Lab  5
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Lab 5






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Lab  5 Lab 5 Document Transcript

  • November 17, 2013 PRACTICAL CELL BIOLOGY LAB 5 Routine Transmission Electron Microscopy (TEM) 9- Sectioning Is the process of making sections (thick, semithin and thin section), for plastic block a special instrument used to make a section which called ultramicrotome. Glass knife is a disposable knife which can be made by glass knife maker, while diamond knife also can be used which it is permanent and can be sharpened. Sections Thick section:- (1-2 micrometer) for light microscope before making thin section for electron microscope. Semithin section:-(0.1-1 micrometer) for light and electron microscope. Ultrathin section:- (<0.1micrometer) for electron microscope, (600-900) A˚ thickness of the section is the best one for transmission electron microscope.
  • November 17, 2013 PRACTICAL CELL BIOLOGY A sample block and a knife are set on a microtome. LAB 5
  • November 17, 2013 PRACTICAL CELL BIOLOGY LAB 5
  • November 17, 2013 PRACTICAL CELL BIOLOGY LAB 5
  • November 17, 2013 PRACTICAL CELL BIOLOGY LAB 5 10- Staining For light microscopy, one micrometer thick section were stained with 1% toluidine blue in 1% borax while for electron microscopy, the plastic block were cut into 600-900 Aº thin sections. The ultrathin sections were mounted on copper grids and stained by uranyl acetate and lead citrate and examined by transmission electron microscope.
  • November 17, 2013 PRACTICAL CELL BIOLOGY LAB 5 Toluidine blue for light microscopy Put a drop of distil water on a clean slide. Put a loop within distil water to make the membrane. Pick up the thick section 1micrometer by loop. Put sections on the droop water on slide. Allow to dry in the air or by using hoot plate. Cover with toluidine blue for half minute. Wash gently with distil water. Dry and examine with light microscope. It can also facilitate the trimming of unwanted material from the block before ultrathin sectioning. Staining for electron microscopy Place a piece of dental wax in the petridish. Put a drop of uranyl acetate on dental wax. Hold the grid gently and pick up the thin section 600-900 Aº . Put the grid which holds the section in the uranyl acetate for 15 min. Wash gently by using boiled distil water. Transfer the grid into a drop of lead citrate on dental wax in the petridish and leave it for 5 min. Wash and examine by transmission electron microscope. Note:- CO2 contamination may occur Source of CO2 and prevention from contamination 1. Air :- put KOH around the drop of lead citrate 2. Worker: -don’t speak and avoid direct breath upon the working area. 3. Water: -boil the water and the use.