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ISC 11> PRACTICAL

Aim : To study the mitotic cell division from onion root tip by preparing temporary wet mount of root tip squash
Materials:

Onion,Razor blades,Microscope slides,Safety glasses,Gloves,Paper towel,Fixative (1:3 Glacial acetic acid : ethanol),.5% toluidine blue in Mcilvaine buffer solution pH 3 to 4,Coverslips,Microscope,Glycerin,Lab coat
Procedure:

  1. Obtain an onion bulb that is just beginning to show the emergence of roots. Cut off a root and lay it on a microscope slide.  Cut off the first approx 1cm of the root tip. Discard the rest of the root.  Mitotic cells are in the tip, so extra root tissue will only interfere with finding mitotic cells. 
     2.   Wear safety glasses and gloves and lab coat.
     3.   Cover the root tip with fixative solution; Let the slide stand for 30 minutes to one hour. After some time of placing the tip in fixative solution you will              see some part of root become transparent or translucent. Remove that cleared (transparent) part of the root by cutting it off as that part is the                   region where cells are elongated (Do not show mitotic cell division).Now you would have a very small piece of root tip which may look pale white or             dirty white in color (a dot-sized piece of root tip is all you need).
     4.  Carefully blot around the root to remove excess fixative.  Add one drop of fresh toluidine blue stain to the slide let the root tip be stained for 2-3                   minutes; after that remove or drain the excess stain (you can even use blotting paper for removing the stain).
     5.  Put one drop of glycerin on the stained root tip and then place cover slip over that.  Place the slide between two layers of paper towel on your                       laboratory bench. Using your finger apply gentle pressure to the cover slip in order to squash and spread the root tip tissue. 
       CAUTION: The applied pressure should be such that it does not break the cover slip.
    6.   Using your microscope (10x), locate the meristematic region of the root tip.  Examine the slide at 40x magnification and identify chromosomes at the             various stages of mitosis.
    7.   Locate cells in prophase, metaphase, anaphase, telophase, and interphase.  Look for evidence of cytokinesis.  If the slide is not satisfactory, repeat               the procedure.  Click the photographs of cells under microscope showing  cell division using your smart phone or other suitable devic
    8.   Paste the photographs of these stages of mitosis below and tag them with the name of the stage and write down the magnification of picture.
          Stage of Mitosis: _______________________                                          Total Magnification:  _______________________                                        

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