JP2018506708A - 整形外科的外傷用外植片における細菌コロニー形成およびバイオフィルム形成の視覚化 - Google Patents
整形外科的外傷用外植片における細菌コロニー形成およびバイオフィルム形成の視覚化 Download PDFInfo
- Publication number
- JP2018506708A JP2018506708A JP2017533034A JP2017533034A JP2018506708A JP 2018506708 A JP2018506708 A JP 2018506708A JP 2017533034 A JP2017533034 A JP 2017533034A JP 2017533034 A JP2017533034 A JP 2017533034A JP 2018506708 A JP2018506708 A JP 2018506708A
- Authority
- JP
- Japan
- Prior art keywords
- gram
- antibody
- explants
- negative
- clsm
- 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
- 230000032770 biofilm formation Effects 0.000 title claims abstract description 23
- 208000034309 Bacterial disease carrier Diseases 0.000 title claims description 14
- 230000000399 orthopedic effect Effects 0.000 title abstract description 32
- 238000012800 visualization Methods 0.000 title description 20
- 208000014674 injury Diseases 0.000 title description 13
- 230000008733 trauma Effects 0.000 title description 9
- 238000000034 method Methods 0.000 claims abstract description 88
- 230000001580 bacterial effect Effects 0.000 claims description 104
- 239000002953 phosphate buffered saline Substances 0.000 claims description 63
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 48
- 210000002966 serum Anatomy 0.000 claims description 40
- 238000010790 dilution Methods 0.000 claims description 30
- 239000012895 dilution Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 29
- 238000012360 testing method Methods 0.000 claims description 28
- 230000000903 blocking effect Effects 0.000 claims description 27
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 23
- 229940098773 bovine serum albumin Drugs 0.000 claims description 22
- 239000000872 buffer Substances 0.000 claims description 15
- 238000003384 imaging method Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 14
- 238000009877 rendering Methods 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 230000008832 photodamage Effects 0.000 claims description 2
- 238000004804 winding Methods 0.000 claims 1
- 238000011002 quantification Methods 0.000 abstract description 46
- 238000001514 detection method Methods 0.000 abstract description 23
- 238000011282 treatment Methods 0.000 abstract description 23
- 238000001356 surgical procedure Methods 0.000 abstract description 15
- 230000000813 microbial effect Effects 0.000 abstract description 11
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 238000003556 assay Methods 0.000 description 90
- 238000005516 engineering process Methods 0.000 description 68
- 241000894006 Bacteria Species 0.000 description 62
- 239000000523 sample Substances 0.000 description 55
- 208000015181 infectious disease Diseases 0.000 description 54
- 241000894007 species Species 0.000 description 51
- 238000002360 preparation method Methods 0.000 description 32
- 238000002372 labelling Methods 0.000 description 31
- 241000588626 Acinetobacter baumannii Species 0.000 description 28
- 210000001519 tissue Anatomy 0.000 description 28
- 238000003501 co-culture Methods 0.000 description 26
- 238000010186 staining Methods 0.000 description 26
- 239000007943 implant Substances 0.000 description 22
- 238000004458 analytical method Methods 0.000 description 20
- 241000192125 Firmicutes Species 0.000 description 19
- 230000006872 improvement Effects 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 17
- 210000003527 eukaryotic cell Anatomy 0.000 description 17
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 17
- 238000012512 characterization method Methods 0.000 description 16
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 15
- 108010013639 Peptidoglycan Proteins 0.000 description 15
- 239000002253 acid Substances 0.000 description 14
- 239000002158 endotoxin Substances 0.000 description 13
- 239000002639 bone cement Substances 0.000 description 12
- 229920006008 lipopolysaccharide Polymers 0.000 description 12
- 241000191967 Staphylococcus aureus Species 0.000 description 11
- 239000002131 composite material Substances 0.000 description 11
- 239000012109 Alexa Fluor 568 Substances 0.000 description 10
- 208000027418 Wounds and injury Diseases 0.000 description 10
- 238000011109 contamination Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 208000020154 Acnes Diseases 0.000 description 9
- 241000186427 Cutibacterium acnes Species 0.000 description 9
- 239000007850 fluorescent dye Substances 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 229940055019 propionibacterium acne Drugs 0.000 description 9
- 230000027455 binding Effects 0.000 description 8
- 230000003115 biocidal effect Effects 0.000 description 8
- 210000005255 gram-positive cell Anatomy 0.000 description 8
- 230000008901 benefit Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 7
- 230000006378 damage Effects 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 7
- 210000005256 gram-negative cell Anatomy 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 7
- 239000004926 polymethyl methacrylate Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 206010052428 Wound Diseases 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 210000002421 cell wall Anatomy 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 238000004393 prognosis Methods 0.000 description 6
- 206010017076 Fracture Diseases 0.000 description 5
- 230000005757 colony formation Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 230000002980 postoperative effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241000589291 Acinetobacter Species 0.000 description 4
- 239000012099 Alexa Fluor family Substances 0.000 description 4
- 208000010392 Bone Fractures Diseases 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000011198 co-culture assay Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- 229910001200 Ferrotitanium Inorganic materials 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 3
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 229960003085 meticillin Drugs 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000010936 titanium Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 2
- 208000002565 Open Fractures Diseases 0.000 description 2
- 206010031252 Osteomyelitis Diseases 0.000 description 2
- 206010067268 Post procedural infection Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000181 anti-adherent effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000011948 assay development Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000001804 debridement Methods 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000002262 irrigation Effects 0.000 description 2
- 238000003973 irrigation Methods 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000000963 osteoblast Anatomy 0.000 description 2
- 210000001322 periplasm Anatomy 0.000 description 2
- 238000011897 real-time detection Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 239000001011 safranin dye Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000004872 soft tissue Anatomy 0.000 description 2
- 210000001179 synovial fluid Anatomy 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- VIBDVOOELVZGDU-UHFFFAOYSA-N 4-(1h-indol-2-yl)benzene-1,3-dicarboximidamide Chemical compound NC(=N)C1=CC(C(=N)N)=CC=C1C1=CC2=CC=CC=C2N1 VIBDVOOELVZGDU-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241001316595 Acris Species 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- XYZZKVRWGOWVGO-UHFFFAOYSA-N Glycerol-phosphate Chemical compound OP(O)(O)=O.OCC(O)CO XYZZKVRWGOWVGO-UHFFFAOYSA-N 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 206010066902 Surgical failure Diseases 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- -1 aromatic amino acids Chemical group 0.000 description 1
- 238000011882 arthroplasty Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- ZINFAXPQMLDEEJ-GFVOIPPFSA-N cefoselis Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CN1C=CC(=N)N1CCO ZINFAXPQMLDEEJ-GFVOIPPFSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical group O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 150000004676 glycans Chemical group 0.000 description 1
- 244000000058 gram-negative pathogen Species 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000001989 nasopharynx Anatomy 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000016446 peptide cross-linking Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000000513 rotator cuff Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000005222 synovial tissue Anatomy 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 239000007102 tryptic soy broth medium Substances 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
Classifications
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0028—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders specially adapted for specific applications, e.g. for endoscopes, ophthalmoscopes, attachments to conventional microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/22—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Neisseriaceae (F), e.g. Acinetobacter
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Multimedia (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Ophthalmology & Optometry (AREA)
- Radiology & Medical Imaging (AREA)
- Surgery (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Microscoopes, Condenser (AREA)
Abstract
Description
以下のステップを行った。
1)3×PBS+1%RSで洗浄し、
2)BSA+RSで30分間ブロッキングし、
3)グラム陽性一次[グラム陽性LTA]を1:100で60分間導入し、
4)2×PBSで洗浄+1%RSで洗浄し、
5)グラム陽性二次[ウサギ抗IgG Fc FITC]を1:50で60分間導入し、
6)2×PBS+1%RSで洗浄し、
7)グラム陰性一次[LPSグラム陰性抗ヤギ]を1:1000で60分間導入し、
8)2×PBS+1%RSで洗浄し、
9)グラム陰性二次[Alexa Fluor 568]を1:200で60分間導入し、
10)2×PBS+1%RSで洗浄し、
11)新しいPBSと置き換え、共焦点法の準備が整う。
アシネトバクター・バウマンニ対照120×(7A)、
全細菌範囲:.145±10.88%、赤色閾値:.26%、
アシネトバクター・バウマンニ対照120×(7B)、
全細菌範囲:.367±.0567%、赤色閾値:.52%。
MSSA対照120×(8A)
全細菌範囲:20.88±.582%
緑色閾値:21.32%
アシネトバクター・バウマンニ対照120×(8B)
全細菌範囲:39.268±.462% 赤色閾値:39.17%
黄色ブドウ球菌特異的CLSM調製アッセイ:2時間
1. 3×5分間PBSで洗浄し、
2. BSAで30分間ブロッキングし、
3. [黄色ブドウ球菌/FITCコンジュゲート抗体]を1:200希釈で60分間導入し、
4. 3×5分間のPBS、共焦点法の準備が整う
1. BSAで30分間ブロッキングし、
2. グラム陰性一次抗体[LPSグラム陰性抗ヤギ]を1:600希釈で60分間導入し、
3. 3×5分間PBSで洗浄し、
5. グラム陰性二次抗体[Alexa Fluor 568]を1:200希釈で60分間導入し、
6. 3×5分間PBSで洗浄し、共焦点法の準備が整う。
1)3×5分間PBSで洗浄し、
2)BSAで1時間30分ブロッキングし、
3)グラム陽性一次抗体[グラム陽性LTA]を1:100で60分間導入し、
4)2×10分間PBSで洗浄し、
6)グラム陽性二次[ウサギ抗IgG Fc FITC]を1:50で60分間導入し、
7)DAPIを5分間添加し、
8)新しいPBSと置き換え、共焦点法の準備が整う。
1)3×5分間PBSで洗浄し、
2)BSAで90分間ブロッキングし、
3)グラム陰性一次[グラム陰性内毒素]を1:100で90分間、
4)2×10分間PBSで洗浄し、
5)グラム陰性二次[Alexa Fluor 594]を1:100で60分間、
6)グラム陽性一次[グラム陽性LTA]を1:100で60分間、1:100、
7)2×10分間PBS洗浄し、
8)グラム陽性二次[ウサギ抗IgG Fc FITC]を1:50で60分間、
9)DAPIを5分間添加し、
10)新しいPBSと置き換え、共焦点法の準備が整う。
ネジ側120×(図12A)
全グラム陽性範囲:.378±3.889%
緑色閾値:.47%
全グラム陰性範囲:.048±27.067%
赤色閾値:.08%
全真核生物範囲:.065±5.585%
青色閾値:.09%
ネジ側部120×(図12B)
全グラム陽性範囲:.399±4.235%
緑色閾値:.47%
全グラム陰性範囲:.094±16.532%
赤色閾値:.094%
全真核生物範囲:0%
1)3×5分間PBSで洗浄し、
2)BSAで30分間ブロッキングし、
3)グラム陰性一次[LPSグラム陰性抗ヤギ]を8=1:200、9=1:400で60分間導入し、
4)2×5分間PBSで洗浄し、
5)グラム陰性二次[Alexa Fluor 568]を1:200で60分間導入し、
6)新しいPBSと置き換え、共焦点法の準備が整う。
1:200でグラム陰性FCBAでタグ化したMSSA、120×(14A)
全細菌範囲:5.4±1.040%
赤色閾値:5.59%
1:400でグラム陰性FCBAでタグ化したMSSA、120×(14B)
全細菌範囲:18.43±.538%
赤色閾値:19.06%
1)3×5分間PBSで洗浄し、
2)BSAで30分間ブロッキングし、
3)グラム陰性一次[LPSグラム陰性抗ヤギ]を10=1:200、11=1:400で60分間導入し、
4)2×5分間PBSで洗浄し、
5)グラム陰性二次[Alexa Fluor 568]を1:200で60分間導入し、
6)2×5分間PBSで洗浄し、
7)グラム陽性一次[グラム陽性LTA]を1:100で60分間導入し、
8)2×5分間PBSで洗浄し、
9)グラム陽性二次[ウサギ抗IgG Fc FITC]を1:50で60分間導入し、
10)新しいPBSと置き換え、共焦点法の準備が整う。
MSSA+アシネトバクター・バウマンニ共培養物、120×(15A)
全グラム陽性範囲:.904±1.668%
緑色閾値:.90%
全グラム陰性範囲:1.65±2.87%
赤色閾値:1.82%
MSSA+アシネトバクター・バウマンニ共培養物、120×(15B)
全グラム陽性範囲:.0621±13.8%
緑色閾値:.08%
全グラム陰性範囲:.0289±2.471%
赤色閾値:2.45%。
MSSA+アシネトバクター・バウマンニ共培養物、120×(16A)
全グラム陽性範囲:1.65±.980%
緑色閾値:1.84%
全グラム陰性範囲:8.05±.883%
赤色閾値:8.37%
全体的な細菌範囲:10.84±1.201%
全体的な範囲の閾値:11.12%
MSSA+アシネトバクター・バウマンニ共培養物120×、(16B)
全グラム陽性範囲:1.725±.336%
緑色閾値:3.84%
全グラム陰性範囲:5.57±.345%
赤色閾値:7.33%
全体的な細菌範囲:28.76±.436%
全体的な範囲の閾値:28.87%
MSSA+アシネトバクター・バウマンニ共培養物+20%RS、120×(17A)
全グラム陽性範囲:9.67±.776%
緑色閾値:9.89%
全グラム陰性範囲:6.66%±1.62%
赤色閾値:9.46%
全体的な細菌範囲:14.73±1.12%
全体的な範囲の閾値:16.64
MSSA+アシネトバクター・バウマンニ共培養物+40%RS、120×(18A)
全グラム陽性範囲:6.934±1.57%
緑色閾値:7.23%
全グラム陰性範囲:5.56±1.74%
赤色閾値:8.04%
全体的な細菌範囲:10.46±1.79%
全体的な範囲の閾値:14.01%
1)3×PBS+1%ウサギ血清(RS)で続けて洗浄し、
2)50%BSA+50%RSで15分間ブロッキングし、
3)グラム陽性およびグラム陰性の一次抗体を15分間導入し、
−[グラム陽性LTA]1:100
−[LPSグラム陰性抗ヤギ]1:1000
4)3×PBS+1%RSで洗浄し、
5)グラム陽性およびグラム陰性の両方の二次抗体[ウサギ抗IgG Fc FITC]を1:50で、[Alexa Fluor 568]を1:200で15分間導入し、
6)PBS+1%RSですすぎ、共焦点法の準備が整う。
MSSA+アシネトバクター・バウマンニ+Euk(20%RS)120×:1時間のプロトコル(19A)
グラム陽性菌:12.54±.757%
緑色閾値:12.52%
グラム陰性菌:0.992±3.166%
赤色閾値:1.43%
真核生物細胞:9.629±.773%
青色閾値:9.75%
範囲面積パーセント:18.013±.643%
全体の閾値:18.21%
MSSA+アシネトバクター・バウマンニ+Euk(50%RS)120×:1時間のプロトコル(20A)
グラム陽性菌:11.52±.647%
緑色閾値:11.51%
グラム陰性菌:0.356±4.716%
赤色閾値:0.68%
真核生物細胞:9.53±1.169%
青色閾値:9.57%
範囲面積パーセント:16.23±.975%
全体の閾値:16.31%
1)3×PBS+1%RSで洗浄し、
2)BSA+RSで30分間ブロッキングし、
3)グラム陽性一次[グラム陽性LTA]を1:100で60分間導入し、
4)2×PBSで洗浄し、1%RSで洗浄し、
5)グラム陽性二次[ウサギ抗IgG Fc FITC]を1:50で60分間導入し、
6)2×PBS+1%RSで洗浄し、
7)グラム陰性一次[LPSグラム陰性抗ヤギ]を1:1000で60分間導入し、
8)2×PBS+1%RSで洗浄し、
9)グラム陰性二次[Alexa Fluor 568]を1:200で60分間導入し、
10)2×PBS+1%RSで洗浄し、CLMの準備が整う
ネジ頭120×(21B)
グラム陽性菌:4.95±.989%
緑色閾値:5.19%
真核生物細胞:.581±1023%
青色閾値:.65%
範囲面積パーセント:6.21±1.17%
全体の閾値:6.49%
ネジ頭、全アッセイ120×(21C)
グラム陽性菌:1.98±1.22%
緑色閾値:2.40%
真核生物細胞:.895±.364%
青色閾値:1.09%
範囲面積パーセント:3.79±.722%
全体の閾値:4.30%
ネジ頭、全アッセイ120×(22B)
グラム陰性菌:2.74±1.19%
赤色閾値:3.48%
真核生物細胞:9.63±.977%
青色閾値:10.09%
範囲面積パーセント:6.27±.77%
全体の閾値:6.86%
定量化:
ネジ頭120×(23B):
グラム陽性菌:6.49±1.67%
緑色閾値:7.10%
真核生物細胞:1.25±1.15%
青色閾値:12.62%
範囲:20.05±1.11%
全体の閾値:20.38%
PMMA骨セメント高速アッセイ120x(24B):
グラム陽性菌:0.285±.508%
緑色閾値:0.46%
グラム陰性菌:0.381±.626%
赤色閾値:0.57%
真核生物細胞:1.248±3.63%
青色閾値:1.33%
範囲面積パーセント:3.36±.867%
全体の閾値:3.72%
1)3×PBS+1%ウサギ血清(RS)で続けて洗浄し、
2)50%BSA+50%RSで15分間ブロッキングし、
3)グラム陽性およびグラム陰性の両方の一次抗体、グラム陽性:[グラム陽性LTA]を1:100で、グラム陰性:[LPSグラム陰性抗ヤギ]を1:1000で15分間導入し、
4)3×PBS+1%RSで続けて洗浄し、
5)グラム陽性およびグラム陰性の両方の二次抗体、グラム陽性:[ウサギ抗IgG Fc FITC]を1:50で、グラム陰性:[Alexa Fluor 568]を1:200で15分間導入し、
6)PBS+1%RSですすぎ、共焦点法の準備が整う。
7)
ネジ頭高速アッセイ120x(25B):
グラム陽性菌:0.028±.6424%
緑色閾値:0.18%
グラム陰性菌:3.185±.575%
赤色閾値:5.44%
真核生物細胞:3.196±1.023%
青色閾値:3.21%
範囲面積パーセント:4.58±.522%
全体の閾値:6.37%
ネジ頭高速アッセイ120x(26B):
グラム陽性菌:0.451±1.364%
緑色閾値:0.59%
グラム陰性菌:1.167±1.1003%
赤色閾値:1.63%
真核生物細胞:1.465±2.309%
青色閾値:1.51%
範囲面積パーセント:2.843±.89%
全体の閾値:3.03%
Claims (15)
- 外植片における細菌コロニー形成およびバイオフィルム形成を視覚化および定量化するための方法であって、
1)前記外植片を、PBSブロッキング緩衝液および1%ウサギ血清(RS)の第1の溶液で続けて少なくとも1回洗浄するステップと、
2)第1の期間、グラム陽性およびグラム陰性の両方のコンジュゲート抗体を適用するステップと、
3)前記外植片を、PBSブロッキング緩衝液および1%RSで続けて少なくとも1回洗浄するステップと、
4)第2の期間、グラム陽性およびグラム陰性の両方の二次抗体を導入するステップと、
5)PBSブロッキング緩衝液および1%RSですすぐステップと、
6)前記外植片をグラム陽性およびグラム陰性の付着に関して、共焦点レーザー走査顕微鏡(CLSM)で確認するステップと、を含む、方法。 - ステップ1)および3)の前記少なくとも1回の洗浄が、それぞれ3回であり、
前記第1および第2の期間が、それぞれおよそ15分である、請求項1に記載の方法。 - ステップ(1)〜(5)の間、光誘起損傷および光退色を予防するために、前記外植片の周囲にスズ箔を巻き付けるステップをさらに含む、請求項1に記載の方法。
- 細菌プレートにおいて、グラム陽性およびグラム陰性の一次および二次の抗体型の間で確実に蛍光のバランスを取ってぼやけを最小限に抑えるために、複数回、抗体希釈試験を反復するステップをさらに含む、請求項1に記載の方法。
- 前記外植片を、30rpmでの緩徐な洗浄効果のためにロッキング装置に載置するステップをさらに含む、請求項1に記載の方法。
- 前記CLSMからの図を前記外植片の表面の三次元レンダリングに配置するステップをさらに含む、請求項1に記載の方法。
- 外植片における細菌コロニー形成およびバイオフィルム形成を視覚化および定量化するための方法であって、
前記外植片を、PBSブロッキング緩衝液および1%ウサギ血清(RS)の第1の溶液で続けて少なくとも1回洗浄するステップと、
第1の期間、50%ウシ血清アルブミン(BSA)および50%RSの第2の溶液でブロッキングするステップと、
第2の期間、グラム陽性およびグラム陰性の両方の一次抗体を導入するステップと、
前記外植片を、PBSブロッキング緩衝液および1%RSで続けて少なくとも1回洗浄するステップと、
第3の期間、グラム陽性およびグラム陰性の両方の二次抗体を導入するステップと、
PBSブロッキング緩衝液および1%RSですすぐステップと、
グラム陽性およびグラム陰性の付着に関して、前記外植片を、共焦点レーザー走査顕微鏡で確認するステップと、を含む、方法。 - ステップ1)および3)の前記少なくとも1回の洗浄が、それぞれ3回であり、
前記第1、第2、および第3の期間が、それぞれおよそ15分間である、請求項7に記載の方法。 - 共焦点レーザー走査顕微鏡において撮像するための試料を調製するための方法であって、
a)前記試料を、第1のウサギ血清溶液で3回すすぐステップと、
b)前記試料を、第1の期間、第2のウサギ血清溶液でブロッキングするステップと、
c)前記第2のウサギ血清から前記試料を取り出すステップと、
d)抗体溶液を添加して第2の期間インキュベートするステップと、を含む、方法。 - ブロッキング中に室温で前記試料をかき混ぜるステップをさらに含む、請求項9に記載の方法。
- 前記第1のウサギ血清溶液が、25%のウサギ血清を含有するリン酸緩衝食塩水の溶液であり、
前記第1の期間が、およそ15分間であり、
前記第2のウサギ血清溶液が、50%のウサギ血清および50%のウシ血清アルブミン(BSA)であり、
前記抗体溶液が、コンジュゲートしたグラム陽性抗体およびグラム陰性抗体のカクテルである、請求項9に記載の方法。 - 前記抗体溶液が、グラム陽性抗体比が1:200であり、グラム陰性抗体量が1:750であることを有する、請求項11に記載の方法。
- 前記抗体溶液が、1%ウサギ血清を有する、請求項11に記載の方法。
- 前記抗体溶液が、摂氏およそ37度であり、前記試料が、暗所でインキュベートされ、前記第2の期間が、およそ15分間である、請求項15に記載の方法。
- インキュベートした後に、前記試料が、摂氏およそ4度でリン酸緩衝食塩水中に保管される、請求項9に記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462094004P | 2014-12-18 | 2014-12-18 | |
US62/094,004 | 2014-12-18 | ||
PCT/US2015/066460 WO2016100712A1 (en) | 2014-12-18 | 2015-12-17 | Visualization of bacterial colonization and biofilm formation on orthopaedic trauma explants |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2018506708A true JP2018506708A (ja) | 2018-03-08 |
JP6660392B2 JP6660392B2 (ja) | 2020-03-18 |
Family
ID=56127624
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2017533034A Active JP6660392B2 (ja) | 2014-12-18 | 2015-12-17 | 整形外科的外傷用外植片における細菌コロニー形成およびバイオフィルム形成の視覚化 |
Country Status (5)
Country | Link |
---|---|
US (1) | US10352933B2 (ja) |
EP (1) | EP3234681B1 (ja) |
JP (1) | JP6660392B2 (ja) |
CA (1) | CA2970678C (ja) |
WO (1) | WO2016100712A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7371913B2 (ja) | 2019-12-09 | 2023-10-31 | 国立研究開発法人物質・材料研究機構 | 微生物汚染の評価方法、洗浄方法の評価方法、洗浄方法、プログラム、微生物汚染の評価装置、洗浄方法の評価装置、及び、洗浄装置 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997035614A1 (en) * | 1996-03-27 | 1997-10-02 | Crc For Biopharmaceutical Research Pty. Ltd. | The use of antibodies against cd48 for the treatment of t and b cell lymphomas and leukemias |
JP2002047211A (ja) * | 2000-08-04 | 2002-02-12 | Japan Science & Technology Corp | 脂溶性物質と生理活性高分子物質結合体および細胞核内への導入方法 |
US20050214279A1 (en) * | 2003-09-19 | 2005-09-29 | Silverstein Samuel C | Methods for stimulating human leukocytes to kill bacteria, yeast and fungi in biofilms that have formed in/on prosthetic devices, catheters, tissues and organs in vivo |
US20080057055A1 (en) * | 2004-10-07 | 2008-03-06 | Fisher Paul B | OLD-35 as an inflammatory agent |
US20110236306A1 (en) * | 2010-03-29 | 2011-09-29 | University Of Southern California | Compositions and methods for the removal of biofilms |
US20130064762A1 (en) * | 2011-09-13 | 2013-03-14 | The Rockefeller University | Methods of detecting and treating cancers using autoantibodies |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10055176B4 (de) | 2000-11-08 | 2007-05-03 | Leica Microsystems Cms Gmbh | Anordnung zur visuellen und quantitativen 3-D-Untersuchung von Proben |
US8323903B2 (en) | 2001-10-12 | 2012-12-04 | Life Technologies Corporation | Antibody complexes and methods for immunolabeling |
EP3501384A3 (en) | 2008-05-20 | 2019-10-16 | University Health Network | Method for fluorescence-based imaging and monitoring |
US20110305717A1 (en) | 2010-06-11 | 2011-12-15 | Jean-Pierre Gorvell | Cultured myeloid dendritic cells isolated from peyer's patches and uses thereof |
WO2012103257A2 (en) * | 2011-01-25 | 2012-08-02 | The Regents Of The University Of California | Transcutaneous multimodal delivery systems |
US10513546B2 (en) * | 2013-12-18 | 2019-12-24 | President And Fellows Of Harvard College | CRP capture/detection of gram positive bacteria |
-
2015
- 2015-12-17 CA CA2970678A patent/CA2970678C/en active Active
- 2015-12-17 EP EP15871100.2A patent/EP3234681B1/en active Active
- 2015-12-17 JP JP2017533034A patent/JP6660392B2/ja active Active
- 2015-12-17 US US14/899,972 patent/US10352933B2/en active Active
- 2015-12-17 WO PCT/US2015/066460 patent/WO2016100712A1/en active Application Filing
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997035614A1 (en) * | 1996-03-27 | 1997-10-02 | Crc For Biopharmaceutical Research Pty. Ltd. | The use of antibodies against cd48 for the treatment of t and b cell lymphomas and leukemias |
JP2000509015A (ja) * | 1996-03-27 | 2000-07-18 | シーアールシー フォー バイオファーマシューティカル リサーチ ピーティーワイ.リミテッド | T細胞及びb細胞リンパ腫及び白血病の治療のためのcd48に対する抗体の使用 |
JP2002047211A (ja) * | 2000-08-04 | 2002-02-12 | Japan Science & Technology Corp | 脂溶性物質と生理活性高分子物質結合体および細胞核内への導入方法 |
US20050214279A1 (en) * | 2003-09-19 | 2005-09-29 | Silverstein Samuel C | Methods for stimulating human leukocytes to kill bacteria, yeast and fungi in biofilms that have formed in/on prosthetic devices, catheters, tissues and organs in vivo |
US20080057055A1 (en) * | 2004-10-07 | 2008-03-06 | Fisher Paul B | OLD-35 as an inflammatory agent |
US20110236306A1 (en) * | 2010-03-29 | 2011-09-29 | University Of Southern California | Compositions and methods for the removal of biofilms |
JP2013529893A (ja) * | 2010-03-29 | 2013-07-25 | ユニバーシティー オブ サザン カリフォルニア | バイオフィルムの除去のための組成物および方法 |
US20130064762A1 (en) * | 2011-09-13 | 2013-03-14 | The Rockefeller University | Methods of detecting and treating cancers using autoantibodies |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7371913B2 (ja) | 2019-12-09 | 2023-10-31 | 国立研究開発法人物質・材料研究機構 | 微生物汚染の評価方法、洗浄方法の評価方法、洗浄方法、プログラム、微生物汚染の評価装置、洗浄方法の評価装置、及び、洗浄装置 |
Also Published As
Publication number | Publication date |
---|---|
EP3234681A1 (en) | 2017-10-25 |
US20160370367A1 (en) | 2016-12-22 |
JP6660392B2 (ja) | 2020-03-18 |
CA2970678C (en) | 2023-07-04 |
EP3234681B1 (en) | 2020-02-26 |
WO2016100712A1 (en) | 2016-06-23 |
EP3234681A4 (en) | 2018-12-26 |
CA2970678A1 (en) | 2016-06-23 |
US10352933B2 (en) | 2019-07-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging | |
Becerra et al. | An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue | |
US20210325308A1 (en) | Artificial flourescent image systems and methods | |
Serena et al. | Real-time bacterial fluorescence imaging accurately identifies wounds with moderate-to-heavy bacterial burden | |
Bernardi et al. | Streptococcus spp. and Fusobacterium nucleatum in tongue dorsum biofilm from halitosis patients: A fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) study | |
WO2021113821A1 (en) | Systems and methods for high throughput drug screening | |
JP2015108549A (ja) | 細胞検査装置および細胞検査方法 | |
Tan et al. | Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis | |
Nistico et al. | Imaging bacteria and biofilms on hardware and periprosthetic tissue in orthopedic infections | |
Khajotia et al. | Concurrent quantification of cellular and extracellular components of biofilms | |
US10955322B2 (en) | Methods and devices for soft and osseous tissue clearing and fluorescent imaging | |
Mukherjee et al. | An improved DAPI staining procedure for visualization of polyphosphate granules in cyanobacterial and microlagal cells | |
Wollman et al. | An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy | |
López-Álvarez et al. | Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections | |
JP6660392B2 (ja) | 整形外科的外傷用外植片における細菌コロニー形成およびバイオフィルム形成の視覚化 | |
Kikhney et al. | Quality control in diagnostic fluorescence in situ hybridization (FISH) in microbiology | |
Yuehua et al. | Current advances in diagnostic methods ofAcanthamoebakeratitis | |
Gunasekaran et al. | Exploratory use of fluorescent SmartProbes for the rapid detection of microbial isolates causing corneal ulcer | |
Ehrlich et al. | Culture-negative infections in orthopedic surgery | |
Szmacinski et al. | Imaging hydroxyapatite in sub-retinal pigment epithelial deposits by fluorescence lifetime imaging microscopy with tetracycline staining | |
WO2018016501A1 (ja) | 汗腺の動態の観察方法 | |
Schwartz et al. | Characterization of biofilm formed by human-derived nanoparticles | |
CN113640520A (zh) | 一种组织透明方法和组织学方法联合用于检测肿瘤内细菌的应用 | |
Garcia et al. | Fluorescent‐conjugated antibodies as rapid ex vivo markers for bacterial presence on orthopedic surgical explants and synovium: A pilot study | |
RU2425384C2 (ru) | Способ диагностики течения сальмонеллёзно-протозойных острых кишечных инфекций у детей |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20181211 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20190925 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20191105 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20191227 |
|
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: 20200110 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20200207 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6660392 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |