JP2021108573A - Coloring culture medium for differentiation of legionella bacteria - Google Patents
Coloring culture medium for differentiation of legionella bacteria Download PDFInfo
- Publication number
- JP2021108573A JP2021108573A JP2020002424A JP2020002424A JP2021108573A JP 2021108573 A JP2021108573 A JP 2021108573A JP 2020002424 A JP2020002424 A JP 2020002424A JP 2020002424 A JP2020002424 A JP 2020002424A JP 2021108573 A JP2021108573 A JP 2021108573A
- Authority
- JP
- Japan
- Prior art keywords
- medium
- color
- phosphate
- aldol
- legionella spp
- 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.)
- Granted
Links
- 241000589248 Legionella Species 0.000 title claims abstract description 70
- 239000001963 growth medium Substances 0.000 title abstract description 11
- 230000004069 differentiation Effects 0.000 title abstract description 7
- 238000004040 coloring Methods 0.000 title abstract 3
- HSJKGGMUJITCBW-UHFFFAOYSA-N 3-hydroxybutanal Chemical compound CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 claims abstract description 82
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 42
- 239000010452 phosphate Substances 0.000 claims abstract description 42
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 40
- 208000007764 Legionnaires' Disease Diseases 0.000 claims description 67
- 229920001817 Agar Polymers 0.000 claims description 36
- 239000008272 agar Substances 0.000 claims description 36
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 239000007991 ACES buffer Substances 0.000 claims description 11
- 239000004471 Glycine Substances 0.000 claims description 11
- 108010093965 Polymyxin B Proteins 0.000 claims description 11
- 108010059993 Vancomycin Proteins 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 11
- VTYGSWGUBDXCRA-UHFFFAOYSA-L dipotassium;2-oxopentanedioate Chemical compound [K+].[K+].[O-]C(=O)CCC(=O)C([O-])=O VTYGSWGUBDXCRA-UHFFFAOYSA-L 0.000 claims description 11
- CADNYOZXMIKYPR-UHFFFAOYSA-B ferric pyrophosphate Chemical compound [Fe+3].[Fe+3].[Fe+3].[Fe+3].[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O CADNYOZXMIKYPR-UHFFFAOYSA-B 0.000 claims description 11
- 229920000024 polymyxin B Polymers 0.000 claims description 11
- 229960005266 polymyxin b Drugs 0.000 claims description 11
- 229960003165 vancomycin Drugs 0.000 claims description 11
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 11
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 8
- 229920000858 Cyclodextrin Polymers 0.000 claims description 7
- 239000001116 FEMA 4028 Substances 0.000 claims description 7
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 7
- 229960004853 betadex Drugs 0.000 claims description 7
- 238000012850 discrimination method Methods 0.000 claims description 6
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims 1
- 229960003942 amphotericin b Drugs 0.000 claims 1
- 239000002609 medium Substances 0.000 description 111
- 238000011161 development Methods 0.000 description 21
- 239000000758 substrate Substances 0.000 description 18
- 238000012360 testing method Methods 0.000 description 18
- 239000006152 selective media Substances 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 15
- 239000006160 differential media Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000007793 ph indicator Substances 0.000 description 8
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 208000004023 Legionellosis Diseases 0.000 description 5
- 230000007613 environmental effect Effects 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 241000589242 Legionella pneumophila Species 0.000 description 4
- 229940115932 legionella pneumophila Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 206010035664 Pneumonia Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 239000003429 antifungal agent Substances 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- IUBOLLVEZYFKQN-UHFFFAOYSA-N (6-chloro-1h-indol-3-yl) hydrogen phosphate;(4-methylphenyl)azanium Chemical compound CC1=CC=C(N)C=C1.ClC1=CC=C2C(OP(O)(=O)O)=CNC2=C1 IUBOLLVEZYFKQN-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000007609 bcye-agar Substances 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- YKJYKKNCCRKFSL-RDBSUJKOSA-N (-)-anisomycin Chemical compound C1=CC(OC)=CC=C1C[C@@H]1[C@H](OC(C)=O)[C@@H](O)CN1 YKJYKKNCCRKFSL-RDBSUJKOSA-N 0.000 description 1
- SUWPNTKTZYIFQT-XZINFULNSA-N 5-bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 SUWPNTKTZYIFQT-XZINFULNSA-N 0.000 description 1
- 108010055851 Acetylglucosaminidase Proteins 0.000 description 1
- YKJYKKNCCRKFSL-UHFFFAOYSA-N Anisomycin Natural products C1=CC(OC)=CC=C1CC1C(OC(C)=O)C(O)CN1 YKJYKKNCCRKFSL-UHFFFAOYSA-N 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 241001135524 Legionella anisa Species 0.000 description 1
- 241000589239 Legionella erythra Species 0.000 description 1
- 241000589259 Legionella feeleii Species 0.000 description 1
- 241000589264 Legionella longbeachae Species 0.000 description 1
- 241000186780 Listeria ivanovii Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 241000415842 Morinda royoc Species 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 206010054161 Pontiac fever Diseases 0.000 description 1
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- LUQUBIOHRRJPBW-UHFFFAOYSA-N [1-[2-[4-(dimethylamino)benzoyl]phenyl]indol-3-yl] dihydrogen phosphate Chemical compound CN(C)C1=CC=C(C=C1)C(=O)C2=CC=CC=C2N3C=C(C4=CC=CC=C43)OP(=O)(O)O LUQUBIOHRRJPBW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000004378 air conditioning Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
本発明は、環境検体あるいは臨床検体からレジオネラ症の原因菌であるレジオネラ属菌(Ｌｅｇｉｏｎｅｌｌａ ｓｐｐ．)を迅速に鑑別するためのレジオネラ属菌鑑別用発色培地に関する。 The present invention relates to a color-developing medium for differentiating Legionella spp. To rapidly discriminate Legionella spp., Which is the causative bacterium of Legionella disease, from an environmental sample or a clinical sample.
レジオネラ属菌は、劇症型肺炎と一過性のポンティアック熱レジオネラ症の二つの病型に分類されるレジオネラ症の原因菌である。 Legionella spp. Is the causative agent of legionellosis, which is classified into two types, fulminant pneumonia and transient Pontiac fever legionellosis.
レジオネラ属菌は、本来、土壌や淡水等の環境中に生息する環境細菌であるが、クーリングタワー等の空気調和設備、噴水等の水景施設、公衆浴場の給湯設備やシャワーヘッド、ジャグジー、加湿器等の人工環境水においても発育し、エアロゾルを介した気道感染により肺炎を引き起こすことが知られている。高齢者、新生児や免疫不全の患者等がレジオネラ症のリスクグループとされており、特に免疫不全患者では肺炎の劇症化と多臓器不全の併発の可能性が高い。 Legionella spp. Are originally environmental bacteria that live in the environment such as soil and fresh water, but air conditioning equipment such as cooling towers, waterscape facilities such as fountains, hot water supply equipment in public baths, shower heads, jacuzzis, humidifiers, etc. It is known that it grows in artificial environmental water and causes pneumonia due to respiratory tract infection via aerosol. Elderly people, newborns, immunocompromised patients, etc. are considered to be a risk group for legionellosis, and especially immunodeficient patients are highly likely to have fulminant pneumonia and multiple organ failure.
臨床検査や環境検査において、レジオネラ属菌の分離は、主要な検査法の一つであるが、レジオネラ属菌の発育条件は一般の細菌に比較して極めて厳しく、レジオネラ属菌用の培養培地として種々の培地が考案されているものの、発育至適ｐＨ は６．９０±０．０５、培養温度は３６±１℃、酸素が十分存在する環境において初代分離に３日以上を要する。このため、分離培養の結果が患者発生時の原因（起炎菌）の特定や治療方針の決定に反映されにくいが、疫学的観点からは依然として重要である。 Isolation of Legionella spp. Is one of the main test methods in clinical tests and environmental tests, but the growth conditions of Legionella spp. Are extremely strict compared to general bacteria, and as a culture medium for Legionella spp. Although various media have been devised, the optimum pH for growth is 6.90 ± 0.05, the culture temperature is 36 ± 1 ° C., and it takes 3 days or more for the primary separation in an environment where sufficient oxygen is present. For this reason, it is difficult for the results of isolation culture to be reflected in the identification of the cause (causing bacteria) at the time of patient development and the determination of treatment policy, but it is still important from an epidemiological point of view.
また、微生物検査の分野においては、人、資源、時間面でのコスト低減が厳しく要求されており、検出対象の細菌の選択分離培養と鑑別培養を同時に行うことのできる選択鑑別培地（又は選択分離鑑別培地ともいう）が求められる傾向が強まっており、レジオネラ属菌においても多分に漏れない。 Further, in the field of microbiological examination, cost reduction in terms of human resources, resources, and time is strictly required, and a selective differential culture medium (or selective separation) capable of simultaneously performing selective isolation culture and differential culture of the bacterium to be detected. There is an increasing tendency to require a differential medium), and even Legionella spp. Will probably not leak.
レジオネラ属菌用の培養培地としては、ＣＹＥ寒天培地、ＢＣＹＥ寒天培地、ＢＣＹＥα寒天培地等の非選択培地、ＧＶＰＣ寒天培地、ＷＹＯα寒天培地、ＰＡＶ寒天培地、ＣＣＶＣ寒天培地等のレジオネラ属菌の選択分離培地（或は単に選択培地、又は、分離培地ともいう）が挙げられるほか、選択分離と同時に、ｐＨ指示薬、発色基質、蛍光基質等の添加剤を利用して検出対象である細菌の生化学的性状を指標として鑑別までを行う選択鑑別培地が挙げられる。 As the culture medium for Regionella spp., Non-selective media such as CYE agar medium, BCYE agar medium, BCYEα agar medium, GVPC agar medium, WYOα agar medium, PAV agar medium, CCVC agar medium and other non-selective media, selective isolation of Regionella spp. A medium (or simply a selective medium or a separation medium) can be mentioned, and at the same time as the selective separation, an additive such as a pH indicator, a color-developing substrate, or a fluorescent substrate is used to biochemically detect the bacterium. Examples thereof include a selective differentiation medium that performs differentiation using properties as an index.
これまでに知られている選択鑑別培地としては、ｐH指示薬を利用したＭＷＹ寒天培地がある（非特許文献１）。ｐH指示薬を利用した鑑別の機構は、検出対象の細菌（菌種）の糖の資化能を利用したｐＨ指示薬の変化によるコロニー色の呈色、すなわち発色であるが、元来、レジオネラ属菌には糖の資化能はない。ＭＷＹ寒天培地においては、レジオネラ属菌に僅かに認められるアミノ酸分解能を利用してｐＨ指示薬を呈色させているのであるが、その発色の程度は非常に弱く、十分な時間（通常３〜５日）培養を行ってもコロニー色の視認性が低いことが難点であった。 As a selective differential medium known so far, there is a MWY agar medium using a pH indicator (Non-Patent Document 1). The mechanism of discrimination using a pH indicator is colony coloration, that is, color development, due to changes in the pH indicator using the sugar assimilation ability of the bacterium (species) to be detected, but originally, Legionella spp. Has no ability to assimilate sugar. In the MWY agar medium, the pH indicator is colored using the amino acid resolution slightly observed in Legionella spp., But the degree of color development is very weak, and it takes a sufficient time (usually 3 to 5 days). ) The problem was that the visibility of the colony color was low even after culturing.
また、ＭＷＹ寒天培地では、Ｌｅｇｉｏｎｅｌｌａ ｐｎｅｕｍｏｐｈｉｌａ、Ｌ．ｂｏｓｅｍａｎｉｉ、Ｌ．ｍｉｃｄａｄｅｉの3菌種を鑑別するのみであるが、レジオネラ属菌には多数の菌種が知られており、レジオネラ属菌は潜在的に病原性があると考えられていることから、先の3菌種の鑑別だけでは不十分と言わざるを得ない。 In addition, in MWY agar medium, Legionella pneumophila, L. bosemani, L. Only three species of micdadei are differentiated, but since many species of Legionella are known and Legionella is considered to be potentially pathogenic, the above three It must be said that the discrimination of bacterial species is not enough.
他方、発色基質を利用したコロニーの発色による標的細菌の鑑別方法では、細菌の持つ酵素の作用（酵素活性）を利用して発色基質を加水分解することで遊離される発色団化合物および／ または蛍光発色団化合物が発色および／または蛍光を呈すること、すなわち呈色反応（発色反応ともいう）の原理を用いており、この呈色反応がコロニー色として現れることで、標的細菌コロニーの目視による視認性を高めたり、装置による簡易かつ高感度な測定を可能にしたりする。また、呈色反応は、生化学、免疫学、分子生物学、微生物学など幅広い分野においても、酵素活性検出指標として応用されている。特に微生物学分野では、酵素活性を測定することにより微生物を検出または識別する方法に応用されており、コロニー所見の鑑別において検査者の熟練を要さず、煩雑な微生物確認試験を省略できることから、簡易で迅速な方法として汎用されている。 On the other hand, in the method for distinguishing target bacteria by color development of colonies using a color-developing substrate, a chromophore compound and / or fluorescence released by hydrolyzing the color-developing substrate by utilizing the action (enzyme activity) of an enzyme possessed by the bacteria. The chromophore compound exhibits color development and / or fluorescence, that is, it uses the principle of a color reaction (also called a color reaction), and this color reaction appears as a colony color, so that the target bacterial colony is visually visible. It enables simple and highly sensitive measurement by the device. The color reaction is also applied as an enzyme activity detection index in a wide range of fields such as biochemistry, immunology, molecular biology, and microbiology. Especially in the field of microbiology, it is applied to a method of detecting or identifying microorganisms by measuring enzyme activity, and it does not require the skill of an inspector to distinguish colony findings, and a complicated microorganism confirmation test can be omitted. It is widely used as a simple and quick method.
例えば、特許文献１ には、発色基質である５−ブロモ−６−クロロ−３−インドキシルホスフェートを培地に含有させ、スタフィロコッカス・アウレウス（ Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ、以下、黄色ブドウ球菌という。） のホスファターゼ活性を検出する方法が記載されている。 For example, in Patent Document 1, 5-bromo-6-chloro-3-indoxyl phosphate, which is a color-developing substrate, is contained in a medium, and Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus) phosphatase. Methods for detecting activity are described.
また、特許文献２ には、５−ブロモ−４−クロロ−３−インドキシル−β−グルコシドを培地に含有させてエンテロコッカス（Ｅｎｔｅｒｏｃｏｃｃｕｓ）属細菌のβ− グルコシダーゼ活性を検出する方法、特許文献３ には、５−ブロモ−４−クロロ−３−インドリル−Ｎ−アセチル−β−Ｄ−グルコサミニドを培地に含有させてカンジダ・アルビカンス（Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ）のＮ−アセチル−β−Ｄ−グルコサミニダーゼ活性を検出する方法が記載されている。 Further, Patent Document 2 describes a method for detecting β-glucosidase activity of Enterococcus bacteria by containing 5-bromo-4-chloro-3-indoxyl-β-glucoside in a medium. Detects the N-acetyl-β-D-glucosaminidase activity of Candida albicans by including 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-glucosaminide in the medium. The method is described.
さらに特許文献４ には、５−ブロモ−４−クロロ−３−インドリルホスファチジルミオイノシトールを培地に含有させてリステリア・モノサイトジェネス（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ） およびリステリア・イヴァノヴィ（Ｌｉｓｔｅｒｉａ ｉｖａｎｏｖｉｉ） のホスファチジルイノシトール特異的ホスホリパーゼＣ活性を検出する方法が記載されている。 Further, Patent Document 4 describes that 5-bromo-4-chloro-3-indrill phosphatidylinositol is contained in a medium and is specific to Listeria monocytogenes and Listeria ivanovii. Methods for detecting phospholipase C activity have been described.
しかし、レジオネラ属菌のコロニーを発色しうる適当な発色基質はこれまで見いだされていない。 However, no suitable color-developing substrate that can color the colonies of Legionella spp. Has been found so far.
本発明の目的は、短い培養期間で、Ｌｅｇｉｏｎｅｌｌａ ｐｎｅｕｍｏｐｈｉｌａおよびその他のレジオネラ属菌のコロニー色を明瞭に発色させ、レジオネラ属菌の鑑別性に優れたレジオネラ属菌の鑑別用発色培地を提供することにある。 An object of the present invention is to provide a color-developing medium for differentiating Legionella spp., Which clearly develops the colony color of Legionella pneumophila and other Legionella spp. be.
本発明者は、前記目的を達成するために鋭意検討を行った結果、レジオネラ属菌用培養培地において、発色基質としてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを用いることで、培養２日目からレジオネラ属菌を特異的且つ明瞭に発色させることができることを見出し、本発明を完成した。 As a result of diligent studies to achieve the above object, the present inventor specifically uses Aldol 515 phosphate as a color-developing substrate in the culture medium for Legionella spp. The present invention has been completed by finding that the color can be clearly developed.
すなわち、本発明の構成は、以下の［１］から［８］の通りである。
［１］Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含有することを特徴とするレジオネラ属菌の鑑別用発色培地。
［２］Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅの含有量が０．０１ｇ／Ｌ以上である［１］に記載の培地。
［３］培地１Ｌあたり、酵母エキス１０ｇ 、α‐ケトグルタル酸カリウム１ｇ 、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、活性炭２ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する［１］または［２］に記載のレジオネラ属菌の鑑別用発色培地。
［４］培地１Ｌあたり、酵母エキス１０ｇ、α‐ケトグルタル酸カリウム１ｇ、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、β-シクロデキストリン１．０〜２５．０ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する［１］または［２］に記載のレジオネラ属菌の鑑別用発色培地。
［５］Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含むレジオネラ属菌鑑別用発色培地を用いるレジオネラ属菌の鑑別方法。
［６］Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅの含有量が０．０１ｇ／Ｌ以上である［５］に記載の鑑別方法。
［７］培地１ Ｌ あたり、酵母エキス１０ｇ 、α‐ケトグルタル酸カリウム１ｇ 、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、活性炭２ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する培地を用いる［５］または［６］に記載の鑑別方法。
［８］培地１ Ｌ あたり、酵母エキス１０ｇ 、α‐ケトグルタル酸カリウム１ｇ 、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、β-シクロデキストリン１．０〜２５．０ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する培地を用いる［５］または［６］に記載の鑑別方法。
That is, the configuration of the present invention is as follows [1] to [8].
[1] A color-developing medium for differentiating Legionella spp., Which contains Aldol 515 phosphate.
[2] The medium according to [1], wherein the content of Aldol 515 phosphate is 0.01 g / L or more.
[3] Yeast extract 10 g, α-ketoglutarate potassium 1 g, ACES (buffer) 10 g, glycine 3 g, activated carbon 2 g, soluble iron pyrophosphate 0.2 g, vancomycin 1 to 5 mg, polymyxin B 5,000 to 100 per 1 L of medium. The color-developing medium for differentiating Legionella spp. According to [1] or [2], which contains 000 unit, 20 to 80 mg of amhotericin B and 15 to 18 g of agar.
[4] Yeast extract 10 g, α-ketoglutarate potassium 1 g, ACES (buffer) 10 g, glycine 3 g, β-cyclodextrin 1.0 to 25.0 g, soluble iron pyrophosphate 0.2 g, vancomycin 1 per 1 L of medium. The color-developing medium for differentiating Legionella spp. According to [1] or [2], which contains ~ 5 mg, polymyxin B 5,000 to 100,000 units, amhotericin B 20 to 80 mg, and agar 15 to 18 g.
[5] A method for differentiating Legionella spp. Using a color-developing medium for discriminating Legionella spp. Containing Aldol 515 phosphate.
[6] The discrimination method according to [5], wherein the content of Aldol 515 phosphate is 0.01 g / L or more.
[7] Yeast extract 10 g, α-ketoglutarate potassium 1 g, ACES (buffer) 10 g, glycine 3 g, activated carbon 2 g, soluble iron pyrophosphate 0.2 g, vancomycin 1 to 5 mg, polymyxin B 5,000 to 1 L of medium. The discrimination method according to [5] or [6], which uses a medium containing 100,000 units, 20 to 80 mg of amhotericin B, and 15 to 18 g of agar.
[8] Yeast extract 10 g, α-ketoglutarate potassium 1 g, ACES (buffer) 10 g, glycine 3 g, β-cyclodextrin 1.0 to 25.0 g, soluble iron pyrophosphate 0.2 g, vancomycin per 1 L of medium. The discrimination method according to [5] or [6], which uses a medium containing 1 to 5 mg, polymyxin B 5,000 to 100,000 units, amhotericin B 20 to 80 mg, and agar 15 to 18 g.
本発明により、レジオネラ属菌用の培養培地において、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを用いることで、培養２日目から、Ｌｅｇｉｏｎｅｌｌａ ｐｎｅｕｍｏｐｈｉｌａおよびその他のレジオネラ属菌のコロニー色を明瞭に発色させ、レジオネラ属菌の鑑別性に優れたレジオネラ属菌の鑑別用発色培地が得られる。 According to the present invention, by using Aldol 515 phosphorate in a culture medium for Legionella spp., From the second day of culture, the colony color of Legionella pneumophila and other Legionella spp. Is clearly developed, and the distinctiveness of Legionella spp. An excellent color-developing medium for differentiating Legionella spp. Can be obtained.
「Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ」は、１−（２−（４−ジメチルアミノベンゾイル）フェニル）−１Ｈ−インドール−３−イルリン酸（ＣＡＳ Ｎｏ．１６３０９４０−４６−３）であり、Ｂｉｏｓｙｎｔｈ社製Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅのナトリウム塩として入手可能である。 "Aldol 515 phosphate" is 1- (2- (4-dimethylaminobenzoyl) phenyl) -1H-indole-3-yl phosphate (CAS No. 1630940-46-3) of Aldol 515 phosphate manufactured by Biosynth. It is available as a sodium salt.
Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅは、黄色ブドウ球菌や偏性嫌気性菌の発色基質としての使用が推奨されているが、これまでにレジオネラ属菌の鑑別培地に用いられた例はない。 Aldol 515 phosphate is recommended for use as a color-developing substrate for Staphylococcus aureus and obligate anaerobic bacteria, but has never been used as a differential medium for Legionella spp.
レジオネラ属菌鑑別用発色培地におけるコロニー発色に適したＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅの濃度は、０．０１ｇ／Ｌ以上、好ましくは０．０２ｇ／以上、より好ましくは０．０５ｇ／Ｌ以上である。
また、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅは、その濃度 ０．４ｇ／Ｌまでは、レジオネラ属菌の発育への影響、すなわち、発育阻害が認められず、レジオネラ属菌のコロニーを明瞭に発色させることができる。
The concentration of Aldol 515 phosphate suitable for colony color development in the color-developing medium for Legionella genus differentiation is 0.01 g / L or more, preferably 0.02 g / or more, and more preferably 0.05 g / L or more.
In addition, Aldol 515 phosphate has no effect on the growth of Legionella spp., That is, growth inhibition is not observed up to a concentration of 0.4 g / L, and colonies of Legionella spp. Can be clearly colored.
更に、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含むレジオネラ属菌用の鑑別培地では、培養２日目からレジオネラ属菌のコロニーに明瞭な発色がみられ、また、２日以上培養を続けても、レジオネラ属菌のコロニーの発色に変化はみられず、安定的に明瞭な発色がみられる。 Furthermore, in the differential medium for Legionella spp. Containing Aldol 515 phosphate, clear color development was observed in the colonies of Legionella spp. From the second day of culture, and even if the culture was continued for 2 days or more, the colonies of Legionella spp. There is no change in the color development of, and stable and clear color development is observed.
Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含むレジオネラ属菌用の鑑別培地における発色は、実体顕微鏡によるコロニー観察、すなわち斜光法によるレジオネラ属菌のカットグラス様あるいはモザイク様とも呼ばれる特徴的外観構造の観察を妨げない。 Color development in the differential medium for Legionella spp., Containing Aldol 515 phosphate, does not interfere with colony observation with a stereomicroscope, i.e., observation of the characteristic appearance structure of Legionella spp. Also called cut glass-like or mosaic-like by the oblique light method.
本発明の鑑別培地によれば、従来は、斜光法によるレジオネラ属菌の特徴的外観構造の観察のみを指標としていたところに、明瞭な発色という指標が加わることで、培地上に発育したコロニーがレジオネラ属菌であるか否かについて、より確度の高い結果が得られる。 According to the differential medium of the present invention, conventionally, only the observation of the characteristic appearance structure of Legionella spp. By the oblique light method was used as an index, but by adding an index of clear color development, colonies grown on the medium can be obtained. More accurate results can be obtained as to whether or not it is a Legionella spp.
本発明の鑑別培地のベースとして使用可能なレジオネラ属菌用の培養培地としては、ＣＹＥ寒天培地、ＢＣＹＥ寒天培地、ＢＣＹＥα寒天培地等の非選択培地、ＧＶＰ寒天培地、ＷＹＯα寒天培地等のレジオネラ属菌の選択分離培地（或は単に選択培地、又は、分離培地ともいう）が挙げられるが、レジオネラの培養を目的とする寒天培地であれば、特に限定されず、本明細書においては、特に断りの無い限り、レジオネラ属菌用の培養培地は、非選択培地と選択分離培地を含む。その他、ＭＷＹ寒天培地等のｐＨ指示薬を用いるレジオネラ属菌用の鑑別培地において、ｐＨ指示薬の代わりに、或いは、ｐＨ指示薬と共にＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを用いることができる。つまり、本発明の鑑別培地は、選択分離培養と鑑別培養を同時に行う選択鑑別培地であってもよく、鑑別培養のみを行う鑑別培地であってもよい。
また、非選択培地、選択培地を問わず、レジオネラ属菌用の培養培地において、活性炭を用いる場合（黒色培地）、活性炭の代わりにβ-シクロデキストリンを含む場合（無色透明培地）、いずれの場合においても、レジオネラ属菌のコロニーを明瞭に発色させることができる。
As the culture medium for Legionella spp. That can be used as the base of the differential medium of the present invention, non-selective media such as CYE agar medium, BCYE agar medium, BCYEα agar medium, GVP agar medium, WYOα agar medium and the like. (Although it is also simply referred to as a selective medium or a separate medium), the medium is not particularly limited as long as it is an agar medium for the purpose of culturing Regionella, and is not particularly specified in the present specification. Unless otherwise specified, culture medium for Legionella spp. Includes non-selective medium and selective isolation medium. In addition, in a differential medium for Legionella spp. Using a pH indicator such as MWY agar medium, Aldol 515 phosphate can be used instead of the pH indicator or together with the pH indicator. That is, the differential medium of the present invention may be a selective differential culture in which selective separation culture and differential culture are performed at the same time, or may be a differential medium in which only differential culture is performed.
In addition, regardless of whether it is a non-selective medium or a selective medium, when active charcoal is used (black medium) or β-cyclodextrin is contained instead of active charcoal (colorless transparent medium) in the culture medium for Legionella spp. Also, the colonies of Legionella spp. Can be clearly colored.
例えば、現在、レジオネラ属菌用の非選択培地のうち、レジオネラ属菌の生育能に最も優れた培地として知られているのがＢＣＹＥα寒天培地であり、ＢＣＹＥα寒天培地は、その組成成分として、酵母エキス、α‐ケトグルタル酸カリウム、ＡＣＥＳ（緩衝剤）、グリシン、活性炭、可溶性ピロリン酸鉄、および寒天を含有する。また、レジオネラ属菌の培養検査においてよく用いられている各種の選択培地（ＢＭＰＡα寒天培地、ＧＶＰＣ寒天培地、ＷＹＯ寒天培地、ＷＹＯα寒天培地、ＰＡＶ寒天培地、ＣＣＶＣ寒天培地、ＭＷＹ寒天培地）は、いずれもＢＣＹＥα寒天培地の組成成分をベースにして、それぞれ種々の抗菌薬と抗真菌剤を含有しており、抗菌薬としては、ポリミキシンＢ、セファマンドール、バンコマイシン、セファロチン、コリスチン、抗真菌剤としては、アニソマイシン、シクロヘキシミド、アムホテリシンＢが、それぞれの選択培地において所定の組合せと所定の濃度で用いられている。本発明の鑑別培地のベースに用いるレジオネラ属菌培養用の培地として、これらの非選択培地や選択培地をベースとする非選択培地の組成成分のまま、選択培地それぞれの抗菌薬および抗真菌剤の組合せと濃度で用いることもできるが、ベースとする非選択培地の各種組成成分の濃度や、選択培地に添加する抗菌薬および抗真菌剤の組合せと濃度を変えた改良培地を用いることもできる。 For example, among the non-selective media for Legionella spp., BCYEα agar medium is currently known as the medium having the best growth ability of Legionella spp., And BCYEα agar medium is a constituent of yeast. Contains extract, potassium α-ketoglutarate, ACES (buffer), glycine, activated charcoal, soluble iron pyrophosphate, and agar. In addition, any of the various selective media (BMPAα agar medium, GVPC agar medium, WYO agar medium, WYOα agar medium, PAV agar medium, CCVC agar medium, MWY agar medium) that are often used in the culture test of Legionella spp. Also contains various antibacterial agents and antifungal agents based on the composition components of BCYEα agar medium. , Anisomycin, cycloheximide, and amhotericin B are used in the respective selective media in a predetermined combination and in a predetermined concentration. As a medium for culturing Legionella spp. Used as the base of the differential medium of the present invention, the compositional components of these non-selective media and the non-selective medium based on the selective medium are used as the antibacterial agents and antifungal agents of each of the selective media. It can be used in combination and concentration, but it is also possible to use an improved medium in which the concentration of various composition components of the non-selective medium as a base and the combination and concentration of the antibacterial agent and the antifungal agent added to the selective medium are changed.
本発明の実施の形態の一例として、例えば、培地１Ｌあたり、酵母エキス１０ｇ、α‐ケトグルタル酸カリウム１ｇ、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、活性炭２ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する請求項１または２に記載のレジオネラ属菌の鑑別用発色培地が挙げられる。 As an example of the embodiment of the present invention, for example, 10 g of yeast extract, 1 g of potassium α-ketoglutarate, 10 g of ACES (buffer), 3 g of glycine, 2 g of activated charcoal, 0.2 g of soluble iron pyrophosphate, and 1 vancomycin per 1 L of medium. The color-developing medium for differentiating Legionella spp. According to claim 1 or 2, which contains ~ 5 mg, polymyxin B 5,000 to 100,000 units, amhotericin B 20 to 80 mg, and agar 15 to 18 g.
また、別の例としては、例えば、培地１Ｌあたり、酵母エキス１０ｇ、α‐ケトグルタル酸カリウム１ｇ、ＡＣＥＳ（緩衝剤）１０ｇ、グリシン３ｇ、β-シクロデキストリン１．０〜２５．０ｇ、可溶性ピロリン酸鉄０．２ｇ、バンコマイシン１〜５ｍｇ、ポリミキシンＢ５，０００〜１００，０００ｕｎｉｔ、アムホテリシンＢ２０〜８０ｍｇおよび寒天１５〜１８ｇを含有する請求項１または２に記載のレジオネラ属菌の鑑別用発色培地が挙げられる。 As another example, for example, 10 g of yeast extract, 1 g of potassium α-ketoglutarate, 10 g of ACES (buffer), 3 g of glycine, 1.0 to 25.0 g of β-cyclodextrin, and soluble pyrophosphate per 1 L of medium. The color-developing medium for differentiating Legionella spp. According to claim 1 or 2, which contains 0.2 g of iron, 1 to 5 mg of vancomycin, 5,000 to 100,000 units of polymyxin B, 20 to 80 mg of amhotericin B and 15 to 18 g of agar. ..
本発明は、上述したＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含むレジオネラ属菌鑑別用発色培地とともに、当該レジオネラ属菌鑑別用発色培地を用いてレジオネラ属菌を鑑別する方法を提供する。 The present invention provides a method for differentiating Legionella spp. Using the Legionella spp. Differentiation color-developing medium containing the above-mentioned Aldol 515 phosphate.
本発明の鑑別培地において培養の対象となる微生物は、鑑別培地に生育し得るＬｅｇｉｏｎｅｌｌａ属菌であるＬ．ｐｎｅｕｍｏｐｈｉｌａ、Ｌ．ｂｏｚｅｍａｎｉｉ、Ｌ．ｌｏｎｇｂｅａｃｈａｅ、Ｌ．ｍｉｃｄａｄｅｉ、Ｌ．ｇｏｒｍａｎｉｉ、Ｌ．ｆｅｅｌｅｉｉ、Ｌ．ｊｏｒｄａｎｉｓ、Ｌ．ｃｉｎｃｉｎｎａｔｉｅｎｓｉｓ、Ｌ．ｏａｋｒｉｄｇｅｎｓｉｓ、Ｌ．ｃｉｎｃｉｎｎａｔｉｅｎｓｉｓ、Ｌ．ａｎｉｓａ、Ｌ．ｐａｒｉｓｉｅｎｓｉｓ等が具体例として挙げられるが、特にこれらに限定されない。 The microorganism to be cultured in the differential medium of the present invention is L. legionella, which can grow in the differential medium. pneumophila, L .; bozemanii, L .; longbeachae, L. et al. mickadei, L. et al. gormanii, L. et al. feeleii, L. et al. Jordanis, L. et al. cincinnitiensis, L. et al. oakridgensis, L .; cincinnitiensis, L. et al. anisa, L. et al. Specific examples include, but are not limited to, parisiensis and the like.
以下に本発明を実施例により具体的に説明するが、本発明は、実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the Examples.
レジオネラ属菌を発色させる化合物の検討
レジオネラ属菌を発色させる各種発色基質を検索するための実験を行った。
Examination of compounds that develop color of Legionella spp. Experiments were conducted to search for various color-developing substrates that develop color of Legionella spp.
（１）培地の調製
表１に示す改良ＷＹＯα培地（活性炭の代わりにβ−シクロデキストリン（表中β−ＣＤ）を８ｇ／Ｌで含むＷＹＯα培地）の組成に、細菌の持つ種々の酵素活性を利用してコロニーを発色させる発色基質として知られる各種化合物をＸ−ｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ、Ｘ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ、ｍａｇｅｎｔａ ｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ（Ｍａｇｅｎｔａ−Ｇａｌ、Ｒｅｄ−Ｇａｌ、５−Ｂｒｏｍｏ−６−ｃｈｌｏｒｏ−３−ｉｎｄｏｙｌ β−Ｄ−ｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅに同じ）、Ｓａｌｍｏｎ ｐｈｏｓｐｈａｔｅ（６−Ｃｈｌｏｒｏ−３−ｉｎｄｏｘｙｌ ｐｈｏｓｐｈａｔｅに同じ）、Ｘ−ｐｈｏｓｐｈａｔｅ、ＯＮＰＣ（２−Ｎｉｔｒｏｐｈｅｎｙｌ−β−Ｄ−ｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅに同じ）、 Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ、Ａｌｄｏｌ ５１４ ｉｎｏｓｉｔｏｌ ｐｈｏｓｐｈａｔｅ、Ａｌｄｏｌ ４７０ ｃｈｏｌｉｎｅ ｐｈｏｓｐｈａｔｅ、ＴＴＣ（２，３，５−Ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ ｃｈｌｏｒｉｄｅに同じ）をそれぞれ添加した培地を調製した。これら各種化合物の培地への添加量については、ＴＴＣを除く9種類の化合物は０．１ｇ／Lとなるように調製し、ＴＴＣのみ０．０３ｇ／Lとなるように調製した。また、対照としていずれの発色基質も含まない培地を調製した。
具体的には、表１（Ａ）の各培地成分を精製水９９０ｍＬに溶解し、１２１℃で１５分間高圧蒸気滅菌した後、５５℃の温水浴槽中にて保温し、これに表１（Ｂ）の成分であるピロリン酸鉄、バンコマイシン、ポリミキシンＢ、各種の発色基質は精製水で溶解し、アムホテリシンＢは１Ｎ ＮａＯＨ溶液で溶解し、それぞれの濾過滅菌済み溶液を調製して（対照用の培地の場合は、滅菌水を用いた）２ｍＬずつ添加し、よく攪拌した後、各培地をシャーレに２０ｍＬずつ分注し、放冷して固化させた。
(1) Preparation of medium The composition of the improved WYOα medium shown in Table 1 (WYOα medium containing β-cyclodextrin (β-CD in the table) at 8 g / L instead of activated charcoal) was added to the composition of various enzymatic activities of bacteria. Various compounds known as color-developing substrates that utilize colonies to develop color are used as X-galactopylanoside, X-glucopylanoside, magenta galactopylanoside (Magenta-Gal, Red-Gal, 5-Bromo-6-chloro-3-indext). (Same as), Salmon phosphate (same as 6-Chloro-3-indoxyl phosphate), X-phosphate, ONPC (same as 2-Nitrophenyl-β-D-galactopylanoside), Aldol 515 phosphate, Aldol A medium was prepared by adding phosphate and TTC (same as 2,3,5-Triphenyltetrazolium chloride), respectively. The amount of these various compounds added to the medium was adjusted so that 9 kinds of compounds excluding TTC were 0.1 g / L, and only TTC was 0.03 g / L. In addition, as a control, a medium containing no chromogenic substrate was prepared.
Specifically, each medium component of Table 1 (A) was dissolved in 990 mL of purified water, sterilized by high-pressure steam at 121 ° C. for 15 minutes, and then kept warm in a hot water bath at 55 ° C., and Table 1 (B) was added to this. ), Iron pyrophosphate, vancomycin, polymyxin B, and various color-developing substrates are dissolved in purified water, amhotericin B is dissolved in 1N NaOH solution, and each filter-sterilized solution is prepared (medium for control). In the case of, 2 mL each (using sterile water) was added, and after stirring well, 20 mL of each medium was dispensed into a chalet and allowed to cool to solidify.
（２）実験方法
試験菌としてＬｅｇｉｏｎｅｌｌａ ｐｎｅｕｍｏｐｈｉｌａ ５菌株（社内保存株、菌株番号：ＥＫＮ３６７７、ＥＫＮ３６７８、ＥＫＮ３６７９、ＥＫＮ３６８０、ＥＫＮ３６８２）およびＬｅｇｉｏｎｅｌｌａ ｆｒａｓｅｒｉ １菌株（社内保存株、菌株番号：ＥＫＮ３６８１）を用いた。
それぞれの菌株をＢＣＹＥα培地に接種し、３７℃、２日間培養した菌体を生理食塩水に懸濁し、ＭｃＦａｒｌａｎｄ Ｎｏ．１の菌懸濁液を調製した。この菌懸濁液１白金耳を各培地に画線塗抹接種し、３７℃、２日間培養した。
(2) Experimental method Legionella pneumophila 5 strains (in-house preserved strain, strain number: EKN3677, EKN3678, EKN3679, EKN3680, EKN3682) and Legionella Fraseri 1 strain (in-house preserved strain, strain number: EKN3681) were used as test bacteria.
Each strain was inoculated into BCYEα medium, and the cells cultured at 37 ° C. for 2 days were suspended in physiological saline to obtain McFarland No. 1 bacterial suspension was prepared. This bacterial suspension 1 platinum loop was inoculated into each medium with a stroke smear and cultured at 37 ° C. for 2 days.
（３）結果
表２に示すように、対照用改良ＷＹＯα培地、Ｓａｌｍｏｎ ｐｈｏｓｐｈａｔｅおよびＸ−ｐｈｏｓｐｈａｔｅ以外の発色基質を含む改良ＷＹＯα培地では、試験菌６菌株すべてが発育し、これらのうち、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅおよびＴＴＣを含む改良ＷＹＯα培地において、試験菌コロニーの発色（赤色）が観察された。Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅでは、コロニーが密集している部分（以下、ローン部分）および単独コロニーにおいて発色が観察され、ＴＴＣではローン部分においてのみで発色が確認された。
(3) Results As shown in Table 2, in the improved WYOα medium for control, the modified WYOα medium containing a color-developing substrate other than Salmon phosphate and X-phosphate, all 6 test strains grew, and among these, Aldol 515 phosphate was developed. And in the improved WYOα medium containing TTC, the color development (red) of the test bacterial colonies was observed. In Aldol 515 phosphate, color development was observed in the part where the colonies were dense (hereinafter referred to as the lawn part) and in a single colony, and in TTC, color development was confirmed only in the lawn part.
Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ添加量の検討
発色基質としてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ添加量の検討する実験を行った。
Examination of the amount of Aldol 515 phosphate added An experiment was conducted to examine the amount of Aldol 515 phosphate added as a color-developing substrate.
（１）培地の調製
表１に示す改良ＷＹＯα培地の組成にＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを０．０１、０．０２、０．０３、０．０４、０．０５、０．０６、０．０７、０．０８、０．１０、０．１５、０．２０、０．４０ｇ／Ｌで含む培地を調製した。また、対照としてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含まない改良ＷＹＯα培地とＷＹＯα培地（組成は表１を参照）をそれぞれ調製した。
(1) Preparation of medium Aldol 515 phosphate was added to the composition of the improved WYOα medium shown in Table 1 at 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0. A medium containing 08, 0.10, 0.15, 0.20, 0.40 g / L was prepared. In addition, as a control, an improved WYOα medium and a WYOα medium (see Table 1 for the composition) containing no Aldol 515 phosphate were prepared, respectively.
（２）実験方法
実施例１と同じ試験菌株を用い、実施例１と同様にそれぞれの菌株の菌懸濁液を調製し、各培地に接種し、３７℃、５日間培養した。
(2) Experimental Method Using the same test strain as in Example 1, a bacterial suspension of each strain was prepared in the same manner as in Example 1, inoculated into each medium, and cultured at 37 ° C. for 5 days.
（３）結果
表３に示すように、対照用改良ＷＹＯα培地およびＷＹＯα培地、発色基質Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを各種濃度で含む改良ＷＹＯα培地のすべての培地において、試験菌６菌株すべてが発育した。培養２日目で、対照用改良ＷＹＯα培地およびＷＹＯα培地では試験菌６菌株すべてにおいて発色は観察されず、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含む改良ＷＹＯα培地では試験菌６菌株すべてでＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ ０．０１ｇ／Ｌで淡桃色、０．０２ｇ／Ｌから０．０４ｇ／Ｌで桃色、０．０５ｇ／Ｌから４．００ｇ／Ｌで赤色の発色が観察された。また、これらの発色は、２日以上培養を継続しても、発色の程度が培養時間の経過により濃くなるが、変色はみられなかった。
(3) Results As shown in Table 3, all 6 test strains grew in all the mediums of the improved WYOα medium for control, the WYOα medium, and the improved WYOα medium containing the color-developing substrate Aldol 515 phosphate at various concentrations. On the second day of culture, no color development was observed in all 6 test strains in the control improved WYOα medium and WYOα medium, and Aldol 515 phosphate 0.01 g / L in all 6 test strains in the improved WYOα medium containing Aldol 515 phosphate. Light pink, pink at 0.02 g / L to 0.04 g / L, and red at 0.05 g / L to 4.00 g / L were observed. In addition, even if the culture was continued for 2 days or more, the degree of color development became darker with the lapse of the culture time, but no discoloration was observed.
異なる培地におけるＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅによるレジオネラ属菌の発色の検討
従来用いられているレジオネラ属菌用培地として、活性炭を含むＷＹＯα培地においてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを発色基質としてレジオネラ属菌を発色できるか検討する実験を行った。
Examination of color development of Legionella spp. By Aldol 515 phosphate in different medium An experiment to examine whether Aldol 515 phosphate can be used as a color-developing substrate in WYOα medium containing activated charcoal as a conventionally used medium for Legionella spp. went.
（１）培地の調製
表１に示すＷＹＯα培地および改良ＷＹＯα培地の組成にＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを０．０５ｇ／Ｌで含む培地をそれぞれ調製した。また、対照としてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含まない培地もＷＹＯα培地および改良ＷＹＯα培地のそれぞれで調製した。
(1) Preparation of medium A medium containing Aldol 515 phosphate at 0.05 g / L in the composition of the WYOα medium and the improved WYOα medium shown in Table 1 was prepared. In addition, as a control, a medium containing no Aldol 515 phosphate was also prepared as a WYOα medium and an improved WYOα medium, respectively.
（２）実験方法
実施例１と同じ試験菌株を用い、実施例１と同様にそれぞれの菌株の菌懸濁液を調製し、各培地に接種し、３７℃、２日間培養した。
(2) Experimental Method Using the same test strain as in Example 1, a bacterial suspension of each strain was prepared in the same manner as in Example 1, inoculated into each medium, and cultured at 37 ° C. for 2 days.
（３）結果
表４に示すように、ＷＹＯα培地および改良ＷＹＯα培地に対する発色基質Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅ添加の有無にかかわらず、すべての培地において、試験菌６菌株はすべて発育した。Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含まないＷＹＯα培地および改良ＷＹＯα培地ではすべての試験菌株において発色は観察されず、Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含むＷＹＯα培地および改良ＷＹＯα培地ではすべての試験菌株において赤色の発色が観察された。
(3) Results As shown in Table 4, all 6 strains of the test bacteria grew in all the media regardless of the addition of the color-developing substrate Aldol 515 phosphate to the WYOα medium and the improved WYOα medium. No color development was observed in all test strains in WYOα medium and improved WYOα medium containing Aldol 515 phosphate, and red color development was observed in all test strains in WYOα medium and improved WYOα medium containing Aldol 515 phosphate.
Ｌ．ｐｎｅｕｍｏｐｈｉｌａ以外のレジオネラ属菌の発色の確認
実施例１から３までで用いた試験菌株とは異なるレジオネラ属菌種、菌株についてＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを発色基質として発色できるかを確認する実験を行った。
L. Confirmation of color development of Legionella spp. Other than pneumophila An experiment was conducted to confirm whether Aldol 515 phosphate could be used as a color-developing substrate for Legionella spp. Strains and strains different from the test strains used in Examples 1 to 3.
（１）培地の調製
表１に示す改良ＷＹＯα培地の組成にＡｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを０．０５ｇ／Ｌで含む培地を調製した。
(1) Preparation of medium A medium containing Aldol 515 phosphate at 0.05 g / L in the composition of the improved WYOα medium shown in Table 1 was prepared.
（２）実験方法
試験菌として、Ｌｅｇｉｏｎｅｌｌａ ａｎｉｓａ（社内保存株、菌株番号：ＥＫＮ５２８２）、Ｌｅｇｉｏｎｅｌｌａ ｅｒｙｔｈｒａ（社内保存株、菌株番号：ＥＫＮ５８８７）、Ｌｅｇｉｏｎｅｌｌａ ｆｅｅｌｅｉｉ（社内保存株、菌株番号：ＥＫＮ５８３１）、Ｌｅｇｉｏｎｅｌｌａ ｆｒａｓｅｒｉ（社内保存株、菌株番号：ＥＫＮ３６８３）、Ｌｅｇｉｏｎｅｌｌａ ｌｏｎｇｂｅａｃｈａｅ（社内保存株、菌株番号：ＥＫＮ３６８９）の５菌株を用いた。実施例１と同様にそれぞれの菌株の菌懸濁液を調製し、培地に接種し、３７℃、２日間培養した。
(2) Experimental method As test bacteria, Legionella anisa (in-house preserved strain, strain number: EKN5282), Legionella erythra (in-house preserved strain, strain number: EKN5887), Legionella feeleii (in-house preserved strain, strain number: EKN5831), Legionella. (In-house preserved strain, strain number: EKN3683) and Legionella longbeachae (in-house preserved strain, strain number: EKN3689) were used. A bacterial suspension of each strain was prepared in the same manner as in Example 1, inoculated into a medium, and cultured at 37 ° C. for 2 days.
（３）結果
表５に示すように、発色基質Ａｌｄｏｌ ５１５ ｐｈｏｓｐｈａｔｅを含む改良ＷＹＯα培地で、試験菌５菌株すべてが発育し、赤色の発色が観察された。
(3) Results As shown in Table 5, all 5 strains of the test bacteria grew in the improved WYOα medium containing the color-developing substrate Aldol 515 phosphate, and red color development was observed.
本発明は、培養２日目からレジオネラ属菌のコロニーを明瞭に発色させるレジオネラ属菌の鑑別用発色培地を提供できる。レジオネラ症の原因菌を迅速に検出することができるため、臨床検査においては治療方針、浴槽水や温泉水、或いは冷却水等の環境水検査においては各種設備の清掃・消毒等の対応方針の早期決定を可能にする The present invention can provide a color-developing medium for differentiating Legionella spp. That clearly develops a color of Legionella spp. Colonies from the second day of culture. Since the causative bacteria of legionellosis can be detected quickly, the treatment policy is early in clinical examinations, and the response policy such as cleaning and disinfection of various facilities is early in environmental water inspections such as bathtub water, hot spring water, or cooling water. Make decisions possible
Claims (8)
10 g of yeast extract, 1 g of potassium α-ketoglutarate, 10 g of ACES (buffer), 3 g of glycine, 1.0 to 25.0 g of β-cyclodextrin, 0.2 g of soluble iron pyrophosphate, 1 to 5 mg of vancomycin, per 1 L of medium. The discrimination method according to claim 5 or 6, wherein a medium containing polymyxin B 5,000 to 100,000 units, amhotericin B 20 to 80 mg, and agar 15 g to 18 is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020002424A JP7503384B2 (en) | 2020-01-09 | 2020-01-09 | Chromogenic medium for Legionella species identification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020002424A JP7503384B2 (en) | 2020-01-09 | 2020-01-09 | Chromogenic medium for Legionella species identification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2021108573A true JP2021108573A (en) | 2021-08-02 |
| JP7503384B2 JP7503384B2 (en) | 2024-06-20 |
Family
ID=77058179
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020002424A Active JP7503384B2 (en) | 2020-01-09 | 2020-01-09 | Chromogenic medium for Legionella species identification |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7503384B2 (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006280219A (en) * | 2005-03-31 | 2006-10-19 | Aquas Corp | Selective culture medium for determining legionella, and method for culturing legionella bacterium |
| JP2010525803A (en) * | 2007-04-30 | 2010-07-29 | ビオメリュー | Reaction medium for identifying / detecting microorganisms |
| US20160046976A1 (en) * | 2013-04-03 | 2016-02-18 | bioMérieux | Use of at least one chromogenic and/or fluorogenic phosphatase substrate for the detection and/or enumeration of enterobacteria in a sample |
| JP2019050751A (en) * | 2017-09-13 | 2019-04-04 | Jnc株式会社 | Culture medium for clostridium bacteria selective separation |
| JP2019058088A (en) * | 2017-09-25 | 2019-04-18 | アクアス株式会社 | Method of examination of legionella bacteria |
| WO2019124904A1 (en) * | 2017-12-19 | 2019-06-27 | 주식회사 바이오맥스 | Microfluidic paper chip for detecting micro-organism, method for preparing the same and method for detecting micro-organism using the same |
| JP2021090403A (en) * | 2019-12-12 | 2021-06-17 | 栄研化学株式会社 | Transparent culture medium for culturing legionella spp. |
-
2020
- 2020-01-09 JP JP2020002424A patent/JP7503384B2/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006280219A (en) * | 2005-03-31 | 2006-10-19 | Aquas Corp | Selective culture medium for determining legionella, and method for culturing legionella bacterium |
| JP2010525803A (en) * | 2007-04-30 | 2010-07-29 | ビオメリュー | Reaction medium for identifying / detecting microorganisms |
| US20160046976A1 (en) * | 2013-04-03 | 2016-02-18 | bioMérieux | Use of at least one chromogenic and/or fluorogenic phosphatase substrate for the detection and/or enumeration of enterobacteria in a sample |
| JP2019050751A (en) * | 2017-09-13 | 2019-04-04 | Jnc株式会社 | Culture medium for clostridium bacteria selective separation |
| JP2019058088A (en) * | 2017-09-25 | 2019-04-18 | アクアス株式会社 | Method of examination of legionella bacteria |
| WO2019124904A1 (en) * | 2017-12-19 | 2019-06-27 | 주식회사 바이오맥스 | Microfluidic paper chip for detecting micro-organism, method for preparing the same and method for detecting micro-organism using the same |
| JP2021090403A (en) * | 2019-12-12 | 2021-06-17 | 栄研化学株式会社 | Transparent culture medium for culturing legionella spp. |
Non-Patent Citations (1)
| Title |
|---|
| 日本細菌学雑誌, vol. 50, no. 3, JPN6023047321, 1995, pages 745 - 764, ISSN: 0005198993 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7503384B2 (en) | 2024-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cook et al. | Selective medium for culture of Mycoplasma hyopneumoniae | |
| CA2250174C (en) | Test media and quantitative method for identification and differentiation of biological materials in a test sample | |
| JPH09512438A (en) | Microbiological medium | |
| CA2718477C (en) | Detection and enumeration of legionella pneumophila | |
| US6228606B1 (en) | Culture medium for detecting pathogenic bacteria of the genus Listeria and method for identifying said bacteria | |
| RU2342435C2 (en) | Selective culture medium for separatng and detecting types of streptococcus | |
| US8709746B2 (en) | Reaction medium for detecting and/or identifying bacteria of the Legionella genus | |
| Gomez‐Gil et al. | Isolation, enumeration, and preservation of the Vibrionaceae | |
| CN101874118A (en) | Detect and/or identify the reaction culture medium of streptococcus aureus | |
| US9404141B2 (en) | Method for detecting the presence or absence of a target microbe in a test sample | |
| JP7314230B2 (en) | H. influenzae screening method and screening medium | |
| JP7503384B2 (en) | Chromogenic medium for Legionella species identification | |
| KR101824588B1 (en) | A culture medium for screening or enrichment of methicillin-resistant s. aureus | |
| Magalhães et al. | Traditional methods of analysis for Listeria monocytogenes | |
| EP3732300B1 (en) | A novel medium for growth and antibiotic susceptibility test of mycobacteria | |
| JP3380956B2 (en) | Staphylococcus aureus detection medium | |
| RU2827840C1 (en) | Elective-differential nutrient medium for klebsiella species recovery | |
| RU2778997C1 (en) | Nutrient medium for differentiation of parahemolytic vibrios | |
| JP7254698B2 (en) | Medium for detection of Listeria | |
| Juthi | Comparative study of microbiological qualities between tap water of old Dhaka and new Dhaka with emphasis on the presence of fecal coliform bacteria | |
| Roberts | Neisseria species and Moraxella catarrhalis | |
| Konica | Isolation of multiple antibiotic resistant bacterial isolates from potable and non-potable drinking water sold in tea-stalls | |
| JP2017079733A (en) | Coloring culture medium for distinguishing candida | |
| JP2014533517A (en) | Medium and method for detecting pathogenic Yersinia enterocolitica bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20221026 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20221026 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20230105 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20231115 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231117 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20240112 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20240312 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20240312 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240531 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240610 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7503384 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |