Biotechnique (BIOL 410) Histology

Fixation and Histology 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

Fixatives Prevent decay Decay is a natural process Bacteria, and Fungi degrade organic molecules in deceased organisms Fixatives remove decomposers Modify molecules

Fixatives Formaldhyde/Formalin Alcohol Ice Dessicants (e.g. Salt)

Formaldehyde Formalin (10 % Formaldehyde) Most commonly used preservative Osmium tetroxide (Alt) Crosslink molecules, creating additional covalent bonds Stabilize membrane proteins Dangerous for living tissue

Lenin’s Tomb Samples can last a very long time Lenin’s Mausoleum Vladimir Lenin 1870 – 1924 (dead 91 Years!)

Formaldehyde Tissue looses pigment over time. Does not bind smaller pigments, which continue to break down.

Alcohol Ethanol, Methanol and Isoproponyl are commonly used preservatives Dehydrate tissue Removal of water prevents decay Some structural change to the resulting molecule/organism.

Alcohol Loss of pigment Molecules not cross-linked Protein Staining/Detection DNA preservation

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

Desiccants Similar to alcohol remove water Mummification Alcohol replaces water Desiccation - air replaces water Mummification Not helpful for examining animal tissue, but very useful in Botany Etamology

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

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

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

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

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

Histology Once we have effectively preserved specimen we can observe and study them Dissection Thin section Tissue digestion/removal

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

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

Microtome Similar principle using in MRI of live tissue 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

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

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

Today’s Lab Prep samples for Alizerin Red staining Observe stained specimen Cleared and stained Mounted In Vitro Embryo Staining Vital Staining E. coli Yeast Human Cheek Elodea Gram Staining Bacteria