Histology Introduction

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  • 1.HISTOLOGY: INTRODUCTION Whatisgoingon ? Pulling it together Regions Organs Molecules Tissues Connections Cells Parts Organelles Development Functions Systems

2. Noon talks for Internal-Medicine residents Board prep Tworecurringthemes-- Isitwhatitappearstobe ? Does the treatment/procedure do what is claimed for it ? What is theevidence ? 3. MEDICINE:Some aspects What gives with this patient ? Regions Molecules Tissues Connections Cells Parts Organelles Development Functions Regions Systems Systemsabnormal Parts Connections Development Tissues Cells Organelles Molecules Functions Microbes Medicines Age Populations Costs ? Gender Organs OrgansAbnormal 4. Abnormal variants for all the earlier fields of knowledge Developing judgment-weighing various contributions for relevance & quality of evidence Foretaste of the pulling it together in the PBL experiences, but much omitted, e.g., therapy, follow-up, cost;likewiseforclinicalcorrelations This doubling, plus more fields, e.g. microbes, is why medical training takes several years Any twit can lay hands on an LCD projector, and push images at youremindsone that the story may be faulty;it is one of many;and there are omissions Feel for the aspects that yield valid risk factors inthis particular diagnosis ? 5. PORNOGRAPHY&THE REAL THING Images versus REALITY What is theevidencefor the real? 6. Images versus REALITY -Functional Anatomy REALITY istheliving person, often via images Surface anatomy Palpation Endoscopy+ Radiology PET scans Ultrasound Doppler flowsGait & Reflexes etc Biopsies Fine-Needle Aspiration Cervical, Blood, etcSmears Flow cytometry & cell sorting Cell culture & grafting etc (Bits cut or sucked outfor microscopy) 7. REALITY is thedeadperson DISSECTION [ Surface anatomy Endoscopy Palpation Radiology Ultrasound aresometimes usefulasadjunctstoautopsy & histology correlations] Organs and large pieces cut out, examined, & prepared for MICROSCOPY-histology & histopathology(normal & altered side-by-side) 8. Images versus REALITY -Anatomy In Anatomy,the source of the evidence - the essentialpoint of reference - is thecadaverfor Gross&themicroscope slidefor Histo As the physician is knowledgeably comfortable with thepatients gross&microscopicstructureandits implications, you will become confident at the cadaver &themicroscope,andwiththeresulting imagesTESTS focus on the cadaver, the slides, and interpreting images -identification, interpretation, & synthesis Bed-rock 9. MICROSCOPIC SLIDESide view of slide Glass coverslip Glass slide1X3 Tissue Section Mounting medium Mounting medium:permeates section; fastens coverslip to slide; is clear; has refractive index as for glass Label 10. SLIDE USE - Cautions GLASS IS FRAGILE ! Take care with individual slides & especially with the boxes of slides The slide must go on the stagecoverslip up The high-dry & oil objectives cannot focus through the thickness of the slide to the section The label may have been put on the non-coverslip side, as shown ~ Slides & Microscope remain in the teaching Lab, always! Label 11. SLIDE PREPARATION ISteps Excise &Fix(preserve) the tissue in fixative Remove the water &replacewith wax-solvent Imbedthe oriented specimen in molten wax After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides When dry, remove the wax, &stainthe section Remove surplus stain & water;mount coverslip When mounting medium has set, domicroscopy 12. SLIDE PREPARATION ISteps Excise &Fix(preserve) the tissue in fixative Remove the water &replacewith wax-solvent Imbedthe oriented specimen in molten wax After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides When dry, remove the wax, &stainthe section Remove surplus stain & water;mount coverslip When mounting medium has set, domicroscopy 13. 50 % ethanol 70 % ethanol 95 % ethanol 100 % ethanol benzene/xylene Dehydrating series paraffinwax Remove the water &replacewith wax-solvent Imbedthe oriented specimen in molten wax Miscible with ethanol; dissolves wax Fresh tissue 10% Formalinfixative label 14. MICROTOME - a fancy meat-slicer - holds the wax block, & cuts off thin slices, as the block is slowly advanced mechanically Block Knife Section Glass slide Water-bath After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides Lift out floating section on the slide 15. FREEZING MICROTOMEholds the frozen tissue, & cuts off thin slices, as the block is slowly advanced mechanically Blockis the tissue Knife Section Water-bath Glass slide For fast biopsy, imbedding is omitted -frozen sections Mountthe thin slices (sections) on slides Lift out section on the slide 16. Dissolve paraffin wax Stain with Hematoxylin -blue Wash Stain with eosin -red Nuclei - blue Cytoplasm- red Wash When dry, remove the wax, &stainthe section 17. Dissolve paraffin wax Stain with Hematoxylin -blue Wash Stain with eosin -red Nuclei - blue Cytoplasm- red Wash When dry, remove the wax, &stainthe section Potassium + eosinate -stain + charged amine, etc,groups on proteins bind - eosinAcidophilic staining Basophilic 18. SLIDE PREPARATION IIISteps Excise &Fix(preserve) the tissue in fixative Remove the water &replacewith wax-solvent Imbedthe oriented specimen in molten wax After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides When dry, remove the wax, &stainthe section Remove surplus stain & water;mount coverslip When mounting medium has set, domicroscopy 19. SLIDE PREPARATION IIISteps Excise &Fix(preserve) the tissue in fixative Remove the water &replacewith wax-solvent Imbedthe oriented specimen in molten wax After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides When dry, remove the wax, &stainthe section Remove surplus stain & water;mount coverslip When mounting medium has set, domicroscopy 20. Images versus REALITY Artifactsare appearances not true to the original state of the tissue SLIDE PREPARATIONIVArtifacts Excise &Fix(preserve) the tissue in fixative Imbedthe oriented specimen in molten wax After it is solid, hold thewax block &cutslices Mountthe thin slices (sections) on slides When dry, remove the wax, &stainthe section Remove surplus stain & water;mount coverslip When mounting medium has set, domicroscopy Knife scores, chatter Bruising/splitting from cutting;Poor preservation, e.g., gutlining,enzymes, lost fat Wrinkles, section not flat, splits Weak/unbalanced staining Dirt,hair,bubbles Dirt on lenses, badillumination Misleading orientation, Shrinkage & distortion, Mislabeled 21. CLASS LIGHT MICROSCOPE Max MAGNIFICATION Eyepiece (10X) times Oil Objective (100X) =1000X Base Eyepiece/Ocular Stage Slide Light source Body Objectivelenses Condenser 22. CLASS LIGHT MICROSCOPEControlsI Base Condenser Eyepiece/Ocular Slide Light Body Inter-ocular distance Moving stage Iris diaphragm Field diaphragm Coarse & Fine focus Light intensity On/Off Objective selection left rear 23. CLASS LIGHT MICROSCOPEControlsII Condenser Eyepiece/Ocular Slide Light Body Stage clip for slide Condenser focusing Condensercentering Ocular focusing left-side Base 24. OPERATIONI Without looking down the eyepieces , plug in the cordTurn the light-intensity knob back counterclockwise, Switch on the light, turn the intensity up (about a 90 oturn) while observing the light via the field opening Open the field diaphragm wide Move the condenser assembly to its top positionSwitch the shortest objective lens (X4) into the working positionOpen the iris diaphragm wideSelect any well-stained slide Base Condenser Eyepiece/Ocular Slide Light Body Inter-ocular distance Moving stage Iris diaphragm Field diaphragm Coarse & Fine focus Light intensity On/Off Objective selection 25. OPERATIONII Field diaphragm Pull back theclip& placeslide, cover-slip up, on the stage Use the stage controls to bring the stained section over the light Focus,using coarse, then fine adjustments Close the iris diaphragm to take the glare out of the viewPush (pull) the eyepieces together to match your eye spacingShut one eye, focus with thefine focus ; then shut that eye, open the other, and focus for it with theocular focus(turningthe eyepiece knurled ring)Switch in the next higher objective, and focus, using the main focusing controls & testing for binocular fusion Base Condenser Eyepiece/Ocular Slide Light Body Inter-ocular distance Moving stage Iris diaphragm Coarse & Fine focus Light intensity On/Off Objective selection 26. SMEAR - another method of preparation Drop of blood Slide 1 Slide 2 On contact, slide 2 extends the drop along its 1 side Slide 2 Slide 2 Pushingangled slide 2 along #1 smears the line of blood acrossslide 1 Lift away slide 2; dry #1 ; stain; coverslip Smear A few cells are damaged; smear is not evenly thick; & staining is uneven. Same apply to SPREADS 27. TEASING - a method of preparation Lumbo-sacral cord Roots Terminal thread A technique you know from using a needle to separate out the connective-tissuefilum terminalefrom the nervouscauda equinaof dorsal & ventralroots On the MICROSCOPE SLIDE, with a needle point one cantease apartindividual nerve or muscle fibers from their bundles in nerve or muscle When tissue is already thin, it can be draped - SPREAD - over the slide like a tablecloth (Filum terminale) 28. Cut across BONE shaft twice Saw out a sector Lay sector flat & grind thin Wash ground section Dry ;placeunstainedon slide Coverslip for viewing GROUNDPREPARARTION 29. HISTOLOGY SOURCES 303 Human Structure Syllabusnext to last section p.8 Powerpoints- Comments &Standing assignment Histo PowerpointsHistology Full-text * & Histology Lab Guide http://wberesford.hsc.wvu.eduhttp://www.geocities.com/Athens/Academy/1575 * Recommendat