Feulgen Squash Technique PDF

It is a staining technique discovered by Robert Feulgen. It‘s used in cyto-histology to identify chromosomal material or DNA in dividing cells (mitotic cell division) depending on acid hydrolysis of DNA and be visualized with optical microscopy. The mitotic cell division takes place...

It is a staining technique discovered by Robert Feulgen. It‘s used in cyto-histology to identify chromosomal material or DNA in dividing cells (mitotic cell division) depending on acid hydrolysis of DNA and be visualized with optical microscopy. The mitotic cell division takes place in meristematic tissue. In plants, meristematic tissue present in special regions: Shoot apex or Root tips Axillary buds  Principale of Feulgin reaction Demonstration of DNA in cells is done, after the cells are hydrolysed by HCL. By time, aldehyde groups from DNA are released. Feulgen reagent react with this aldehyde group to form magenta color complex product (purple color) which is visible under microscope as stained DNA chromatin. Practical Session  Soaking in tap water Soaked seeds in irrigated sand or sawdust 24 hr Dry broad bean (uniform in size and shape) Why sand or sawdust? for 3 days Germinated seeds with straight roots (2 cm long) were root tips (0.5-1 cm) were cut off collected The collected root tips should immediately transfer in fixative solution (acetic acid : alcohol = 1 : 3 ) for 24 hrs, then store them in 70% ethanol in refrigerator. Why do this step? Acetic acid : alcohol 1 : 3 Ø The combination of these two chemicals leaves the chromosomes in a highly precipitated form suitable for staining and microscopic study by preserving their structural and/or chemical components i.e. stop the cell division and growth.  Wash with distilled water To remove excess fixative Root tips HCL with 1N (1N) HCL Wash with distilled water Water bath at 60ºC for 6,7,8 min Transfere the hydrolyzed roots to dry test tube  Why Hydrolysis step is very important Step? Optimum hydrolysis Less hydrolysis Release aldehyde gp (CHO) from the Excess hydrolysis Cause less liberated deoxypentose sugar of DNA which in Cause destructive effect aldehydic groups. turn, react with the staining reagent on the structure of the giving rise to the max. results of the nucleic acid. purple dye.  Remove the root tip from stain &transfer it to a slide 2ml feulgen reagent For 30 min Squash the root tip with the pencil eraser and add Examin under 1-2 drops of 45% acetic microscope acid  Results Under Microscope  Stages of Mitosis 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase Illustrate your results with drawing with full labels