JPH06319594A - Determination of bio-activity - Google Patents

Abstract

PURPOSE:To provide a process for the determination of bio-activity to enable the estimation of the activity of living cell and the determination of the concentration in a short time. CONSTITUTION:A specimen solution and a maleimide derivative are charged to a reaction tank 3 and mixed. The mixed specimen liquid containing a component emitting light with organism is retained for a prescribed period in the reaction tank 3 and transferred to a flow cell 5. Light having a definite wavelength is transmitted through a filter 9 and a condenser lens 10 and applied to the specimen in the flow cell 5. Light emitted from the specimen is passed through a condenser lens 11 and a filter 12, detected with a photomultiplier 13 and counted with a counter 14 to determine the number of living cells and the activity of each organism in the specimen solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the activity of living organisms (microorganisms) used in the food industry, wastewater treatment, etc. and living organisms (microorganisms) contained in drinking water and the like.

[0002]

2. Description of the Related Art Hitherto, in order to measure the activity of a living body, the growth rate of the living body, the rate at which the living body converts from substance A to substance B, etc. have been grasped. It took time.

Therefore, the conventional methods for measuring the activity of a living body (microorganism) have the following drawbacks. It is necessary to grasp the change over time in the biological concentration (cells / ml) in order to capture the activity from the growth rate of the biological body, but since the growth of the biological body is generally slow, it takes half a day to several days to know the change in the biological concentration. .

Further, in order to obtain the rate at which a living body changes from substance A to substance B, it is necessary to grasp the change from substance A to substance B over time, and the measurement requires several hours to several days. .

In the present invention, the activity of a living body can be measured within a short time of at least 1 hour by measuring the fluorescence intensity of the fluorescent substance after reacting with the living body.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a measuring method capable of evaluating the activity of living cells and measuring the concentration thereof in a short time.

Another object of the present invention is to provide a method capable of measuring biological activity with a simple operation and without requiring skill.

[0008]

In the present invention, a non-fluorescent substance reacts directly with living cells to convert a non-fluorescent substance into a fluorescent substance, and a maleimide derivative is added to a sample to give fluorescence from a maleimide derivative bound to a living body. We adopted a method to detect the intensity with a photomultiplier tube and to evaluate the activity quickly.

N- (Pyrene) maleimide, which is a type of maleimide derivative, is used as the luminescent probe.

[0010]

[Function] Maleimide derivatives are generally non-fluorescent,
It reacts with SH groups of living cells and binds to form fluorescent substances. FIG. 1 shows a reaction mechanism in which a maleimide derivative is converted into a fluorescent substance as described above when the maleimide derivative is allowed to act on a living body. The present invention, N- (Pyrene) maleimide, which is one of the maleimide derivatives, is applied to bacteria with different activities and its fluorescence intensity is measured, and the fluorescence intensity from the highly active bacteria is high, and the bacterial activity is reduced. It was found that the fluorescence intensity decreased as the temperature increased, and this effect was used.

[0011]

EXAMPLE An embodiment of the measuring method according to the present invention will be described with reference to FIG. A sample solution as a sample is flown into the reaction tank 3 through the inflow line 1, and a maleimide derivative such as N- (Pyrene) maleimide is added as a fluorescent probe through the chemical injection line 2 and mixed. The amount of chemicals is approximately 0.01 to 25 mg / ml. The sample solution containing the luminous body by the living body mixed therein is continuously sent to the flow cell 5 after a certain residence time in the reaction tank 3.

When the sample in the flow cell 5 is irradiated with the excitation light from the light source 7 through the slit 8 for limiting the width of the light, the filter 9 for obtaining light of a constant wavelength, and the condenser lens 10, the light emitted by the living body is emitted. The body emits light of a specific wavelength. This light is detected by a photomultiplier tube 13 through a condenser lens 11 and a filter 12 for detecting a specific emission wavelength,
By counting this number with the counter 14, the number of living bodies in the sample liquid and the activity of each living body can be measured.

Next, an example of the measuring method according to the present invention carried out according to the above-mentioned embodiment will be described. In this example, N- (Pyrene) maleimide (malei) was used as a fluorescent probe.
mide) was used. The excitation fluorescence spectrum is shown in FIG.
Each living body (microorganism) can be identified by using a wavelength between 300 and 350 nm as an excitation wavelength or a part of this wavelength range and detecting a fluorescence wavelength of 360 to 450 nm or a part of this wavelength range. Figure 3 shows N- (Pyrene) maleimide (malei
An example of measuring E. coli using mide) is shown. Live bacteria 0hr in the figure
The sample is a sample immediately after collecting Escherichia coli cultured in a culture medium and suspending it in physiological saline, that is, after 0 hour. After 24 hours of refrigerating and storing the 0 hr sample of live cells thus obtained,
90 hours later, live bacteria 24hr, live bacteria 90, respectively
hr. The dead cells are those obtained by sterilizing live cells for 0 hr in an autoclave.

N- (Pyrene) maleimide (maleimid) was added to each sample.
The result of applying e) is shown in FIG. Since there are no nutrients that E. coli can sustain life activity in physiological saline,
It gradually loses its bioactivity during storage. The fluorescence intensity also decreased with this decrease in activity, and the activity of the living body (bacteria) could be detected from the change in fluorescence intensity.

[0015]

As described in detail above, according to the present invention, it is possible to evaluate the activity of living cells within 1 hour, which conventionally required a long time of several hours to several days. The concentration of living cells in a sample (cells / ml) can be measured simultaneously. Therefore, according to the present invention, the activity and concentration of the living body (microorganism) contained in the sample obtained from the steps of food production and wastewater treatment can be measured quickly, so that process control and quality control can be performed more quickly than ever. It is possible to obtain economical and technical effects such as labor saving of measurement and improvement of quality control.

[Brief description of drawings]

FIG. 1 is a chemical formula showing a reaction mechanism between a maleimide derivative used in a method for measuring biological activity according to the present invention and a living body.

FIG. 2 is a device layout view for explaining an embodiment of the method for measuring bioactivity according to the present invention.

FIG. 3 is a graph showing the relationship between cell activity and fluorescence intensity obtained by the measurement method according to one example of the present invention.

FIG. 4 is a graph showing a fluorescence spectrum of excitation light used in the measurement method according to the embodiment of the present invention.

[Explanation of symbols]

 1 sample solution inflow line 2 fluorescent chemical injection line 3 reaction vessel 4 sample solution inflow line 5 flow cell 6 sample solution discharge line 7 excitation light source 8 slit 9 filter 10 condenser lens 11 condenser lens 12 filter 13 photomultiplier tube 14 counter

Claims (1)

[Claims] 1. Fluorescence generated from the maleimide derivative bound to a living body by adding a maleimide derivative that reacts with living cells in the specimen to convert a non-fluorescent substance into a fluorescent substance, and then irradiating the specimen with excitation light A method for measuring bioactivity, which comprises detecting intensity.