measurement time is much faster than the conventional agar culture method, which required tens of hours at 10 hours, to less than 10 minutes. By establishing a means of detection and measurement, it has become possible to directly and automatically count viable bacteria, allowing rapid control of sterilization equipment and rapid management of product quality.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an apparatus used to detect and measure viable bacteria in an example of the present invention, and FIG. 2 is a chart showing the measurement results of viable bacteria measured using the apparatus shown in FIG.

Claims (1)

[Claims] A chemical substance that reacts with enzymes or coenzymes present in living cells of microorganisms to produce fluorescent substances within the cells is applied to the measurement target sample containing microorganisms, and after a certain period of mixed contact, the fluorescent substances are produced within the cells. A method for measuring living microbial cells, characterized by irradiating a sample with light of a wavelength necessary to excite a fluorescent substance, and measuring the light emitted from individual microorganisms in the sample as a number of points.