Transcript Fixation
Biotechnique (BIOL 410) Histology Fixation and Histology Fixation • Process by which biological samples are preserved – Retaining many of the natural characteristics • Preventing Decay Staining • Using pigments to enhance features Histology • Study of preserved specimen 2 Fixatives • Prevent decay • Decay is a natural process – Bacteria, and Fungi degrade organic molecules in deceased organisms • Fixatives remove decomposers – Modify molecules 3 Fixatives • Formaldhyde/Formalin • Alcohol • Ice • Dessicants (e.g. Salt) 4 Formaldehyde • Formalin (10 % Formaldehyde) – Most commonly used preservative – Osmium tetroxide (Alt) • Crosslink molecules, creating additional covalent bonds • Stabilize membrane proteins • Dangerous for living tissue 5 6 Lenin’s Tomb • Samples can last a very long time • Lenin’s Mausoleum – Vladimir Lenin 1870 – 1924 (dead 91 Years!) 7 Formaldehyde • Tissue looses pigment over time. – Does not bind smaller pigments, which continue to break down. 8 Alcohol • Ethanol, Methanol and Isoproponyl are commonly used preservatives • Dehydrate tissue – Removal of water prevents decay • Some structural change to the resulting molecule/organism. 9 Alcohol • Loss of pigment • Molecules not crosslinked • Protein Staining/Detection • DNA preservation 10 Ice • Freezing will stabilize water molecules, preventing decay • However freezing expands water, which can damage cells – But now while frozen in place – Thawing • Repeated freeze-thaw events will degrade tissue rapidly. – Dehydration 11 12 13 Desiccants • Similar to alcohol remove water – Alcohol replaces water – Desiccation - air replaces water • Mummification • Not helpful for examining animal tissue, but very useful in – Botany – Etamology 14 A History of Preservation • Ancient Egyptians practiced fixation of remains – Ensuring they passed on to the afterlife • Removed vital organs – Sealed in jars with Alcohol • Desiccant wrapped bodies – Salts 15 16 Staining • Using chemicals that bind to molecules in specific tissue to enhance view. • Common Stains for this course – – – – – – – Eosin Toluidine Blue Orcein or Acid-Orecin Methylene Blue Neutral Red Nile Blue Alizarin Red 17 Staining • Protocol for exposure to stain – Allows it to bind • Washes used to remove excess stain – Remove stain from surrounding tissue • Vital Stains can be used on living tissue – Very low concentration, not to mildly toxic to cells 18 Gram Staining • Used to identify types of bacterial • Two major Taxonomic Groups – Gram Positive & Gram Negative – Gram (-) have additional extra cellular layers • Common detection method in research & medicine 19 20 Hans Christian Gram • Developed the technique in 1884 while working in the Berlin City Hospital Morgue • Originally developed to make bacterial more visible in lung tissue. – But only worked on some bacteria • Gram Positive bacteria bind the violet stain – Gram negative only bind the counterstain, and appear Pink 21 Histology • Once we have effectively preserved specimen we can observe and study them • Dissection • Thin section • Tissue digestion/removal 22 Dissection • Dissection allows you to remove specific tissue for investigation • Or to observe internal tissue more closely – Including stained tissue, which can be further observed 23 Thin Section • Takes micrometers thick sections of tissue for observation – Cell structure – Overall structure • Including stained samples, allowing for identification of specific cells within the larger structure 24 Microtome • Machine for cutting thin sections. – Very sharp blade • Requires sample be fixed and mounted for successful sectioning – Usually in parafin wax or frozen • Similar principle using in MRI of live tissue – Lower resolution 25 Microtome • First microtome developed in 1770 • Designed refined during the 1800 to create cleaner samples • Late 20th century gave us higher quality blades capable of even thinner samples – Ultramicrotome for Electron Microscopy 26 27 Tissue removal • Often you want to observe a subset of tissue in place, and need to remove surrounding tissue – Articulated skeleton • Scavengers • Enzyme digestion R. C. Albertson 28 Today’s Lab 1. Prep samples for Alizerin Red staining 2. Observe stained specimen – Cleared and stained – Mounted 3. In Vitro Embryo Staining 4. Vital Staining – – – – E. coli Yeast Human Cheek Elodea 5. Gram Staining Bacteria 29