JPH02286096A - Estimation of bacteria concentration - Google Patents
Estimation of bacteria concentrationInfo
- Publication number
- JPH02286096A JPH02286096A JP10948389A JP10948389A JPH02286096A JP H02286096 A JPH02286096 A JP H02286096A JP 10948389 A JP10948389 A JP 10948389A JP 10948389 A JP10948389 A JP 10948389A JP H02286096 A JPH02286096 A JP H02286096A
- Authority
- JP
- Japan
- Prior art keywords
- gram
- negative bacteria
- substance
- cells
- surfactant
- 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.)
- Pending
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 22
- 210000004027 cell Anatomy 0.000 claims abstract description 38
- 239000000126 substance Substances 0.000 claims abstract description 22
- 239000004094 surface-active agent Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 14
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 12
- 238000011088 calibration curve Methods 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 23
- 241000192125 Firmicutes Species 0.000 claims description 10
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 abstract description 16
- 238000000605 extraction Methods 0.000 abstract description 12
- 238000005259 measurement Methods 0.000 abstract description 6
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 abstract description 4
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 abstract description 4
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 abstract description 4
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- 108060001084 Luciferase Proteins 0.000 abstract description 4
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- 230000029918 bioluminescence Effects 0.000 abstract description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 239000003945 anionic surfactant Substances 0.000 description 7
- 239000002736 nonionic surfactant Substances 0.000 description 5
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- 229960003964 deoxycholic acid Drugs 0.000 description 3
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- 230000000694 effects Effects 0.000 description 3
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- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 2
- ZORQXIQZAOLNGE-UHFFFAOYSA-N 1,1-difluorocyclohexane Chemical compound FC1(F)CCCCC1 ZORQXIQZAOLNGE-UHFFFAOYSA-N 0.000 description 2
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- 239000004380 Cholic acid Substances 0.000 description 2
- IYFATESGLOUGBX-YVNJGZBMSA-N Sorbitan monopalmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O IYFATESGLOUGBX-YVNJGZBMSA-N 0.000 description 2
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000019416 cholic acid Nutrition 0.000 description 2
- 229960002471 cholic acid Drugs 0.000 description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
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- 229940035044 sorbitan monolaurate Drugs 0.000 description 2
- 235000011069 sorbitan monooleate Nutrition 0.000 description 2
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- 238000010186 staining Methods 0.000 description 2
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- FFJCNSLCJOQHKM-CLFAGFIQSA-N (z)-1-[(z)-octadec-9-enoxy]octadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCCCCCCC\C=C/CCCCCCCC FFJCNSLCJOQHKM-CLFAGFIQSA-N 0.000 description 1
- AJDONJVWDSZZQF-UHFFFAOYSA-N 1-(2,4,4-trimethylpentan-2-yl)-4-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]benzene Chemical compound C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OC1=CC=C(C(C)(C)CC(C)(C)C)C=C1 AJDONJVWDSZZQF-UHFFFAOYSA-N 0.000 description 1
- LTSWUFKUZPPYEG-UHFFFAOYSA-N 1-decoxydecane Chemical compound CCCCCCCCCCOCCCCCCCCCC LTSWUFKUZPPYEG-UHFFFAOYSA-N 0.000 description 1
- CMCBDXRRFKYBDG-UHFFFAOYSA-N 1-dodecoxydodecane Chemical compound CCCCCCCCCCCCOCCCCCCCCCCCC CMCBDXRRFKYBDG-UHFFFAOYSA-N 0.000 description 1
- FDCJDKXCCYFOCV-UHFFFAOYSA-N 1-hexadecoxyhexadecane Chemical compound CCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC FDCJDKXCCYFOCV-UHFFFAOYSA-N 0.000 description 1
- OUUCZGCOAXRCHN-UHFFFAOYSA-N 1-hexadecoxyoctadecane Chemical compound CCCCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC OUUCZGCOAXRCHN-UHFFFAOYSA-N 0.000 description 1
- CKQAOGOZKZJUGA-UHFFFAOYSA-N 1-nonyl-4-(4-nonylphenoxy)benzene Chemical compound C1=CC(CCCCCCCCC)=CC=C1OC1=CC=C(CCCCCCCCC)C=C1 CKQAOGOZKZJUGA-UHFFFAOYSA-N 0.000 description 1
- HBXWUCXDUUJDRB-UHFFFAOYSA-N 1-octadecoxyoctadecane Chemical compound CCCCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCCCC HBXWUCXDUUJDRB-UHFFFAOYSA-N 0.000 description 1
- PIIRYSWVJSPXMW-UHFFFAOYSA-N 1-octyl-4-(4-octylphenoxy)benzene Chemical compound C1=CC(CCCCCCCC)=CC=C1OC1=CC=C(CCCCCCCC)C=C1 PIIRYSWVJSPXMW-UHFFFAOYSA-N 0.000 description 1
- HANWHVWXFQSQGJ-UHFFFAOYSA-N 1-tetradecoxytetradecane Chemical compound CCCCCCCCCCCCCCOCCCCCCCCCCCCCC HANWHVWXFQSQGJ-UHFFFAOYSA-N 0.000 description 1
- CSHOPPGMNYULAD-UHFFFAOYSA-N 1-tridecoxytridecane Chemical compound CCCCCCCCCCCCCOCCCCCCCCCCCCC CSHOPPGMNYULAD-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 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
- 241000254158 Lampyridae Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000000981 basic dye Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 description 1
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 description 1
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
Ａ、産業上の利用分野
本発明は細菌内の物質を抽出して測定することにより菌
体濃度（菌体数）を推定する方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to a method for estimating bacterial cell concentration (bacterial cell number) by extracting and measuring substances within bacteria.
Ｂ１発明の概要 。B1 Overview of the invention.
本発明はグラム陰性菌またはグラム陽性菌の細胞膜を界
面活性剤により破壊して細胞内の被測定物質を抽出し、
その後この物質を光学的手法により測定して菌体濃度を
推定する方法において、界面活性剤の他にクロロポルム
を添加することによって、
抽出効率を高めると共に、室温においてら十分な抽出が
できるようにしたものである。The present invention destroys the cell membrane of Gram-negative bacteria or Gram-positive bacteria with a surfactant to extract the substance to be measured inside the cells,
Later, in the method of estimating the bacterial cell concentration by measuring this substance using an optical method, we added chloroporum in addition to a surfactant to increase extraction efficiency and enable sufficient extraction even at room temperature. It is something.
Ｃ１従来の技術
グラム染色法と呼ばれる細菌の染色法は、細菌を大きく
２群に分類することのできる方法として古くから利用さ
れている。この方法は、熱固定した細菌をまず塩基性の
色素で染めた後、Ｉｔ Ｋ■混合溶液で処理し、次い
でアセトンまたはアルコールで脱色した後異なった色素
で後染色する方法であり、最初に染めた色素で染まるも
のをグラム陽性菌といい、後染色で染まるものをグラム
陰性菌という。グラム陰性菌は細胞膜が薄く、その外側
にリボ多糖を持つものが多いが、グラム陽性菌は細胞膜
が厚く、その外側にリボ多糖を欠くものが多い。C1 Prior Art A bacterial staining method called Gram staining has been used for a long time as a method that can roughly classify bacteria into two groups. In this method, heat-fixed bacteria are first dyed with a basic dye, then treated with an It K mixed solution, decolorized with acetone or alcohol, and then post-stained with a different dye. Bacteria that stain with post-staining are called gram-positive bacteria, and those that stain with post-staining are called gram-negative bacteria. Gram-negative bacteria often have thin cell membranes and have ribopolysaccharides on the outside, while Gram-positive bacteria often have thick cell membranes and lack ribopolysaccharides on the outside.
このように分類される菌群について夫々その菌体濃度を
測定することにより有効な知見を得ろことができ、その
測定方法として、菌体内に存在する物質を測定すること
により菌体濃度を推定する方法がある。Effective knowledge can be obtained by measuring the bacterial cell concentration of each bacterial group classified in this way, and the method for this measurement is to estimate the bacterial cell concentration by measuring the substances present inside the bacterial cells. There is a way.
例えば生体細胞中には、生体のリン酸代謝及びエネルギ
ー代謝の役割を果たしているアデノシン三リン酸（ＡＴ
Ｐ）が必ず存在し、死細胞中にはＡＴＰが存在しないこ
とが知られている。また１個の細胞中に存在するＡＴＰ
量は、同一細胞では同一濃度のＡ ’ｌ’ Ｉ）が存在
する。従ってＡ ’ｒ Ｐが定量できれば、菌体の数か
測定できることになる。For example, in living cells, adenosine triphosphate (AT), which plays a role in phosphate metabolism and energy metabolism, is present in living cells.
P) is always present, and it is known that ATP is not present in dead cells. Also, ATP present in one cell
In the same cell, the same concentration of A'l'I) is present. Therefore, if A'rP can be quantified, the number of bacterial cells can also be determined.
このように細胞内に存在する物質を測定するためには、
多くの場合細胞に何らかの処理を施し、細胞膜を破壊し
、細胞外にその物質を放出させること、いわゆる抽出操
作を行う必要がある。In order to measure the substances present in cells in this way,
In many cases, it is necessary to perform some kind of treatment on cells to destroy the cell membrane and release the substance outside the cell, a so-called extraction operation.
一方最近において、可溶化することが困難な非常に疎水
性の強い蛋白質を比較的温和な条件で可溶化できる界面
活性剤の種類が増えてきた。On the other hand, recently, the number of types of surfactants that can solubilize highly hydrophobic proteins that are difficult to solubilize under relatively mild conditions has increased.
界面活性剤はその電気的な性質に基づいて、陰イオン性
界面活性剤、陽イオン性界面活性剤９両性界面活性剤及
び非イオン性界面活性剤に分類される。また電気的な性
質とは無関係にコール酸。Based on their electrical properties, surfactants are classified into anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Also cholic acid independent of electrical properties.
デオキシコール酸−どのようなステロイド骨格を持つ界
面活性剤も存在する。この中で膜蛋白質の可溶化には、
陰イオン性界面活性剤、非イオン性界面活性剤及び胆汁
酸（コール酸、デオキシコール酸）がよく利用されてい
る。Deoxycholic acid - Surfactants with any steroid backbone also exist. Among these, for membrane protein solubilization,
Anionic surfactants, nonionic surfactants and bile acids (cholic acid, deoxycholic acid) are commonly used.
こうしたことから界面活性剤を用いて細胞内の物質を抽
出することが試みられている。For this reason, attempts have been made to extract intracellular substances using surfactants.
Ｄ１発明が解決しようとする課題
ところが、界面活性剤は表面張力を著しく低下させる作
用を有し、細胞の膜破壊にも実際にいくつかの界面活性
剤が使用されているらのの、従来の界面活性剤を用いた
方法では、体細胞のような比較的Ｉｌ！破壊の容易な細
胞を対象とした場合には、良好な抽出を行うことかでき
るが、微生物細胞の場合には、膜破壊作用が弱く、抽出
効率が低下する欠点があった。さらに界面活性剤を用い
た抽出方法は、一般に時間がかかることも問題点となっ
ていた。D1 Problem to be solved by the invention However, surfactants have the effect of significantly lowering surface tension, and although some surfactants are actually used to destroy cell membranes, conventional In the method using a surfactant, relatively Il! When targeting cells that are easy to destroy, good extraction can be performed, but in the case of microbial cells, the membrane-destroying effect is weak, resulting in a reduction in extraction efficiency. Another problem with extraction methods using surfactants is that they generally take a long time.
本発明の目的は、グラム陰性菌またはグラム陽性菌の菌
体敢を推定するにあたって細胞内の被測定物質の抽出効
率が高く、しかも操作が簡単で、短時間で分析を行うこ
とのできる方法を提供することにある。The purpose of the present invention is to provide a method for estimating the bacterial cell strength of Gram-negative or Gram-positive bacteria that has high extraction efficiency of intracellular analyte substances, is easy to operate, and can be analyzed in a short time. It is about providing.
Ｅ、課題を解決するための手段
本発明は、グラム陰性菌群またはグラム陽性菌群と界面
活性剤及びクロロホルムとを混合し、その混合液を振盪
することにより菌の細胞膜を破壊して細胞内の被測定物
質を抽出し、次いでこの被測定物質を光学的手法により
測定し、その測定値と予め作成した検量線とにもとづい
て菌体濃度を推定することを特徴とする。E. Means for Solving the Problems The present invention involves mixing gram-negative bacteria or gram-positive bacteria with a surfactant and chloroform, and shaking the mixture to destroy the cell membrane of the bacteria and destroy the inside of the cells. This method is characterized by extracting a substance to be measured, then measuring the substance to be measured using an optical method, and estimating the bacterial cell concentration based on the measured value and a calibration curve prepared in advance.
Ｆ、実施例
（実施例！；陰イオン性界面活性剤の１つであるドデシ
ル硫酸ナトリウム（ＳＤＳ）によるＰｓｅ−ｎｄｏ＠ｏ
ｎａｓ ｆｌｕｏｒｅｓｃｅｎｓ （グラム陰性菌の代
表種）中のＡＴＰの測定）
上記の菌群を含む懸濁菌液１１Ｑを試験管に分取してク
ロロホルム０．２村を加え、更に界面活性剤である１％
のドデシル硫酸すトリウム（ＳＤＳ）溶液を０ 、２
ｍＱ加える。なおＳＤＳの濃度単位の「％」については
、１％が０．０１９ＩＲＱの意味である。次いでこの試
験管をポルテックスミキサー等の振盪器を用いて２０秒
間撹拌振盪し、これにより細胞膜を破壊する。次にＡＴ
Ｐと他の細胞成分を分離するために遠心分離機により３
゜００ ｒｐｍの回転数で５分間遠心分離し、その後上
澄成分をピペットで静かに別の試験管に分取し、これを
０．１ｍｏＣ／１２ ）リス／Ｅ Ｄ Ｔ Ａ緩衝液
（ｐＨ７，７５）で１０倍に希釈する。そしてこの反応
液０ 、５ ｍＱをピペットで測定装置専用のバイヤル
ビンに分取し、発光試薬を含む反応試薬を０．５ｍＱ自
動的に分注して、分注直後から３０秒間の発光型を計測
する。F, Example (Example!; Pse-ndo@o with sodium dodecyl sulfate (SDS), one of the anionic surfactants
Measurement of ATP in Nas fluorescens (representative species of Gram-negative bacteria)) A bacterial suspension containing the above bacterial group 11Q was taken into a test tube, 0.2 ml of chloroform was added, and 1 ml of surfactant was added. %
Sodium dodecyl sulfate (SDS) solution of 0 and 2
Add mQ. Note that regarding the SDS concentration unit "%", 1% means 0.019 IRQ. Next, this test tube is agitated and shaken for 20 seconds using a shaker such as a portex mixer, thereby disrupting the cell membrane. Next, A.T.
3 by centrifuge to separate P and other cellular components.
Centrifugation was performed at a rotational speed of 0.000 rpm for 5 minutes, and then the supernatant component was gently pipetted into another test tube and added to 0.1moC/12) Lis/EDT A buffer (pH 7, Dilute 10 times with 75). Then, pipette 0.5 mQ of this reaction solution into a vial dedicated to the measuring device, automatically dispense 0.5 mQ of the reaction reagent containing the luminescent reagent, and measure the luminescence type for 30 seconds immediately after dispensing. do.
以上において上記の発光法は、生物発光法と呼ばれ、以
下に示すようにＡＴＰとルシフェリンとルシフェラーゼ
との反応により発生する光の儀にもとづいてＡＴＰの量
を知る方法であり、簡便で高感度な測定法である。なお
ホタルの光はこれに相当する。The luminescence method described above is called the bioluminescence method, and as shown below, it is a simple and highly sensitive method for determining the amount of ATP based on the light produced by the reaction of ATP, luciferin, and luciferase. It is a measurement method. The light of fireflies corresponds to this.
ルシフエラーゼ
ＡＴＰ ＋ ルシフェリン
→ γｆニルルシ７五リす１ｆニルルンフ１リン＋
Ｏｔ ７７’
ニルオキジルシフ工リン＋光種々の菌体濃度において、
本実施例に従ってＡＴＰを抽出した結果を第１図に示す
。Luciferase ATP + Luciferin
→ γf Nirrushi 75 Risu 1f Nirrunfu 1 Lin+
Ot 77'
At various bacterial cell concentrations,
The results of extracting ATP according to this example are shown in FIG.
（実施例２；陰イオン性界面活性剤の１つであるドデシ
ル硫酸ナトリウム（ＳＤＳ）によるＢａｃ−ｉｌｌｕｓ
ｍｅｇａｔｅｒｉｕ＠（グラム陽性菌の代表＆）中の
ＡＴＰの測定）
上記の菌群を含む閑懸蜀液０ 、５ ｍＱを試験管に分
取して実施例１と同様の試験を行ったところ、第２図の
結果が得られた。(Example 2; Bacillus by sodium dodecyl sulfate (SDS), one of the anionic surfactants.
Measurement of ATP in megateriu (representative of gram-positive bacteria) 0.5 mQ of the suspension containing the above bacteria group was taken into test tubes and the same test as in Example 1 was conducted. The results shown in Figure 2 were obtained.
第１図及び第２図のグラフから、本実施例による測定に
よれば、ＡＴ Ｐｉ１１度と菌体濃度とは直線関係にあ
り、良好な抽出が行われていることがわかる。従ってこ
れらのグラフを検量線として用い、ＡＴＰｉｌｌＩ度を
測定することによって、菌体濃度を正確に推定できる。From the graphs in FIG. 1 and FIG. 2, it can be seen that according to the measurements made in this example, there is a linear relationship between AT Pi 11 degrees and the bacterial cell concentration, indicating that good extraction is being performed. Therefore, by using these graphs as a calibration curve and measuring the ATPill degree, the bacterial cell concentration can be estimated accurately.
抽出すべき物質としては、元から細胞内に存在する物質
及び外部からの刺激により生成する物質のいずれをも含
む。前者の元から細胞内に存在する物質とは、代謝物質
、蛋白質、核酸あるいは脂質等であり、このうち代謝物
質とは、アデノシン三リン酸等のヌクレオチド、ヌクレ
オシド、アミノ酸あるいはホルモン等である。後者の物
質とは、β−Ｄ−ガラクトシダーゼ等の酵素である。Substances to be extracted include both substances originally existing within cells and substances produced by external stimulation. The former substances originally existing in cells include metabolites, proteins, nucleic acids, lipids, etc. Among these, metabolites include nucleotides such as adenosine triphosphate, nucleosides, amino acids, hormones, and the like. The latter substance is an enzyme such as β-D-galactosidase.
界面活性剤としては、陰イオン性界面活性剤、非イオン
性界面活性剤あるいはステロイド骨格をもつ界面活性剤
等を用いることができる。As the surfactant, an anionic surfactant, a nonionic surfactant, a steroid skeleton-containing surfactant, etc. can be used.
陰イオン性界面活性剤としては、ドデシル硫酸ナトリウ
ム、テトラデシル硫酸ナトリウム、ドデシルスルホン酸
ナトリウム、テトラデシルスルホン酸ナトリウム、ドデ
シルベンゼンスルホン酸ナトリウム、ドデシル−Ｎ−サ
ルコシン酸ナトリウム等が挙げられる。Examples of the anionic surfactant include sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium dodecyl sulfonate, sodium tetradecyl sulfonate, sodium dodecylbenzenesulfonate, sodium dodecyl-N-sarcosinate, and the like.
非イオン性界面活性剤としては、デシルエーテル、ドデ
シルエーテル、トリデシルエーテル、テトラデシルエー
テル、セチルエーテル、ステアリルエーテル、オレイル
エーテル、セチル−ステアリルエーテル、ｐ−ｔ−オク
チルフェニルエーテル、ｐ−オクチルフェニルエーテル
、ｐ−ノニルフェニルエーテル、モノラウリン酸ソルビ
タン、モノパルミチン酸ソルビタン、モノステアリン酸
ソルビタン、モノオレイン酸ソルビタン、モノラウリン
酸ソルビタン、モノパルミチン酸ソルビタン、モノステ
アリン酸ソルビタン、モノオレイン酸ソルビタン、ラウ
リルジメヂルアミンアキシド等を挙げることができる。Examples of nonionic surfactants include decyl ether, dodecyl ether, tridecyl ether, tetradecyl ether, cetyl ether, stearyl ether, oleyl ether, cetyl-stearyl ether, p-t-octylphenyl ether, and p-octylphenyl ether. , p-nonylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, lauryl dimedylamine amine Sid et al.
ステロイド骨格をもつ界面活性剤としては、コール酸ナ
トリウム、デオキシコール酸ナトリウム、ケノデオキシ
コール酸ナトリウム、タウロコール酸ナトリウム、タウ
ロデオキシコール酸ナトリウム、ジギトニン等を挙げる
ことができる。Examples of the surfactant having a steroid skeleton include sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium taurocholate, sodium taurodeoxycholate, digitonin, and the like.
抽出した物質については、生物発光法や化学発光法を利
用して、その発光量を吸光光度法や蛍光光度法により測
定することができる。The amount of luminescence of the extracted substance can be measured by absorbance photometry or fluorescence photometry using bioluminescence or chemiluminescence.
Ｇ１発明の効果
本発明によれば、界面活性剤とクロロホルムとを組み合
わせて菌体の細胞膜を破壊し、細胞内の被測定物質を抽
出するようにしているため、実施例からも明らかなよう
に抽出効率が高くて精度の高い測定を行うことができ、
従ってグラム陰性菌群あるいはグラム陽性菌群の菌体濃
度を正確に推定できる。また陰イオン性界面活性剤や非
イオン性界面活性剤等種々のものを使用することができ
、界面活性剤に制約がない。しかも室温で抽出すること
ができ、その操作も簡単なため、自動化が容易である。G1 Effects of the Invention According to the present invention, a surfactant and chloroform are combined to destroy the cell membrane of the bacterial cells and extract the substance to be measured inside the cells. It has high extraction efficiency and can perform highly accurate measurements,
Therefore, the bacterial cell concentration of the Gram-negative bacteria group or the Gram-positive bacteria group can be estimated accurately. Moreover, various surfactants such as anionic surfactants and nonionic surfactants can be used, and there are no restrictions on the surfactants. Furthermore, extraction can be performed at room temperature, and the operation is simple, making automation easy.
その上抽出時間が短いため作業上有利である。Furthermore, the extraction time is short, which is advantageous in terms of work.
第１図及び第２図は、各々菌体濃度とＡＴＰ濃度との関
係を示すグラフである。
外２名
手続補上書（１）
６．補正の内容
平成−１年９月８
日
明細書第１
３頁第１行および第２行を「光FIG. 1 and FIG. 2 are graphs showing the relationship between bacterial cell concentration and ATP concentration, respectively. Supplementary statement of procedures for two other persons (1) 6. Contents of the amendment dated September 8, 1999, the first and second lines of page 13 of the specification were changed to
Claims (2)
を混合し、その混合液を振盪することによりグラム陰性
菌の細胞膜を破壊して細胞内の被測定物質を抽出し、次
いでこの被測定物質を光学的手法により測定し、その測
定値と予め作成した検量線とにもとづいてグラム陰性菌
の菌体濃度を推定することを特徴とする菌体濃度推定方
法。(1) Mix Gram-negative bacteria, a surfactant, and chloroform, and shake the mixture to destroy the cell membrane of the Gram-negative bacteria and extract the substance to be measured inside the cells. A method for estimating bacterial cell concentration, characterized in that the bacterial cell concentration of Gram-negative bacteria is estimated based on the measured value and a calibration curve prepared in advance.
を混合し、その混合液を振盪することによりグラム陽性
菌の細胞膜を破壊して細胞内の被測定物質を抽出し、次
いでこの被測定物質を光学的手法により測定し、その測
定値と予め作成した検量線とにもとづいてグラム陽性菌
の菌体濃度を推定することを特徴とする菌体濃度推定方
法。(2) Mix Gram-positive bacteria, a surfactant, and chloroform, and shake the mixture to destroy the cell membrane of the Gram-positive bacteria and extract the substance to be measured inside the cells. A method for estimating bacterial cell concentration, characterized in that the bacterial cell concentration of Gram-positive bacteria is estimated based on the measured value and a calibration curve prepared in advance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10948389A JPH02286096A (en) | 1989-04-28 | 1989-04-28 | Estimation of bacteria concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10948389A JPH02286096A (en) | 1989-04-28 | 1989-04-28 | Estimation of bacteria concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02286096A true JPH02286096A (en) | 1990-11-26 |
Family
ID=14511386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10948389A Pending JPH02286096A (en) | 1989-04-28 | 1989-04-28 | Estimation of bacteria concentration |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02286096A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007502967A (en) * | 2003-08-20 | 2007-02-15 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Method for extraction and concentration of hydrophilic compounds diffused or dispersed in a hydrophobic liquid matrix |
| JP2009112199A (en) * | 2007-11-02 | 2009-05-28 | Toyobo Co Ltd | Method for detecting multicolor luciferase |
| WO2014061785A1 (en) * | 2012-10-19 | 2014-04-24 | 株式会社日立製作所 | Biological material collection method and biological material collection device |
-
1989
- 1989-04-28 JP JP10948389A patent/JPH02286096A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007502967A (en) * | 2003-08-20 | 2007-02-15 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Method for extraction and concentration of hydrophilic compounds diffused or dispersed in a hydrophobic liquid matrix |
| JP2009112199A (en) * | 2007-11-02 | 2009-05-28 | Toyobo Co Ltd | Method for detecting multicolor luciferase |
| WO2014061785A1 (en) * | 2012-10-19 | 2014-04-24 | 株式会社日立製作所 | Biological material collection method and biological material collection device |
| JP2014082946A (en) * | 2012-10-19 | 2014-05-12 | Hitachi Ltd | Biological material collection method and biological material collection apparatus |
| US9988663B2 (en) | 2012-10-19 | 2018-06-05 | Hitachi, Ltd. | Method for collecting biological material and device for collecting biological material |
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