Practical training in Histology Physiotherapy (bc.) Organization issues •Beginning - strictly •Change your shoes - you cannot enter the hall with outdoor shoes (slippers) •Locker room – shoes, jackets, coats, bags etc. (use padlock) •Cell phone – switched off or in silent mode •Microscopic hall = laboratory • – eating, drinking, smoking not allowed • – students have to follow the instructions • – academic misconducts or inappropriate behavior result in excluding • from the lesson or course • •Follow safety rules •You have dedicated working place •You are responsible for microscope, slide set, EM atlas •Practical lesson -Introduction, presentation (demonstrated topic) -Your individual work = study of the slides, schematic but precise drawing of tissue structure, careful description. The result of your work is PROTOCOL in each practice. •Students come prepared for practices - programmes on pin-boards or dpt. webpage • •Attendance -100% attendance -Substitution only in exceptional cases, after permissions from both the teacher of your group and the lesson where you plan to substitute -Make a protocol during substitution, let it check and sign by the chief teacher Registration of substitution: Datum Jméno Ročník Skupina Č. praktika Č. místa Vyučující - podpis Date Name Year Group Nr. of practice Nr. of place Teacher- signature Each absence must be excused via study department and in IS (medical report from doctor, Official invitation, etc. Is necessary). •Protocols •you have to make paper protocols (no tablets, laptops) •A4 size, blank, without lines, according to the template •pencil handdrawings (no pen) •complete set of your protocols is required for getting the credits •the quality of the protocol is approved by your teacher’s signature at the end of practical lesson •Low-quality protocols cannot be approved and you have to substitute the respective practical lesson • •Testing your knowledge •Credit test in the last practice of semester (20 questions – max. 20 points, result 12 – 20 points = P (passed), 0 – 11 N (not passed), one resit is possible. •Exam in exam period: 2 questions from histology topics of lectures and practices (marks A – F). •3 terms: 1 regular and 2 resits. The english form of protocol: on www of department •Credits • - 100% attendance • - complete set of signed protocols from all lessons • - Passed credit test (written in practice before the last) • •End of practice: • - The practice is closed by teacher • http://www.med.muni.cz/histology ü ü ü protocol •Recommended literature + Lectures Protocols HISTOLOGY • •structure and ultrastructure of normal cells and tissues, •cytology and general histology •special histology = microscopic anatomy of individual organs • • •relevance: oncology, surgery, hematology, pathology, forensic,… • Histology •Resolution of naked eye – 0,1 mm •Resolution of light microscopy – 10 nm •Resolution of electron microscopy – 0,1 nm frozensections Tissue processing for the light microscopy (LM) (making of permanent preparations – slides) •SAMPLING (obtaining of material – cells, tissue pieces) •FIXATION of samples (tissue blocks) •RINSING (washing) of samples •EMBEDDING of samples - embedded blocks •CUTTING of blocks - sections •AFFIXING of sections •STAINING of sections •MOUNTING of sections SAMPLING •A small piece of organ (tissue) is sampled and quickly put into the fixative medium. • •Biopsy during surgical dissection of organs in living organism • = excision • = puncture (liver or kidney parenchyma, bone marrow) • = curettage (uterine endometrium, adenoid vegetation) •Necropsy from dead individual (sections); in experiments laboratory animals are used and tissue have to be sampled as soon as possible after the break of blood circulation • •The specimens shouldn't be more than 5 – 10 mm3 thick and fixation should follow immediately. FIXATION •Definition: denaturation and stabilization of cell proteins with minimum artifacts) • •The reason of fixation: freshly removed tissues are chemically unstable – dry, shrink, undergo hypoxia, autolysis and bacteriological changes • •To stop or prevent these changes and preserve the structure tissue samples have to be fixed. During the fixation, all tissue proteins are converted into inactive denaturized (stable) form. • •3 main requirements on fixatives: • - good preservation of structure • - quick penetration into tissue block • - no negative effects on tissue staining •Fixatives: solutions of different chemicals • - organic fixatives – ALDEHYDES – formaldehyde (most frequently used for LM) • – glutaraldehyde (used for EM) • – ALCOHOLS – 96 – 100 % (absolute) ethylalcohol • – ORGANIC ACIDS – glacial acetic acid, picric acid, • trichloracetic acid • - inorganic fixatives – INORGANIC ACIDS – chromic acid, osmium tetraoxide (OsO4) • – SALTS OF HEAVY METALS – mercuric chloride HgCl2 • - compound fixatives – mixtures (two or more chemical components to offset • undesirable effects fo indiviual (simple) fixatives. • FLEMMING´s fluid – with OsO4 • ZENKER´s and HELLY´s fluid, SUSA fluid – with HgCl2 • BOUIN´s fluid – with picric acid • CARNOY´s fluid – with alcohol • • Performance: fixatives are carried out at room temperature, the duration varies • between 12 – 24 hours, specimen must be covered by 20 – 50 times fixative volume: • Ratio of tissue block volume to fixative volume 1 cm3 : 20 – 50 cm3 RINSING and EMBEDDING •All samples should be washed to remove the excess of fixative; the choice of rinsing medium is determined by type of fixative: running tap-water or 70-80% ethanol • •Relevance of embedding: tissues and organs are brittle and unequal in density, they must be hardened before cutting • Embedding media §water soluble – gelatine, celodal, water soluble waxes §anhydrous – paraffin, celoidin •EMBEDDING into PARAFFIN • •dehydration – to remove water from fixed samples by ascending series of ethanol is used (50%, 70%, 90%, 96%. each step - 2 – 6 hours •clearing – the ethanol must be replaced with organic solvatant that dissolves paraffin – benzene or xylene •infiltration – melted paraffin wax (56°C) is used; 3 x 6 hours. • •casting (blocking out) – moulds (plastic, paper or metal chambers) are used for embedding. - -The moulds are filled with melted paraffin, tissue samples are then placed inside and immediately immersed in cold water to cool paraffin quickly down. -These paraffin blocks are ready for trimming autotechnikon Leica Automated device for tissue dehydration Leica TP 1020 http://www.schuco.co.uk/images%5Cembedding-moulds.jpg http://www.emsdiasum.com/microscopy/products/histology/imaages2/62527__6.jpg Tissue-Tek® Embedding Rings Tissue-Tek® Process/Embedding Cassette http://www.cmhd.ca/enu_mutagenesis/assets/kidney_tissue_block.jpg Paper chambers - metal CUTTING •Microtome – a machine with automatic regulation of section thickness: 5 – 10 μm is optimum. groot_acc_11 rotary sliding microtome – block is fixed in holder, knife or razor moves horizontally rotary microtome – knife is fixed, block holder moves vertically ERM-230L ESM-150S Rotary microtome Sliding microtome fig3b_2 Freezing microtome (cryostat) = rotary microtome housed in freezing box (– 60º C) Cutting of frozen tissue without the embedding Type-Freezing-Microtome botany_histology7 Cutting a slice of mussel Tweezers drop a piece of mussel into a dehydration cassette Several yellow dehydration cassettes lying in a beaker containing (clear) ethanol Shaving thin slices using a microtome Microtome shaving a slice off a wax block A metal basin filled with warm water Slipping a slide under wax floating in a water bath 1 2 3 4 5 6 7 sampling fixation fixation cutting cutting section affixing section affixing oyster_blockribbon_brush image002 http://www.siumed.edu/~dking2/bluehist/epithel3.gif AFFIXING • •Mixture of glycerin and egg albumin or gelatin • •Section are transferred from microtome razor or knife on the level of warm water (45º C), where they are stretched; then they are put on slides coated with adhesive mixture; excess of water is drained and slides are put in incubator (thermostat, 37º C) over night to affixing of sections. HIST005 Medite TFP 40 RTB Stretching of sections on warm water Stretching on a warm plate STAINING •Different cell or tissue structures are not apparent without staining. •Cellular structures exhibit different affinity to staining dyes •alkaline dyes (basic or nuclear) – react with anionic groups of cell and tissue components •basophilia – basophilic structures in the cell • •acid dyes (cytoplasmic) – react with cationic groups • acidophilia – acidophilic structures in the cell • neutrophilia – no reaction Staining methods: routine – HE, AZAN (demonstrate all components of tissue) special visualizes only special structures impregnation by silver salt for detection of nerve or reticular fibers N_UR_KD_06 Masson-Trichrome-g HistologySlidesOriginal004 DSCN0909 HE – the most frequent used method 1144-3i Lipid droplets detected by oil red ROUTINE STAINING with HEMATOXYLINE – EOSIN (HE) •Hematoxyline – basic (nuclear) dye •Eosin – acid (cytoplasmic dye • •Staining procedure: •paraffin must be removed (dissolved) by xylene •sections are rehydrated in descending series of ethanol (100% Ò96% Ò80%) •staining with hematoxyline •differentiation in acid ethanol and water (excess of dye is removed) •staining with eosin •rinsing in water (excess of dye is removed) •dehydration in graded ethanol series (80% Ò96% Ò100%) •clearing in xylene vertical staining jar http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif Xylen I Xylen II 100% 96% H2O hematoxyline acidic ethanol ethanol ethanol Xylen IV xylen III 100% 96% H2O eosin H2O ethanol ethanol HEMATOXYLINE – EOSIN (HE) Deparaffination Rehydration Washing Staining Differentiation Clearing Dehydration Washing Staining Washing Staining results: •HE = Hematoxyline – Eosin • nuclei – bright clear blue or dark violet • cytoplasm and collagen fibers – pink • muscle tissue – red • • •HES = Hematoxyline – Eosin – Safron • connective tissue – yellow • • •AZAN = AZocarmin – ANiline blue – orange G • nuclei – red • erythrocytes – orange • muscle – red • collagen fibers – blue A hand holding a slide with a red-stained sample on it mhe_staining tray_of_mhe_oyster_slides vertical staining jar cuvette http://homer.hsr.ornl.gov/CBPS/Arraytechnology/WashSTD.jpg flask http://www.bio-optica.it/gfx/set%203.jpg slides holder (basket) Staining tools: Automatic slide stainer SHANDON LIPSHAW VARISTIAN 24-4 vertical staining jar staining set of boxes with media MOUNTING •Finally, preparates are closed with coverslip (coverglass) to form a permanent preparate. Small amount of mounting medium must be placed between stained section and the coverslip. • • • • • • • • • • • • • • •Mounting media: soluble in xylene – canada balsam soluble in water – glycerin-gelatine, arabic gum http://www.canemco.com/images5/4470.jpg http://nationaldiagnostics.com/images/h1_7a.gif 275496597_bc08febb4b Hematoxyline and eosin (HE) uri04 Hematoxyline and eosin (HE) N_UR_KD_09 cell cytoplasm cell nuclei collagenous connective tissue Hematoxyline and eosin (HE) • 74 basophilic cytoplasm of glandular cells (contains ribosomes with RNA) acidophilic cytoplasm of epithelial cells Hematoxyline, eosin and saffron (HES) • 109 cartillage Collagenous fibers of connective tissue are yellow after staining with saffron Azocarmine and aniline blue (AZAN) • • aza_005 112 Kidney – collagen connective tissue Impregnation of tissue with silver • • 6 Lien - reticular fibers 197%20cortex Cerebellum – nerve fibers •Visit us at: • •http://www.med.muni.cz/histology