Cytology Lab.1 تربية خاص PDF

Cell Biology Practical Session One By: Rehab Nady The light microscope allows us to magnify cells up to 1000 times and to resolve details as small as 0.2 μm.  Inverted Microscope A microscope with its light source and condenser on the top (above the stage), while the...

Cell Biology Practical Session One By: Rehab Nady The light microscope allows us to magnify cells up to 1000 times and to resolve details as small as 0.2 μm.  Inverted Microscope A microscope with its light source and condenser on the top (above the stage), while the objectives are below the stage.  Stereo Microscope (Dissecting or Low Power Microscope) Used to view specimens visible to the naked eye such as insects.  Phase-Contrast Microscope Used for observing transparent specimens that have not been stained and are in their natural states such as living cells (usually in culture), microorganisms, thin tissue slices, fibers, and subcellular particles (including nuclei and other organelles). The same unstained, living animal cell in culture viewed with: (A) light microscope (B) phase-contrast.  http://www.finddiagnostics.org/export/sites/default/programs/tb/images/m_tuberculosis_bacteria.jpg Fluorescent microscope Xenon arc lamp or Mercury-vapor lamp* (UV generating lamps) Power control box AP Biology  Confocal Microscope AP Biology  An electron source (cathode, electrongun) such as a heated tungsten filament, emits electrons. The electrons are attracted toward an anode. An electrical difference between the cathode and the anode imparts an accelerating voltage of between 20,000 and 200,000 volts to the electrons, creating the Electron beam. The beam then passes through a series of electromagnetic lenses that serve the same function as the glass lenses of a light microscope. TEM SEM SEM TEM  Sample Preparation for Transmission Electron Microscopy 1- Fixation ❖ Prevent autolysis preservation of sub- cellular structures. ❖Primary fixation with an aldehyde (usually glutaraldehyde 2–3%, for 3–6 hours) to stabilize proteins, followed by secondary fixation in 1–2% aqueous osmium tetroxide (OsO4 for 1–2 hours) to retain lipids. AP Biology 2- Washing - 0.1M phosphate buffer (Sorensen’s phosphate buffer). 3- Dehydration - By ascending series of ethanol. 4- Infiltration - Propylene oxide (1,2-epoxypropane) as a transition solvent facilitates resin infiltration. 5- Embedding - Epoxy resin is used as the embedding medium in TEM.  6- Sectioning Block trimming Removal of excess resin to expose the tissue for sectioning. Semi-thin sections - Commonly, semi-thin sections are cut at 0.5–1 μm using the ultra- microtome and a glass knife. - Sections are stained by 1% toluidine blue. Ultra-thin sections Between 60–90 nm from using the ultramicrotome and a Diamond knife. Diamond knife Glass knife Diamond knives Ultra-microtome  7- Staining Uranyl acetate (2%-5%) Combines with phosphate groups in nucleic acids and phosphate and carboxyl groups on the cell membrane (Hayat 2000). Lead citrate stain Increase the contrast of tissue components (Stains protein and glycogen). 1.33 g of lead nitrate, 1.76 g of sodium citrate, and 30 mL of distilled water are mixed. 8 ml of 1N NaOH. Distilled water is added so that the whole quantity is 50 ml. REFERENCES 1- PREPARATION OF BIOLOGICAL SPECIMENS FOR ELECTRON MICROSCOPY (JEOL). 2- SUVARNA, S. K., LAYTON, C., AND BANCROFT, J. D. (7TH ED), BANCROFT'S THEORY AND PRACTICE OF HISTOLOGICAL TECHNIQUES. LONDON, UK, CHURCHILL LIVINGSTONE ELSEVIER. 3- WWW.OLYMPUSMICRO.COM/PRIMER/TECHNIQUES/CONFOCAL/CONFOCALINTRO.HTML 4- WWW.CYTO.PURDUE.EDU/CDROMS/MICRO1/10_EDU/524LEC4/SLD006.HTM 5- HTTP://WWW.SFB1064.MED.UNI-MUENCHEN.DE/IRTG/IRTG-DOWNLOADS/LIGHT- SHEET-SPINNING-DISK.PDF 6- HTTPS://WWW.SCIENTIFICA.UK.COM/LEARNING-ZONE/A-GUIDE-TO-PHASE-CONTRAST