JP2948405B2 - Quick method for measuring microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an inspection method for quickly counting the number of living organisms (microorganisms) present in a liquid such as food, waste water, industrial pure water and the like.

[0002]

2. Description of the Related Art As a method for detecting microorganisms , a method using agar culture has been conventionally used. However, this method requires a culture operation, and thus requires a long time of 24 hours to several days for measurement. For this reason, attempts have been made to shorten the microorganism measurement time. For example, 5 (6) -carboxyfluorescein diacetate ｛5 (and-6) -CARBOXYFLUORESCEIN DIA
CETATE) 方法 (hereinafter referred to as FDA) has been proposed. In the FDA method, when FDA acts on microbial cells, FDA passes through the cell membrane, and is then decomposed by esterases (enzymes) in the cells and converted into fluorescein, which is a fluorescent substance, as shown in Reaction Formula 1. I do. When excitation light is applied to fluorescein remaining in the cells, it emits fluorescence, and this fluorescence is detected to detect microorganisms . Degradation of FDA by esterase occurs in living cells but does not occur in dead cells, so that only live cells can be detected and dead cells cannot be detected. In addition, most of the fluorescein generated in the living cells remains in the cells. However, since there is leakage to the outside of the cells, the difference in the fluorescence intensity between the living cells and the surrounding cells is reduced, and the S / N on the measurement is reduced. There is a problem that the ratio decreases.

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[0003]

DISCLOSURE OF THE INVENTION The present invention rarely reduces the S / N ratio in measurement due to leakage of a fluorescent substance generated after interacting with a cell, and reduces the S / N ratio in the measurement. An object of the present invention is to provide a method for obtaining a method for detecting a bacterium capable of obtaining a clear difference in the amount of luminescence.

[0004]

SUMMARY OF THE INVENTION The present invention irradiates a substance that directly reacts with a microorganism in a sample and changes from a non-luminescent substance to a luminescent substance such as fluorescence or phosphorescence with excitation light. A method for counting the number of microorganisms in a specimen by detecting light generated, wherein the substance uses N- (7-dimethylamino-4-methylcoumarinyl) maleimide having a maleimide functional group. This is a rapid method for measuring microorganisms .

In the present invention, a maleimide derivative capable of directly reacting with microbial cells and converting a non-fluorescent substance into a fluorescent substance is added to a specimen, instead of being converted into a fluorescent substance through the indirect action of an enzymatic reaction. A means for detecting and counting the light emission of the sample with a photomultiplier tube was employed. As the luminescent probe, N- (7-dimethylamino-4-methylcoumarinyl) maleimide [N- (7-
Dimethylamino-4-methy lcoumarinyl) maleimide] (hereinafter referred to as DACM).

[0006]

[Action] Maleimide derivatives are generally non-luminescent,
It reacts with and binds to SH groups present in microbial cells to form a fluorescent substance. Therefore, since the degree of leakage of a substance converted into a fluorescent substance like the above-mentioned FDA out of living cells is small,
This is advantageous in measurement because the N ratio can be increased.

The following reaction formula 2 converts a maleimide derivative into a fluorescent substance when it acts on a microorganism ( living body ) .

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[0008]

FIG. 1 shows an embodiment of the present invention.
To the reaction vessel 3 by flowing the sample solution is a subject in the sample solution inlet line 1, where via the fluorescent chemical injection line 2 fluorescent probes are mixed with to a KazuSatoshi DACM. The amount of the chemical is approximately 0.01 to 1 mg / ml. The sample liquid mixed and containing the luminous substance caused by the microorganisms passes through a certain retention time in the reaction tank 3 and then continuously reaches the flow cell 5 through the sample liquid inflow line 4. Here, when the excitation light is irradiated to the sample in the flow cell 5 through the slit 8 for limiting the width of light from the excitation light source 7, the filter 9 for obtaining light of a certain wavelength, and the condenser lens 10, the luminous body by the microorganism is identified. It emits light of a wavelength. This light is detected by a photomultiplier tube 13 via a condenser lens 11 and a filter 12 for detecting a specific emission wavelength, and the number thereof is counted by a photon counter 14, whereby the number of microorganisms in the sample liquid can be measured.

In this embodiment, DAC is used as a fluorescent probe.
M was used. Microbial cells can be identified with a high S / N ratio by detecting excitation wavelength of 428 nm and detection of fluorescence wavelength of 470 nm. 2 to 5 show examples of measurement of microorganisms (Escherichia coli) using DACM. Fig. 2 shows the excitation spectrum, Fig. 3 shows the fluorescence spectrum when live E. coli is in the sample, Fig. 4 shows the fluorescence spectrum when dead E. coli is in the sample, and Fig. 5 shows the case where no bacteria are present in the sample solution. The spectrum of is shown. Comparing the intensities at the fluorescence wavelength of 470 nm, the presence of live bacteria (FIG. 3): the presence of dead bacteria (FIG. 4): the absence of bacteria (FIG. 5) = 5.00: 2.84: 0.62 = 8.1: 4.1.
6: 1.0, viable bacteria (FIG. 3): dead bacteria (FIG. 4) = 1.8: 1.0, dead bacteria (FIG. 4): no bacteria (FIG. 5) = 4. 6: 1.0, and sufficiently viable and dead bacteria can be distinguished by using DACM as a luminescent probe in a solution containing the bacteria.

[0010]

According to the present invention, microbial cell counting and measurement, which conventionally required a long time of 24 hours to several days, can be performed continuously in 5 to 40 minutes, and live and dead cells can be counted. Can be measured separately from the difference in fluorescence intensity. Therefore, according to the present invention, it is possible to quickly detect and count microorganisms contained in water or a solution used in food production and also in pure water for cleaning in semiconductor production, and to separate and count live microorganisms and dead microorganisms. It can be used for quick check of the sterilization efficiency of the process, and economic and technical effects such as labor saving of inspection and improvement of quality control can be obtained.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of an apparatus used in one embodiment of the present invention.

FIG. 2 is a table showing an excitation spectrum of DACM used in the present invention.

FIG. 3 is a chart showing a fluorescence spectrum when living Escherichia coli is present in a specimen when DACM is applied.

FIG. 4 is a chart showing a fluorescence spectrum when dead E. coli is present in a sample when DACM is applied.

FIG. 5 is a chart showing a fluorescence spectrum when no bacteria are present in a sample when DACM is applied.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12Q 1/06 G01N 21/78 C12Q 1/44 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method in which a substance that directly reacts with a microorganism in a sample and changes from a non-luminescent substance to a luminescent substance such as fluorescence or phosphorescence is irradiated with excitation light, and the light generated by the excitation light is detected. a method of measuring the number of microorganisms, rapid method of measuring microorganisms above substances, characterized by using a maleimide functionality comprises a group N-(7- dimethylamino-4-methyl coumarin sulfonyl) maleimide.