Method for preparing samples for testing acid-tolerant microorganisms

Abstract

To enable easy preparation of a highly reliable test sample necessary for testing acid-resistant microorganisms contained in test water such as Legionella species.SOLUTION: The pH of test water is adjusted to pH 3.5 which is an acidic region that Legionella species are shown to tolerate. When test water adjusted to pH 3.5 is passed through a filter material such as glass fiber filter paper through which Legionella species can pass and which has a negative charge in high pH regions including pH 3.5, Legionella species which is positively charged in the test water is captured by the filter material due to electrostatic attraction. Then, when a buffer solution with a pH higher than about pH 4 to 5, which is the isoelectric point of Legionella species, for example, a pH 9.5 buffer solution, is passed through the filter material, the Legionella species captured by the filter material is negatively charged by the pH environment of the buffer solution so as to be repelled from the filter material and to be eluted into the buffer solution. As such, a test sample can be obtained by securing buffer solution that has passed through the filter material.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a test sample for microorganisms, and more particularly to a method for preparing a test sample for acid-resistant microorganisms contained in test water. [Background technology]

[0002] Drinking water, bath water, and other water used for daily life can contain trace amounts of various microorganisms due to various factors, such as the water source and the environment. If the amount exceeds a certain level, it may cause infectious diseases. For example, Legionella bacteria, which are often found in circulating hot spring water and bath water at public baths and lodging facilities, or in circulating cooling water in cooling towers, are acid-resistant microorganisms that are the cause of diseases such as Pontiac fever and serious pneumonia (Legionella pneumonia). Therefore, local ordinances stipulate that the Legionella bacteria limit for bath water, such as hot spring water, must be less than 10 CFU / 100 mL, and annual testing for Legionella bacteria is mandatory. For circulating cooling water in cooling towers, a separate recommended limit of less than 100 CFU / 100 mL is set, and similar testing is recommended.

[0003] Non-Patent Document 1 specifies an official method for testing bath water and circulating cooling water for Legionella bacteria. In this method, a test sample is prepared by concentrating sample water collected from bathtub water or the like and then sterilizing the contaminants. A fixed amount of the test sample is then applied to selective medium and cultured for 5 to 7 days. If moist, pale-white or grayish-white colonies appear, it is tentatively determined that the test water contains Legionella bacteria at levels above the regulatory limit, and a confirmation test is then performed. In the confirmation test, the colonies that appear on the selective medium are applied to L-cysteine-free medium and BCYEα medium and further cultured for 2 to 7 days. If colonies form only on BCYEα medium, the colonies are subjected to Gram staining. Gram-negative bacilli are identified as Legionella bacteria, and the test water is finally determined to contain Legionella bacteria at levels above the regulatory limit.

[0004] Official methods for concentrating test water and preparing test samples include membrane filtration or centrifugation. The filtration method involves suction-filtering the test water using a filter fitted with a polycarbonate membrane filter with a pore size of 0.2 μm or 0.22 μm. The membrane filter is then removed from the filter and transferred to a container. Sterile distilled water is added to the container and vigorously shaken using a test tube mixer, resulting in a homogeneous suspension. The suspension is then treated with acid or heat to kill any bacteria present, and the test sample is prepared.

[0005] On the other hand, in the centrifugation method, the test water is placed in multiple centrifuge tubes, centrifuged, and the supernatant liquid is removed from each tube. Next, the tubes are shaken using a test tube mixer to resuspend the precipitate in the remaining liquid, and the contents of each tube are combined into one centrifuge tube. This centrifuge tube is then centrifuged again, and the supernatant liquid is removed and the remaining liquid is shaken using a test tube mixer to resuspend the precipitate. The resulting suspension is then treated with acid or heat to kill any bacteria, and the test sample is prepared.

[0006] The filtration and centrifugation methods require complex manual handling and processing of membrane filters and centrifuge tubes, respectively, making it necessary to transport the collected test water to a facility equipped with the necessary equipment. Furthermore, the reliability of the test sample may be affected by the skill of the operator. While the reliability of the test sample can be ensured by automating either method, such automated equipment inevitably becomes complex and large because it must incorporate the complex steps of each method. Therefore, using the filtration and centrifugation methods, it is difficult to easily prepare reliable test samples on-site at public baths, lodging facilities, etc. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Japanese Industrial Standards JIS K 0350-50-10:2006, "Testing method for Legionella in industrial water and industrial wastewater" Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to enable the easy preparation of a highly reliable test sample required for testing acid-resistant microorganisms, such as Legionella bacteria, contained in test water. [Means for solving the problem]

[0009] The present invention relates to a method for preparing a test sample for acid-tolerant microorganisms contained in test water. This preparation method includes step 1 of adjusting the pH of the test water to an acidic range to which the acid-tolerant microorganisms are resistant, step 2 of passing the test water whose pH has been adjusted in step 1 through a filter material that has a negative charge in a high pH range that includes the pH value of the test water adjusted in step 1 and that allows the acid-tolerant microorganisms to pass through, and step 3 of passing a buffer solution with a pH higher than the isoelectric point of the acid-tolerant microorganisms through the filter material that has been subjected to step 2.

[0010] In the test water whose pH has been adjusted in step 1, acid-tolerant microorganisms stably inhabit in a state of having a positive charge, whereas microorganisms other than acid-tolerant microorganisms cannot inhabit the acidic environment of the test water and are eliminated. In step 2, when the test water that has been subjected to step 1 is passed through a filter, the filter becomes negatively charged and attracts and captures the positively charged acid-tolerant microorganisms in the test water by electrostatic attraction. As a result, the acid-tolerant microorganisms remain on the filter and are separated from the test water. Then, in step 3, when a buffer solution is passed through the filter, the acid-tolerant microorganisms acquire a negative charge due to the pH environment of the buffer, and are repelled by the negatively charged filter, detaching from the filter and migrating into the buffer. Therefore, by collecting the buffer solution that has passed through the filter in step 3, a test sample for acid-tolerant microorganisms is obtained.

[0011] In one embodiment of the preparation method of the present invention, the filter medium is heated under the heat-resistant conditions of the acid-tolerant microorganisms between steps 2 and 3. In another embodiment of the preparation method of the present invention, an acidic solution adjusted to a pH in the acidic range to which the acid-tolerant microorganisms are resistant and lower than the pH of the test water adjusted in step 1 is applied to the filter medium between steps 2 and 3. The operations according to these embodiments can also be used in combination.

[0012] The filter used in the preparation method of the present invention is formed using, for example, glass fiber, glass wool, or a mixture of these fibers, or a cation exchange resin.

[0013] In the preparation method of the present invention, for example, in step 4, a buffer solution containing a protein, a sugar, an alcohol, or a surfactant is used.

[0014] Acid-tolerant microorganisms from which test samples can be prepared by the method of the present invention include, for example, bacteria, molds, and yeasts. [Effects of the Invention]

[0015] According to the preparation method of the present invention, a highly reliable test sample required for testing acid-tolerant microorganisms, such as Legionella bacteria, contained in test water can be easily prepared. DETAILED DESCRIPTION OF THE INVENTION

[0016] The water from which test samples are prepared by the method of the present invention is not particularly limited, and examples include environmental waters such as river water, lake water, and groundwater, tap water, industrial water, and sewage, as well as various types of water, such as circulating hot spring water, bath water, and circulating cooling water in cooling towers. The acid-tolerant microorganisms to be tested in the test water are acid-tolerant microorganisms, typically bacteria such as Legionella or Salmonella, mold, or yeast. Acid-tolerant microorganisms also include acidophilic microorganisms.

[0017] In preparing a test sample, a sample of water is collected from the target water, and the pH of the sample is adjusted to an acidic range to which the acid-tolerant microorganisms to be tested are resistant, preferably to a pH lower than the isoelectric point of the acid-tolerant microorganisms to be tested (Step 1). For example, if the acid-tolerant microorganisms to be tested are Legionella bacteria (it is known that the isoelectric point of general bacteria is approximately 4 to 5, and the isoelectric point of Legionella bacteria is also within this range), the pH of the sample is preferably adjusted in this step to a range of 2 to 6, particularly 3.5 to 4.5.

[0018] The pH of the test water can be adjusted using an acidic agent to lower the pH of the test water. Examples of the agent include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and hydrofluoric acid, and organic acids such as citric acid, acetic acid, formic acid, and oxalic acid. Two or more of these agents may be used in combination.

[0019] By adjusting the pH in this process, among the various microorganisms contained in the test water, acid-tolerant microorganisms can stably survive in a state with a positive charge, while microorganisms other than acid-tolerant microorganisms (hereinafter sometimes referred to as "non-acid-resistant microorganisms") cannot survive in the acidic environment of the test water and are therefore eliminated.

[0020] Next, the test water whose pH has been adjusted in step 1 is passed through a filter (step 2). The filter used in this step has a negative charge in the high pH range that includes the pH value of the test water adjusted in step 1. Such a filter can be formed using a material that is negatively charged in the high pH range that includes the pH value of the test water adjusted in step 1. Examples of such materials include glass fiber, glass wool, and mixed fibers thereof, quartz wool, and cation exchange resin. Of these, it is preferable to use glass fiber, glass wool, or mixed fibers thereof, or cation exchange resin.

[0021] The filter material is not limited in form as long as it is made of the above-mentioned material and is formed to allow the test water to pass through. For example, the material may be formed into a filter paper shape, or the material may be filled into a water passage through which the test water passes in this step. However, since acid-resistant microorganisms must be eluted from the filter material in step 3 described below, the filter material must be designed to allow the acid-resistant microorganisms to be tested to pass through. For example, when the acid-resistant microorganisms to be tested are Legionella bacteria and a filter paper-shaped filter material is used, it is preferable that the particle diameter of the filter material be set to 0.2 μm or more, particularly 0.4 μm or more.

[0022] When the test water, the pH of which was adjusted in step 1, is passed through the filter in this step, the filter becomes negatively charged by being exposed to the pH environment of the test water. In contrast, the acid-tolerant microorganisms in the test water passing through the filter are positively charged, so they are attracted to the filter by electrostatic attraction and captured by the filter. As a result, the acid-tolerant microorganisms in the test water are separated from the test water onto the filter.

[0023] After this step and before proceeding to the next step, the filter material that has captured the acid-tolerant microorganisms may be subjected to a heat treatment or an acid solution treatment, if necessary, in order to enhance the extermination effect of other microorganisms captured in the filter material along with the acid-tolerant microorganisms to be tested.

[0024] In the heat treatment of the filter material, the filter material is heated under the heat resistance conditions of the acid-resistant microorganisms to be tested. For example, when the acid-resistant microorganisms to be tested are bacteria of the genus Legionella, the heating temperature of the filter material is preferably set to 40°C to below the heat resistance temperature of the bacteria of the genus Legionella, particularly below 60°C.

[0025] In the treatment of applying an acidic solution to the filter material, an acidic solution is used whose pH has been adjusted to fall within the acidic range to which the acid-tolerant microorganisms to be tested are resistant, and whose pH is lower than the pH of the test water adjusted in step 1. For example, when the acid-tolerant microorganisms to be tested are Legionella bacteria, the lower limit of the acidic range to which they are resistant is pH 2. Therefore, for example, if the pH of the test water is set to 3.5 in step 1, it is preferable to use an acidic solution whose pH has been adjusted to fall within the range of about 2 to 3.

[0026] Generally, various acidic solutions can be used as long as the pH is adjusted as described above. For example, hydrochloric acid-potassium chloride buffer solution, glycine-hydrochloric acid buffer solution, citrate buffer solution, acetate buffer solution, phosphate buffer solution, etc. can be used as the acidic solution here.

[0027] Next, a buffer solution is supplied to the filter, and the buffer solution that has passed through the filter is collected (Step 3). The buffer solution used here is adjusted to a pH higher than the isoelectric point of the acid-tolerant microorganism to be tested. Examples of buffer solutions whose pH can be adjusted in this way include phosphate buffer, citrate buffer, carbonate buffer, bicarbonate buffer, and potassium chloride-sodium hydroxide buffer.

[0028] In this process, when the buffer solution is passed through the filter, the acid-tolerant microorganisms captured on the filter become negatively charged by being exposed to the pH environment of the buffer solution, and are repelled by the filter, which is also negatively charged. As a result, the acid-tolerant microorganisms detach from the filter and migrate into the buffer solution. Therefore, by collecting the buffer solution that has passed through the filter, a test sample for acid-tolerant microorganisms is obtained.

[0029] In this step, the greater the total amount of buffer solution passed through the filter, the higher the elution rate of the acid-tolerant microorganisms captured on the filter (i.e., the recovery rate of the acid-tolerant microorganisms captured on the filter). On the other hand, using a buffer solution containing proteins, sugars, alcohols, or surfactants can promote the elution of acid-tolerant microorganisms from the filter, thereby increasing the elution rate of acid-tolerant microorganisms while reducing the total amount of buffer solution passed through the filter.

[0030] In buffer solutions containing the above-mentioned additives, the concentration of each additive is preferably adjusted to fall within the ranges described below. If the additive concentration is lower than this range, it may be difficult to obtain the additive's effect of promoting the elution of acid-tolerant microorganisms. If the additive concentration is higher than this range, the reliability of the analytical results for acid-tolerant microorganisms using test samples may be impaired by the additive's influence. Two or more types of additives may be used in combination.

[0031] Examples of proteins that can be added to the buffer solution include beef extract, bovine serum albumin, and bovine gamma globulin. Two or more proteins may be used in combination. The concentration of the protein added to the buffer solution is usually set to preferably 0.1 to 5.0 W / V%, more preferably 1.0 to 3.0 W / V%.

[0032] The sugars that can be added to the buffer solution are not particularly limited, and specific examples include heptasaccharide ketoses such as sedoheptulose and coriose, hexose aldoses such as allose, talose, gulose, glucose, altrose, mannose, galactose, and idose, hexose ketoses such as psicose, fructose, sorbose, and tagatose, pentose aldoses such as ribose, lyxose, xylose, arabinose, and apiose, pentose ketoses such as ribulose and xylulose, tetraose aldoses such as erythrose and threose, tetraose ketoses such as erythrulose, triose aldoses such as glyceraldehyde, and triose ketoses such as dihydroxyacetone. Two or more sugars may be used in combination.

[0033] The concentration of sugars added to the buffer solution is usually preferably set to 0.1 to 5.0 W / V %, more preferably 0.5 to 3.0 W / V %.

[0034] The alcohols that can be added to the buffer solution are not particularly limited, but specific examples include monohydric lower alcohols such as methanol, ethanol, and isopropanol, dihydric alcohols such as ethylene glycol, and trihydric alcohols such as glycerin. Two or more types of alcohols may be used in combination. The concentration of the alcohol added to the buffer solution is usually preferably set to 5.0 to 15.0 W / V%, and more preferably set to 8.0 to 10.0 W / V%.

[0035] The surfactant that can be added to the buffer solution is not particularly limited, and soaps and various synthetic surfactants can be used. Specific examples of usable synthetic surfactants include anionic surfactants such as carboxylic acid types, sulfonic acid types, sulfate ester types, and phosphate ester types; cationic surfactants such as quaternary ammonium salt types, alkylamine salt types, and pyridine derivatives; amphoteric surfactants such as alkyl betaine types, fatty acid amidopropyl betaine types, alkyl imidazole types, amino acid types, and amine oxide types; and nonionic surfactants such as ester types, ether types, ester ether types, alkanolamide types, alkyl glycosides, and higher alcohols. Two or more types of surfactants may be used in combination.

[0036] The concentration of the surfactant added to the buffer solution is usually preferably set to 0.01 to 5.0 W / V %, more preferably 0.1 to 1.0 W / V %.

[0037] The preparation method of the present invention enables the preparation of test samples for acid-tolerant microorganisms contained in test water through a relatively simple operation using a filter material. This allows for the easy preparation of highly reliable test samples on-site, such as at a cooling tower or in a public bath. Furthermore, unlike the filtration and centrifugation methods prescribed by official methods, this method is easily automated. When preparing test samples on-site at a cooling tower or when automating test sample preparation, it is preferable to select surfactants or alcohols, particularly surfactants, as additives to be added to the buffer solution in step 3 in terms of storage stability at room temperature. When proteins or sugars are used as additives, these or the buffer solution to which they are added must be stored refrigerated to prevent spoilage. However, when surfactants or alcohols are used as additives, this is less necessary and the solution is easier to store.

[0038] The test sample obtained by the preparation method of the present invention can be tested by the method specified in the official method, but can also be tested by other methods. For example, if the acid-resistant microorganism to be tested is Legionella, the test sample can be applied to an immunochromatographic test paper for testing for Legionella. If the immunochromatographic test paper to which the Legionella test sample is applied shows a positive result, it can be determined that the test water contains Legionella bacteria at or above the regulated level. Conversely, if the immunochromatographic test paper shows a negative result, it can be determined that the test water does not contain Legionella bacteria at or above the regulated level.

[0039] Commercially available immunochromatographic test papers for testing for Legionella can be used, such as "Check Legionella" (trade name of SA Scientific, Inc.) and "Q-Line Kyokuto Legionella" (trade name of Kyokuto Pharmaceutical Industries Co., Ltd.), which are reagents for detecting Legionella pneumophila serotype 1 antigen (LPS) in urine.

[0040] The test sample obtained by the preparation method of the present invention can also be applied to test methods other than official methods and test methods using immunochromatographic test paper. For example, the test sample prepared by the present invention can be applied to the LAMP method (genetic testing method), the PCR method (polymerase chain reaction method), or the ELISA method, which is one of the immunoassay methods.

[0041] [Experimental Example] <Preparation of test sample water> Legionella pneumophila grown on BCYEα agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was suspended in phosphate buffer (pH 7.2) to a concentration of approximately 100,000 CFU / mL to prepare a bacterial suspension, which was used as the test water sample. The actual number of Legionella bacteria contained in the test water sample was confirmed by culturing using BCYEα agar medium (36°C for 4 days).

[0042] <Experimental Example 1> Step 1: The test water samples were diluted 100-fold with 50 mM citrate buffer (pH 3.5) to adjust the pH of the test water samples to 3.5. The number of Legionella bacteria in the pH-adjusted test water samples was then confirmed using a culture method (36°C, 4 days) using BCYEα agar medium.

[0043] Step 2: 10 mL of the test water after pH adjustment in step 1 was passed through a glass fiber filter paper (Advantec Corporation model number "GD-120" / diameter 25 mm, particle size 0.9 μm, thickness 0.51 mm) held in a holder at a flow rate of 200 mL / min, and the passed-through water (filtrate) was collected.

[0044] Step 3: 10 mL of 50 mM bicarbonate buffer solution at pH 9.5 was passed through the glass fiber filter paper that had been subjected to step 2 at a flow rate of 200 mL / min, and the passing-through liquid was secured.

[0045] <Experimental Example 2> The procedure was the same as in Experimental Example 1, except for the following points. (1) In step 2, 10 mL of the test sample water, pH adjusted in step 1, was passed through a 9 mm inner diameter column packed with 0.025 g of glass wool (manufactured by Masuda Rika Kogyo Co., Ltd.) at a flow rate of 0.33 mL / min, ensuring that the water passed through. (2) In step 3, 10 mL of 50 mM bicarbonate buffer solution at pH 9.5 was passed through the column that had been subjected to step 2 at a flow rate of 0.33 mL / min, and the flow-through liquid was secured.

[0046] <Experimental Example 3> The procedure was the same as in Experimental Example 1, except for the following points. (1) In step 2, 10 mL of the test water, pH adjusted in step 1, was passed through a 9 mm inner diameter column packed with 0.1 g of quartz wool (Azwan Corporation's "B grade") at a flow rate of 0.5 mL / min, ensuring that the water passed through. (2) In step 3, 10 mL of 50 mM bicarbonate buffer solution at pH 9.5 was passed through the column that had been subjected to step 2 at a flow rate of 0.5 mL / min, and the flow-through liquid was secured.

[0047] <Experimental Example 4> The procedure was the same as in Experimental Example 1, except for the following points. (1) In step 2, 10 mL of the test sample water, pH adjusted in step 1, was passed through a 9 mm inner diameter column packed with 2.5 g of cation exchange resin (UBK10 manufactured by Mitsubishi Chemical Corporation) at a flow rate of 1.7 mL / min, ensuring that the water passed through. (2) In step 3, 10 mL of 50 mM bicarbonate buffer solution at pH 9.5 was passed through the column that had undergone step 2 at a flow rate of 1.7 mL / min, and the flow-through liquid was secured.

[0048] <Experimental Examples 5-10> The procedure was the same as in Experimental Example 1, except for the following points. (1) In step 2, 10 mL of the test water after pH adjustment in step 1 was passed through a glass fiber filter paper (Advantech Co., Ltd. model number "GA-100" / diameter 25 mm, particle size 1 μm, thickness 0.44 mm) held in a holder at a flow rate of 200 mL / min, and the amount of water passing through was ensured. (2) In step 3, 10 mL of 50 mM bicarbonate buffer solution (pH 9.5) containing the additives shown in Table 1 at the concentrations shown in the table was passed through the glass fiber filter paper that had been subjected to step 2 at a flow rate of 200 mL / min, and the passing-through liquid was secured.

[0049] <Experimental Examples 11-14> The procedures were the same as in Experimental Examples 5 to 10, except for the following points. (1) In step 2, after passing the test sample water through the glass fiber filter to ensure that the water had passed through, 5 mL of 50 mM hydrochloric acid-potassium chloride buffer solution at pH 2.2 was passed through the glass fiber filter at a flow rate of 200 mL / min and left for 5 minutes before proceeding to the next step 3. Before making this change, it was confirmed that Legionella bacteria survive in a 50 mM hydrochloric acid-potassium chloride buffer solution at pH 2.2. Furthermore, the Legionella count in the flow-through liquid of the 50 mM hydrochloric acid-potassium chloride buffer solution at pH 2.2 was confirmed to be essentially zero using a culture method (36°C for 4 days) using BCYEα agar medium, confirming that no Legionella bacteria had leaked from the glass fiber filter into the flow-through liquid. (2) In step 3, 10 mL of 50 mM bicarbonate buffer solution (pH 9.5) containing the additives shown in Table 1 at the concentrations shown in the table was passed through the glass fiber filter at a flow rate of 200 mL / min, and the passing-through liquid was secured.

[0050] <Evaluation> (1) Legionella capture rate in process 2 The number of Legionella bacteria contained in the passing water collected in step 2 of each experimental example was confirmed by culture using BCYE α agar medium (36°C for 4 days). The Legionella capture rate by the glass fiber filter paper or column was then calculated using the following formula. The results are shown in Table 1.

[0051]

number

[0052] (2) Legionella elution rate in process 3 The number of Legionella bacteria contained in the flow-through liquid collected in step 3 of each experiment was confirmed by culturing it on BCYE α agar medium (36°C for 4 days). The elution rate of Legionella bacteria from the glass fiber filter paper or column was then calculated using the following formula. The results are shown in Table 1.

[0053]

number

[0054] (3) Results [Table 1]

[0055] In Experimental Examples 2 and 3, the elution rate in step 3 was less than 50%, but this elution rate can be improved by increasing the total amount of buffer solution used in this step. In Experimental Examples 3 and 4, the collection rate in step 2 was lower than in other Experimental Examples, but this can be improved by changing the pH value of the test sample water in step 1 or adjusting the flow rate of the test sample water in step 2. Comparing Experimental Example 5 with Experimental Examples 6 to 14, it can be seen that adding an additive to the buffer solution used in step 3 significantly increases the elution rate in this step, even if the total amount of the buffer solution is the same.

Claims

1. 1. A method for preparing a test sample for acid-tolerant microorganisms contained in test water, comprising: Step 1: adjusting the pH of the test water to an acidic range in which the acid-tolerant microorganisms can stably live in a state of having a positive charge; a step 2 of passing the test water, the pH of which has been adjusted in the step 1, through a filter material that has a negative charge in a high pH range including the pH value of the test water adjusted in the step 1 and that allows the acid-tolerant microorganisms to pass through; Step 3: Passing a buffer solution having a pH higher than the isoelectric point of the acid-tolerant microorganism through the filter material that has been subjected to step 2, and collecting the buffer solution that has passed through the filter material; A method for preparing a sample for testing acid-tolerant microorganisms, comprising:

2. 2. The method for preparing a sample for testing acid-tolerant microorganisms according to claim 1, wherein the filter medium is heated under heat-resistant conditions for the acid-tolerant microorganisms between steps 2 and 3.

3. 3. The method for preparing a sample for testing acid-tolerant microorganisms according to claim 1 or 2, wherein, between step 2 and step 3, an acidic liquid is applied to the filter material, the acidic liquid being in an acidic range in which the acid-tolerant microorganisms can stably live in a state of having a positive charge and being adjusted to a pH lower than the pH of the test water adjusted in step 1.

4. 4. The method for preparing a sample for testing acid-tolerant microorganisms according to claim 1, wherein the filter medium is made of glass fiber, glass wool, or a mixture of these fibers, or a cation exchange resin.

5. 5. The method for preparing a sample for testing acid-tolerant microorganisms according to claim 1, wherein in step 3, the buffer solution contains a protein, a sugar, an alcohol, or a surfactant.

6. 6. The method for preparing a sample for testing acid-tolerant microorganisms according to claim 1, wherein the acid-tolerant microorganisms are bacteria, fungi, or yeasts.

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