ANAT 261 Study Guide- Theory.docx

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McGill University
Anatomy & Cell Biology
ANAT 261
Craig A.Mandato

ANAT 261 STUDY GUIDE THEORY HISTOLOGICAL METHOD What is Dynamic HistologyMedical Histology identifying tissues and cellsDynamic Histology is an extension where we not only identify the tissues but we examine functions of the cells and molecules that make up that tissueSystemic Human Anatomy macroscopic level eyes onlyDynamic Histology microscopic level sections and scopes Structure Type of Structure Type of Basic Tissue Skin Organ Epithelial and Connective Fascia Tissue Connective Tissue Tendons Tissue Connective Tissue Aponeurosis Tissue Connective Tissue Muscle OrganTissue Muscular Tissue Bone OrganTissue Variety of CT Blood Vessels OrgansTissues Epithelial CT Muscular Nerves TissueOrganSystem Nervous Tissue CHAPTER 1 OF THE TEXT BOOK REVIEW Plane of section is something that is very important in this courseSections can be in cross section oblique section or in longitudinal sectionImportant measurements o 1 cm10 mm o 1 mm1000 micrometers o 1 micrometer1000 nm o 1 nm10 AWhat does this mean o Thickness of a section5 micrometers o Diameter of a cell10 micrometers o Diameter of a lysosome200 nm o Thickness of a PM75 A 75 nm Preparing a tissue section MC HINTThe left side of the picture is for light microscopy while the right side is for EM1 CHEMICAL FIXATION o There are two ways to fix the tissueImmersion anaesthetize the animal and take a section of tissue The tissue gets fixed and the chemicals must fix the tissue from the outside in The cells around the tissue fix right away and look good but as the fixative moves in you are fighting the clock against death to get a good fix Diffusion rate36 mmhourPerfusion inject the animal with the fix and fixation occurs from the inside out This is the preferred way of fixation 1Alternatively you can place the tissue in liquid nitrogen this is FREEZING o Light microscopy Perfusion ImmersionFormalin formaldehydewaterMercuric Chloride poisonousPicric Acid very explosiveBouins Fluid acqueous less dangerousAcetic acid picric acid formalin formaldehyde water o Electron microscopy preferable perfusionGlutaraldehyde 255 better than Bouins fluid needs to be since we are looking at subcellular structure2 DEHYDRATION ethanol o The sample is in fixative which is 100 water You put it into 50 ethanol then 60 all the way until 100 At this point the sample is completely dehydrated o Since ethanol dissolves fat this causes empty spaces in place of fat cells3 CLEARING AND INFILTRATION o For use of the LM clearing occurs with xylene in paraffin Paraffin Solvent while for EM you use propylene glycol in epoxy Epoxy Solvent To do this you dip the tissue into the solvent paraffin or epoxy and the xylene or propylene glycol will rush into the dehydrated sections going where the water was4 EMBEDDINGo For the LM you pump more paraffin in pushing the xylene out until you are in 100 paraffin wax For the EM you pump in plastico Then you harden it by cooling it down5 SECTIONING o For the LM you can use a microtome for the wax block or a cryostat for a frozen section o For the EM you use a diamondedge knife ultramicrotome o It is almost impossible to get a perfect cross section6 MOUNTING o For LM you use a slide and for EM you use a grid7 REMOVAL OF PARAFFIN o You need to remove the paraffin in the case of a LM because the stain is waterbased Adding xylene will make a paraffinxylene mixture8 REHYDRATION o Now we need to get rid of the xylene because it does not mix well with water Again this is only in the case of LM Using 100 ethanolwater ETOHHO add it to the 2mixture then remove it and add 90 then 80 etc until it is all water9 STAINING o The stain used for LM is HE hematoxylin and eosin andor histochemical reaction and the stain used for EM is lead citrate andor histochemical reaction10 MICROSCOPY 2StainingThe most common stain used in histology is HE o Basic dye hematoxylin stains basophilic structures with bluepurple hue and stains cell nucleus and other acidic structures o Acidic dye eosin colours eosinic structures bright pink it stains cytoplasm and collagenTerms o Basophilic structures are usually the ones containing nucleic acids such as the ribosomes and the chromatinrich cell nucleus and the cytoplasmatic regions rich in RNA o Eosinophilic structures are generally composed of intracellular or extracellular protein Most of the cytoplasm is eosinophilic NOTE Structures do not have to be acidic or basic to be called basophilic or eosinophilic The terminology is based on affinity to the dyesFor the LM you have a light source at the bottom Some of the light is collected and passes through the condenser which will focus the light on the sample The light hits the specimen bounces up crosses and goes through the objective It passes through and goes through another objective called the ocular This puts it into the right orientation and focuses the light so that it hits your eye The EMs source is coming from a cathode ray shooting electrons The specimen is bombarded with electrons which culminate on the condenser coil and then hit the sample They go through the sample and hit the back focal plane Then they go through the condenser coil and hit the 3
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