FI91977C - Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi - Google Patents
Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi Download PDFInfo
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- FI91977C FI91977C FI910358A FI910358A FI91977C FI 91977 C FI91977 C FI 91977C FI 910358 A FI910358 A FI 910358A FI 910358 A FI910358 A FI 910358A FI 91977 C FI91977 C FI 91977C
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 244000005700 microbiome Species 0.000 title claims abstract description 17
- 238000000926 separation method Methods 0.000 title description 2
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 230000002452 interceptive effect Effects 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims 2
- CWRVKFFCRWGWCS-UHFFFAOYSA-N Pentrazole Chemical compound C1CCCCC2=NN=NN21 CWRVKFFCRWGWCS-UHFFFAOYSA-N 0.000 claims 2
- 230000001376 precipitating effect Effects 0.000 claims 2
- 210000002700 urine Anatomy 0.000 claims 2
- YJTKZCDBKVTVBY-UHFFFAOYSA-N 1,3-Diphenylbenzene Chemical group C1=CC=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 YJTKZCDBKVTVBY-UHFFFAOYSA-N 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
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- 230000001404 mediated effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 16
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 5
- 108020005199 Dehydrogenases Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 4
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 4
- 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 3
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- 239000000975 dye Substances 0.000 description 3
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 3
- 238000011045 prefiltration Methods 0.000 description 3
- 238000006479 redox reaction Methods 0.000 description 3
- 125000003831 tetrazolyl group Chemical group 0.000 description 3
- WYFYSTBFFDOVJW-UHFFFAOYSA-L 2-[4-[4-(3,5-diphenyltetrazol-2-ium-2-yl)phenyl]phenyl]-3,5-diphenyltetrazol-2-ium;dichloride Chemical compound [Cl-].[Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC(=CC=2)C=2C=CC(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=NN1C1=CC=CC=C1 WYFYSTBFFDOVJW-UHFFFAOYSA-L 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 241000588915 Klebsiella aerogenes Species 0.000 description 2
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- HWYNRVXFYFQSID-UHFFFAOYSA-M benzo[a]phenoxazin-9-ylidene(dimethyl)azanium;chloride Chemical compound [Cl-].C1=CC=C2C(N=C3C=CC(C=C3O3)=[N+](C)C)=C3C=CC2=C1 HWYNRVXFYFQSID-UHFFFAOYSA-M 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- JORABGDXCIBAFL-UHFFFAOYSA-M iodonitrotetrazolium chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C=CC=CC=2)=N1 JORABGDXCIBAFL-UHFFFAOYSA-M 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- MUUHXGOJWVMBDY-UHFFFAOYSA-L tetrazolium blue Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 MUUHXGOJWVMBDY-UHFFFAOYSA-L 0.000 description 2
- MASUWVVNWALEEM-UHFFFAOYSA-M 1-methoxy-5-methylphenazin-5-ium;methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2N=C3C(OC)=CC=CC3=[N+](C)C2=C1 MASUWVVNWALEEM-UHFFFAOYSA-M 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 240000007108 Fuchsia magellanica Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000219470 Mirabilis Species 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 241001147691 Staphylococcus saprophyticus Species 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 239000011358 absorbing material Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000008953 bacterial degradation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011152 fibreglass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
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- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 235000012711 vitamin K3 Nutrition 0.000 description 1
- 239000011652 vitamin K3 Substances 0.000 description 1
- 229940041603 vitamin k 3 Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/528—Atypical element structures, e.g. gloves, rods, tampons, toilet paper
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2304/00—Chemical means of detecting microorganisms
- C12Q2304/20—Redox indicators
- C12Q2304/24—Tetrazolium; Formazan
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/92—Nitro blue tetrazolium chloride, i.e. NBT
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Farming Of Fish And Shellfish (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
MENETELMA MIKRO-ORGANISMIEN MAARITTÅMISEKSI JA EROTTAMISEK-
SI
91977 5 Keksinnon kohteena on menetelmå, jolla voldaan nopeasti osolttaa mikro-organismien kokonaismaarå nestemåisestå naytteesta. Keksinnolle on tunnusomaista se, mika måaritel-låån patenttivaatimuksissa.
10 Mikro-organismit voidaan osoittaa biologisista naytteista suoraan konsentroimalla nåytetta erilaisilla suodatinsys-teemeillå ja vårjaamållå konsentroitunut bakteerikasvusto erilaisilla mikrobien pintarakenteisiin tarttuvilla våri-aineilla kuten safraniinilla tai fuksiinilla. Tållaisia 15 menetelmia kuvataan mm. patenteissa US-A-4336337 ja EP-A-288621.
Bakteerien nopeaan tunnistamiseen biologisista naytteista voidaan kayttaa myos biokemiallisia reaktioita kuten 20 entsymaattisia osoituksia ja sokerien kåyttokykyyn perustu-via menetelmia. Entsymaattisista reaktioista ovat erityisen nopeita sytokromien ja entsyymien, kuten dehydrogenaasien katalysoimat hapetus-pelkistysreaktiot, joita esiintyy kaikissa soluissa, seka elainsoluissa, etta bakteereissa.
25 Dehydrogenaasit ovat mitattavissa bakteereja hajottamatta ennestaan tunnetulla histokemiallisella menetelmalla, jota on kaytetty dehydrogenaasien paikantamiseen mm. leuko-syyteista ja histologisista kudosleikkeista (Michel, G. et al., Methods of Enzymatic Analysis, p. 197-232, vol. 1, 30 1983). Talloin luonnollinen vetyakseptori dehydrogenaasi- reaktiossa korvataan esim. tetratsoliumsuolalla, joka vetya vastaanottaessaan pelkistyy vMrilliseksi sinisenvioletiksi ja samalla saostuu liukenemattomaksi. Dehydrogenaasien maåritysseokseen voidaan lisata myos vålittajå, elektronin-35 siirtaja, kuten fenatsiinimetosulfaatti (PMS), joka nopeut-taa ja vahvistaa reaktiota. PMS valittaa NADrlta vedyn tetratsoliumsuolalle, joka puolestaan saostuu varillikseksi 2 91977 formatsaaniksi. PMS voidaan myds korvata muilla vetyaksep-toreilla kuten menadionilla, meldola bluella (C.I.51175; Basic blue 6), tai metoksifenatsiinimetosulfaatilla.
5 Edel lå kuvattua menetelmåå on kåytetty myos bakteerien osoittamisessa. Kun bakteeripitoiseen nåytteeseen lisåtåån hapetus-pelkistysreaktiota mittaava yhdiste kuten trifenyy-litetratsoliumkloridi (TTC) se pelkistyy ja yhdiste muuttuu vårittomåstå punaiseksi, mikå voidaan todeta visuaalisesti 10 tai kolorimetrisesti mittaamalla. TTC:n lisåksi voidaan kayttåa myos muita,nopeampia, tetratsoliumsuoloja kuten jodonitrotetratsoliumia (INT)ja neotetratsoliumkloridia (NTC), seka siniseksi muuttuvia: blue tetratsoliumia (BT) ja nitroblue tetratsoliumia (NBT) (Histological and His-15 tochemical Methods, Theory and Practice, Chapter 16, p. 258, second edition 1990).
Menetelmå on erityisen sovelias kåytettåvaksi laitteessa, jossa bakteerit mååritetåån suoraan biologisista nåytteistå 20 kerååmallå bakteerit suotimelle, jonka alia on imukykyinen materiaali, kuten US-patentissa 3741877 on esitetty. Jos nåytteesså on håiritseviå aineita, niitå ei voida US-patentissa 3741877 esitetyllå menetelmållå poistaa, pese-mållå bakteereja ennen maåritystå, koska ne valuisivat pois 25 suotimelta. Siksi edellå mainitun patentin mukaista mååri-tysmenetelmåå on kehitetty edelleen yhdiståmållå siihen bakteereja kerååvå ja bakteerien pesua edesauttava keruuosa kuten WO-patentissa 89/01966 aikaisemmin on esitetty. Patenttihakemuksessamme esitetyn keksinnon mukaisessa mene-30 telmåsså bakteereja ei tarvitse kasvattaa pesåkkeiksi, kuten US-patentissa 3741877 on esitetty, eika suodatinta tarvitse irrottaa maåritystå vårten kuten WO-patentissa 89/01966 on esitetty, vaan bakteerit voidaan måårittåå vålittdmåsti suoraan siltå suotimelta, jolle ne on keråtty.
35 Patenttihakemuksessamme esitetty laite koostuu huokoisesta suotimesta, tai useista erilaisen huokoskoon omaavista suotimista, jolle nåyte imeytetåån ja mikro-organismit
II
91977 3 todetaan valittomasti hapetus-pelkistysreaktiota mittaavan vårin avulla suoraan silta suotimelta, jonka pinnalle bakteerit jåSvat. Talloin muut eri partikkelikoon omaavat solut, kuten leukosyytit, jotka pelkistyvat samoissa 5 olosuhtelssa, voidaan erottaa eri suotimelle tal useaminalle suotimelle ja mSårittåS erikseen kyseiselta suotimelta. NaytteessS mahdollisesti vapaana olevat varia pelkistavat aineet kuten liukoiset entsyymit, askorbiinihappo, låakeai-neet, pienimolekyyliset sulfhydryyliryhmiå sisaltavat 10 molekyylit, kuten glutationi, ja muut mahdolliset pelkistavat aineet menevat sen suotimen lapi, jolle bakteerit jSavåt.
Seuraavassa keksintoa kuvataan yksityiskohtaisesti viittaa-15 malla oheisiin piirustuksiin, joissa kuva 1 esittåa laitet-ta, jossa nayte keratMan suotimelle ja kuva 2 toista lai-tetta, jossa nayte imeytetaån suotimeen. Molemmilta lait-teilta mikro-organismien kokonaismååra voidaan todeta suoraan dehydrogenaasien aktiivisuutta mittaavan vårin avulla.
20
Kuvassa 1 on esitetty lohkokaavio laitteesta, jossa neste-mainen nayte pipetoidaan esisuotimen 1 paalle, jos nayte on rakeinen, tai sisaltaa runsaasti leukosyytteja, muuten esi-suodinta 1 ei tarvita. Keruuosa 2 keskittaa naytteen mikro-25 organismit suotimelle 3, jolle bakteerit jaavat. Nayte voi 4 olla etukateen puskuroitu, tai bakteerit voidaan pesta suo-timella 3 puskuriliuoksella ja valittomasti vårjata dehydrogenaasien osoitusseoksella, jolloin mikro-organismien ymparille muodostuu sinista formatsaania. Suotimen 3 alla 30 on imukykyista materiaalia 4, johon naytteen nesteylimaara imeytyy. Kaikki osat voidaan liittMa pohja-osaan 5, jolloin laitteesta muodostuu koottu rasia.
Kuvassa 2 on naytteen kiinteåt osat sitova esisuodatin 1 35 ja suodin 3, jota mikro-organismit eivat lapaise, seka nes-tetta imevå materiaali 4 asetettu kaikki tukialustalle 2, joka voi olla esimerkiksi muovia. Suotimet 1 ja 3 ja imeva « 91977 4 materiaali 4 voidaan kiinnittåå esimerkiksi kaksipuolisella teipillå tukialustaan 2. Puskuroitu nayte imeytetåån esi-suotimeen 1 ja bakteerit kerååntyvåt sen ja suotixnen 3 rajakohtaan, josta ne voidaan todeta imeyttåmållå esisuo-5 timeen 1 dehydrogenaasien osoitusseoksella, jolloin mikro-organismien ympårille muodostuu sinistå formatsaania.
10 ESIMERKKI 1
Bakteerit olivat virtsatieinfektioissa esiintyviå ATCC:n tyyppikantoja tai kliinisia kantoja, jotka oli saatu Hel-15 singin yliopiston serobakteriologian laitokselta. Gram-negatiivisia bakteereja: Escherichia coli (ATCC 25922) ja useita kliinisia kantoja, Klebsiella aerogenes (ATCC 13883), Proteus mirabilis, Pseudomonas aeruginosa, Entero-bacter aerogenes (ATCC 13048). Gram-positiivisia bakteere-20 ja: Staphylococcus aureus (ATCC 25923), Staphylococcus saprophyticus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus faecium (ATCC 9790), Streptococcus beta-haemolyticus B-ryhma, Enterococcus sp., Corynebac-terium sp. Kaikki kannat tarkistettiin biokemiallisten 25 reaktioiden ja sokerifermentaatioiden avulla (API-10S, API-Staph, API-Strep., Bio Merieux, Montalieu-Vercieu). Hiivoja oli: Candida albicans (ATCC 28367 ja 36802). Bakteerit kas-vatettiin yli yon BHI:ssa (Brain Heart Infusion, Difco, Detroit), 37 °C ja bakteeritiheys såadettiin BHI:sta laimen-30 tamalla 10® kpl/ml steriilillM 0.9 % NaCl, joka vastasi op-tista tiheyttå 0.400, 650 nm mitattaessa (Perkin Elmer
Spectrophotometer, Norwalk). Pesåkkeiden maara/ml tarkistettiin tekemållå laimennussarja ja viljelemalla verimal-joilla. Hiivoja kasvatettiin kaksi vuorokautta 37 °C Sa-35 bouraud-maljoilla ja maljalta keratyista pesakkeista teh- tiin suspensio 10® kpl/ml kuten edella.
*
II
5 91977 Måårityksesså 100 μΐ bakteereja lisattiin 100 μΐ 0.2 Μ Tris-HCl puskurissa, pH 6.5, pH 7.2, pH 8.5 (Sigma, St. Louis), johon lisattiin 1 % :ksi glukoosia (BDH, Poole), tai fruktoosia (BDH, Poole), tai asetaattia (Merck, Darms-5 tadt), tai glutamiinihappoa (Merck, Darmstadt). Nayte siir-rettiin pipetillå suodatuslaitteistolle, johon bakteerit keskittyivat noin halkaisijaltaan 3 mm alueelle suotimen påålle (Schleicher & Schuell lasikuitusuodatin No 8, Das-sel) , tai suodatin (leukosyyttifiltteri, Pali BioSupport 10 Company, Glen Cove) kastettiin nåytteeseen ja bakteerit imeytyivåt siihen ja kulkeutuivat suodattimella rajakoh-taan, jossa on toinen suodatin (Schleicher & Schuell, No 8, Dassel). Bakteerit osoitettiin lisaåmalla 100 ul NBT-liuosta (1 mg/ml, Sigma, St Louis), johon oli lisatty 10 ul 15 PMS (lmg/ml, Sigma, St.Louis).
Vårin muodostuminen luettiin visuaalisesti eri aikojen kuluttua seuraavasti: 0= våriton, 1= heikko vari, 2= selva våri, 3= hyvå våri, 4= tumma våri, 5= hyvin tumraa våri.
20 ESIMERKKI 2 25
Mikro-organismien osoitus suotimelta eri pitoisuuksissa tehtiin esimerkin 1 mukaan. Lyhyesti, mikro-organismeja se-koitettiin ravistelijassa hetki ja inkuboitiin 30 sekunttia 0.2 M Tris-HCl-puskurissa, eri pH:ssa, jossa oli 1 % 30 glukoosia, siirrettiin suotimelle ja lisåttiin NBT-PMS-liuos ja luet-tiin sinisen formatsaanin intensiteetti 30 min kuluttua.
35 91977 6
Escherichia Escherichia Escherichia Escherichia 5 coli coli a coli b 2956 (ATCC 25922) pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 10 bakt.
/ml 107 233 443 444 231 106 222 222 023 111 105 0 0 0 0 0 0 0 0 0 0 0 0 15 _ 20 Escherichia Escherichia Klebsiella Enterobacter coli 3338 coli 110515 aerogenes aerogenes (ATCC 13883) (ATCC 13048) pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 25_____________ bakt.
/ml 107 4 5 5 2 3 3 5 5 5 5 5 5 106 222 222 445 455 30 105 000 000 233 333
II
35 7 91977
Proteus Pseudomonas Enterococcus Streptococcus 5 mirabilis aerogenes sp. agalactiae pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 bakt.
10 /ml 107 5 5 5 4 4 4 5 5 5 5 5 5 106 334 222 333 323 10s 112 122 222 121 15
Streptococ- Streptococ- Staphylococ- Staphylococ-20 cus beta- cus faecium cus aureus cus epider- haemol. B (ATCC 9790) midis pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 25 bakt.
/ml 107 5 5 5 4 4 4 3 5 5 4 4 4 106 344 333 233 223 10s 122 122 012 012 3 0 _ 35 91977 8
Corynebacte- Corynebacte- Corynebacte- Corynebacte-5 rium sp. rium sp. rium sp. riuxn sp.
pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 bakt.
10 /ml 107 555 444 555 555 106 334 222 333 323 105 111 122 111 111 15
Candida Candida Candida Candida
20 albicans albicans albicans sp. albicans B
(ATCC 28367) (ATCC 36802) pH 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 6.5 7.2 8.5 25 hiiva /ml 107 555 444 355 444 106 233 333 233 223 30 105 122 122 012 Oil
II
35
Claims (6)
1. Menetelmå mikro-organismien kokona i småårån osoittami-seksi nestemåisestå nåytteestå tunnettu sii- 5 tå, ettå a) mikro-organismit erotetaan suspensiosta suodatinma-teriaalin (3) avulla, jonka huokoskoko on sellainen, ettå mikrobien låpåisy estyy jolloin ne konsentroitu- 10 vat optimaalisen herkkyyden saavuttamiseksi, mutta riittåvå håiritsevien vapaana olevien pelkiståvien yhdisteiden poistamiseksi, ja b) osoitetaan mikro-organismien dehydrogenaasientsyy- 15 mien låsnåolo organismeja hajoittamatta kåyttåmållå dehydrogenaasientsyymin substraattina varittomåstå vårillisiksi muuttuvia saostuvia redox-yhdisteitå suo-raan siltå suotimelta, jolle mikro-organismit suoda tettaessa jååvåt. 20
2. Patenttivaatimuksen 1 mukainen menetelmå t u η n e t-t u siitå, ettå nestemåinen nåyte on virtsa tai pro-sessineste.
3. Patenttivaatimuksen 2 mukainen menetelmå tunnet tu siitå, ettå nestemåinen nåyte on virtsa.
4. Patenttivaatimuksen 1 mukainen menetelmå t u η n e t-t u siitå, ettå redox-yhdiste on saostuva trifenyyli- 30 tetratsoliumkloridi (TTC), jodonitrotetratsolium (INT), neotetratsoliumkloridi (NTC), blue tetratsolium (BT) tai nitroblue tetratsolium (NBT).
5. Patenttivaatimuksen 1 mukainen menetelmå t u η n e t- 35 tu siitå, ettå suotimen (3) huokoskoko on 0.75-1.2 μτα. 91977
6. Patenttivaatimuksen 1 mukainen menetelma t u η n e t-t u siita, etta kaytetty suodatin on kiinnitetty laitteeseen siten, etta se rajoittuu imukykyiseen adsorbenttiin joka imee naytteen nesteylimaaran sekå 5 liukoiset hairitsevat tekijat. 15 20 25 30 II 35 91977
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI910358A FI91977C (fi) | 1991-01-24 | 1991-01-24 | Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi |
| US07/740,320 US5420017A (en) | 1991-01-24 | 1991-08-05 | Method and kit for the detection of microorganisms |
| EP92101133A EP0496410B1 (en) | 1991-01-24 | 1992-01-24 | A method for the detection of microorganisms |
| JP4032921A JPH078293A (ja) | 1991-01-24 | 1992-01-24 | 微生物検出方法 |
| DE69214474T DE69214474T2 (de) | 1991-01-24 | 1992-01-24 | Ein Verfahren zum Nachweis von Mikroorganismen |
| AT92101133T ATE144289T1 (de) | 1991-01-24 | 1992-01-24 | Ein verfahren zum nachweis von mikroorganismen |
| US08/450,630 US5714343A (en) | 1991-01-24 | 1995-07-31 | Method and kit for the detection of microorganisms |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI910358 | 1991-01-24 | ||
| FI910358A FI91977C (fi) | 1991-01-24 | 1991-01-24 | Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi |
Publications (4)
| Publication Number | Publication Date |
|---|---|
| FI910358A0 FI910358A0 (fi) | 1991-01-24 |
| FI910358L FI910358L (fi) | 1992-07-25 |
| FI91977B FI91977B (fi) | 1994-05-31 |
| FI91977C true FI91977C (fi) | 1994-09-12 |
Family
ID=8531791
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| FI910358A FI91977C (fi) | 1991-01-24 | 1991-01-24 | Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0496410B1 (fi) |
| JP (1) | JPH078293A (fi) |
| AT (1) | ATE144289T1 (fi) |
| DE (1) | DE69214474T2 (fi) |
| FI (1) | FI91977C (fi) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6818417B2 (en) * | 2002-08-06 | 2004-11-16 | Nalco Company | Method for locating hidden microorganism contaminated surfaces in industrial water systems |
| GB2409519B (en) * | 2003-12-22 | 2005-11-09 | Prail Price Richardson Diagnos | Microorganism detector |
| CH703678B1 (de) * | 2004-04-06 | 2012-03-15 | Empa Testmaterialien Ag | Verfahren und Einrichtung zum Testen bakterizider Wirkung von Substanzen. |
| JP5771934B2 (ja) * | 2010-10-01 | 2015-09-02 | コニカミノルタ株式会社 | 細胞染色方法及び細胞染色用キット |
| CZ2013456A3 (cs) * | 2013-06-14 | 2014-12-29 | Metacell, S.R.O. | Způsob separace sporadických buněk z tělních tekutin a zařízení pro provádění tohoto způsobu |
| JP6724333B2 (ja) * | 2015-10-26 | 2020-07-15 | 大日本印刷株式会社 | 微生物培養シート及び微生物の検出方法 |
| WO2021190340A1 (zh) * | 2020-03-27 | 2021-09-30 | 康博医创股份有限公司 | 一种用于富集和检测生物样品中微生物的方法和装置 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4525453A (en) * | 1982-10-26 | 1985-06-25 | Eastman Kodak Company | Process for rapidly differentiating between gram-negative and gram-positive microorganisms |
| DE3784702T2 (de) * | 1987-04-27 | 1993-09-16 | Texas Bioresource Corp | Verfahren und vorrichtung zum nachweis und zur mengenbestimmung von bakterien. |
-
1991
- 1991-01-24 FI FI910358A patent/FI91977C/fi active IP Right Grant
-
1992
- 1992-01-24 AT AT92101133T patent/ATE144289T1/de not_active IP Right Cessation
- 1992-01-24 DE DE69214474T patent/DE69214474T2/de not_active Expired - Fee Related
- 1992-01-24 JP JP4032921A patent/JPH078293A/ja active Pending
- 1992-01-24 EP EP92101133A patent/EP0496410B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE69214474D1 (de) | 1996-11-21 |
| DE69214474T2 (de) | 1997-05-22 |
| FI910358L (fi) | 1992-07-25 |
| JPH078293A (ja) | 1995-01-13 |
| EP0496410B1 (en) | 1996-10-16 |
| FI910358A0 (fi) | 1991-01-24 |
| EP0496410A1 (en) | 1992-07-29 |
| ATE144289T1 (de) | 1996-11-15 |
| FI91977B (fi) | 1994-05-31 |
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