JPH0669393B2 - Stabilized color enhancement reagent - Google Patents
Stabilized color enhancement reagentInfo
- Publication number
- JPH0669393B2 JPH0669393B2 JP20800388A JP20800388A JPH0669393B2 JP H0669393 B2 JPH0669393 B2 JP H0669393B2 JP 20800388 A JP20800388 A JP 20800388A JP 20800388 A JP20800388 A JP 20800388A JP H0669393 B2 JPH0669393 B2 JP H0669393B2
- Authority
- JP
- Japan
- Prior art keywords
- color
- reagent
- naphthol
- enhancing
- mineral acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 31
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 12
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 12
- 239000011707 mineral Substances 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- RSEOPLGIFDMYKN-UHFFFAOYSA-N ethanol;naphthalen-1-ol Chemical compound CCO.C1=CC=C2C(O)=CC=CC2=C1 RSEOPLGIFDMYKN-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明はフォーゲス・プロスカウエル試験に用いる安定
化された発色増強試薬に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a stabilized color-enhancing reagent for use in the Forges-Proskouer test.
フォーゲス・プロスカウエル試験は細菌がグルコースを
発酵して中性の終末産物であるアセチルメチルカルビノ
ール（アセトイン）を産生する能力を検定する試験であ
り、微生物の生化学的性状検査のひとつとして病原菌の
同定の際に実施される。The Forges-Proschauer test is a test that tests the ability of bacteria to ferment glucose to produce acetyl methyl carbinol (acetoin), which is a neutral end product, and is one of the biochemical characterization tests of microorganisms. It is carried out at the time of identification.
［先行技術およびその問題点］ フォーゲス・プロスカウエル試験は、培地で培養した試
験菌にフォーゲス・プロスカウエル試薬をかけることに
よって行なわれ、かけた試薬溶液が桃赤色に呈色する場
合は陽性（アセトインが存在）、黄色あるいは無色（試
薬の色と同じ）の場合は陰性とされる。フォーゲス・プ
ロスカウエル試薬は一般的にはα‐ナフトールをエタノ
ールに５〜６％（ｗ／ｖ）濃度に溶かした発色増強試薬
と、40％（ｗ／ｖ）の水酸化カリウム（またはナトリウ
ム）水溶液の酸化試薬からなる。ところが、上記発色増
強試薬はα−ナフトールの安定性が悪いため室温で保存
すると経時的に褐色に着色し、呈色の判定が困難となり
誤判定の原因となった。そのため発色増強試薬は冷蔵す
る必要があり、長期間の保存は避けなければならなかっ
た。[Prior Art and Problems Therefor] The Forges-Proschauer test is carried out by applying a Forges-Pros Powell reagent to a test bacterium that has been cultured in a medium, and a positive (acetoin Is present), yellow or colorless (same as the color of the reagent), it is considered negative. The Forges-Proscher's reagent is generally a color-enhancing reagent prepared by dissolving α-naphthol in ethanol at a concentration of 5 to 6% (w / v) and a 40% (w / v) aqueous solution of potassium (or sodium) hydroxide. It consists of an oxidizing reagent. However, since the above-mentioned color-enhancing reagent has poor stability of α-naphthol, when it is stored at room temperature, it is colored brown over time, and it becomes difficult to judge the coloration, which causes an erroneous judgment. Therefore, it was necessary to refrigerate the color-enhancing reagent and avoid long-term storage.
［問題点を解決するための手段］ 本発明はフォーゲス・プロスカウエル試験に用いられる
安定化された発色増強試薬を提供することを目的とす
る。[Means for Solving Problems] It is an object of the present invention to provide a stabilized color-enhancing reagent for use in the Forges-Proskouer test.
上記目的を達成するため、本発明の発色増強試薬は、α
‐ナフトールを含むアルコール溶液に鉱酸を添加してな
る。鉱酸としてはリン酸、塩酸、硫酸等が用いられるが
特にリン酸または塩酸が好ましい。鉱酸の添加量はα‐
ナフトール１モルに対して約0.14〜６モル当量が適当で
ある。In order to achieve the above object, the color-enhancing reagent of the present invention comprises α
-Adding a mineral acid to an alcohol solution containing naphthol. As the mineral acid, phosphoric acid, hydrochloric acid, sulfuric acid or the like is used, and phosphoric acid or hydrochloric acid is particularly preferable. The amount of mineral acid added is α-
About 0.14 to 6 molar equivalents are suitable for 1 mol of naphthol.
アルコール溶液中のα‐ナフトールは２〜10％（ｗ／
ｖ）とするのが適当であり、好ましくは４〜７％（ｗ／
ｖ）である。2-10% (w / α-naphthol in alcohol solution)
v) is suitable, and preferably 4 to 7% (w /
v).
本発明の発色増強試薬は、常法に従ってエタノール等の
アルコールに計算量のα‐ナフトールおよび鉱酸を加え
ることによって容易に調製される。The color-enhancing reagent of the present invention is easily prepared by adding a calculated amount of α-naphthol and a mineral acid to an alcohol such as ethanol according to a conventional method.
次に実施例および試験例を示して本発明をさらに具体的
に説明する。Next, the present invention will be described more specifically by showing Examples and Test Examples.
実施例 １ 下記表１の組成物をエタノールに加えて溶解し、発色増
強試薬（Ａ）〜（Ｄ）を得た。また、比較のために鉱酸
を添加しない発色増強試薬（Ｅ）を得た。Example 1 The compositions shown in Table 1 below were added to ethanol and dissolved to obtain color-enhancing reagents (A) to (D). For comparison, a color-enhancing reagent (E) without addition of mineral acid was obtained.
上記発色増強試薬（Ａ）〜（Ｅ）を30℃で３ケ月間保存
し、着色の有無を調べた結果、Ｅは褐色に着色したが
（Ａ）〜（Ｄ）は着色しなかった。 The color-enhancing reagents (A) to (E) were stored at 30 ° C. for 3 months and examined for coloration. As a result, E was colored brown, but (A) to (D) were not colored.
試験例 １ 下記組成の培地を調整し、オートクレーブ滅菌した。多
穴プレートの各ウェルに50μｌずつ分注し、25℃で乾燥
した。Test Example 1 A medium having the following composition was prepared and autoclaved. 50 μl was dispensed into each well of the multiwell plate and dried at 25 ° C.
（培 地） ペプトン 7.0g グルコース 5.0g リン酸二カリウム 5.0g 蒸留水 1,000ml 次に、寒天培地上で各種細菌を37℃で18〜20時間培養
し、滅菌蒸留水に１×108個／mlとなるように懸濁し、
各ウェルに50μｌずつ接種し、37℃18〜20時間培養し
た。培養後、30℃３ケ月間保存した（Ａ）〜（Ｅ）の各
発色増強試薬を各ウェルに50μｌずつ分注しさらに酸化
試薬として40％（ｗ／ｖ）水酸化カリウムを各ウェルに
50μｌずつ添加し呈色の有無を判定した。対照として、
実施直前に調整した発色試薬〔５％（ｗ／ｖ）α‐ナフ
トール‐エタノール溶液〕を用いた。(Cultivation site) Peptone 7.0 g Glucose 5.0 g Dipotassium phosphate 5.0 g Distilled water 1,000 ml Next, various bacteria were cultivated on agar medium at 37 ° C for 18 to 20 hours, and then sterilized in distilled water 1 × 10 8 / Suspend to make ml
50 μl of each well was inoculated and cultured at 37 ° C. for 18 to 20 hours. After culturing, 50 μl of each of the color-enhancing reagents (A) to (E) stored at 30 ° C. for 3 months was dispensed into each well, and 40% (w / v) potassium hydroxide was added to each well as an oxidizing reagent.
The presence or absence of coloration was determined by adding 50 μl each. As a control
A coloring reagent [5% (w / v) α-naphthol-ethanol solution] prepared just before the execution was used.
結果を表２に示す。The results are shown in Table 2.
上記の結果から明らかなように本発明の発色増強試薬
（Ａ）〜（Ｄ）は３ケ月30℃で保存したにもかかわらず
着色せず、新鮮な発色増強試薬と同じ判定結果を与えた
のに対して、鉱酸を添加しなかった発色増強試薬（Ｅ）
は着色し、誤まった判定結果を与えた。 As is apparent from the above results, the color-enhancing reagents (A) to (D) of the present invention did not stain even though they were stored at 30 ° C. for 3 months, and gave the same judgment result as the fresh color-enhancing reagent. On the other hand, the color-enhancing reagent (E) to which no mineral acid was added
Colored and gave an incorrect judgment result.
実施例 ２ ５％（ｗ／ｖ）α‐ナフトールのエタノール溶液にリン
酸0.0868M（Ｆ）、0.0496M（Ｇ）、0.0386M（Ｈ）を加
えた発色増強試薬（Ｆ）〜（Ｈ）を調製した。上記のリ
ン酸対α−ナフトールのモル比はそれぞれ1:4,1:7およ
び1:9であった。上記の各発色増強試薬（Ｆ）〜（Ｈ）
を30℃で３ケ月保存したところ（Ｆ）および（Ｇ）は着
色しなかったが（Ｈ）は僅かに着色した。Example 2 Color-enhancing reagents (F) to (H) obtained by adding 0.0868M (F), 0.0496M (G) and 0.0386M (H) phosphoric acid to an ethanol solution of 5% (w / v) α-naphthol were added. Prepared. The above molar ratios of phosphoric acid to α-naphthol were 1: 4, 1: 7 and 1: 9, respectively. Each of the above-mentioned color-enhancing reagents (F) to (H)
Was stored at 30 ° C. for 3 months, (F) and (G) were not colored, but (H) was slightly colored.
実施例 ３ ペプトン7.0g、グルコース5.0g、リン酸二カリウム5.0
g、蒸留水1000mlの組成の培地を調整し、オートクレー
プ滅菌した。多穴プレート各ウェルに50μｌずつ分注
し、25℃で乾燥した。次に寒天培地上で、エンテロバク
ター・アエロジェネス（RIMD 050200）を37℃で18〜20
時間培養し、滅菌蒸留水に１×108個／mlとなるように
懸濁し、各ウェルに50μｌずつ接種し、37℃18〜20時間
培養した。別に、塩酸を2.08M（Ｉ）,3.47M（Ｊ）加え
た５％（ｗ／ｖ）α‐ナフトール（エタノール溶液）
（Ｉ）および（Ｊ）を調整した。上記の塩酸対α‐ナフ
トールのモル比はそれぞれ6:1（Ｉ）および10:1（Ｊ）
であった。培養したプレートに各発色増強試薬（Ｉ）お
よび（Ｊ）を各ウェル50μｌずつ分注し、さらに酸化試
薬として40％（ｗ／ｖ）水酸化カリウムを50μｌずつ添
加し、呈色を判定したところ、発色増強試薬（Ｉ）を用
いた場合は陽性、（Ｊ）を用いた場合は弱い陽性であっ
た。Example 3 Peptone 7.0 g, glucose 5.0 g, dipotassium phosphate 5.0
A medium having a composition of g and 1000 ml of distilled water was prepared and autoclaved. 50 μl was dispensed into each well of a multi-well plate and dried at 25 ° C. Next, enterobacter aerogenes (RIMD 050200) at 37 ℃ for 18-20 on agar medium.
After culturing for a period of time, the cells were suspended in sterile distilled water at 1 × 10 8 cells / ml, 50 μl of each well was inoculated, and the cells were cultured at 37 ° C. for 18 to 20 hours. Separately, hydrochloric acid was added to 2.08 M (I) and 3.47 M (J), and 5% (w / v) α-naphthol (ethanol solution)
(I) and (J) were adjusted. The above molar ratios of hydrochloric acid to α-naphthol are 6: 1 (I) and 10: 1 (J), respectively.
Met. 50 μl of each color-enhancing reagent (I) and (J) was dispensed to each well of the cultured plate, and 50 μl of 40% (w / v) potassium hydroxide was added as an oxidizing reagent to determine the coloration. , Using the color-enhancing reagent (I) was positive, and using (J) was weakly positive.
以上の試験結果から本発明における好ましい鉱酸の使用
量は、α‐ナフトール１モルに対して0.14〜６モル当量
であることが明らかである。From the above test results, it is apparent that the preferable amount of the mineral acid used in the present invention is 0.14 to 6 molar equivalents relative to 1 mol of α-naphthol.
［発明の効果］ α‐ナフトールを含むエタノール溶液に鉱酸を添加して
なる本発明の発色増強試薬は、室温で長期間保存しても
着色せず、また添加した鉱酸が呈色反応を阻げることも
ないのでフォーゲス・プロスカウエル試験に用いた場合
に呈色の判定を容易にし、正しい菌の同定を可能にす
る。このように、本発明は室温で長期間保存可能な発色
増強試薬を提供するものである。[Effects of the Invention] The color-enhancing reagent of the present invention obtained by adding a mineral acid to an ethanol solution containing α-naphthol does not color even when stored at room temperature for a long time, and the added mineral acid causes a color reaction. Since it does not prevent it, it facilitates the determination of coloration when used in the Forges-Proskouer test and enables the correct identification of bacteria. Thus, the present invention provides a color-enhancing reagent that can be stored at room temperature for a long period of time.
Claims (3)
酸を添加してなる安定化された発色増強試薬。1. A stabilized color-development-enhancing reagent obtained by adding a mineral acid to an alcohol solution containing α-naphthol.
記載の発色増強試薬。2. The color-enhancing reagent according to claim 1, wherein the mineral acid is phosphoric acid or hydrochloric acid.
して0.14〜６モルである請求項１または２に記載の発色
増強試薬。3. The color-enhancing reagent according to claim 1, wherein the amount of mineral acid added is 0.14 to 6 mol per mol of α-naphthol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20800388A JPH0669393B2 (en) | 1988-08-24 | 1988-08-24 | Stabilized color enhancement reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20800388A JPH0669393B2 (en) | 1988-08-24 | 1988-08-24 | Stabilized color enhancement reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0257196A JPH0257196A (en) | 1990-02-26 |
| JPH0669393B2 true JPH0669393B2 (en) | 1994-09-07 |
Family
ID=16549060
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20800388A Expired - Lifetime JPH0669393B2 (en) | 1988-08-24 | 1988-08-24 | Stabilized color enhancement reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0669393B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0710494U (en) * | 1993-07-14 | 1995-02-14 | 株式会社鎌倉製作所 | Blower swinging device |
-
1988
- 1988-08-24 JP JP20800388A patent/JPH0669393B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0710494U (en) * | 1993-07-14 | 1995-02-14 | 株式会社鎌倉製作所 | Blower swinging device |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0257196A (en) | 1990-02-26 |
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