Histotechnology 2023 with complete solution

Fixation First and most critical step Primary purpose of Fixation is: preserve the morphological and chemical integrity of the cell Secondary purpose of Fixation is: harden and protect the tissue , thus easier to cut Size of tissue to be processed ideal size is 3mm Amount of fixative 10-20 times volume of specimen (except Osmium tetroxide) Ideal ratio of tissue:fixative 1:10 Hydrogen ion concentration pH 6.0-8.0 Temperature for routine RT Temperature for electron microscopy and histochemistry 0-4°C Osmolality isotonic solution MICROANATOMICAL general microscopic study CYTOLOGICAL preserve particular microscopic elements of the cell Nuclear contain glacial acetic acid pH 4.6 or less Cytoplasmic does not contain glacial acetic acid pH 4.6 or above Histochemical preserve chemical constituents of cells and tissues Glutaraldehyde For light microscopy and electron microscopy Formaldehyde Satisfactory for routine paraffin sections Most common, widely used 10% recommended percentage of formaldehyde 10% Formol saline Recommended for CNS tissues and post-mortem tissues for histochemical exam 10% neutral buffered formalin (NBF) preservation and storage of surgical, post mortem and research specimens Formol Corrosive (Formol Sublimate) Recommended for routine post-mortem tissues Formol-Calcium Best fixative for phospholipids Unbuffered Zinc Formalin alternatives to mercuric chloride formulations Alcoholic Formalin (Gendre's) Can be used to fix sputum Kardasewitch's Method Place in mixture of 70% ETOH and 28% Ammonia water for 5mins-3hrs Lilie's Method Place in mixture of Acetone, H2O2, 28% Ammonia water for 1-5 mins Wash with 70% ETOH Picric Acid Method Place in saturated alcoholic picric acid for 5mins-2hrs Wash in running water for 10-15mins 2.5% Glutaraldehyde needle biopsies 2 to 4 hours at RT 4% Glutaraldehyde larger tissues 6 to 24 hours at RT Mercuric chloride Most common metallic fixative Mercuric chloride produces black precipitates of mercury, remedy: remove by 0.5% iodine solution in 70% ethanol then decolorize iodine using absolute alcohol Zenker's Fluid Recommended for liver, spleen, CT fibers, nuclei recommended for trichrome staining Zenker-Formol/Helly's Solution Excellent micro anatomic fixative for pituitary, BM, spleen, liver; Zenker-Formol produces brown pigment if fixed more than 24 hrs, remedy: remove by saturated picric acid or NaOH Heidenhain's Susa for skin biopsies Heidenhain's Susa produces black precipitate, remedy: immerse in alcoholic iodine Schaudinn's Fluid/Sublimated Alcohol for making smears of loose cells on slides B-5 Fixative for hematopoietic and lymphoid tissue 1-2% Chromic Acid preserves CHO Potassium Dichromate preserves lipids, mitochondria Regaud's/Moller's Fluid for chromatin, mitochondria, Golgi, RBC, colloid, mitotic figures; slow, not for fats Orth's Fluid for Rickettsia Recommended for early degenerative process and tissue necrosis Lead fixatives For acid MPS Bouin's for embyros, glycogen Brasil's Alcoholic Picroformol good for glycogen Hollande's Fixative for GIT specimens and endocrine tissues Alcoholic fixatives both fixative and dehydrating agent Methanol for BM/blood smears Ethanol strong reducing agent Carnoy's Fluid most rapid Alcoholic Formalin/Gendre's Fixative sputum Newcomer's Fluid for MPS Clarke's Solution for frozen section and smear Flemming's Solution Nuclear fixative - Flemming's Solution without Acetic Acid - Champy's Fluid Cytoplasmic Fixatives glacial acetic acid Solidifies at 17ºC For nucleoproteins, chromosomes Trichloroacetic acid (TCA) Weak decalcifying agent Acetone Fixes brain - for rabies For enzyme studies Secondary Fixation (Post-mordanting) process of placing a fixed tissue in a second fixative Post-chromatization form of secondary fixation which utilizes 2.5% to 3% potassium dichromate as mordant Freeze-drying Preserving tissue by rapid freezing (quenching) and removing water (dessication) by a physical process, without the use of any chemical fixative Freeze substitution fixed in Rossman's fluid or Osmium tetroxide in 1% acetone for 1-6 days at temperature of -60°c to -70°c and dehydrated in 70% absolute alcoho