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Biotechnique (BIOL 410) Histology
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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
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Fixatives Prevent decay Decay is a natural process
Bacteria, and Fungi degrade organic molecules in deceased organisms Fixatives remove decomposers Modify molecules
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Fixatives Formaldhyde/Formalin Alcohol Ice Dessicants (e.g. Salt)
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Formaldehyde Formalin (10 % Formaldehyde)
Most commonly used preservative Osmium tetroxide (Alt) Crosslink molecules, creating additional covalent bonds Stabilize membrane proteins Dangerous for living tissue
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Lenin’s Tomb Samples can last a very long time Lenin’s Mausoleum
Vladimir Lenin 1870 – 1924 (dead 91 Years!)
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Formaldehyde Tissue looses pigment over time.
Does not bind smaller pigments, which continue to break down.
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Alcohol Ethanol, Methanol and Isoproponyl are commonly used preservatives Dehydrate tissue Removal of water prevents decay Some structural change to the resulting molecule/organism.
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Alcohol Loss of pigment Molecules not cross-linked
Protein Staining/Detection DNA preservation
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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
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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
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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
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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
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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
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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
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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
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Histology Once we have effectively preserved specimen we can observe and study them Dissection Thin section Tissue digestion/removal
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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
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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
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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
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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
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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
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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
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