JPH0586196B2 - - Google Patents
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- Publication number
- JPH0586196B2 JPH0586196B2 JP9803386A JP9803386A JPH0586196B2 JP H0586196 B2 JPH0586196 B2 JP H0586196B2 JP 9803386 A JP9803386 A JP 9803386A JP 9803386 A JP9803386 A JP 9803386A JP H0586196 B2 JPH0586196 B2 JP H0586196B2
- Authority
- JP
- Japan
- Prior art keywords
- parts
- nitrate
- medium
- culture
- bacteria
- 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 - Fee Related
Links
- 238000012360 testing method Methods 0.000 claims description 26
- 229910002651 NO3 Inorganic materials 0.000 claims description 17
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 17
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- 239000013028 medium composition Substances 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 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 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 6
- 244000052616 bacterial pathogen Species 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical group [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 description 17
- 239000002609 medium Substances 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 2
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- -1 nitrite ions Chemical class 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000589513 Burkholderia cepacia Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910001514 alkali metal chloride Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
．発明の背景
技術分野
本発明は微生物の硝酸塩還元能検査用培地組成
物に関する。
さらに詳しくは本発明はグルコース非発酵性グ
ラム陰性桿菌の硝酸塩還元能検査用培地組成物に
関するものである。
細菌感染症に対して適切な治療を施すためには
病原菌を同定し、感受性試験を行い、その病原菌
に有効な薬剤を決定することが重要である。この
ような病原菌の同定に際しては多項目にわたる生
化学的性状検査が行われ、その項目の１つとして
グルコース非発酵性グラム陰性桿菌について硝酸
塩還元能の検査が行われる。これは、上記菌が有
する硝酸塩還元能を利用し、菌の同定を行うもの
である。本発明の培地組成物はこのような試験に
好適に使用される。
先行技術およびその問題点
硝酸塩還元反応は培地中の硝酸塩を還元して亜
硝酸塩にする反応であり、従来下記の組成を有す
る硝酸塩ブイヨン培地が用いられている。
硝酸塩ブイヨン培地
肉 エ キ ス 3g
ペ プ ト ン 5g
硝酸カリウム 1g
蒸 留 水 1000ml
PH6.8
検査に際しては上記硝酸塩ブイヨン培地に検査
菌を接種し、30℃で20〜48時間培養する。
検査菌の硝酸塩還元能の有無は培養液に、スル
フアニル酸およびα−ナフチルアミンを添加し、
亜硝酸塩が存在すれば生成される赤色物質パラ−
スルホベンゼン−アゾ−α−ナフチルアミンを検
出することによつて判定される。
上記従来培地によれば、短時間の培養では判定
の信頼性が低く、正確な判定のためには長時間の
培養を必要とする。
細菌感染症の早期治療のためには、菌の同定は
できるだけ速やかに行うことが必要であり、より
短かい時間で菌の同定が可能な培地の出現が望ま
れていた。しかし、一方においては、検査作業の
都合上、判定を翌日に行なわざるを得ない場合も
生じ、このような場合には培養期間が厳格に規制
されておらず、都合により培養時間を延長しても
反応過剰になつたりせず、正確な判定が可能な培
地が望ましい。
．発明の目的
本発明は短時間の培養で同定が可能であるとと
もに長時間培養しても正確な同定が可能であるグ
ルコース非発酵性グラム陰性桿菌の硝酸塩還元能
の検査用培地組成物を提供することを目的とす
る。
かかる目的を達成するため、本発明は硝酸カリ
ウム1.0部（重量部、以下同じ）に対し、ペプト
ン3.125〜50部、酵母エキス0.625〜10部およびリ
ン酸一水素二アルカリ金属塩0.313〜５部の組成
よりなるグルコース非発酵性グラム陰性桿菌の硝
酸塩還元能検査用培地組成物からなる。
さらに本発明はリン酸一水素二アルカリ金属塩
がリン酸一水素二ナトリウム塩である硝酸塩還元
能検査用培地組成物からなる。
．発明の具体的説明
本発明の培地組成物において硝酸カリウムは基
質であり、これが検査菌によつて分解されると培
養液中で亜照酸イオンを生成し、この変化によつ
て検査菌の硝酸塩還元能を判定する。ペプトンお
よび酵母エキスは養分として用いられる。リン酸
一水素二アルカリ金属塩はPH調整剤であり、菌の
発育に最適なPH6.9〜7.1になるような範囲に設定
されている。本発明の培地組成物にはさらに浸透
圧調整剤としてアルカリ金属塩1.25〜20部が使用
されるのが望ましく、塩化ナトリウム、塩化カリ
ウムのようなアルカリ金属塩化物が好適である。
本発明の培地組成物の前述した成分割合は臨界
的であり、硝酸カリウム1.0部に対し、ペプトン
3.125〜50部（好ましくは0.25〜25部）、酵母エキ
ス0.625〜10部（好ましくは1.25〜５部）および
リン酸一水素二アルカリ金属塩0.313〜５部（好
ましくは0.625〜2.5部）である。特にペプトン濃
度は重要であり、ペプトン濃度が低すぎると偽陽
性の判定となりやすく、逆に高すぎると偽陰性の
判定となりやすい。本発明の培地では、上述した
偽陽性や偽陰性の誤まつた判定を下す危険が少な
く、かつ、接種菌数を適宜選択することにより短
時間培養（４〜５時間）または一昼夜培養（18〜
22時間）のいずれによつても正しい判定が可能な
ようにペプトン濃度が設定されている。
本発明の培地組成物は常法に従つて硝酸カリウ
ム１部当り625〜2500部の滅菌蒸留水に溶解して
使用される。近年生化学的性状検査は多数の試験
を多穴プレート上で同時に行うのが普通であり、
このような場合には本発明培地組成物の水溶液を
試験用プレートのウエルに注入し、乾燥させて乾
燥培地とする。試験に際しては該ウエルに試験菌
の懸濁水を所定量分注し、30℃で所定時間培養す
る。４〜５時間の培養で判定することが望まれる
場合は、液体培地50μ当り約7.5×107個の菌を
接種し、18〜22時間培養後の判定が望まれる場合
は約1.5×107個の菌を接種するのが好ましい。こ
のように接種菌数を調整することにより、短時間
でもまた翌日でも検査が行える。
次に本発明の培地組成物を使用して細菌の硝酸
塩還元能を検査した実施例を示す。
実施例
表１に記載の組成を有する本発明培地組成物を
常法に従つて蒸留水１に溶解し、菌同定用培地
１〜３を得た。[Detailed description of the invention]. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium composition for testing the nitrate reducing ability of microorganisms. More specifically, the present invention relates to a medium composition for testing the nitrate reducing ability of glucose non-fermenting Gram-negative bacilli. In order to provide appropriate treatment for bacterial infections, it is important to identify the pathogen, conduct susceptibility tests, and determine effective drugs against the pathogen. When identifying such pathogenic bacteria, a multi-item biochemical property test is performed, and one of the items is a test for nitrate-reducing ability of glucose non-fermenting Gram-negative rods. This method utilizes the nitrate-reducing ability of the above-mentioned bacteria to identify the bacteria. The medium composition of the present invention is suitably used for such tests. Prior Art and its Problems The nitrate reduction reaction is a reaction in which nitrate in a medium is reduced to nitrite, and a nitrate broth medium having the following composition has conventionally been used. Nitrate bouillon medium Meat extract 3g Peptone 5g Potassium nitrate 1g Distilled water 1000ml PH6.8 For testing, inoculate the test bacteria into the above nitrate bouillon medium and culture at 30°C for 20 to 48 hours. The presence or absence of nitrate reducing ability of the test bacteria is determined by adding sulfanilic acid and α-naphthylamine to the culture solution.
A red substance produced when nitrite is present
Determined by detecting sulfobenzene-azo-α-naphthylamine. According to the above-mentioned conventional culture medium, the reliability of determination is low in short-time culture, and long-time culture is required for accurate determination. For early treatment of bacterial infections, it is necessary to identify bacteria as quickly as possible, and there has been a desire for a culture medium that can identify bacteria in a shorter time. However, on the other hand, due to the convenience of testing work, there are cases where the judgment has to be made the next day, and in such cases, the culture period is not strictly regulated and the culture time may be extended for convenience. It is desirable to use a medium that does not overreact and allows accurate determination. ．． Purpose of the Invention The present invention provides a medium composition for testing the nitrate reducing ability of glucose non-fermenting Gram-negative bacilli, which can be identified in a short time culture and can be accurately identified even in a long time culture. The purpose is to In order to achieve such an object, the present invention has a composition of 3.125 to 50 parts of peptone, 0.625 to 10 parts of yeast extract, and 0.313 to 5 parts of a dialkali metal monohydrogen phosphate to 1.0 part of potassium nitrate (part by weight, the same applies hereinafter). A medium composition for testing the nitrate reducing ability of glucose non-fermenting Gram-negative bacilli. Furthermore, the present invention comprises a medium composition for testing nitrate reducing ability, in which the dialkali metal salt of monohydrogen phosphate is disodium monohydrogen phosphate. ．． Detailed Description of the Invention Potassium nitrate is a substrate in the culture medium composition of the present invention, and when it is decomposed by the test bacteria, it produces nitrite ions in the culture solution, and this change reduces the nitrate of the test bacteria. Determine ability. Peptone and yeast extract are used as nutrients. Dialkali metal monohydrogen phosphate is a pH regulator, and is set at a pH range of 6.9 to 7.1, which is optimal for bacterial growth. It is desirable that the medium composition of the present invention further contains 1.25 to 20 parts of an alkali metal salt as an osmotic pressure regulator, with alkali metal chlorides such as sodium chloride and potassium chloride being preferred. The above-mentioned component ratio of the culture medium composition of the present invention is critical, and peptone is added to 1.0 part of potassium nitrate.
3.125 to 50 parts (preferably 0.25 to 25 parts), yeast extract 0.625 to 10 parts (preferably 1.25 to 5 parts), and 0.313 to 5 parts (preferably 0.625 to 2.5 parts) of dialkali metal monohydrogen phosphate. . In particular, the peptone concentration is important; if the peptone concentration is too low, a false positive determination is likely to occur, whereas if the peptone concentration is too high, a false negative determination is likely to occur. The culture medium of the present invention has a low risk of making false positive or false negative judgments as described above, and can be cultured for a short time (4 to 5 hours) or overnight (18 to 5 hours) by appropriately selecting the number of inoculated bacteria.
The peptone concentration is set so that correct judgment can be made regardless of the time (22 hours). The medium composition of the present invention is used by dissolving 625 to 2,500 parts of potassium nitrate in sterile distilled water according to a conventional method. In recent years, it has become common for biochemical property testing to perform multiple tests simultaneously on multi-hole plates.
In such a case, an aqueous solution of the culture medium composition of the present invention is injected into the wells of a test plate and dried to obtain a dry culture medium. During the test, a predetermined amount of suspension water of test bacteria is dispensed into the well, and cultured at 30°C for a predetermined time. If it is desired to make a judgment after culturing for 4 to 5 hours, inoculate about 7.5 x 107 bacteria per 50μ of liquid medium, and if you want to make a judgment after culturing for 18 to 22 hours, about 1.5 x 107 bacteria. It is preferable to inoculate the bacteria. By adjusting the number of inoculated bacteria in this way, testing can be carried out in a short period of time or even the next day. Next, an example will be shown in which the nitrate reducing ability of bacteria was tested using the medium composition of the present invention. Example The culture medium composition of the present invention having the composition shown in Table 1 was dissolved in distilled water 1 according to a conventional method to obtain culture media 1 to 3 for bacterial identification.
【表】
上記培地１〜３を多穴プレート各ウエルに50μ
ずつ分注し、40℃で乾燥した。次に寒天培地上
で各種検査細菌を30℃で18〜24時間培養し、1.0
mlの滅菌蒸留水中に培養時間が４時間の場合は
1.5×109個／mlまた20時間の場合３×108個／ml
となるように懸濁し各ウエルに50μずつ接種
し、30℃で所定時間培養した。比較試験として、
前出の硝酸塩ブイヨン培地を用いる従来法により
培養を行なつた。
所定時間培養したのち、各培地について亜硝酸
塩の検出試験を行うことにより硝酸塩還元能の有
無を判定した。結果を表２に示す。[Table] Add 50μ of the above medium 1 to 3 to each well of a multi-well plate.
The solution was divided into portions and dried at 40°C. Next, various test bacteria were cultured on agar medium at 30°C for 18 to 24 hours, and 1.0
If the incubation time is 4 hours in ml of sterile distilled water,
1.5×10 9 pieces/ml or 3×10 8 pieces/ml for 20 hours
The cells were suspended in such a manner that 50μ of each well was inoculated, and cultured at 30°C for a predetermined period of time. As a comparative test,
Culture was carried out by a conventional method using the nitrate broth medium described above. After culturing for a predetermined period of time, each medium was subjected to a nitrite detection test to determine the presence or absence of nitrate reducing ability. The results are shown in Table 2.
【表】
．発明の具体的作用および効果
表２から明らかなように、グルコース非発酵性
グラム陰性桿菌の硝酸塩還元能検査において、本
発明の培地を使用すると、培養時間の長短を問わ
ず正しい菌の同定が可能である。従つて検査作業
の都合により即日同定、翌日同定のいずれも選択
することができる。これに対して対照培地は少く
とも20時間の培養が必要であり、即日判定は不可
能である。例えば、シユードモナス・アエルギノ
ーザATCC10145、シユードモナス・セパシア
ATCC27515、シユードモナス・スタツチエリ
ATCC17588およびシユードモナス・アンドボラ
ンスATCC15688は対照培地では５時間培養では
陰性であり、22時間培養で陽性になる。これに対
して本発明の培地を使用すると５時間培養で正し
い判定が可能である。【table】 . Specific Actions and Effects of the Invention As is clear from Table 2, when the medium of the present invention is used in testing the nitrate reducing ability of glucose non-fermenting Gram-negative bacilli, it is possible to correctly identify the bacteria regardless of the length of the culture time. It is. Therefore, either same-day identification or next-day identification can be selected depending on the convenience of the inspection work. On the other hand, the control medium requires cultivation for at least 20 hours, and same-day determination is not possible. For example, Pseudomonas aeruginosa ATCC10145, Pseudomonas cepacia
ATCC27515, Pseudomonas stutscheri
ATCC17588 and Pseudomonas andvorans ATCC15688 are negative after 5 hours of culture and become positive after 22 hours of culture in control medium. On the other hand, when the medium of the present invention is used, correct determination can be made after 5 hours of culture.
Claims (1)
対し、ペプトン3.125〜50部、酵母エキス0.625〜
10部およびリン酸一水素二アルカリ金属塩0.313
〜５部の組成よりなるグルコース非発酵性グラム
陰性桿菌の硝酸塩還元能検査用培地組成物。 ２ リン酸一水素二アルカリ金属塩がリン酸一水
素二ナトリウム塩である特許請求の範囲第１項記
載の培地組成物。[Claims] 1. 1.0 parts by weight of potassium nitrate (parts by weight, the same applies hereinafter), 3.125 to 50 parts of peptone, and 0.625 to 0.625 parts of yeast extract.
10 parts and 0.313 parts of dialkali metal monohydrogen phosphate
A medium composition for testing the nitrate reducing ability of glucose non-fermenting Gram-negative bacilli, comprising 5 parts. 2. The medium composition according to claim 1, wherein the dialkali metal salt of monohydrogen phosphate is disodium monohydrogen phosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9803386A JPS62257399A (en) | 1986-04-30 | 1986-04-30 | Medium composition for testing nitrate reducing ability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9803386A JPS62257399A (en) | 1986-04-30 | 1986-04-30 | Medium composition for testing nitrate reducing ability |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62257399A JPS62257399A (en) | 1987-11-09 |
| JPH0586196B2 true JPH0586196B2 (en) | 1993-12-10 |
Family
ID=14208706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9803386A Granted JPS62257399A (en) | 1986-04-30 | 1986-04-30 | Medium composition for testing nitrate reducing ability |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62257399A (en) |
-
1986
- 1986-04-30 JP JP9803386A patent/JPS62257399A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62257399A (en) | 1987-11-09 |
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