JP4414515B2 - Identification method of microorganisms - Google Patents

Description

【表１】に示す如く各菌種の持つ生化学的性状は一致した。なお、表中の＋は陽性、−は陰性を示す。
【００３１】
実施例２：スクリーニング試薬としての有用性確認
（Ａ）材料および方法
供試株は臨床および食品からの分離株ですでにＡＴＢ（ｂｉｏＭｅｒｉｒｕｓ）で同定済みの株を用いた。
１．チフス菌を含むサルモネラ・スピーシーズ ３５血清型（Ｓａｌｍｏｎｅｌｌａ ｓｐｐ．３５血清型）８０株およびサルモネラ・アリゾナ（Ｓａｌｍｏｎｅｌｌａ Ａｒｉｚｏｎａ）１株
２．エスケリチア・コリー Ｏ１５７：Ｈ７（ＶＴ＋）１４株
３．エスケリチア・コリー Ｏ１５７：Ｈ７（ＶＴ−）４株
４．エスケリチア・コリー Ｏ１５７：Ｈ−（ＶＴ＋）１株
５．サイトロバクター・フレウンディー（Ｃｉｔｒｏｂａｃｔｅｒ ｆｒｅｕｎｄｉｉ）４株
６．プロテウス・ミララビリス（Ｐｒｏｔｅｕｓ ｍｉｒａｂｉｌｉｓ）７株
７．クレブシエラ・ニューモニア（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ）２株
８．エンテロバクター・クロアカ（Ｅｎｔｅｒｏｂａｃｔｅｒ ｃｌｏａｃａｅ）５株
９．セラチア・マルセッスセンス（Ｓｅｒｒａｔｉａ ｍａｒｃｅｓｃｅｎｓ）５株
【００３２】
（Ｂ）実施した発光酵素基質試験と試験菌株
１．β−ガラクトシダーゼテスト：大腸菌群
２．β−グルクロニダーゼテスト：エスケリチア・コリー
３．β−キシロシダーゼテスト：クレブシエラ−エンテロバクター
【００３３】
（Ｃ）実施した発色酵素基質試験と試験菌株
４．Ｌ−ピロリドニルペプチダーゼテスト：サルモネラ−サイトロバクター
５．インドールテスト、トリプトファンデアミナーゼテスト：プロテウス−プロビデンシア−モルガネーラ
【００３４】
（Ｄ）試験菌の培養と試験法
試験菌はＴＳＢ（ＯＸＯＩＤ社）で３７℃１８時間静置培養した供試株をＢＨＩＡ及びＣＨＲＯＭａｇａｒＯ１５７ＴＡＭ（関東化学：以下ＣＨＲＯＭ）培地に画線塗抹し、培地上に発育した集落を基質綿棒で直接釣菌後、０．０６７Ｍリン酸緩衝液３０μｌ滴下した小試験管内で３７℃、１０分間反応させた後、実施例１と同様な方法で処理判定した。
【００３５】
（Ｅ）結果
ＢＨＩＡ及びＣＨＲＯＭに発育したエスケリチア・コリー Ｏ１５７、サルモネラとも予測された酵素活性を示した。
供試したエスケリチア・コリー Ｏ１５７はβ−ガラクトシダーゼテスト、トリプトファナーゼテスト全てに陽性を示し、β−グルクロニダーゼテストは陰性であった。
サルモネラ・スピーシーズはＬ−ピロリドニルペプチダーゼテスト全てに陰性を示し、陽性株のサイトロバクターと鑑別できた。
サルモネラ・スピーシーズはトリプトファンデアミナーゼテストが全て陰性を示し、陽性のプロテウスとの鑑別が可能である。
β−グルクロニダーゼテストでＢＨＩＡ及びＣＨＲＯＭ発育菌がサルモネラ・アリゾナ、サルモネラ・スピーシーズのサルモネラ・ブランデンブルグ（Ｓ．Ｂｒａｎｄｅｎｂｕｒｇ）、サルモネラ・グルンペンシス（Ｓ．Ｇｒｕｍｐｅｎｓｉｓ）、サルモネラ・モンテビデオ（Ｓ．Ｍｏｎｔｅｖｉｄｅｏ）、サルモネラ・ムエンスター（Ｓ．Ｍｕｅｎｓｔｅｒ）、サルモネラ・オスマルシェン（Ｓ．Ｏｔｈｍａｒｓｃｈｅｎ）、サルモネラ・シュバルツェングランド（Ｓ．Ｓｃｈｗａｒｚｅｎｇｒｕｎｄ）は陽性を示した。（Ｓ．は、サルモネラを、次にくるＡｒｉｚｏｎａ等の語は血清型を表している。）β−グルクロニダーゼは志賀毒素産生性大腸菌Ｏ１５７を除く大腸菌が有する酵素であるが、一部のシゲラおよびサルモネラの特定の血清型が本酵素を有し、本試験成績は既に報告のあるβ−グルクロニダーゼ陽性のサルモネラ血清型と一致した。
【００３６】
以上の結果から、本発明の発色または発光酵素基質担体（綿棒）は、サルモネラおよびエスケリチア・コリー Ｏ１５７同定過程でのスクリーニングとして有用である事が確認できた。
【００３７】
【発明の効果】
以上説明したように、本発明によって得られた成績は、従来の細菌同定に用いられていた生化学的性状と完全に一致するものである。従って、臨床材料或いは食品検査において、分離培地上に発育した集落の鑑別をする場合、従来行なわれている方法では多数の培地と培養ステップが必要で結果判定まで数日必要であったが、本発明は少なくとも、分離培地上に菌の発育が認められたら十数分以内には菌種が鑑別することを可能とするものである。また、本発明の担体は長期間安定的に保存でき、コスト面でも効果がある。この迅速性、簡易性および経済性は、細菌検査を治療、予防、安全食品の供給等のためにより一般的な試験方法として適用することを可能にする。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a discrimination method based on biochemical properties of microorganisms using color development or luminescence.
[0002]
[Prior art]
Bacteria discrimination in clinical examinations or food examinations is usually performed by purely cultivating isolated colonies on a separation medium and then comparing the biochemical properties with a discrimination table. For example, if a colony suspected of Salmonella arises on the isolation medium, a biochemical identification must be made to ensure that it is Salmonella. The decision is based on biochemical properties. Therefore, in order to distinguish bacteria, dozens of kinds of biochemical property test mediums corresponding to the target microorganism are required, and after inoculating the test material into the medium, the test items are also tested for 18 to 72 hours. In some cases, it is necessary to carry out the culture for one week, and it takes a long time to obtain the result of the test. In addition, depending on the inspection item, the result of the bacteria property test may be unclear, and skill is required for the determination.
[0003]
[Problems to be solved by the invention]
As mentioned above, conventional techniques for diagnosis of infections and food bacteria testing require many biochemical characterization tests after isolation of bacteria, which requires many types of culture media and days, and the operation is complicated. In some cases, there is a risk of misidentification of the discrimination results. At present, this fact is a major obstacle to the diagnosis of infectious diseases and the testing of food bacteria. It is well known that bacterial identification is performed by biochemical properties, but each of these properties is a manifestation of each enzyme, and one property test is based on the action of several enzymes. This takes a long time for discrimination. Therefore, it is difficult to say that the test results are always reflected in the early determination of infectious disease treatment and preventive measures, or safe food supply. The object of the present invention is to solve various problems associated with such microorganism testing.
[0004]
[Means for Solving the Problems]
The present invention relates to the identification of microorganisms based on biochemical properties, a carrier that has a water absorption property that contains a substance that develops or emits light by the enzyme activity of the microorganism, or an enzyme substrate that binds the coloring substance or the luminescent substance, and a specimen. The present invention relates to a method for distinguishing microorganisms, characterized by detecting color development or luminescence of a carrier after direct contact with a treated product or an isolated colony grown on a medium. Hereinafter, the present invention will be described in detail.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a simple and rapid method for distinguishing bacteria in clinical tests or food tests. Specifically, based on the determination of the isolated bacteria by examining the presence or absence of the enzyme activity of a specific bacterium that forms a solitary colony on a specimen treatment product (for example, centrifugal sedimentation of urine, etc.) or on a separation medium. To do. As a reagent for confirming this specific enzyme, the chromogenic substance or luminescent substance is used as it is, or the chromogenic substance or luminescent substance is bound to the enzyme substrate. This reagent is contained in a carrier having water absorbency, and the isolated colony on the separation medium is brought into direct contact with the carrier so that it can be changed or released by bacterial enzyme activity under appropriate conditions. The signal of is detected. At this time, a coloring reagent is used as necessary.
[0006]
A number of known substances can be used as the coloring substance. For example, derivatives such as 4-nitrophenol, 2-nitrophenol, 5-bromo-4-chloro-3-indolyl, 6-chloro-3-indolyl, 4-nitroaniline, β-naphthylamine, and tryptophan, gelatin, hippuric acid Examples include substances used for conventional biochemical property tests such as salts, dopamine, tetramethylparaphenylenediamine, sodium citrate, nitrate, lysine, ornithine, arginine, α-naphthol, and various sugars.
[0007]
As the luminescent material, many known materials can be used. Examples include 4-methylumbelliferone, 7-amido-4-methylcoumarin, 4-methoxy-2-naphthylamine, 7-amino-4-trifluoromethylcoumarin, and derivatives thereof.
[0008]
Examples of the substance serving as an enzyme substrate to which the coloring substance or the luminescent substrate is bound include, for example, L-pyroglutamic acid, β-D-galactopyranoside, α-D-galactopyranoside, β-D-glucopyranoside, α- D-glucopyranoside, β-D-xylopyranoside, α-L-arabinopyranoside, β-D-cellopyranoside, β-D-glucuronide, α-D-glucuronide, β-D-fucopyranoside, N-acetyl-β- D-galactosaminide, N-acetyl-β-D-glucosaminide, α-D-mannopyranoside, β-D-mannopyranoside, butyrate, β-N-acetylglucosaminide, β-N-acetylgalactosaminide, caprylic acid, L -Aspartic acid, L-glutamic acid, glycyl-proline, L-histidine, L-proline, seryl-trypsi L-valine, L-arginine, L-lysine, L-leucine, L-methionine, L-phenylalanine, L-arginyl-L-arginine, N-succinyl-L-alanyl-L-prolyl-L-alanine, L -Glutarylglycine-L-arginine, L-glutamyl-L-lysyl-L-lysine, L-lysine-L-alanine, L-proglutamic acid, L-valine, L-alanine, sulfuric acid, urea, phosphoric acid, L -Pyrrolidone carboxylylic acid, deoxyriboscranic acid, etc. can be mentioned. In order to bind the coloring substance or the luminescent substance to the enzyme substrate, it may be bound by a covalent bond such as a peptide bond, an ester bond or a glycoside bond by a known means. Or a commercial item can be used.
[0009]
The carrier having water absorbability containing the coloring substance or luminescent substance obtained as described above or the enzyme substrate to which the coloring substance or luminescent substance is bound is not particularly limited as long as it has the function. Examples thereof include paper, filter paper, cellulose, non-woven fabric, and cotton. This carrier may be used as it is, or it may be used in a suitable member such as glass, plastic, wood, metal, etc. for easy handling. Its shape, length, and thickness are not particularly limited.
[0010]
In order to include (impregnate) a coloring substance or a luminescent substance, or an enzyme substrate bound with a coloring substance or a luminescent substance, in a carrier having water absorption, these substances are dissolved in a suitable solvent, and the liquid is used as a carrier. It should be included. At this time, a solvent that does not affect the performance of the coloring substance, the luminescent substance, or the enzyme substrate is selected. For example, N, N, -dimethylformamide, dimethyl sulfoxide, ethanol, methanol, acetone, toluene, butanol, hydrochloric acid, phosphate buffer, purified water and the like can be used. Specifically, a 10 to 100 μl solution of 0.01 to 20% of a coloring substance or luminescent substance or an enzyme substrate to which the chromogenic substance or luminescent substance is bound is impregnated, natural drying, air drying, vacuum drying, freeze drying It is used after being dried.
[0011]
Samples in the present invention include blood, urine, feces, cerebrospinal fluid, skin or throat wipes, sputum, all clinical materials, milk and dairy products, meat and meat products, seafood and seafood products, frozen food, Japanese food Use bacteria isolated from foods such as food distribution, drinking water, feed and feed products, confectionery, and spices.
[0012]
The general test procedure for pathogenic bacteria in clinical materials or foods is to isolate bacteria by culturing from a sample, and then distinguish the results determined after culturing for 1 to 7 days in many biochemical property test media. It goes through the process of checking the bacterial species against the table. In contrast, according to the present invention, a sample processed material such as centrifugal sedimentation of urine is directly contacted with an isolated colony on a separation medium, and the color or luminescence of the carrier itself is visually observed after instant to 60 minutes, for example, 10 minutes. The presence or absence and discrimination of bacteria are performed by determining in step (b). Alternatively, color development or luminescence may be detected mechanically.
[0013]
For example, when the isolated bacteria are identified as Salmonella, when MLCB agar is used as the separation medium, the grown black colony (due to hydrogen sulfide production) is mixed with 95% ethanol in L-pyroglutamic acid-2-naphthylamide. For example, an industrial cotton swab (Nippon Cotton Swab Co., Ltd.) was impregnated with 30 to 50 μl, and contacted with a carrier (part containing a reagent) dried at 37 ° C. for 2 hours. In a state where the carrier portion is wet with the buffer solution in a small test tube in which 30 μl of a cotton swab is dispensed in 0.03-0.1 M phosphate buffer (pH 4.0-10.0) (the buffer solution is small The mixture is allowed to react at 37 ° C. for 10 minutes, and a coloring reagent (1.0% 4-dimethylaminocinnamaldehyde solution) is added to determine the color change of the cotton swab with the naked eye (PYR test). A black colony is shown on the separation medium. If the PYR test is negative (no color change or changes color to blue), it can be identified as Salmonella.
[0014]
In the present invention, the chromogenic or luminescent substance, or the kind of enzyme substrate that binds it or the combination thereof, can be used to select various bacteria. For example, Salmonella, Cytrobacter, Klebsiella, Escherichia coli, Serratia, Proteus, orenidenia M, Providencia Enterobacter, Streptococcus, Enterococcus, Neisseria, Bacteroides, Candida, Staphylococcus, Staphylococcus, Staphylococcus Differentiation of Listeria etc. can be performed.
[0015]
Carrier prepared as described above, the material, regardless of the shape, six months to as least as long as cold storage (2 to 10 ° C.) reagent performance (color, luminescent) deterioration of not observed It can be used stably. Surprisingly, when a cotton swab was used as a carrier having water absorbency, as a result of conducting a storage test at 4 ° C., 25 ° C., and 37 ° C., the performance did not change over 24 months at any temperature, It was extremely excellent in storage stability. Therefore, there is a possibility that it can be used even if a period of 24 months or more elapses. This demonstrates the exceptional effect that the use range and test conditions of the present invention are greatly expanded.
[0016]
[Reference example]
Examples of reagent preparation and carrier production of the present invention are shown below.
(A) 4-methylumbelliferyl-β-D-galactoside (source: Sigma) for β-galactosidase test (MGAL test) is dissolved in NN-dimethylformamide, and the enzyme substrate is used as a final concentration. A solution prepared by diluting with 0.03-0.1M phosphate buffer (pH 4.0-10.0) so that the solvent is 0.05-0.5% and the solvent is 10-50% is an industrial swab (stock) Company Japan swab) was soaked in 30-50 μl, dried at 37 ° C. for 18 hours, and then stored in a light-shielding container with a desiccant.
[0017]
(B) 4-methylumbelliferyl-β-D-glucuronide (source: Nacalai Tesque) for β-glucuronidase test (MUG test) is dissolved in NN-dimethylformamide, and the enzyme substrate is used as the final concentration. A solution prepared by diluting with a 0.03-0.1 M phosphate buffer (pH 4.0-10.0) so that the solvent is 0.05-0.5% and the solvent is 10-50% is applied to an industrial cotton swab. It was impregnated with ˜50 μl, dried at 37 ° C. for 18 hours, and stored in a light-shielding container containing a desiccant.
[0018]
(C) 4-methylumbelliferyl-β-D-xylopyranoside (source: Fluka) for β-xylosidase test (MXYL test) was dissolved in NN-dimethylformamide, and the enzyme substrate was adjusted to a final concentration of 0. 30 to 50 μl of an industrial cotton swab containing a solution prepared by diluting with 0.03 to 0.1 M phosphate buffer (pH 4.0 to 10.0) so that the solvent becomes 10 to 50% It was soaked and dried at 37 ° C. for 18 hours, and then stored in a light-shielding container containing a desiccant.
[0019]
(D) 0.05-0.5% L-pyrrologlutamic acid-2-naphthylamide (source: Fluka) is added to an industrial cotton swab in a 95% ethanol solution for L-pyrrolidonyl peptidase test (PYR test). It was impregnated with 30 to 50 μl, dried at 37 ° C. for 2 hours, and stored in a shading bottle containing a desiccant.
(Coloring reagent)
A solution obtained by dissolving 4-dimethylaminocinnamaldehyde (source: Sigma) in 1% hydrochloric acid in 1% was put in a light-shielding bottle and stored.
[0020]
(E) 0.05-0.3% L-tryptophan (source: sum) in heated 0.03-0.1 M phosphate buffer (pH 4.0-10.0) for indole test (IND test) The solution was soaked in an industrial cotton swab in an amount of 30 to 50 μl, dried at 37 ° C. for 18 hours, and then stored in a shading bottle containing a desiccant.
(Color disc)
5 g of paradimethylbenzaldehyde was dissolved in a mixed solution of 10 ml of phosphoric acid and 50 ml of methanol, 30 μl of the solution was soaked in a filter paper (Advantech) disk having a diameter of 7 mm, air-dried at room temperature, and stored in a shading bottle containing a desiccant. .
[0021]
(F) 0.05-0.3% L-tryptophan was dissolved in 0.03-0.1 M phosphate buffer (pH 4.0-10.0) heated for tryptophan deaminase test (TDA test), The solution was impregnated with 30 to 50 μl of an industrial cotton swab, dried at 37 ° C. for 18 hours, and stored in a shading bottle containing a desiccant.
(Coloring reagent)
A solution prepared by dissolving ferric chloride in 6N hydrochloric acid so as to be 10% was put in a light-shielding bottle and stored.
[0022]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
Example 1: Identification of laboratory-preserved strains (A) Culture of test bacteria and test bacteria The following laboratory-preserved strains that had already been identified were used.
1. Escherichia coli O157: H7 (Escherichia coli O157: H7)
2. Escherichia coli O157: H7 (Escherichia coli O157: H7)
3. Escherichia coli O26: H11 (Escherichia coli O26: H11)
4). Escherichia coli O1: H7 (Escherichia coli O1: H7)
5). Salmonella, serotype Enteritidis (Salmonella, serotype Enteritidis)
6). Salmonella, serotype Typhimurium (Salmonella, serotype Typhimurium)
7). Cytobacter freundii
8). Proteus vulgaris
9. Proteus mirabilis
10. Klebsiella pneumoniae
11. Enterobacter cloacae
12 Serratia marcescens
The test bacteria were cultured on a brain heart infusion agar medium (BBL) at 37 ° C. for 6 hours, and then subjected to the following tests.
[0023]
(B) An MGAL test test bacterium was brought into contact with an MGAL test substrate swab and put in a small test tube into which 0.067 M phosphate buffer (pH 8.0) was dispensed, and reacted at 37 ° C. for 10 minutes. The presence or absence of light emission from a cotton swab (positive: blue fluorescence, negative: no fluorescence) was determined with the naked eye.
[0024]
(C) A MUG test test bacterium was brought into contact with a MUG test substrate swab and placed in a small test tube into which 30 μl of 0.067M phosphate buffer pH 8.0 was dispensed. After reacting at 37 ° C. for 10 minutes, UV at 366 nm The presence or absence of light emission from a cotton swab (positive: blue fluorescence, negative: no fluorescence) was determined with the naked eye.
[0025]
(D) The MXYL test test bacterium was brought into contact with the MXYL test substrate swab and placed in a small test tube into which 30 μl of 0.067M phosphate buffer pH 8.0 was dispensed. After reacting at 37 ° C. for 10 minutes, 366 nm UV The presence or absence of light emission from a cotton swab (positive: blue fluorescence, negative: no fluorescence) was determined with the naked eye.
[0026]
(E) A PYR test test bacterium was brought into contact with a PYR test substrate swab and placed in a small test tube into which 0.067 M phosphate buffer pH 8.0 was dispensed, and reacted at 37 ° C. for 10 minutes. The color change of the cotton swab (positive: red, negative: unchanged) was determined with the naked eye.
[0027]
(F) An IND test test bacterium is brought into contact with an IND test substrate swab and put in a small test tube into which 30 μl of 0.067 M phosphate buffer pH 8.0 is added. After reacting for a minute, the change in color of the disc (positive: pink to red, negative: no change) was determined with the naked eye.
[0028]
(G) A TDA test test bacterium is brought into contact with a TDA test substrate swab and placed in a small test tube into which 0.067 M phosphate buffer pH 8.0 is dispensed, and reacted at 37 ° C. for 10 minutes. A cotton swab color change (positive: reddish brown, negative: no change) was determined with the naked eye.
[0029]
(H) Results Table 1 shows the biochemical properties of each test strain using substrate swabs.
[0030]
[Table 1]

As shown in [Table 1], the biochemical properties of each bacterial species were consistent. In addition, + in a table | surface shows positive and-shows negative.
[0031]
Example 2: Confirmation of usefulness as a screening reagent (A) Materials and methods The test strains used were isolates from clinical and foods that had already been identified by ATB (bioMerirus).
1. 1. Salmonella sp. 35 serotype 35 serotype containing Salmonella typhi and Salmonella Arizona 1 strain 2. 2. Escherichia coli O157: H7 (VT +) 14 strain 3. Escherichia coli O157: H7 (VT-) 4 strain 4. Escherichia coli O157: H- (VT +) 1 strain 4. Citrobacter freundii 4 strains 6. Proteus mirabilis 7 strains 7. Klebsiella pneumoniae 2 strains Enterobacter cloacae 5 strains9. Serratia marcescens 5 strains [0032]
(B) Conducted luminescent enzyme substrate test and test strain 1. β-galactosidase test: coliform group 2. β-glucuronidase test: Escherichia coli β-xylosidase test: Klebsiella enterobacter
(C) The chromogenic enzyme substrate test performed and the test strain 4. 4. L-pyrrolidonyl peptidase test: Salmonella-cytolobacter Indole test, tryptophan deaminase test: Proteus-Providencia-Morganera
(D) Culture of test bacteria and test method The test bacteria were smeared on BHIA and CHROMagarO157TAM (KANTO CHEMICAL: CHROM) medium after stir culture at 37 ° C for 18 hours in TSB (OXOID) The colony grown on the plant was directly picked with a substrate swab, reacted at 37 ° C. for 10 minutes in a small test tube dropped with 30 μl of 0.067M phosphate buffer, and then treated in the same manner as in Example 1.
[0035]
(E) Results The predicted enzyme activities of Escherichia coli O157 and Salmonella developed in BHIA and CHROM were shown.
The tested Escherichia coli O157 was positive for all β-galactosidase tests and tryptophanase tests, and the β-glucuronidase test was negative.
Salmonella sp. Was negative for all L-pyrrolidonyl peptidase tests and could be distinguished from positive strains of Cytobacter.
Salmonella species are all negative for tryptophan deaminase tests and can be differentiated from positive Proteus.
In the β-glucuronidase test, the BHIA and CHROM-growing bacteria are Salmonella Arizona, Salmonella sp. (S. Muenster), Salmonella Osmarschen, Salmonella Schwarzengrand (S. Schwarzengrand) were positive. (S. stands for Salmonella, and the following words such as Arizona et al. Represent serotypes.) Β-glucuronidase is an enzyme possessed by Escherichia coli except Shiga toxin-producing Escherichia coli O157, but some Shigella and Salmonella Specific serotypes have this enzyme, and the test results are consistent with the previously reported β-glucuronidase-positive Salmonella serotype.
[0036]
From the above results, it was confirmed that the chromogenic or luminescent enzyme substrate carrier (cotton swab) of the present invention was useful as a screening in the identification process of Salmonella and Escherichia coli O157.
[0037]
【The invention's effect】
As described above, the results obtained by the present invention are completely consistent with the biochemical properties used for conventional bacterial identification. Therefore, in the clinical material or food inspection, when distinguishing a colony that has grown on a separate medium, the conventional method requires a large number of mediums and culture steps, and it takes several days to determine the result. The invention makes it possible to distinguish bacterial species within at least ten minutes when growth of bacteria is observed on the separation medium. Further, the carrier of the present invention can be stored stably for a long period of time, and is effective in terms of cost. This rapidity, simplicity and economy make it possible to apply bacterial testing as a more general test method for treatment, prevention, safe food supply and the like.

Claims (5)

In the identification of microorganisms by biochemical properties, they developed on a cotton swab containing a substance that develops or emits light by the enzyme activity of the microorganism, or an enzyme substrate to which the coloring substance or luminescent substance is bound, and a specimen treatment product or medium. A method for distinguishing microorganisms that detect color development or luminescence of a cotton swab after directly contacting an isolated settlement, wherein the swab dissolves the substance or enzyme substrate in a solvent, and soaks the solution into the swab, The method for distinguishing microorganisms described above, wherein the microorganism or the enzyme substrate is contained by drying.   The method for distinguishing microorganisms according to claim 1, wherein the color development or luminescence reaction is carried out in a state where a cotton swab is wetted with a buffer solution.   The solvent is NN-dimethylformamide, and the solution is a solution in which the substance or enzyme substrate is dissolved in NN-dimethylformamide and then diluted with a phosphate buffer. Item 3. A method for identifying a microorganism according to Item 1 or 2. The cotton swab for a microorganism identification method according to any one of claims 1 to 3, further comprising a substance that develops or emits light by an enzyme activity possessed by a microorganism, or an enzyme substrate to which a coloring substance or a luminescent substance is bound. The swab is obtained by dissolving the substance or enzyme substrate in a solvent, soaking the solution in the swab, and drying to contain the substance or enzyme substrate, Cotton swab.   The solvent is NN-dimethylformamide, and the solution is a solution in which the substance or enzyme substrate is dissolved in NN-dimethylformamide and then diluted with a phosphate buffer. Item 5. A cotton swab according to Item 4.