JP2005102645A - Method for determining sterilization effect on microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly and exactly determining the sterilization effect on microorganisms by determining the proliferation activity of individual microorganisms contained in a specimen. <P>SOLUTION: In this method for determining the sterilization effect on microorganisms, a sterilized sample is collected from a sterilized object and the sample is previously cultured for a prescribed time, then the proliferation activity of individual microorganisms are measured electrically or optically and the results obtained by the determination are compared electrically or optically with the results obtained by treating an untreated sample in the same way. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for rapidly measuring the sterilization effect of microorganisms by measuring the proliferation activity of individual microorganisms contained in a sample.

  In various industrial products such as foods, pharmaceuticals, and cosmetics, and in the industrial field, quality deterioration of products due to the growth of microorganisms and food poisoning cases have become problems. For this reason, sterilization / disinfection treatment methods using heating, ultraviolet irradiation, radiation irradiation, ozone treatment, ethylene oxide gas treatment, electrolyzed water, and various chemicals have been studied and developed for the purpose of suppressing the growth of microorganisms.

  In order to determine the effects of these sterilization / disinfection methods, (1) sterilization / disinfection methods are applied to samples containing microorganisms, and (2) the treated samples are treated with an appropriate growth medium such as a standard agar medium. After mixing, the cells were cultured for 24 to 48 hours under appropriate conditions (temperature, aerobic / anaerobic atmosphere, etc), and (3) the number of colonies formed by the growth of microorganisms was counted. (4) Determine the effect of the sterilization / disinfection treatment method by comparing with the number of viable bacteria in the unsterilized control sample.

  In addition, by using B. subtilis spores or the like as an index for determining whether sterilization is sufficient or not, by comparing multi-angle light scattering of sterilized spores with non-sterilized multi-angle light scattering A method for detecting the effectiveness of sterilization is known (for example, see Patent Document 1).

Further, a method is known in which a bacterial sample is fluorescently stained specifically for viable bacteria or dead bacteria, and then the viable or dead bacteria are selectively counted with a flow cytometer or a fluorescence microscope.
Special table 2002-542836 gazette

  In the method of counting colonies, it takes a long time for the colonies to be formed, so it takes time to determine the effect of the sterilization / disinfection treatment.

  In the method for comparing the multi-angle light scattering of the spore, there is a problem that the difference in multi-angle light scattering does not necessarily coincide with the difference in sterilization rate.

  In the method using a live or dead fungus-specific fluorescent dye, there is a problem that, for example, in order to determine the viability of a microorganism based on cell membrane permeability, the dyeability of the fluorescent dye does not necessarily match the viability of the microorganism. .

  The present invention has been made in view of such circumstances, and provides a method for measuring the sterilization effect of microorganisms quickly and accurately by measuring the proliferation activity of individual microorganisms contained in a sample. To do.

  The present invention is characterized by collecting a sterilized sample from a sterilization target, culturing the sample in advance for a predetermined time, and then measuring the proliferation activity of individual microorganisms contained in the sample electrically or optically. Provided is a method for measuring the sterilization effect of microorganisms.

  The inventor changes the growth activity of microorganisms by culturing a sample after sterilization treatment, and effectively detects and counts the number of living microorganisms by measuring the growth activity electrically or optically. I found that I can do it. Thereby, it became possible to measure the sterilization effect of microorganisms from the number of living microorganisms, and the present invention was completed. For example, it is possible to measure whether or not the effect of the sterilization treatment is sufficient by examining whether or not the number of detected live microorganisms is equal to or less than a predetermined value.

  The time required for the sample culturing step in the present invention is sufficient for changing the growth activity of the microorganism. Since this time is much shorter than the time required for the microorganisms to form colonies, the present invention makes it possible to measure the sterilization effect in a short time.

  In the method of the present invention, by measuring the growth activity of individual microorganisms electrically or optically, live microorganisms can be directly counted and the bactericidal treatment effect can be measured. Compared to the method of indirectly measuring the sterilization effect from the difference, the reliability is high.

  Since the method of the present invention utilizes the fact that a predetermined growth activity is different between a living microorganism and a dead microorganism by culturing a sample, a conventional fluorescent dye specific to a living or dead microorganism is used. Like the method used, there is no error due to inconsistency between the cell membrane permeability of the fluorescent dye and the life and death of the microorganism, and the reliability is high.

  The method for measuring the effect of sterilization treatment of microorganisms according to the first embodiment of the present invention involves collecting a sample sterilized from a sterilization target, culturing the sample in advance for a predetermined time, and then proliferating individual microorganisms contained in the sample. The activity is measured electrically or optically.

  First, a process of collecting a sterilized sample from a sterilization target and culturing the sample for a predetermined time in advance will be described.

  The sterilization treatment refers to killing or inactivating microorganisms by chemical / physical means, for example. Specifically, for example, (1) irradiating the sample with ultraviolet rays / radiation, (2) adding a chemical substance having a bactericidal effect to the sample, and (3) heating the sample are included.

  The type of microorganism is not limited, and one or more types of microorganisms may be included in the sterilization target. Microorganisms refer to those having a diameter or length of about 0.1 μm to 500 μm, for example.

  Incubating a sample in advance for a predetermined time refers to placing the sample in an environment suitable for the growth of microorganisms contained in the sample for a predetermined time in advance.

  The environment suitable for the growth of microorganisms differs depending on the sample and microorganism to be measured, and the embodiment is not particularly limited. For example, when the microorganism to be measured is a bacterium, it is preferable to use a liquid medium. Specifically, for example, a normal bouillon medium, a heart infusion broth medium, a tryptic soy bouillon medium, or the like is preferably used for growing general bacteria.

  When the measurement sample is liquid, it is only necessary to mix the measurement sample and the liquid medium as they are. When the measurement sample is solid or semi-solid, it is preferable to mix the sample and a liquid medium after mixing the sample with an appropriate diluent such as physiological saline and peptone water and homogenizing using a stomacher or the like.

  When the homogenized sample contains particles other than microorganisms having a minimum diameter of 100 μm or more, it is preferable to perform filtration using a mesh with a staining solution in order to reduce measurement accuracy. The size of the mesh is preferably about 100 μm. Filtration is preferably performed before the start of culture, preferably before measurement with a flow cytometer. When homogenizing, the process can be simplified if a liquid medium used for culture is used instead of the diluent.

  Although there is a temperature range in which culture can be performed depending on the type of microorganism, culture is preferably performed at an optimal growth temperature. For example, about 27 ° C. is suitable for thermophilic bacteria, 37 ° C. is suitable for mesophilic bacteria, and 55 ° C. is suitable for thermophilic bacteria. For example, many pathogenic bacteria that cause food poisoning or spoilage / degradation bacteria are mesophilic bacteria, and actively proliferate around 37 ° C.

  In addition to these conditions, special bacteria may require various conditions as growth conditions such as salt, pH, and oxygen concentration. The growth conditions of the microorganisms to be tested have been studied in detail, and various documents and textbooks are readily available.

  The predetermined time refers to the time required to change the growth activity of the microorganism. “Varying” refers to, for example, causing a difference between live and dead microorganisms that can be distinguished by electrical or optical means. A culture time in which the growth activity of microorganisms approaches the maximum is suitable, and the culture time is preferably cultured immediately before entering the logarithmic phase or until the end of the induction phase.

  For example, in E. coli, it is preferable to culture at around 37 ° C. for about 1 to 2 hours.

  Next, the step of measuring the growth activity of microorganisms contained in the sample electrically or optically will be described.

  The growth activity of microorganisms refers to the characteristics of microorganisms that change by culturing a sample and are measured optically or electrically. More specifically, it is detected by a particle detection and counting device such as a flow cytometer. The characteristics of microorganisms. Specifically, there are, for example, the size, length, and volume of microorganisms, and intracellular contents of microorganisms such as nucleic acid content.

  “Electrically or optically measuring the growth activity of microorganisms” means, for example, based on the growth activity of microorganisms, by distinguishing between live and dead microorganisms using electrical or optical means. Refers to individual detection and counting. Thereby, the bactericidal treatment effect can be measured from the number of living microorganisms in the sample.

  Electrical or optical measurements can be made using, for example, a flow cytometer. Flow cytometers can obtain electrical or optical signals from individual particulates. As the flow cytometer, for example, an optical flow cytometer, an electric resistance type flow cytometer, or the like can be used. The flow cytometer is preferably capable of measuring fine particles of about 1 to 500 microns.

  As the optical flow cytometer, BACTANA released from Sysmex Corporation, EPICS series released from Beckman Coulter, and FACS series released from Becton Dickinson can be used.

  The electric resistance type flow cytometer is a particle measuring device using a measurement principle well known as a Coulter principle. For example, devices such as SD-2000 and CDA-500 sold by Sysmex Corporation can be suitably used. .

  When measuring the nucleic acid content of a microorganism as the growth activity of the microorganism, the nucleic acid content of the microorganism can be measured based on the intensity of fluorescence emitted from the fluorescently stained nucleic acid.

  For example, (a) a microorganism in a sample is stained with a nucleic acid-specific fluorescent dye, (b) the obtained sample is introduced into an optical flow cytometer, and light is applied to each stained microorganism ( c) Based on the intensity of fluorescence emitted from individual microorganisms and representing the nucleic acid content of the microorganisms, the microorganisms can be individually measured and counted by an optical flow cytometer. Further, instead of staining with a nucleic acid-specific fluorescent dye, it may be labeled with a fluorescently labeled nucleic acid probe.

  As the nucleic acid-specific fluorescent dye, one that specifically stains nucleic acid (RNA, DNA) in a microorganism, absorbs light, and emits fluorescence is used. Here, “specifically stain nucleic acid” mainly refers to staining nucleic acid, and within a range that does not depart from the purpose of the present invention, for example, a protein in a microorganism that is increased or activated during culture, It does not exclude the case of staining something other than nucleic acids, such as enzymes. Therefore, the “nucleic acid-specific fluorescent dye” includes, for example, fluorescent dyes that stain other than nucleic acids such as proteins and enzymes in microorganisms without departing from the object of the present invention. As such fluorescent dyes, for example, dyes described in JP-A-9-104683 and JP-A-2001-258590 can be used. When this dye is used, the nucleic acid of the microorganism can be stained within 1 minute, which is preferable.

  Microbial nucleic acid staining is basically performed using a staining solution that is an aqueous solution containing a nucleic acid-specific fluorescent dye and a buffer. Dilute the sample with diluent before staining. It is preferable to add an agent, for example, a surfactant, for damaging the cell membrane of the microorganism and facilitating the dye to enter the cell or for improving the dispersion of the fine particles. In addition, when the stability of the fluorescent dye is poor, it is preferable to store the dye in a solution such as ethylene glycol and mix it with a diluent at the time of use.

  Microorganisms that have been stained with nucleic acid absorb light such as laser from the flow cytometer, and emit fluorescence with an intensity that represents the amount of stained nucleic acid. The flow cytometer detects fluorescence and measures the intensity of fluorescence. To do. Based on the difference in fluorescence intensity, live and dead microorganisms can be distinguished and counted, and the bactericidal treatment effect can be measured from the number of live microorganisms.

  When measuring the size of a microorganism as the growth activity of the microorganism, the size of the microorganism can be measured based on the intensity of scattered light emitted from the microorganism.

  For example, (a) a sample is introduced into an optical flow cytometer, each individual microorganism is irradiated with light, and (b) a microorganism based on the intensity of scattered light representing the size of the microorganism scattered by the individual microorganism. Can be measured by a process of individually detecting and counting with an optical flow cytometer.

  When a microorganism is irradiated with light, the light is scattered by the microorganism, and the optical flow cytometer detects scattered light indicating the size of the microorganism and measures the intensity of the scattered light. Based on the difference in the size of the measured microorganism, live microorganisms and dead microorganisms can be distinguished and counted, and the sterilization effect can be measured from the number of live microorganisms. The “scattered light” includes forward scattered light, side scattered light, back scattered light, and the like.

  For microorganisms whose length changes due to growth, such as yeast and natto, the pulse width of the scattered light may be used instead of the intensity of the scattered light. The pulse width of the scattered light can be measured from the time when the scattered light is detected. Based on the difference in the measured pulse widths of microorganisms, live microorganisms and dead microorganisms can be distinguished and counted, and the sterilization effect can be measured from the number of living microorganisms.

  When measuring the size (volume) of a microorganism as the growth activity of the microorganism, the size (volume) of the microorganism can be measured based on the inter-electrode electrical resistance value that changes due to the presence of the microorganism.

  For example, (a) a sample is introduced into an electric resistance type flow cytometer comprising an orifice and a pair of electrodes electrically connected through the orifice with an electrolyte, and (b) individual microorganisms in the electrolyte The microorganisms can be measured by a process of individually detecting and counting microorganisms with an electric resistance type flow cytometer based on the inter-electrode electrical resistance value representing the volume of the microorganisms when passing through the orifice.

  An orifice is a fine through-hole, for example, those having a diameter of about 30 μm to 200 μm can be used, and those having a diameter of about 100 μm are preferable.

  A pair of electrodes are electrically connected by an electrolyte solution through an orifice, and a change in the inter-electrode electrical resistance value that occurs when particles in the electrolyte solution pass through the orifice is detected as an electric pulse. Can accurately measure the volume. Further, the microorganisms and the particles other than the microorganisms can be distinguished and counted by the difference in volume.

  Based on the measured difference in the volume of microorganisms, live microorganisms and dead microorganisms can be distinguished and counted, and the sterilization effect can be measured from the number of living microorganisms.

  The method of the present invention may further include a step of comparing the results obtained by performing the same treatment and measurement on the sample before the sterilization treatment and the sample subjected to the sterilization treatment.

  By comparing the results obtained for the sterilized and non-sterilized samples, it is possible to obtain a relative value for the number of microorganisms that are still alive after sterilization. Can be measured.

  That is, by obtaining the relative value, the sterilization effect can be accurately measured without being influenced by the number of microorganisms included in the sterilization target. In addition, when the effect of sterilization treatment is measured by the absolute value of the number of living microorganisms after sterilization treatment, a certain variation occurs in the measurement result due to fluctuations in the culture time. When measured, the number of viable microorganisms in the two samples to be compared increases at approximately the same rate, so that the variation in measurement results due to fluctuations in the culture time is small.

Next, a second embodiment of the present invention will be described.
The method for measuring the effect of sterilization treatment of microorganisms according to the second embodiment of the present invention collects the first sample and the second sample from the sterilization target, sterilizes the first sample, and performs the first sample and the second sample. After culturing with each of the two samples for a predetermined time, the proliferation activity of the microorganisms contained in the first and second samples is measured electrically or optically, respectively.

At least the first sample may be sterilized, and the second sample may or may not be sterilized. In other words, the second sample may be subjected to the sterilization treatment without performing the sterilization treatment, and the second sample may be measured with respect to the first sample. Different sterilization treatments may be applied and the effects of the two sterilization treatments may be compared.
Regarding the description of the common parts of the first and second embodiments, the description of the first embodiment is applied to the second embodiment.

  In addition, this invention is applicable also when proliferation activity does not change clearly by culture | cultivation of a sample. In this case, it is impossible to detect and count live microorganisms and dead microorganisms separately, but if the live microorganisms contained in the first and second samples are sufficiently grown by culturing, the number of dead microorganisms is substantially reduced. Can be ignored.

  As a result, it can be said that the ratio of the number of microorganisms after culturing reflects the ratio of the number of living microorganisms before culturing, and the sterilizing effect can be measured by comparing the number of microorganisms after culturing.

  The reason is that the smaller the number of living microorganisms before culturing, the smaller the number of microorganisms after culturing, and the higher the sterilization effect, the smaller the number of microorganisms after culturing. For example, it is desirable to culture for a period of time during which the difference between the number of viable microorganisms contained in the first sample and the viable microorganism contained in the second sample becomes clear as the microorganism enters the logarithmic growth phase.

In the present invention, when a flow cytometer is used, the bacterial concentration in the sample is preferably 10 ² / mL to 10 ³ / mL or more, and the initial bacterial concentration in the measurement sample is low. For example, in order to grow E. coli having an initial concentration of 10 ⁰ / mL to a concentration of 10 ³ / mL or more, a culture time of about 4-5 hours is required.

  Examples of the present invention are shown below. The embodiments of the present invention are not limited to the following examples.

Preparation of Sample for Measurement A sample obtained by culturing E. coli in a heart- infusion broth medium overnight was diluted with physiological saline to prepare a sample with an initial concentration of 10 ⁵ / mL. This sample was sterilized by heating at 45-80 ° C. for 10 minutes. 1 mL of the sample after sterilization treatment and 9 mL of heart infusion broth medium were mixed and cultured at 37 ° C. for 2 hours.

    Next, after adding 340 μL of diluent to 50 μL of the sample after incubation for a certain period of time and stirring, add 10 μL of staining solution and incubate at 40 ° C. for 20 seconds to stain with a nucleic acid-specific fluorescent dye. The measurement sample was prepared.

Reagents having the following composition were used.
(Diluted solution)
Citric acid dihydrate 21.6 g / L
Myristyltrimethylammonium bromide 1g / L
Purified water 1.0L
Adjusted to pH 2.5 with NaOH (staining solution)
50 mg of dye having the following structure

エチレングリコール １.０Ｌ

Ethylene glycol 1.0L

Measurement Method Using a flow cytometer using a red semiconductor laser of 633 nm as a light source, the intensity of red fluorescence (650 nm or more) and the intensity of forward scattered light were measured for the prepared sample.

  1 and 2 are histograms of fluorescence intensity measured for a sample that has not been sterilized. FIG. 1 is before culture, and FIG. 2 is after culture.

  3 and 4 are histograms of fluorescence intensities measured for samples that have been sterilized by heating at 55 ° C. for 10 minutes. FIG. 3 is the one before culture, and FIG. 4 is the one after culture.

  1 to 4, the horizontal axis represents fluorescence intensity (FLP), the vertical axis represents the frequency (ALL) of microorganisms emitting fluorescence of each intensity, and the vertical broken line represents the discreet level.

  Comparing FIGS. 1 and 2, the intensity of the fluorescence emitted by most microorganisms was smaller than the discreet level before culturing (FIG. 1), whereas most of the fluorescence after culturing (FIG. 2). You can see that it is above the discretion level. This is because the amount of nucleic acid in the living microorganism increased due to the culture.

  Comparing FIGS. 3 and 4, it can be seen that the intensity of fluorescence emitted by most microorganisms is smaller than the discreet level both before (FIG. 3) and after (FIG. 4). This is because most of the microorganisms were killed by the sterilization treatment, and the number of living microorganisms became very small.

  Depending on the presence or absence of the sterilization treatment, the number of microorganisms emitting fluorescence with an intensity higher than the discrete level differs in the measurement after culturing, which can be said to reflect the effect of the sterilization treatment.

The number of microorganisms above the discrete level in the histogram of FIG. 4 was 2.5 × 10 ³ / mL, and the number of microorganisms above the discrete level of the histogram in FIG. 2 was 4.63 × 10 ⁶ / mL. . By removing both, the suppression rate of microorganisms by sterilization treatment can be obtained.
Inhibition rate = suppressed sample measured value / control sample measured value = 2.50 × 10 ³ /4.63×10 ⁶ = 5.40 × 10 ⁻⁴
It becomes.

  5 to 8 are histograms showing the frequency (ALL) with respect to the forward scattered light intensity (FSCP), and were measured using the same sample as that used in the measurement for FIGS.

  5 and 6 show the results of measurement on a sample that has not been sterilized. FIG. 5 shows the result before culture, and FIG. 6 shows the result after culture.

7 and 8 show the measurement results of a sample that has been sterilized by heating at 55 ° C. for 10 minutes. FIG. 7 shows the result before culture, and FIG. 8 shows the result after culture.
Accordingly, FIGS. 5 to 8 correspond to FIGS. 1 to 4, respectively.

  FIGS. 5-8 shows the same tendency as FIGS. 1-4, and shows that the effect of a sterilization process can be measured even if it uses forward scattered light. That is, in the sample that has not been sterilized (FIGS. 5 and 6), the intensity of the forward scattered light is increased because the size of the live microorganisms is increased by the culture. On the other hand, in the sterilized sample (FIGS. 7 and 8), most of the microorganisms were killed by the sterilization process, and the number of living microorganisms was very small. Not.

Verification of effectiveness In order to verify the effectiveness of the method of the present invention, the inhibition rate was determined using the conventional method and the method of the present invention for samples that were sterilized under the same conditions, and the two were compared.

As a conventional method, the inhibition rate of microorganisms was determined by the following procedure.
First, a sample containing E. coli was prepared under the same conditions as in Example 1, and the sample was sterilized by heating under the same conditions as in Example 1. After 1 mL of the sterilized sample was mixed with about 20 mL of agar medium, it was cultured at 35 ° C. for 48 hours, and the number of colonies was counted.

  The number of colonies was counted in the same manner for the samples that were not sterilized. The inhibition rate of microorganisms was determined by dividing the measured value for the sample that had been sterilized by the measured value for the sample that had not been sterilized.

  The temperature of the heat treatment for sterilization treatment was changed from 45 ° C. to 80 ° C. in increments of 5 ° C., and the inhibition rate at each temperature was determined using the conventional method and the method of the present invention. The results are shown in Table 1.

  FIG. 9 is a graph showing the relationship between the inhibition rate obtained by the conventional method shown in Table 1 and the inhibition rate obtained by the method of the present invention. This graph shows that both are well correlated, and thus the effectiveness of the method of the present invention.

  Also, when looking at the measured value column according to the present invention, the higher the heat treatment temperature, that is, the higher the sterilization effect, the smaller the measured value, and the one subjected to the heat treatment at 80 ° C. Microorganisms were not detected. This result shows that the bactericidal effect can be measured by the method of the present invention without determining the relative value by measuring the absolute value of the number of living microorganisms.

It is the histogram of the fluorescence intensity measured before culture | cultivation about the sample which has not performed sterilization treatment. It is the histogram of the fluorescence intensity measured after culture | cultivation about the sample which has not performed sterilization treatment. It is the histogram of the fluorescence intensity measured before culture | cultivation about the sample which performed the sterilization process. It is a histogram of the fluorescence intensity measured after culture | cultivation about the sample which performed the sterilization process. It is a histogram of the forward scattered light intensity measured before culture | cultivation about the sample which has not performed sterilization treatment. It is a histogram of the forward scattered light intensity measured after culture | cultivation about the sample which has not performed sterilization treatment. It is a histogram of the forward scattered light intensity measured before culture | cultivation about the sample which performed the sterilization process. It is a histogram of the forward scattered light intensity measured after culture | cultivation about the sample which performed the sterilization process. It is a graph which shows the relationship between the suppression rate by the conventional method, and the suppression rate by the method of this invention.

Claims (7)

  Sterilization of microorganisms characterized by collecting a sterilized sample from an object to be sterilized, culturing the sample in advance for a predetermined time, and then measuring the proliferation activity of individual microorganisms contained in the sample electrically or optically Treatment effect measurement method.   The microorganism obtained according to claim 1, further comprising a step of comparing the result obtained by the measurement with the result obtained by performing the same treatment / measurement as the sample subjected to the sterilization treatment on the sample before the sterilization treatment. Method for measuring sterilization effect.   The method for measuring the effect of bactericidal treatment of microorganisms according to claim 1 or 2, wherein the proliferation activity of each microorganism is represented by at least one of the size of the microorganism and the nucleic acid content of the microorganism.   The method for measuring the effect of sterilizing a microorganism according to any one of claims 1 to 3, wherein the electrical or optical measurement is performed by a flow cytometer.   The first sample and the second sample are collected from the sterilization target, the first sample is sterilized, and each of the first sample and the second sample is cultured for a predetermined time, and then the first and second samples are obtained. A method for measuring the effect of bactericidal treatment of microorganisms, wherein the proliferation activity of microorganisms contained therein is measured electrically or optically.   The method for measuring the effect of sterilizing a microorganism according to any one of claims 1 to 5, wherein the predetermined time is a culture time in which the growth activity of the microorganism approaches a maximum.   The method for measuring the effect of sterilizing a microorganism according to any one of claims 1 to 5, wherein the predetermined time is immediately before entering the logarithmic phase of the microorganism.

Images