JP4911423B2 - Microorganism measurement method - Google Patents

Description

  The present invention relates to a method for measuring microorganisms in a sample. The present invention can be used in a wide range of fields such as food, medicine, pharmaceuticals, agriculture, and environmental analysis.

  In recent years, risks caused by microorganisms, such as food poisoning on a large scale and the occurrence of emerging infectious diseases, have become major social problems. One major factor that does not solve these problems is that it takes a long time to test microorganisms. Under these circumstances, rapid microbiological testing is strongly demanded.

  Conventionally, culture methods have been used for microorganism testing. The culture method has been the basis of hygiene management since the beginning of the 20th century. However, since it is necessary to wait for the growth of microorganisms, detection takes a long time. According to official methods established in various fields such as food, it takes approximately 24 hours or more to detect microorganisms. Also, fungi (molds and yeasts) require one week of culture.

  Various methods that do not require culturing have been studied as rapid inspection techniques for solving such problems of the culturing method. A typical example is a technique of staining microorganisms with a reagent and measuring the coloration or fluorescence using a microscope or an automatic detection device. Specific examples are given below.

A. Direct Staining Method Using 4 ′, 6-diamidino-2-phenylindylene chloride, all microbial genes are stained and detected regardless of the type of microorganism.
Using Propidium iodide, dead microorganisms are fluorescently stained and detected regardless of the type of microorganism.

B. Staining Method Utilizing Physiological Activity Using a carboxyfluorescein diacetate (hereinafter abbreviated as CFDA) that emits fluorescence when decomposed by an enzyme contained in the microorganism, the microorganism having the enzyme activity is fluorescently stained and detected.
Using 5-cyano-2,3-diethyltetrazolium chloride that fluoresces by respiratory activity, a microorganism having respiratory activity is fluorescently stained and detected.

C. Method of staining a specific gene sequence This is a method of staining and detecting DNA or RNA in microbial cells. A gene fragment (referred to as a probe) that binds in a complementary manner to a unique sequence of a microorganism to be detected is prepared in advance, and the probe is labeled with a fluorescent dye. By adding this labeled probe to the sample, the target gene, that is, the target microorganism is specifically fluorescently stained and detected.

D. Method for staining a specific antigen A method for staining and detecting an antigen possessed by a microorganism. An antibody that specifically binds to a specific antigen possessed by a microorganism to be detected is prepared in advance, and the antibody is labeled with a fluorescent dye. By adding this labeled antibody to the sample, the target antigen, that is, the target microorganism, is specifically fluorescently stained and detected.

With these technologies, the inspection time was within a few hours, and about 10 minutes for fast ones, the speed was greatly increased.
However, there are still significant problems in applying these techniques to actual samples such as food. That is, substances other than microorganisms to be detected (hereinafter referred to as “contaminants”) are non-specifically stained with reagents. When non-specific staining occurs, microorganisms and contaminants emit fluorescence at the same wavelength during detection. Although it is possible to distinguish between microorganisms and contaminants to some extent by using the size and shape of fluorescent luminescent spots, signal intensity, etc., it is practical for samples that contain a lot of biological contaminants (such as food). It is often difficult to distinguish microorganisms and contaminants to the level. For example, in the sanitary standards for sugar beet, the number of viable bacteria is 100,000 / g as the standard for determining the quality of shipment, but it is difficult to reduce the detection limit of microorganisms to this level by eliminating the influence of foreign substances. .

For these situations, adding two or more types of staining reagents to the sample and combining the specificities of these reagents reduces the effects of contaminants and more accurately detects the target microorganism. A technique called multiple staining has been devised.
Examples of the prior art using multiple staining include the following ac.

a: Y. Hansson et. al. Journal of Immunological Methods, 100 (1987) pp .; 261-267
Fluorescein diacetate is used as a reagent for fluorescent emission of living cells, and hemoglobin is used to quench the fluorescence of fluorescein leaking from the living cells. The sample is double-stained with these reagents, and the stained sample is irradiated with excitation light to detect the intensity of fluorescence emitted from living cells stained with fluorescein diacetate using a photomultiplier connected to a fluorescence microscope. Thus, a method for calculating the number of viable cells from the fluorescence emission intensity is described.

b: U.S. Pat. No. 6,459,805 A sample is added with a fluorescent dye that accumulates only in living cells and a quenching dye that can penetrate dead cells but is excluded from living cells. A method for measuring the number of viable cells by measuring is described.
Specifically, examples include the use of fluorescein diacetate as a fluorescent dye and Eosin Y as a quenching dye, and the example of using Calcein-AM as a fluorescent dye and trypan blue as a quenching dye.

c: JP-A-2002-34594 In a method for detecting a living cell in which a dye or a fluorescent enzyme substrate is added to a sample containing cells to detect or measure the dye or fluorescence, the dye cannot pass through the cell membrane, and the dye Alternatively, a method is described in which the detection or measurement is performed in the presence of an absorber that absorbs the luminescence of the fluorescent enzyme substrate.
Specifically, a fluorescent enzyme substrate Carboxyfluorescein diacetate methyl ester is added to a soil suspension to fluorescently stain live cells, and nonspecific fluorescence emitted by contaminants other than live cells is absorbed by Cytochrome c Has been introduced.
US Pat. No. 6,459,805 JP 2002-34594 A

  Although the above-mentioned prior art is an example in which the influence of impurities is reduced, there are the following problems. That is, in the samples measured by these prior arts, a and b are cultures of human cells and nerve cells, and c is a suspension of soil. The prior arts a to c are methods that can be applied only to samples with little influence of contaminants, and are effective for samples containing a large amount of biological contaminants that are close to microorganisms, such as food. It's not a means.

  In order to solve these problems of the prior art, an object of the present invention is to find an effective multiple staining method for samples containing food and other biological contaminants, and to provide a method for measuring microorganisms using the same.

  In order to achieve the above object, a microorganism measuring method according to the present invention comprises contacting a sample with a first reagent, fluorescently staining the microorganism to be detected in the sample with the first reagent, The second reagent is contacted, the substance other than the microorganism to be detected in the sample is stained with the second reagent, and the sample is irradiated with light having a wavelength that excites the first reagent, Detecting the bright spot emitting fluorescence at the fluorescence wavelength of the first reagent, and determining the presence or number of microorganisms from the information (number, size, shape, distribution) of the detected bright spot, The reagent fluoresces and dissolves in the solvent containing the sample, the second reagent absorbs the fluorescence of the first reagent, dissolves in the solvent containing the sample, and is negative in the sample. It has the group which dissociates in. Here, it is preferable that the second reagent has fat solubility and has a small molecular weight.

In the microorganism measuring method according to the present invention, as the first reagent, at least one reagent that can fluorescently stain a microorganism to be detected in the sample and has a fluorescence wavelength band of 500 to 530 nm. (Excluding those in which the first reagent is impermeable to the cell membrane) and red 102 as the second reagent.
In addition, the method for measuring microorganisms according to the present invention includes, as the first reagent, more specifically, Fluorescein diacetate, Carboxyfluorescein diacetate, Carboxyfluorescein diacetate, Aceticylethylester, Carboxymethycetylacetate Using at least one reagent selected from the group consisting of Green I and Rhodamine 123 , Red No. 102 is used as the second reagent.

  The present inventors have found an effective multiple staining method for samples containing food and other biological contaminants. According to the present invention, a method for measuring microorganisms using the same is provided.

  The detailed contents and examples of the microorganism measuring method according to the present invention will be described below. However, the scope of application of the present invention is not limited to this description.

[Principle of the present invention]
In the living microorganisms, in the water, the cell surface is negatively charged due to dissociation of carboxyl groups and the like, and exists as negatively charged particles. For this reason, a substance having a negative charge is less likely to come into contact with living microorganisms due to electrical repulsion. If a negatively charged reagent is used as a staining reagent, the reagent is difficult to come into contact with microorganisms and does not stain living microorganisms.

  On the other hand, negative charges are not usually localized in contaminants having no physiological activity other than living microorganisms. For this reason, impurities can be stained without being repelled even by a negatively charged reagent. Microorganisms lose their negative charge when they lose their biological activity (when they die), so they become stained with a negatively charged reagent.

Taking these into consideration, a reagent that can fluorescently stain the target microorganism is used as the first reagent, and a reagent that has a negative charge and can absorb the fluorescence of the first reagent is used as the second reagent. In the state where the target microorganism is fluorescently stained, other impurities can be stained with the second reagent. As a result, the target microorganism and other impurities are dyed in different colors. This staining with the second reagent is called mask staining.
Further, in consideration of staining of dead microorganisms, the second reagent preferably has fat solubility and a small molecular weight.

  The kind of microorganism detected in the present invention is not particularly limited. For example, in addition to bacteria such as Escherichia coli, Staphylococcus, Pseudomonas, Bacillus and Serratia, the present invention can also be applied to fungi such as yeast and protozoa such as Cryptosporidium.

  Furthermore, the method of the present invention is not limited to microorganisms, and can also be applied to measurement of animal cells and plant cells.

[reagent]
The first reagent used in the present invention is required to have the following characteristics:
・ Fluorescent staining of the target microorganism.
-Dissolve in the solvent containing the sample.

The second reagent requires the following properties:
Absorb the fluorescence of the first reagent.
-Dissolve in the solvent containing the sample.
-It has a group that dissociates negatively in the sample.
The second reagent also preferably has the following characteristics:
-It has a fat-soluble group.
・ Low molecular weight.

The combinations of the first reagent and the second reagent are shown in Table 1 as suitable combinations.

Table 1 shows a group of reagents in which the fluorescence wavelength band of the first reagent and the absorption wavelength band of the second reagent are 500 to 530 nm.

The following abbreviations were used in these tables.
FDA: Fluorescein diacetate
CFDA: Carboxyfluorescein diacetate
CFDA-AM: Carboxyfluorescein diacetate acetate methyl ester
CMFDA: Carboxymethylfluorescein diacetate

[Measurement procedure]
The measurement flow of the present invention will be described with reference to the microorganism measurement procedure shown in FIG.
In the method of the present invention, first, the sample 1 is subjected to a process for separating microorganisms and substances other than microorganisms (contaminants) such as a stomat, reagent treatment, centrifugation, and filtration (step 2). Subsequently, the first reagent is brought into contact (step 3). The first reagent fluorescently stains microorganisms and remaining contaminants. Next, the second reagent is brought into contact with the sample (step 4). The second reagent masks contaminants.

  Then, the sample is irradiated with light having a wavelength that excites the first reagent, and a bright spot emitting fluorescence at the fluorescence wavelength of the first reagent is detected (step 5). The fluorescent bright spot can be detected visually using a fluorescence microscope. It is also possible to detect a bright spot by combining a fluorescent microscope with a CCD camera or a CMOS camera and acquiring an image and then processing the image. Furthermore, if the inventor applies an automatic detection apparatus as shown in the previous application, it is possible to automate the processes from image acquisition to image processing and bright spot detection.

  From the information (number, size, shape, distribution) of the bright spots detected by these methods, the presence or absence and number of microorganisms can be determined.

  As a sample form, a liquid sample that does not contain solid matter can be measured, and even a sample containing solid matter can be extracted by sterilizing water and pulverized and mixed (stomac) to extract and measure microorganisms. It is possible.

[Example 1]
Examples of measuring potato salad using the microorganism method of the present invention will be described below. Unless otherwise stated in the description, sterilized laboratory equipment was used.

Preparation of Sample An appropriate amount of Pseudomonas bacterium purely cultured in potato salad was added to prepare a sample contaminated with bacteria in a simulated manner.

Stomac / reagent reaction / centrifugation First, 10 g of the sample potato salad was weighed, and 90 mL of sterilized water was added to perform the stomac. The used smack bag is equipped with a mesh filter so that when collecting the smack liquid, large garbage can be separated.
1 mL of Stomack solution was collected in a microtube, and 250 μL of 20% Trypsin aqueous solution and 20 μL of 10% Polyoxyethylene (10) octylphenyl ether aqueous solution were added thereto. The sample solution was stirred with a vortex mixer and immersed in a constant temperature water bath at 42 ° C. for 1 minute.
Subsequently, the sample solution was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was removed.

900 μL of phosphate physiological saline was added to the pellet obtained by redispersion / filtration centrifugation, and the pellet was dispersed by stirring. To the sample solution, 250 μL of 20% Trypsin aqueous solution and 20 μL of 10% Polyoxyethylene (10) octylphenyl ether aqueous solution were added. The sample solution was stirred with a vortex mixer and immersed in a constant temperature water bath at 42 ° C. for 1 minute.
Subsequently, the total amount of the sample solution was filtered with a membrane filter, and microorganisms to be detected were captured on the membrane filter. As the membrane filter, a polycarbonate black membrane manufactured by Whatman, a pore size of 0.4 μm, and a diameter of 25 mm was used.

Fluorescent staining / mask staining In advance, a fluorescent staining stock solution in which CFDA was dissolved in DMSO at 1 to 10 mg / mL was prepared. The fluorescent staining stock solution was mixed with a phosphate buffer solution to obtain a fluorescent staining solution. The fluorescent staining solution was dropped on the membrane filter and allowed to stand for 1 to 5 minutes, and then the fluorescent staining solution was removed by suction through filtration.
Next, a mask solution containing Red No. 102 was prepared. The mask solution was dropped on the membrane filter and allowed to stand for 1 to 5 minutes, and then the fluorescent staining solution was removed by suction through filtration.

[Membrane filter setting, fluorescent image acquisition, bright spot counting by image processing]
The funnel of the filter was removed, the membrane filter was removed with tweezers and placed on a slide glass. During these operations, care was taken not to touch the collection surface of the membrane filter with fingers, etc., and when the fingers touched the collection surface, all operations were repeated.
The membrane filter was observed with a fluorescence microscope and an automatic analyzer.

Results / Analysis In FIG. 2, the left side is an image obtained by observing a sample after CFDA fluorescent staining (before mask staining) using a fluorescent microscope, and the right side is an image obtained by observing the sample after mask staining. In the image before mask staining, it is difficult to distinguish between bacteria (Pseudomonas) and impurities. On the other hand, in the image after mask staining, the contaminants are dyed into a different color (red) from bacteria (green) by the mask reagent, and both can be clearly identified.

[Example 2]
FIG. 3 shows the results of measuring microorganisms by the method of the present invention by adding Enterobacter to onion shredded by the same operation as in Example 1. In this example, an automatic detection device is used to detect microorganisms, the measurement result of the automatic detection device is plotted on the vertical axis, the measurement result of the conventional culture method is plotted on the horizontal axis, and the correlation between the two is graphed. is there. In the conventional rapid inspection method as mentioned in the prior art, it is difficult to distinguish bacteria from contaminants when the number of bacteria detected by the culture method is lower than about 1 to 1 million cells / g (see the figure). Not shown). On the other hand, if the present invention is used, it can be seen from FIG. 3 that the lower limit of quantification of bacteria has reached 10,000 cells / g or less.

[Comparison with conventional multiple staining reagents]
It has been confirmed by the present inventors that Trypan Blue introduced in the prior art b is effective for mask staining of contaminants contained in raw milk. However, when applied to a sugar beet sample as described above, the ability to mask contaminants was weak, and the detection sensitivity of microorganisms could not be lowered.

  In addition, Propidium iodide is said to fluorescently stain dead cells regardless of the type of microorganism, but no effect was seen on the mask of impurities contained in sugar beet samples. On the other hand, in some cases, the affinity for live bacteria was higher than that of impurities. Propidium iodide is considered not necessarily highly specific for staining dead cells.

  By using the present invention, it is possible to measure a sample containing biological contaminants while distinguishing the microorganisms contained therein from the contaminants. In conventional rapid methods such as fluorescent staining, practical detection sensitivity (for example, 100,000 in terms of the number of viable bacteria in the sanitary norms of sugar beet) is used for samples containing a lot of biological solid impurities such as sugar beet. / G is the standard for determining the quality of shipment), but 10,000 live cells / g were detected by the present invention.

With this effect, for example, microbial inspection of food can be performed quickly, and the risk of food poisoning can be reduced by speeding up shipping inspection at a food factory. In addition, since the dynamics of microorganisms can be grasped in real time in a food production process (for example, fermentation), it is possible to control the process more precisely than before, and an improvement in product quality is expected. In addition, in the pharmaceutical field, it is possible to improve the efficiency of drug development by accelerating and improving the evaluation of the effects of chemical substances on microorganisms and cells. Increasing the throughput of pharmaceutical research is also a social request, and is an area where the present invention can contribute. Furthermore, the present invention has many uses such as detection of nosocomial infection-causing bacteria, measurement of microorganisms in soil, and monitoring of microorganisms related to water pollution.

It is a flowchart which shows the procedure of the measurement of microorganisms by this invention. It is a photograph which shows the discrimination effect of the microorganisms and impurities by a 2nd reagent. It is a graph which shows the result of having reduced the influence of a foreign material by this invention and measuring with the automatic detection apparatus.

Claims (2)

Contact the sample with the first reagent,
Fluorescent staining of the microorganisms to be detected in the sample with the first reagent,
Contact the sample with a second reagent,
Staining a substance other than the microorganism that is the detection target in the sample by the second reagent,
Irradiating the sample with light having a wavelength that excites the first reagent,
Detect bright spots emitting fluorescence at the fluorescence wavelength of the first reagent,
Including determining the presence or number of microorganisms from the detected bright spot information,
The first reagent emits fluorescence and is dissolved in a solvent containing the sample;
The second reagent has a group that absorbs the fluorescence of the first reagent, dissolves in the solvent containing the sample, and negatively dissociates in the sample;
As the first reagent, a microorganism to be detected in the sample can be fluorescently stained, and at least one reagent having a fluorescence wavelength band of 500 to 530 nm (the first reagent is impermeable to the cell membrane). And the red reagent No. 102 is used as the second reagent. Contact the sample with the first reagent,
Fluorescent staining of the microorganisms to be detected in the sample with the first reagent,
Contact the sample with a second reagent,
Staining a substance other than the microorganism that is the detection target in the sample by the second reagent,
Irradiating the sample with light having a wavelength that excites the first reagent,
Detect bright spots emitting fluorescence at the fluorescence wavelength of the first reagent,
Including determining the presence or number of microorganisms from the detected bright spot information,
The first reagent emits fluorescence and is dissolved in a solvent containing the sample;
The second reagent has a group that absorbs the fluorescence of the first reagent, dissolves in the solvent containing the sample, and negatively dissociates in the sample;
The first reagent is selected from the group consisting of Fluorescein diacetate, Carboxyfluorescein diacetate, Carboxyfluorescein diacetate acetoxymethyl ester, CarbineMethylfluorescein diacetate, and R A method for measuring microorganisms, comprising using Red No. 102 as a second reagent.

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